Title of Invention

ANTIBODIES FOR DETECTING MICROORGANISMS

Abstract An antibody for detection of microorganisms, a method of detection of microorganisms, and a reagent kit for detection of microorganisms, which is species specific for every species of microorganisms and with which all serotypes within the same species can be detected, are provided. Antibody to intracellular molecules with the same function in each type of microorganism, particularly antibody to ribosomal protein, that is, ribosomal protein L7/L12, is made and antibody that reacts specifically with the microorganism in question is selected.
Full Text DESCRIPTION


DESCRIPTION OF BACKGROUND ART
Field of the Invention
The present invention pertains to antibodies
useful in the detection of various microorganisms,
particularly bacteria, and a method of detecting
microorganisms, reagent kits for detection of
microorganisms that use said antibodies, and a method
for preparing specific antibodies for detecting
microorganisms.
Moreover, the present invention is valuable to
the drug industry, particularly for diagnostic
medicine of microbial infections with an emphasis on
bacteria.
Prior Art
Diagnosis of microbial infection is confirmed by
detection of the causative pathogen form the infected
area or by detection of antibodies to the causative
pathogen in serum and body fluids. Detection of the
causative pathogen is particularly important in the
sense that it makes quick treatment to the patient
possible.
Detection of the causative pathogen of infections
can be generally classified as cultivation and
identification methods, whereby the causative
pathogen is separated and cultivated and then
identified based on its biochemical properties;
genetic diagnosis, whereby amplification by PCR,
etc., is performed based on specific genes of the
causative pathogen and thus the causative pathogen is
detected; or immunological methods, whereby the
causative pathogen is detected using a specific
reaction of antibody with surface antigen marker of
the causative pathogen. However, it takes time to
obtain results by cultivation and identification
methods or genetic diagnosis methods. Therefore,
diagnosis by immunological methods is commonly used
because the causative pathogen can be detected within
a short time with high sensitivity and the patient
can be quickly and appropriately treated.
Depending on the bacterial species, a variety of
marker antigens and antibodies can be used alone or,
in combination to detect the causative patho-gen of
infections by conventional immunological methods.
For instance, it is known that lipopolysaccharide
(LPS), which is a genus-specific antigen of
Chlamydia, is present as an antigen determinant
(Stephens, R., et al.: J. Immunol., 128:1033-89,
1982, Caldwell, M.D.: Inf. Immun. , 44:306-14, 1984),
and antibodies to LPS are used as the reagent
antibody in a variety of diagnostic kits,
particularly for the detection of Chlamydia
trachomatis.
Moreover, Ellena M. Peterson et al., (Infection
and Immunity, 59(11), 4147-4153, 1991) and Byron E.
Batteiger et al. , (Infection and Immunity, 53(3) ,
530-533, 1986) have reported monoclonal antibodies to
major outer membrane protein (MOMP) of the genus
Chlamydia respectively.
Publication of unexamined Japanese patent
application No. 298/1988 discusses an immunodetection
method based on the western blot method that uses a
monoclonal antibody to an approximately 43 kilo
Dalton membrane protein antigen of Mycoplasma
pneumoniae.
Moreover, a method of preparing monoclonal
antibody to Haemophilus influenzae and a diagnostic
method that uses said antibody are presented in
Publication of unexamined Japanese patent application
No. 148859/1987 (Japanese Patent No. 64065/1994) .
Proteins of approximately 20 kilo Dalton isolated
from the sodium cholate extract of the outer membrane
vesicle of Neisseria gonorrhoeae strain BS4 (NCTC
11922)are documented and preparation of hybridomas
using said substance is disclosed in British Patent
Application No. 2,172,704. Moreover, European Patent
Document no., EP 419238 Al describes preparation of a
monoclonal antibody, which can bind to a protein of
approximately 14 kilo Dalton prepared by using
Neisseria gonorrhoeae as an immunogen and a method
for the preparation of such a monoclonal antibody.
Additionally, a method of detecting the same
Neisseria gonorrhoeae using monoclonal antibody to
lipopolysaccharide (LPS) is mentioned in Canadian
Patent Application No. 1,220,147.
However, there are problems with said antibodies
and detection methods based on those antibodies in
that species specificity to microorganisms is
insufficient for proper diagnosis. The antibodies do
not d-etect all serum types as plural surface antigens
present within one species.
Marker antigens used in these prior arts are not
standardized so that microorganisms can be detected
using a same functional molecule (for instance,
protein, LPS or surface polysaccharide component with
the same function) which is generally present in
cells of various microorganisms and which changes
during the course of evolution of the microorganisms
as a marker, and an immunodiagnostic method based on
the concept of detecting the difference in
antigenicity between bacterial species using one
molecule as a standard is not known.
DISCLOSURE OF THE INVENTION
The present invention strives to present
antibodies to the same molecule for different
microorganisms as a standard marker antigen to make
ideal microorganism detection and immunodiagnosis
possible, particularly antibodies -to the molecular
segment in the same intracellular functional
component molecule for all microorganisms to be
detected that changes during the course of evolution
of microorganisms, a method of detecting
microorganisms, which is species specific and can
cover almost all serum types, a reagent kit for
detection of microorganisms that use said antibody,
and a method for preparing a specific antibody used
for detection of microorganisms.
The present inventors discovered a protein with
the same function, preserved in all microorganisms as
a useful antigen protein. Usually, it is expected
that a structural change of such a protein is
extremely small. However, surprisingly it was found
that the antigen epitope(s) of this protein has
specificity to certain species or genus of
microorganisms, and that the antibody for this
protein not only has potentialities of being used for
specifically identifying species or genus of
microorganisms, but also is capable of detecting all
serotypes of the target microorganisms.
»
The inventors focused on intracellular molecule
that are present in all microbial cells and differ
between microorganisms in terms of its amino acid
sequence, particularly ribosomal protein L7/L12,
which is a member of ribosomal proteins . Ribosomal
protein L7/L12 is a protein with a molecular weight
of approximately 13 kilo Daltons and is known to
exist as a ribosomal protein essential in protein
synthesis. Progress has been made in understanding
the complete amino acid sequence of the protein from
several microorganisms including particularly
Escherichia coli and Baccillus subtilus, etc., and 50
% to 65 % homology of the amino acid sequence between
the microorganisms has been confirmed.
The inventors focused on the fact that even
though there are similarities in said molecule
between different microorganisms., this molecule also
has a structural segment that is unique to each
microorganism and discovered that it is possible to
detect various microorganisms with species
specificity and to detect all serotypes within the
same species by using antibody to the protein. As a
result of attempting to develop a technology for
immuno-diagnosis of microorganism species using
antibody specific to, for instance, Haemophilus
influenzae, Streptococcus pneumoniae, and Neisseria
gonorrhoeae, the inventors completed the present
invention upon discovering that antibody specific to
said protein of each species of microorganisms can be
obtained and species-specific detection of different
bacteria is possible using said antibody.
The present invention relates to an antibody used
for detecting microorganisms, a method of detecting;
microorganisms using the antibody, a reagent kit for
detecting microorganisms using the antibody, and a
method for preparing specific antibodies for
detecting microorganisms.
1) Antibodies which are antibodies to ribosomal
protein of microorganisms and which react
specifically with said microorganisms.
2) The antibodies described in 1) above, where
the ribosomal protein of the microorganisms is
ribosomal protein L7/L12.
3) The antibodies described in 1) or 2) above,
where said microorganisms are microorganisms, which
cause a sexually transmitted disease (STD).
4) The antibodies described in 1) or 2) above,
where said microorganisms are microorganisms which
cause- respiratory tract infection.
5) The antibody described in 4) above, where the
causative microorganisms of respiratory tract
infection are microorganisms of Haemophilus
influenzae.
6) The antibody described in 4) above, where the
causative microorganisms of respiratory tract
infection are microorganisms of Streptococcus
pneumoniae.
7) The antibody described in 3) above, where the
causative microorganisms of sexually transmitted
diseases (STD) are microorganisms of Neisseria
gonorrhoeae.
8) The antibody described in 7) above, which is
the antibody to ribosomal protein L7/L12 of Neisseria
gonorrhoeae and which recognizes a continuous amino
acid sequence moiety from 5 to 30 amino acids
including the 115th alanine in the amino acid
sequence of Sequence ID No. 22 of the Sequence Table.
9) A method of detecting microorganisms, which is
characterized by the fact that antibody to
intracellular molecules that have the same function
for a variety of microorganisms is used.
10) A method of detecting microorganisms, which
is characterized by the fact that any antibody
described in 1) to 8) above is used.
11) A reagent kit for detecting microorganisms,
which is characterized: by the fact that antibody to
irrtracellnlar molecules that have the same function
for a variety of microorganisms is used.
12) A reagent kit for detecting microorganisms,
which is characterized by the fact that any antibody
described in 1) to 8) above is used.
13) A method of preparing any antibody described
in 1) to 8) above, characterized by the fact that
ribosomal protein L7/L12 of microorganisms obtained
by a gene manipulation procedure or by isolation from
microorganisms, peptide moiety thereof, or a
synthesized peptide corresponding to the peptide
moiety is used as an immunogen.
The present invention will now be explained in
detail.
Sequences No. 1 and No. 2 in the Sequence Table
are the DNA sequence of the ribosomal protein L7/L12
gene of Haemophilus influenzae and corresponding
amino acid sequence. Sequences No. 3 and No. 4 are
the DNA sequence of the ribosomal protein L7/L12 gene
of Helicobacter pylori and the corresponding amino
acid sequence. Sequences No. 5 and No. 6 show the DNA
sequence and the corresponding amino acid sequence of
the ribosomal protein L7/L12 gene of Streptococcus
pneumoniae. Sequences No. 7. and No. 8 show the DNA
sequence and the corresponding amino acid sequence of
the ribosomal protein L7/L12. gene of Neisseria
gonorrhoeae. Sequence No 9 and No. 10 show the DNA
sequence and the corresponding amino acid sequence of
the ribosomal protein L7/L12 gene of Neisseria
meningitidis. Sequence No. 11 and Sequence No. 12 in
the Sequence Table are the primers DNA for PCR used
to acquire the ribosomal protein L7/L12 gene from
Haemophilus influenzae. Sequences No. 13 and No. 14
in the Sequence Table are the primer DNA for PCR used
to acquire the ribosomal protein L7/L12 gene from
Streptococcus pneumonias. Sequences No. 15 and No.
16 in the Sequence Table are the primer DNA for PCR
used to acquire the ribosomal protein L7/L12 gene
from Neisseria gonorrhoeae. Sequences No. 17 and No.
18 show the DNA sequence and the corresponding amino
acid sequence of the ribosomal protein L7/L12 gene of
Haemophilus influenzae. Sequences No. 19 and No. 2 0
show the DNA sequence and the corresponding amino
acid sequence of the ribosomal protein L7/L12 gene of
Streptococcus pneumoniae. Sequences No. 21 and No. 22
show the DNA sequence and the corresponding amino
acid sequence of the ribosomal protein L7/L12 gene of
Neisseria gonorrhoeae.
Furthermore, the left and right terminals of the
amino acid sequences entered in the Sequence Table
are amino group (referred to below as the N terminal)
and carboxyl group terminals (referred to below as
the C terminal), respectively, and the left terminal
and right terminal of the base sequence is the 5 '
terminal and the 3' terminal, respectively.
Moreover, the series of biomolecular experiments
of gene preparation mentioned in this text can be
performed by methods entered in standard experimental
manuals. "Molecular cloning: A laboratory manual,"
Cold Spring Harbor Laboratory Press, Sambrook, J. et
al. (1989), is an example of the aforementioned
standard experimental manual.
The term microorganism in "the present invention
refers to all species of microorganisms , including
bacteria, yeast, mold, Actinomyces, rickettsia, etc.,
but bacteria in particular, pose a problem in terms
of diagnosis of microbial infections.
The term "antibody which reacts specifically with
microorganisms" as used in the present invention
means an antibody, which reacts specifically with a
species or group of microorganisms. An antibody,
which reacts specifically with a species of
microorganisms, is particularly useful for the
diagnosis of microbial infection diseases.
In the present invention, causative
microorganisms of STD (sexually transmitted disease)
include, but are not limited to, Neisseria
gonorrhoeae, Chlamydia trachomatis, Candida albicans,
Treponema pallidum, and Ureaplasma urealyticum.
In the present invention, causative
microorganisms of respiratory tract infection
include, but are not limited to, Haemophilus
influenzae, Streptococcus pneumoniae, Chlamydia
pneumoniae, Mycoplasma pneumoniae, Klebsiella
pneumoniae, Staphylococcus aureus, Pseudomonas
aeruginosa, Streptococcus, sp, Group—A, Mycobacterium
tuberculosis, Legionella pneumophila, and Aspergillus
spp.
The term antibody in the present invention means
a polyclonal antibody or monoclonal antibody that can
be made using the entire length or only a partial
peptide of said ribosomal protein. Although there are
no special restrictions to the peptide length for
making the antibody, the segment should be of the
length characterizing the ribosomal protein L7/L12,
and a peptide of 5 amino acids or longer,
particularly 8 amino acids or longer, is preferred.
Antiserum containing antibody (polyclonal antibody)
that identifies ribosomal protein L7/L12 can be
obtained by inoculating laboratory animals with
adjuvant and a peptide or the full length protein as
it is or when necessary, after being cross linked
with a carrier protein such as KLH (keyhole-limpet
hemocyanin) and BSA (bovine serum albumin) and
recovering the serum. Moreover, the antibody can be
used after it has been purified from the antiserum.
The laboratory animals that are inoculated include
sheep, horses, goats, rabbits, mice, rats, etc. , and
sheep, rabbits, etc., are particularly preferred for
preparation of polyclonal antibody. Moreover,
monoclonal antibody can also be obtained by
conventional methods of making hybridoma cells, but
mice are preferred in this case. The entire length
of the said protein, or its partial peptide
consisting amino acid residues of 5 or more.
preferably 8 or more, residues that has been fused
with GST (glutathione S-transferase), etc., can be
purified and used as antigen, or it can be used as
antigen without being purified. The antibody can
also be the genetic recombination antibody expressed
cellularly using immunoglobulin genes that have been
separated by a variety of methods in manuals
("Antibodies: A Laboratory manual," E. Harlow et
al., Cold Spring Harbor Laboratory), cloning methods,
etc.
Antibody to ribosomal protein L7/L12 that can be
employed as the marker antigen of the present
invention can be obtained by the following 3 methods,
and other similar methods as well. The methods,
however, are not limited to these.
a) The desired antibody can be acquired by
synthesizing a peptide fragment consisting of 5-30
amino acids for microorganisms with a known ribosomal
protein L7/L12 genetic sequence and amino acid
sequence using the region least similar to the amino
acid sequence of said protein of another
microorganisms and making polyclonal antibody, or
monoclonal antibody, using this peptide fragment as
the immunogen.
Moreover, it is possible to acquire the entire
sequence of said gene by using a conventional genetic
procedure, such as gene amplification by PCR with the
DNA sequence at both terminals of said known genetic
sequence as the probe, or hybridization using the
sequence of a homologous segment as the template
probe.
Then, a fused gene with another protein gene is
constructed and said fused gene is inserted into the
host by conventional gene insertion methods using
Escherichia coli, etc., as the host and expressed in
large quantities. The desired protein antigen can
then be acquired by purifying the expressed protein
by affinity column methods with antibody to the
protein that was used as the fusion protein. In such
a case, even if antibody to the amino acid segment
conserved within microorganisms is acquired, it does
not coincide with the purpose of the present
invention because the full length of ribosomal
protein L7/L12 becomes the antigen. Consequently,
hybridoma that produces monoclonal antibody to the
antigen that has been obtained by this method is
acquired by conventional methods and the desired
antibody can be obtained by selecting a clone that
produces antibody that will react only with the
desired microorganisms.
b) First, since there is 50 to 60% homology
between bacterial species in terms of their ribosomal.
protein L7/L12 amino acid sequence, it is possible to
easily acquire said protein genes for microorganisms
with unknown L7/L12 amino acid sequence by-
conventional genetic procedures, such as gene
amplification of a specific sequence segment by FCR
methods based on the sequence of the homologous
segments of the amino acid sequence, or hybridization
with the homologous segments as the template probe,
using bacteria having a known ribosomal protein
L7/L12 amino acid sequence.
Then, a fused gene with another protein gene is
constructed and said fused gene is inserted into the
host by conventional gene , insertion methods using
Escherichia coli, etc., as the host and expressed in
large quantities. The desired protein antigen can
then be acquired by purifying the expressed protein
by affinity column methods with antibody to the
protein that was used as the fusion protein. In such
a case, even if antibody to the amino acid segment
conserved within microorganisms is acquired, it does
not coincide with the purpose of the present
inv-ention because th-e full length of ribosomal
protein L7/L12 becomes the antigen. Consequently,
hybridoma that produces monoclonal antibody to the
antigen that has been obtained by this method is
acquired by conventional methods and the desired
antibody can be obtained by selecting a clone that
produces antibody that will react only with the
desired microorganisms.
c) Ribosomal protein L7/L12 that has been
purified to a high purity can also be obtained by
another method, in the case where the amino acid
sequence of the ribosomal protein L7/L12 is unknown
whereby a peptide of 5 to 30 amino acids
corresponding to the common sequence segment retained
in the microorganisms is synthesized from the known
amino acid sequence of the ribosomal protein L7/L12,
and polyclonal antibody or monoclonal antibody to
this peptide sequence is made by conventional
methods. Then the highly purified Ribosomal protein
L7/L12 from disrupted microorganisms is obtained by
affinity column chromatography using said antibody.
If purity of the protein is insuf f icieTit, it can
be purified by conventional methods, such as ion
exchange chromatography, hydrophobic chromatography,
gel filtration, etc., after which the eluted fraction
of ribosomal protein L7/L12 is identified by western
blotting, etc., using antibody that was made to
obtain the pure fraction. The desired antibody can be
obtained by acquiring hybridoma or polyclanal
antibody by conventional methods using the pure
ribosomal protein L7/L12 antigen that has been
obtained and selecting hybridoma or polyclonal
antibody that will react specifically with the
desired bacteria, as in b).
The antibody of the present invention specific to
a variety of microorganisms that has been obtained by
the methods in a) through c), etc., can be used in a
variety of immunoassay methods to obtain diagnostic
reagent kits specific to a- variety of microorganisms.
For example, this antibody can be used in aggregation
reactions where antibody is adsorbed on polystyrene
latex particles, ELISA, which is a conventional
technology performed in a microtiter plate,
conventional immunochromatography methods, sandwich
assay, whereby said antibody labeled with colored
particles or particles that have coloring capability,
or with enzyme or phosphor, and magnetic
microparticles coated with capture antibody, etc.,
are used, etc.
The term detection methods for microorganism
using antibody means detection methods that use
conventional immunoassay such as aggregation
reactions where antibody is adsorbed on polystyrene
latex particles, ELISA, which is a conventional
technology performed in a microtiter plate,
conventional immunochromatography methods, sandwich
assay, whereby said antibody labeled with colored
particles or particles that have coloring capability,
or with enzyme or phosphor, and magnetic
microparticles coated with capture antibody, etc.,
are used, etc.
Moreover, the optical immunoassay (OIA)
technology described in International Patent
Application Japanese Laid-open (Toku-Hyou) No.
509565/1995, in which microorganisms are detected by
an optical interference induced by an antibody
reaction on the optical thin film which is formed by
silicone or silicon nitride, is a useful detection
method using an antibody.
Moreover, treatment with an extraction reagent
that uses a variety of surfactants, beginning with
Triton X-100 and Tween-20, enzyme treatment with an
appropriate enzyme, such as protease, etc., known
methods whereby the cell structure is disrupted,
beginning with disruption of the microorganism by
physical methods, can be used to extract the
intracellular marker antigen from the necessary
microorganism in said detection method. However, it
is preferred that the extraction conditions be
designed using a combination of surfactants, etc., so
that the conditions are optimized for extraction of
each microorganism with reagents.
Moreover, the term a reagent kit for detection of
microorganisms using antibody means a reagent kit
that uses said detection method.
For instance, in the case of obtaining the
specific antibody to Haemophilus influenzae, which is
of extreme diagnostic significance as a causative
pathogen of pneumonia, bronchitis, meningitis, etc.,
the amino acid sequence and DNA sequence of ribosomal
protein L7/L12 is entered in data bases, etc.
The amino acid and DNA sequence of ribosomal
protein L7/L12 of Haemophilus influenzae are shown in
"Sequences No. 1 and No. 2."
Consequently, in the case of this bacteria, it is
possible to similarly compare the amino acid sequence
of ribosomal protein L7/L12 with the same protein of,
for instance, Helicobacter pylori, which is shown in
"Sequence No. 3 and No. 4," and synthesize a peptide
of 5 to 30 amino acids for the segment of low
homology and make polyclonal antibody or monoclonal
antibody specific to Haemophilus influenzae using
this peptide.
In the case of a specific polyclonal antibody, it
is preferred that IgG fraction be obtained by
purification of the antiserum of immunized laboratory
animals with a protein A column, etc., and affinity
purification be performed with the synthetic peptide
uses in immunization of the laboratory animals.
Moreover, PCR primers based on the sequences o-f
N-terminal and C-terminal, for example, the PCR
primers shown in Sequences No. 11 and No. 12 in the
Sequence Table, were designed from the DNA sequence
of ribosomal protein L7/L12 of Haemophilus
influenzae. Utilizing homology of the PCR primers,
DNA fragments amplified by the PCR method or the like
using genomic DNA which is extracted from cultivated
Haemophilus influenzae can be acquired by a
conventional method. The entire length of the gene
for ribosomal protein L7/L12 of Haemophilus
influenzae can be acquired by the analysis of the DNA
sequence information of these fragments.
The ribosomal protein L7/L12 gene of Haemophilus
influenzae thus acquired forms a fusion protein gene
with, for instance, GST etc., and expression vector
is constructed using the appropriate expression
plasmid, Escherichia coli is transformed and large
quantities of said protein can be expressed. A
suitable amount of the transformed Escherichia coli
is cultivated and the crushed bacterial fluid that is
recovered is purified by an affinity column using GST
to obtain the ribosomal protein L7/L12 and GST fusion
protein of Haemophilus influenzae. It is also
possible to acquire the target specific monoclonal
antibody by establishing piural hybridojnas using said
protein as it .Ls or GST moiety fragments cut from the
protein by pxcrteas-e- or the like, as an antigen
protein and selecting the antibody which exhibits a
specific response to Haemophilus influenzae bacteria,
or a disrupt fluid of the bacteria, or ribosomal
protein L7/L12 of Haemophilus influenzae.
Moreover, the amino acid sequence and the DNA
sequence of ribosomal protein L7/L12 of Streptococcus
pneumoniae which is also highly significant as a
diagnostic agent for respiratory infection diseases
as well as Haemophilus influenzae, are known from
de-scriptions in data bases and the like. The amino
acid and DNA sequences of ribosomal protein L7/L12 of
Streptococcus pneumoniae are shown in Sequences No. 5
and No. 6 of sequence table.
It is therefore possible to acquire a polyclonal
antibody or monoclonal antibody which is specific to
Streptococcus pneumoniae by designing a PCR primer,
the PCR primer shown by Sequence ID No. 13 or 14 in
the Sequence Table, for example, based on the
sequences of N-terminai and C-terminal of DNA
sequence of ^Ribosomal Proteins L7/L12 of
Streptococcus pneumoniae in the same manner as in the
case Of Haemophilus influenzae, and processing
thereafter in the same manner as in the case of
Haemophilus influenzae.
The hybridoma AMSP-2 which produces the
monoclonal antibody spe-clfi-C to Streptoooccus
pneumoniae rras tee-n deposited with the Rational
Institute of Bioscience and Human-Technology, the
Agency of Industrial Science and Technology, the
Ministry of International Trade and Industry, Japan,
on July 28, 1939, with the deposition number FERM BP-
6807.
Moreover, although the DNA and amino acid
sequences of the ribosomal protein L7/L12 of, for
instance, Neisseria gonorrhoeae, which is the
causative pathogen of gonorrhea and has be-en shown to
have diagnostic significance as a typical causative
pathogen of STD, were unknown, a major part of the
DNA sequence and amino acid sequence, which was
determined by the Neisseria Gonorrhea Genome Project
at Oklahoma University, USA, is disclosed on the
Internet.
When part of the known DNA sequence of ribosomal
protein L7/L12 was used to probe the existence of DNA
fragments with a similar sequence, it was found that
DNA sequence corresponding to the ribosomal protein
L7/L12 gene is present and it was possible to obtain
data on its entire DNA sequence. The entire base
sequence and corresponding amino acid sequence of the
ribosomal protein L7/L12 gene of this Neisseria
gonorrhoeae are shown in Sequences No. 7 and No. 8 of
the sequence table.
It is therefore possible to acquire the target
antibody which is specific to Neisseria gonorrhoeae
having the entire or partial Ribosomal Protein L7/L12
of Neisseria gonorrhoeae as an antigen by designing a
PCR primer, the PCR primer shown by Sequence ID No.
15 or 16 in the Sequence Table, for example, based on
the sequences of N-terminal and C-terminal of DNA
sequence of Ribosomal Protein L7/L12 of Neisseria
gonorrhoeae in the same manner as in the case of
Haemophilus influenzae, and Streptococcus pneumoniae,
and processing thereafter in exactly the s-ame manner
as in the case of Haemophilus influenzae or
Streptococcus pneumoniae.
Particularly, the gene sequence of ribosomal
protein L7/L12 of Neisseria meningitides, which
belongs to the same Neisseria genus as Neisseria
gonorrhoeae is disclosed and readily available on the
Internet. The entire base sequence and the
corresponding amino acid sequence of the ribosomal
protein L7/L12 gene of Neisseria meningitidis are
shown in "Sequences No. 9 and No. 10." Here,
comparing the entire base sequence for ribosomal
protein L7/L12 genes of Neisseria meningitidis and
Neisseria gonorrhoeae, only difference in the amino
acid sequence is that Neisseria gonorrhoeae has
alanine for the 115th amino acid from the N-terminal,
whereas Neisseria meningitidis has glutamic acid.
Therefore, It can be concluded that the antibody for
the ribosomal protein L7/L12 of Neisseria gonorrhoeae
which can specifically detects Neisseria gonorrhoeae
is the antibody which identifies alanine at 115 from
the N-terminal and the amino acid region including
the alanine of ribosomal protein L7/L12 as an
epitope.
Antibody made based on the present invention can
be used in all known types of immunoassay, such as
aggregation whereby said antibody is adsorbed on
polystyrene latex, ELISA, which is a conventional
technology performed in a microtiter plate,
conventional immunochromatography, sandwich assay,
whereby said antibody labeled with colored particles
or particles that have coloring capability, or
enzymes ox phosphor, and magnetic particles coated
with capture antibody are used, etc.
Moreover, antibody that is made based on the
present invention can simultaneously function in any
of these immunoassay methods as a so-called capture
antibody that captures said antigen protein in solid
or liquid phase and a so-called enzyme-labeled
antibody by modification using an enzyme, such as
peroxidase and alkali phosphatase, etc., by
conventional methods.
BEST MODE FOR CARRYING OUT THE INVENTION
The following examples are given to explain the
present invention in actual term-s, the present
invention not being restricted to these examples.
Example 1
Cloning of ribosomal protein L7/L12 genes from
Haemophilus influenzae
After inoculating an appropriate amount of
Haemophilus influenzae strain ATCC9334 (IID984)
(obtained from Tokyo University School of Medicine
Laboratories) in a chocolate agar culture medium, the
strain was cultivated for 24 hours in a CO2 incubator

under conditions of 37oc and 5.0% CO2. The
colonies that grew were suspended in a TE buffer
(manufactured by Wako Pure Chemical Co., Ltd.) to a
final concentration of approximately 5 x 109 CFU/ml.
Approximately 1.5 ml of this suspension was
transferred to a microcentrifuge tube and centrifuged
for 2 minutes at 10,000 rpm. The supernatant was
discarded. The sediment was resuspended in 567 \xl TE
buffer. Then 30 ml 10 % sodium dodecylsulfate (SDS)
and 3 ml 20 mg/ml Proteinase K solution were added
and thoroughly mixed. The suspension was incubated
for another hour at 37°C. Next, after adding 80 ul
10 % cetyl trimethyl ammonium bromide/0.7 M NaCl
solution and thoroughly mixing the product, it was
incubated for 10 minutes at 65 °C. Next, 700 Ml
chloroform-isoamyl alcohol solution at a volume ratio
of 24:1 was added and stirred well. The solution was
centrifuged for 5 minutes (while being kept at 4°C)
at 12,000 rpm using a microcentrifugation device and
the aqueous fraction was transferred to a new
microtube. Isopropanol was added to the fraction at
0.6-times its volume and the tube was vigorously
shaken to form sediment of the DNA. The white DNA
sediment was scooped with a glass rod and transferred
to a different microcentrifuge tube containing 1 ml
70 % ethanol (cooled to -20 °C) .
Next, the product was centrifuged for 5 minutes
at 10,000 rpm and the supernatant was gently removed.
Then another 1 ml 70 % ethanol was added and the
product was centrifuged for 5 more minutes.
Once the supernatant had been removed, the
sediment was dissolved in 100 ml TE buffer to obtain
the DNA solution. The concentration of the genomic
DNA solution was determined quantitatively according
to E5, Spectrophotometric determination of the amount
of DNA or RNA "Molecular cloning: A laboratory
manual," 1989, Eds. Sambrook, J., Fritsch, E.F., and
Maniatis, T., Cold Spring Harbor Laboratory Press.
PCR (polymerase chain reaction) was performed
using 10 ng of this genomic DNA. Taq polymerase
(Takara Co., ltd., code R001A) was employed for PCR.
Then 5 ul of buffer, 4 ul dNTP mixture, and 200 pmol
each of synthetic oligonucleotide-A, shown in
Sequence No. 11 of the Sequence Table and synthetic
oligonucleotide-B, shown in Sequence No. 12 of the
Sequence table, were added to the enzyme to bring the
final volume to 50 ml.
This mixture was cycled 5 times with a TaKaRa PCR
Thermal Cycler 480 for 1 minute at 95 °C, 2 minutes
at 50 °C, and 3 minutes at 72 °C and was then cycled
25 times for 1 minute at 95 °C, 2 minutes at 60 °C,
and 3 minutes at 72 °C. Electrophoresis was performed
in 1.5 % agarose gel using some of this PCR product.
This product was then stained with ethidium bromide
(Ninon Gene Co., ltd.) and observed under ultraviolet.
rays to confirm amplification of approximately 400 bp
cDNA. After digestion treatment with restriction
endonucleases BamHI and Xhol, electrophoresis was
performed in 1.5 % agarose gel and staining with
ethidium bromide was carried out. An approximately
370 bp band was cut out from the gel. This band was
purified with SuprecOl (Takara Co., Ltd.) and then
inserted into pGEX-4T-l (Pharmacia), which is a
commercial vector. This same vector can function as
an expression vector for the desired molecule, which
can express fused protein with GST protein, by
insertion of the desired gene fragment into the
appropriate restriction endonuclease site.
Actually, vector pGEX-4T-l and the previous DNA
were mixed together at a molar ratio of 1:3 and DNA
was inserted into the vector with T4 DNA ligase
(Invitrogen Co.). Vector pGEX-4T-l into which DNA
had been inserted was genetically introduced to
Escherichia coli one-shot competent cells (Invitrogen
Co., Ltd.) and then inoculated in a plate of L-Broth
(Takara Co., Ltd.) semi-sold culture plate containing
50 ug/ml ampicillin (Sigma). The plate was then set-
aside at 37 °C for 12 hours and the colonies that
grew were selected at random and inoculated into 2 ml
L-Broth liquid culture medium containing the same
concentration of ampicillin. Shake cultivation was
performed at 37 °C for 8 hours and the bacteria were
recovered and the plasmid was separated using Wizard
Miniprep (Promega) in accordance with the attached
literature. The plasmid was cleaved with restriction
endonuclease BamHI/XhoI. Insertion of said PCR
product was confirmed by cutting out approximately
370 bp DNA. The base sequence of the DNA that had
been inserted was determined using said clone.
Determination of the base sequence of the
inserted DNA fragment was performed using the
Fluorescence Sequencer of Applied Biosystems. The
sequence sample was prepared using PRISM, Ready
Reaction Dye Terminator Cycle Sequencing Kit (Applied
Biosystems). First, 9.5 ml reaction stock solution,
4.0 ml T7 promoter primer at 0.8 pmol/ml (Gibco BRL)
and 6.5 ml of template DNA for sequencing at 0.16
mg/ml were added to a microtube with a capacity of
0.5 ml, mixed ana superposed with 100 ml mineral oil.
PCR amplification was performed for 25 cycles, where
one cycle consisted, of 30 seconds at 96 °C, 15
seconds at 55 °C, and 4 minutes at 60 °C. The product
was then kept at 4 °C for 5 minutes . After the
reaction was completed, 80 ml sterilized pure water
was added and stirred. The product was centrifuged
and the aqueous layer was extracted 3 times with

phenol-chloroform. 10ml 3 M sodium
acetate (pH 5.2) and 300 ml ethanol were added to 100
ml aqueous Layer- and starred. The product was then
centrifuged for 15 minutes at room temperature and
14,000 rpm and the sediment was recovered. Once the
sediment was washed with 75 % ethanol, it was dried
under a vacuum for 2 minutes to obtain the sequencing
sample. The sequencing sample was dissolved in
formamide containing 4 ml 10 mM EDTA and denatured
for 2 minutes at 90 °C. This was then cooled in ice
and applied to sequencing.
One of the 5 clones obtained had homology of the
sequence with the probe used for PCR. In addition,
DNA sequences extremely similar to the gene sequence
of ribosomal protein L7/L12 gene of the other
microorganisms, for example, Neisseria gonorrhoeae,
were discovered. The entire base sequence and the
corresponding amino acid sequence of the structural
gene moiety are as shown in Sequence ID No. 17 and
No. 18 of the Sequence Table. This gene "fragment
clearly codes for Haemophilus influenzae ribosomal
protein L7/L12.
Example 2
Mass expression in Escherichia coli and purification
of ribosomal protein L7/L12 from Haemophilus
influenzae
Fifty milliliters Escherichia coli into which
expression vector had been inserted were cultivated
overnight in LB at 37°C. Then 500 ml YT medium at
cancentration that was twice that of the
aforementioned culture was heated at 37 °C "for 1
hour. Fifty milliliters of the Escherichia coli
solution that had been cultivated overnight were
introduced to 500 ml of the aforementioned medium.
One hour later, 550 |ul 100 mM isopropyl ($-(D)-
thiogalactopyranoside (IPTG) were introduced and
cultivated for 4 hours. The product was then
recovered and introduced to centrifugatiorr tube at
each 250 ml and centrifuged for 10 minutes at 7,000
rpm.
The supernatant was discarded and dissolved in 25
ml each 50 mM Tris-HCl at a pH of 7.4 and Lysis
buffer containing 25 % sucrose.
Furthermore, 1.25 ml 10 % Nonidet P-40 (NP-40)
and 125 |il 1 M MgCl2 were added and transferred to a
plastic tube. Sonication was performed 1 minute x 5
times while ice cold. The product was centrifuged
for 15 minutes at 12,000 rpm and the supernatant vrars
recovered.
Next, the aforementioned supernatant was adsorbed
on a glutathione agarose column conditioned with
phosphate-buffered saline (PBS).
Then the column was washed with twice the bed
volume using a washing solution containing 20 mM Tris
buffer at a pH of 7.4, 4.2 mM MgCl2, and 1 mM
dithiothreithol (DTT). Elution was performed with 50
mM Tris buffer at a pH of 9.6 containing 5 mM
glutathione. The protein content in the fraction was
determined by the pigment bonding method (Bradford
method; BioRad Co.) and the main fraction was
acquired.
Purity of the purified ribosomal protein
L7/L12/GST fused protein that was obtained was
confirmed by electrophoresis to be approximately 75
%, showing that a purity satisfactory for an
immunogen could be guaranteed.
Example 3
Preparation of monoclonal antibody to ribosomal
protein L7/L12 of Haemophilus influenzae
First, 100 ng fused protein antigen of ribosomal
protein L7/L12/GST of Haemophilus influenzae were
dissolved in 200 Hi PBS and then 200 \il Freund's
complete adjuvant were added and mixed and
emulsif .Lcation was performed. Two-hundred
microliters were injected intraperitoneally to
immunize mice.
Then the same emulsion antigen was
intraperitoneally injected after 2 weeks, after 4
weeks, and after 6 weeks. Two-fold the concentration
of antigen emulsion was injected intraperitoneally
after 10 weeks and after 14 weeks. The spleen was
excised 3 days after the final immunization and
submitted to cell fusion.
After thoroughly mixing 2 x 107 myeloma cells per
108 spleen cells from mice, which had been recovered
aseptically, in a glass tube, the mixture was
centrifuged for 5 minutes at 1,500 rpm and the
supernatant was discarded. The cells were thoroughly
mixed.
The myeloma cells used for cell fusion were
obtained by cultivation of cell strain NS-1 with an
RPMI 1640 culture medium containing 10 % FCS,
cultivating this product beginning 2 weeks before
cell fusion using an RPMI 1640 medium containing 0.13
mM azaguanine, 0.5 ng/ml MC-210, and 10 % FCS for 1
weeks, and then further cultivating the cell strain
for 1 week with an RPMI 1640 medium containing 10 %
FCS .
Thirty milliliters of RPMI 1640 culture medium 50
ml that had been kept at 37 °C were added to the
mixed cell sample and centrifuged at 1,500 rpm.
After removal of the supernatant, 1 ml 50 %
polyethylene glycol that had been kept at 37 °C was
added and stirred for 2 minute. 10 ml RPMI 1640
medium kept at 37 °C were added and the solution was
vigorously mixed for approximately 5 minutes as it
was suctioned and evacuated from a sterile pipette.
After centrifugation for 5 minutes at 1,000 rpm
and removal of the supernatant, 30 ml HAT medium were
added to bring the cell concentration to 5 x 106
cells/ml. This mixture was stirred till uniform and
then poured, 0.1 ml at a time, into a 96-well culture
plate and cultivated at 37 °C under 7 % CO2. HAT
medium was added, 0.1 ml at a time, on Day 1 and at
Week 1 and Week 2.
Then the cells that had produced the desired
antibody were screened by ELISA.
Solutions of GST fusion ribosomal protein L7/L12
and GST protein of Haemophilus influenzae dissolved
in PBS containing 0.05 % sodium azide diluted to 10
mg/ml were poured, each 100 ml at a time, into
separate 96-well plates and adsorbed overnight at 4
°C.
After removal of the supernatant, 200 ml of 1 %
bovine serum albumin solution (in PBS) were added and
reacted and blocked for 1 hour at room temperature.
After removal of the supernatant, the product was
washed with washing, solution (0.02 % Tween 20, PBS).
One-hundred microliters culture solution of fused
cells were added to this and reacted for 2 hours at
room temperature. The supernatant was removed and
washed with washing solution. Next, 100 ml
pero-xidase-labeied goat anti-mouse IgG antibody
solution at a concentration of 50 ng/ml were added
and the solution was reacted for 1 hour at room
temperature. The supernatant was removed and the
product was again washed with washing solution. Then
TMB solution (KPL Co., ltd) was added, 100 ml at a
time, and the mixture was reacted for 20 minutes at
room temperature. After coloration, 100 \xl 1 N
sulfuric acid were added to stop the reaction and
absorbance at 450 nm was determined.
As a result, positive wells that only reacted
with GST fusion ribosomal protein L7/L12 and did not
react with GST protein were detected, and it was
concluded that antibody to ribosomal protein L7/L12
is present.
Therefore, the cells in the positive wells were
recovered and cultivated with HAT medium in a 24-well
plastic plate. The fused medium that had been
cultivated was diluted with HT medium to a cell count
of approximately 20 cells/ml and then mixed with 106
six-week-old mouse thyroid cells suspended in HT
Cultivation medium in a 96-well culture plate.
Culture was performed for 2 weeks at 37 °C under
conditions of 7 % CO2.
Antibody activity in the culture supernatant was
similarly determined by the aforementioned ELISA
method and the cells that showed positive reaction
with ribosomal protein L7/L12 were recovered.
Furthermore, the same dilution test and cloning
procedure was repeated to obtain a total of 5 clones
of hybridoma HIRB-1 ~ 5.
Example 4
Reaction of monoclonal antibody that reacts with
rifoosomal protein L7/L12 of Haemophilus influenzae,
with Neisseria gonorrhoeae and other microorganisms
Monoclonal antibody was produced and recovered in
accordance with standard methods using the positive
hybridoma cells obtained as previously described.
Basically, 5 x 106 cells that had been
subcultured using RPMI 1640 culture medium
«
(containing 10 % FCS) were intraperitoneally injected
into Balb/C mice that had been intraperitoneally
injected with 0.5 ml Pxistane 2 weeks earlier.
Ascites was recovered 3 weeks later and the
centrifugation supernatant was obtained.
The solution containing antibody that was
obtained was adsorbed in a Protein A column (5 ml
bed, Pharmacia) and washed with 3-bed volume of PBS.
Then eluted with citrate buffer at pH 3, the antibody
fraction was recovered and the monoclonal antibody
produced by each hybridoma was obtained.
The monoclonal antibody derived from these 5
strains of hybridoma was used in ELISA.
The sandwich assay method was used to assess the
monoclonal antibody. The monoclonal antibody that
was prepared was used as an enzyme-labeled antibody
by being chemically bound to peroxidase.
That is, enzyme labeling was performed in
accordance with the method in "Analytical
Biochemistry" 132 (1983) , 68-73 with the reagent S-
acetylthioacetic acid N-hydroxysucciaimide for
binding using horseradish peroxidase (Sigma Grade
VI). By means of the ELISA reaction, a solution of
commercial anti-Haemophilus influenzae polyclonal
antibody which dissolved in FBS containing 0.05 %
sodium azide, (Biodesign, rabbit) was diluted to a
concentration of 10 mg/ml and poured,100 ml at a
time, into a separate 96-well plate and adsorbed
overnight at 4°C.
After removal of the supernatant, 200 ml 1 % FCS
solution (in PBS) were added and reacted and blocked
for 1 hour at room temperature. The supernatant was
removed and the product was washed with washing
solution (0.02 % Tween 20, PBS). One-hundred
microliters of antigen solution, which had been
obtained by adding Triton X-10C to culture solutions
of each species of microorganism to a concentration
of 0.3 % and then extracting the solution for 5
minutes at room temperature, were added to this and
reacted for 2 hours at room temperature. The
supernatant was removed and the product was further
washed with washing solution. Then 100 ml
peroxidase-labeled anti-ribosomal protein L7/L12
antibody solution at 5 mg/ml were added and reacted
for 1 hour at room temperature. The supernatant was
removed and the product was washed with washing
solution. 1MB (KPL Co.) solution was added, 100 |il at
a time, and reacted for 2 0 minutes at room
temperature. After coloration, 100 |il 1 N sulfuric
acid were added to stop the reaction. Absorbance at
450 nm was determined.
As a result, as shown in Table 1 it is clear that
when monoclonal antibody derived from hybridoma HIRB-
2 was used as the enzyme-labeled antibody, all
strains of Haemophilus influenzae tested were
detected at a sensitivity of 106 bacteria/ml, while
reactivity of other bacteria belonging ~to "the genus
Neisseria and other microorganisms could not be
detected, even at high concentrations of 108
bacteria/ml and therefore, antibody with specific
reactivity to Haemophilus influenzae can be obtained
by using monoclonal antibody to ribosomal protein
L7/L12.
Table 1
(+: Positive; -: Negative)
Example 5
Cloning of ribosomal protein L7/L12 genes from
Streptococcus pneumponiae, mass expression in
Escherichia coli and purification of the same protein
and preparation of monoclonal antibody to the same
protein
After inoculating an appropriate amount of
Streptococcus pneumoniae strain IID555 (obtained from
Tokyo University School of Medicine Laboratories) in
a blood agar culture medium, the strain was
cultivated for 48 hours in an incubator at 37 °C. The
colonies that grew were suspended in a TE buffer to a
final concentration of approximately 5 x 109 CFU/ml.
Approximately 1.5 ml of this suspension was
transferred to a microcentrifuge tube and centrifuged
for 2 minutes at 10,000 rpm. The supernatant was
discarded. The sediment was resuspended in 567 ul TE
buffer. Then 30 ul 10 % SDS and 3 ul 20 mg/ml
Proteinase K solution were added and thoroughly
mixed. The suspension was incubated for another hour
at 37 °C. Next, after adding 80 ml 10 % cetyl
trimethyl ammonium bromide/0.7 M NaCl solution and
thoroughly mixing the product, it was incubated for
10 minutes at 65 °C. Next, 700 ul chloroform-isoamyl
alcohol solution at a volume ratio of 24:1 was added
and stirred well. The solution was centrifuged for 5
minutes (while being kept at 4 °C) at 12,000 rpm
using a microcentrifugation device and the aqueous
fraction was transferred to a new microtube.
Isopropanol was added to the fraction at 0.6-times
its volume and the tube was vigorously shaken to form
sediment of the DNA. The white DNA sediment was
scooped with a glass rod and transferred to a
different microcentrifuge tube containing 1 ml 70 %
ethanol (cooled to -20 °C)
Next, the product was centrifuged for 5 minutes
at 1.0,000 rpm and the supernatant was gently removed.
Then another 1 ml 7 0 % ethanol was added and the
product was centrifuged for 5 more minutes. Once the
supernatant had been removed, the sediment was
dissolved in 100 ml TE buffer to obtain the DNA
solution. The concentration of the genomic DNA
solution was determined quantitatively in accordance
with E5, Spectrophotometric determination of the
amount of DNA or RNA, "Molecular cloning: A
laboratory manual," 1989, Eds. Sambrook, J., Fritsch,
E.F., and Maniatis, T., Cold Spring Harbor Laboratory
Press.
PCR was performed using 10 ng of this genomic
DNA. Taq polymerase (Takara Co., ltd., code R001A)
was employed for PCR. Then 5 ul of buffer attached
to enzyme, 4 ml dNTP mixture attached to enzyme, and
200 pmol each of synthetic oligonucleotide C shown in
Sequence No. 13 of the Sequence Table and synthetic
oligonucleotide D shown in Sequence No. 14 of the
Sequence table were added to the enzyme to bring the
final volume to 50 ml.
This mixture was cycled 5 times with a TaKaRa PCR
Thermal Cycler 480 for 1 minute at 95 °C, 2 minutes
at 50 °C, and 3 minutes at 72 °C and was then cycled.
25 times for 1 minute at 95 °C, 2 minutes at 60 °C,
and 3 minutes at 72 °C. Electrophoresis was performed
in 1.5 % agarose gel using some of this PCR product.
This product was then stained with ethidium bromide
(Ninon Gene Co., ltd.) and observed under ultraviolet
rays to confirm amplification of approximately 400 bp
cDNA. After digestion treatment with restriction
endonucleases BamHI and XhoI, electrophoresis was
performed in 1.5 % agarose gel and staining with
ethidium bromide was carried out. An approximately
370 bp band was cut out from the gel. This band was
purified with SuprecOl (Takara Co., Ltd.) and then
inserted into pGEX-6P-l (Pharmacia), which is a
commercial vector.
This same vector can function as an expression
vector for the desired molecule, which can express
fused protein with GST protein, by insertion of the
desired gene fragment into the appropriate
restriction endonuclease site. Actually, vector
pGEX-6P-l and the previous DNA were mixed together at
a molar ratio of 1:5 and DNA was inserted into the
vector with T4 DNA ligase (Invitrogen Co.). Vector
pGEX-4T~-l into which ETNA had been inserted was
genetically introduced to Escherichia coli One-Shot
Competent Cells (Invitrogen Co., Ltd.) and then
inoculated in a plate of L-Broth (Takara Co., Ltd.)
semi-sold culture plate containing 50 mg/ml
ampicillin (Sigma). The plate was then set aside at
37°C for 12 hours and the colonies that grew were
selected at random and inoculated into 2 ml L-Broth
liquid culture medium containing the same
concentration of ampicillin. Shake cultivation was
performed at 37°C for 8 hours and the bacteria was
recovered and the plasmid was separated using Wizard
Miniprep (Promega Co.,) in accordance with the
attached literature. The plasmid was cleaved with
restriction endonuclease BamHI/XhoI. Insertion of
said PCR product was confirmed by cutting out
approximately 370 bp DNA. The base sequence of the
DNA that had been inserted was determined using said
clone.
Determination of the base sequence of the
inserted DNA fragment was performed using the
Fluorescence Sequencer of Applied Biosystems. The
sequence sample was prepared using PRISM, Ready
Reaction Dye Terminator Cycle Sequencing Kit (Applied
Biosystems). First, 9.5 ml reaction stock solution,
4.0 ml T7 promoter primer at 0.8 pmol/ml (Gibco BRL)
and 6.5 ml of template DNA for sequencing at 0.16
mg/ml were added to a microtube with a capacity of
0.5 ml, mixed and superposed with 100 ml mineral oil.
PCR amplification was performed for 25 cycles, where
one cycle consisted of 30 seconds at 96 °C, 15
seconds at 55 °C, and 4 minutes at 60 °C. The product
was then kept at 4 °C for 5 minutes . After the
reaction was completed, 80 ul sterilized pure water
was added and stirred. The product was centrifuged
and the aqueous layer was extracted 3 times with
phenol-chloroform. Ten microliters 3 M sodium
acetate (pH 5.2) and 300 ml ethanol were added to 100
ml aqueous layer and stirred. The product was then
centrifuged for 15 minutes at room temperature and
14,000 rpm and the sediment was recovered. Once the
sediment was washed with 75 % ethanol, it was dried
under a vacuum for 2 minutes to obtain the sequencing
sample. The sequencing sample was dissolved in
formamide containing 4 ml 10 mM EDTA and denatured
for 2 minutes at 90 °C. This was then cooled in ice
and submitted to sequencing.
One of the 7 clones obtained had homology of the
sequence with the probe used for PCR. In addition,
DNA sequences extremely similar to the gene sequence
of ribosomal protein L7/L12 gene of the other
microorganisms, for example. Neisseria gonorrhoeae,
were discovered. The entire base sequence and the
corre-sponding amino acid sequence of the structural
gene moiety are as sJaown Ln Sequence ID No. 19 and
No. 20 of the Sequence Table. This gene fragment
clearly codes for Streptococcus pneumoniae ribosomal
protein L7/L12.
50 ml Escherichia coli into which expression
vector had been inserted was cultivated overnight in
a two-fold concentration YT medium at 37 °C. Then,
450 ml of the two-fold concentration YT medium was
heated at 37 °C for 1 hour. 50 ml of the Escherichia
coli solution that had been cultivated overnight was
introduced to 450 ml of the aforementioned medium.
After cultivation for one hour at 37 °C, 100 ml 500
mM IPTG was introduced and cultivated for 4 hours at
25 °C. The product was then recovered and introduced
to centrifugation tube at each 250 ml and centrifuged
for 20 minutes at 5000 rpm. The supernatant was
discarded and dissolved in 25 ml each 50 mM Tris-HCl
at a pH of 7.4 and Lysis buffer containing 25 %
sucrose.
Furthermore, 1.25 ml 10 % NP-40 and 125 ml 1 M.
MgCl2 were added and transferred to a plastic tube.
Sonication was performed 1 minute x 5 times while ice
cold. The product was centrifuged for 15 minutes at
12,000 rpm and the supernatant was recovered.
Next, the aforementioned supernatant was adsorbed
on a glutathione sepharose column (manufactured by
Pharmacia) condLLtioned with. PBS. Then, the column
was washed with PBS three timers the tred volume.
Elution was performed with ,50 mM Tris-HCl at a pH of
8.0 containing 10 mM glutathione. The protein
content in the fraction was determined by the pigment
bonding method (Bradford method; BioRad Co.) and the
main fraction was acquired. The main fraction was
dialyzed three times against 3 L PBS.
1 ml of a cleavage buffer containing 500 mM Tris-
HC1 (pH 7.0), 1.5 M NaCl, 10 mM EDTA, and 10 mM DTT
was added to 10 ml of 1 mg/ml solution of the
resulting GST fusion ribosomal protein L7/L12. 100 |ll
of 2 m/ml PreScission Protease (manufactured by
Pharmacia company) was further added and reacted at 4
°C to separate the GST moiety from ribosomal protein
L7/L12.
The reaction solution was passed through a
glutathione sepharose column which had been
conditioned with PBS. The solution coming out from
the column was recovered. One-bed volume of PBS was
passed through and also recovered. Purity of the
purified ribosomal protein L7/L12 that was obtained
was confirmed by electrophoresis to be approximately
90 %, showing that a purity satisfactory for an
immunogen could be guaranteed.
First, 100 ug protein antigen erf ribosomal
protein L7/L12 of Streptococcus pneumoniae was
dissolved in 2 00 ul PBS and tfcen 200 jol Fxsmrd * s
complete adjuvant was added and mixed axtd
emulsification was performed. 200 ul was
intraperitoneally injected to immunize mice. Then,
the same emulsion antigen was intraperitoneally
injected after 2 weeks, after 4 weeks, and after 6
weeks. A two-fold concentration antigen emulsion was
further injected intraperitoneally after 10 weeks and
after 14 weeks. The spleen was excised 3 days after
the final immunization and submitted to cell fusion.
After thoroughly mixing 2 x 107 myeloma cells per
108 spleen cells from mice,, which had been recovered
aseptically, in a glass tube, the mixture was
centrifuged for 5 minutes at 1500 rpm and the
supernatant was discarded. The cells were thoroughly
mixed.
The myeloma cells used for cell fusion were
obtained by cultivation of cell strain NS-1 with an
RPMI 1640 culture medium containing 10 % FCS,
cultivating this product beginning 2 weeks before
cell fusion using an RPMI 1640 medium containing 0.13
mM azaguanine, 0.5 ug/ml MC-210, and 10 % FCS for 1
weeks, and then further cultivating the cell strain
for 1 week with an RPMI 1640 medium containing 10 %
FCS. 30 ml of RPMI 1640 culture medium 50 ml that had
been kept at 37 °C was added to the mixed cell sample
and centrifuged at 1,500 rpm. After removal of the
supernatant, 1 ml 50 % polyethylene glycol that haad
been kept at 3 7 °C was added and stirred far 2
minute. 10 ml RPMI 1640 medium kept at 37 °C was
added and the solution was vigorously mixed for
approximately 5 minutes as it was suctioned and
evacuated using a sterile pipette.
After centrifugation for 5 minutes at 1,000 rpm
and removal of the supernatant, 30 ml HAT medium were
added to bring the cell concentration to 5 x 106
cells/ml. This mixture was stirred till uniform and
then poured, 0,1ml at a time, into a 96-well culture
plate and cultivated- at 37 °C under 7 % CO2. HAT
medium was added, 0.1 ml at a time, on Day 1 and at
Week 1 and Week 2.
Then, the cells that had produced the desired
antibody were screened by ELISA. Solutions of
ribosomal protein L7/L12 of Streptococcus pneumoniae
dissolved in PBS containing 0.05 % sodium azide
diluted to 10 ug/ml were poured, 100 ml at a time,
into separate 96-well plates and adsorbed overnight
at 4 °C. After removal of the supernatant, 200 ul 1 %
bovine serum albumin solution (in PBS) were added and
reacted and blocked for 1 hour at room temperature.
The supernatant was removed and the product was
washed with a washing solution (0.02 % Tween 20,
PBS). 100 pi of a culture solution of fusion cells
was added and reacted for two hours at room
temperature. The supernatant was removed and tire
product was further washed with a washing solution-.
Then,' 100 ul of a peroxidase-labeled goat anti-mouse
antibody solution at 50 ng/ml was added and reacted
for one hour at room temperature. The supernatant
was removed and the product was washed with a washing
solution. TMB (KPL) solution was added, 100 ml at a
time, and reacted for 20 minutes at room temperature.
After coloration, 100 ml 1 N sulfuric acid were added
to stop the reaction. Absorbance at 450 nm was
determined.
As a result, positive wells that reacted with
ribosomal protein L7/L12 were detected, confirming
presence of the antibody to ribosomal protein L7/L12.
Therefore, the cells in the positive wells were
recovered and cultivated with HAT medium in a 24-well
plastic plate. The fused medium that had been
cultivated was diluted with an HT medium to a cell
count of approximately 20 cells/ml and then mixed
with 106 six-week-old mouse thyroid cells suspended
in the HT medium in a 96-well culture plate. The
cells were cultivated for 2 weeks at 37 °C under the
conditions of 7 % C02. The antibody activity in the
culture supernatant was determined by the
aforementioned ELISA method and the cells that showed
a positive reaction with ribosomal protein L7/L12
were recovered.
Furthermore, the same dilution and cloning
procedure was repeated to obtain a total of 4 clones
of hybridoma AMSP-1 to 4.
Example 6
Reaction of monoclonal antibody that reacts wich
ribosomal protein L7/L12 of Streptococcus pneumoniae,
with Streptococcus pneumoniae and other
microorganisms
A monoclonal antibody was produced and recovered
in accordance with standard methods using the
positive hybridoma cells obtained as previously
described.
Specifically, cells subcultured in RPMI 1640
medium (containing 10 % FCS) was diluted with a
serum-free medium to about 2 x 105 cells/ml, 3.3 x 105
cells/ml, and 5 x 105 cells/ml in 25 cm2 culture
flasks, and the total volume was made 5 ml. After
cells were grown for 3 to 5 days in 7 % CO2 at 37 °C ,
a flask which contains the least number of original
cells was selected among flasks in which cells were
grown. The same procedure was repeated until the
cells diluted to 2 x 105 cells/ml grow to 2 x 106
cells/ml in 3 to 4 days, thereby adapting the cells
with the serum-free medium. Next, cloning was
performed in a 96-well plate for bacteria cultivation
to select cells exhibiting fastest growth and a
highest antibody titer. The selected cells were
grown in a 24-well plate and diluted with a serum-
free medium in a 2 5 cm2 culture flask to a
concentration of about 2 x 105 cells/ml and the total
volume was made 10 ml. After incubation for 3 to 4
days in 7 % C02 at 37 °C to a concentration of 1 x 106
cells/ml, the culture broth 100 ml, 1 x 106 cells/ml
was transferred to a bottle for mass cultivation
which were grown in the same manner in a 75 cm
flask. 100 ml of a serum-free medium was added to
the mixture, which was incubated at 37 °C for two
days while stirring. 200 ml of the serum-free medium
was added again and the mixture was incubated for a
further two days. The culture broth was divided into
four aliquot, the serum-free medium was added to each
portion, followed by incubation for two days. After
further addition of 400 ml of the serum-free medium,
the culture broth was incubated for 6 days. The
culture broth was collected and centrifuged at 10,000
rpm for 15 minutes to obtain a- culture supernatant
that contain the target antibody. After the addition
of sodium azide to final concentration 0.1 %, the
culture supernatant was stored at 4 °C. 100 ml of the
solution containing the antibody that was obtained
was 5-fold diluted with PBS and adsorbed in a Protein
G column (5 ml bed, Pharmacia) and washed with 3-bed
volume of PBS. Then eluted with citrate buffer at a
pH 3, the antibody fraction was recovered, and
monocloaal antibody pxodxiced by each hybridoma was
obtained. The monoclonal antibodies originating from
the four hybridoma clones were evaluated according to
the OIA method described in Publication of
International Patent Application Japanese Laid-open
(Toku-Hyou) No. 509565/1995.
Specifically, the OIA method comprises preparing
a reactive substrate by reacting an antibody for
capture on a silicon wafer having a thin film layer
of silicon nitride, causing this substrate to react
with an antigen which is an extract of microorganisms
for a prescribed period of time, causing the captured
antigen to react with an antibody (an amplification
reagent) which is an enzyme-labeled antibody, and
finally adding a substrate solution to produce a
thin-film precipitate. The antigen-antibody reaction
can be judged visually by a degree of light
interference color produced in the precipitate.
The monoclonal antibody preparation was used and
evaluated as a capture antibody to be immobilized on
a silicon wafer having a silicon nitride thin film
layer in the OIA method. Moreover, peroxidase-
labeled AMGC-1 monoclonal antibody which can non-
specifically react with ribosomal proteins L7/L12
protein of a variety of microorganisms described in
Reference Example was used as the detect antibody.
That is, enzyme labeling was performed in accordance
with the- method in "Artalyticsl Biochemistry" 132
(19-83), 68-73 with the re-agent S—acetyIthioacetic
acid N-hydroxysuccinimide for binding using
horseradish peroxidase (Sigma Grade VI).
In the OIA reaction, monoclonal antibody in a PBS
containing 0.05 % sodium azide was diluted with 0.1 M
HEPES (pH 8.0) to a concentration of 10 mg/ml and
added onto a silicone wafer which has a thin film
layer of silicon nitride, 50 ul at a time, to react
for 30 minutes at room temperature, followed by
washing with distilled water and use.
15 ml of antigen solution, which, had been
obtained by adding 0.5% Triton X-100 to culture
solutions of various species of microorganisms and
then extracting the solution for 5 minutes at room
temperature, was added to the specimen obtained in
the above-described procedure and reacted for 10
minutes at room temperature. Then, 15 ml of 20 mg/ml
peroxidase-labeled AMGC1 anti-body was added and
reacted for 10 minuter. After washing with distilled
wairer, a substrate solution (KPL) was added, 15 ul at
a time, and reacted for 5 minutes at room
temperature. The product was washed with distilled
water to judge the concentration of detection signals
as an intensity of light interference by naked eyes.
As a result, as shown in Table 2 it is clear that
when monoclonal antibody derived from hybridoma AMSP-
2 was used as the capture antibody, all strains of
Str^pxocaccus pneunion.Lae tested were detected at a
sensitivity of 106 bacteria/ml, while reactivity of
other bacteria could not be detected at a higher
concentration of 108 bacteria/ml. Thus, the antibody
with specific reactivity to Streptococcus pneumoniae
was confirmed to have been obtained by using the
monoclonal antibody to ribosomal protein L7/L12.
The hybridoma AMSP-2 which produces the
monoclonal antibody specific to Streptococcus
pneumoniae has been deposited with National Institute
of Bioscience and Human-Technology, the Agency of
Industrial Science and Technology, the Ministry of
International Trade and Industry, Japan, on July 28,
1999, with the disposition number FERM BP-6807.
Table 2
( + : Positive; -: Negative)
Example 7
Cloning of ribosomal protein L7/L12 genes from
Neisseria gonorrhoeae, mass expression in Escherichia
coli and purification of the same protein and
preparation of monoclonal antibody to the same
protein
After inoculating an appropriate amount of
Neisseria gonorrhoeae strain IID821 (obtained from
Tokyo University School of Medicine Laboratories) in
a chocolate agar culture medium, the strain was
cultivated for 24 hours in a CO2 incubator under

conditions of 37 °C and 5.0% CO2. The colonies
that grew were suspended in a TE buffer to a final
concentration of approximately 5 x 109 CFU/ml.
Approximately 1.5 ml of this suspension was
transferred to a microcentrifugation tube and
centrifuged for 2 minutes at 10,000 rpm. The
supernatant was discarded. The sediment was
resuspended in 567 ul TE buffer. Then 30 Hi 10 % SDS
and 3 ml 20 mg/ml Proteinase K solution were added
and thoroughly mixed. The suspension was incubated
for another hour at 37oC.
Next, after adding 80 ml 10 % cetyl trimethyl
ammonium bromide/0.7 M NaCl solution and thoroughly
mixing the product, it was incubated for 10 minutes
at 65 °C. Next, 700 ml chloroform-isoamyl alcohol
solution at a volume ratio of 24:1 was added and
stirred well. The solution was centrifuged for 5
minutes (while being kept at 4 °C) at 12,000 rpm
using a microcentrifugation device and the aqueous
fraction was transferred to a new microtube.
Isopropanol was added to the fraction at 0.6-times
its volume and the tube was vigorously shaken to form
sediment of the DNA. The white DNA sediment was
scooped with a glass rod and transferred to a
different microcentrifugation tube containing 1 ml 70
% ethanol (cooled to -20 °C) .
Next, the product was centrifuged for 5 minutes
at 10,000 rpm and the supernatant was gently removed.
Then another 1 ml 70 % ethanol was added and the
product was centrifuged- for 5 more minutes.
Once the supernatant had been removed, the
sediment was dissolved in 100 ml TE buffer to obtain
the DNA solution. The concentration of the genomic
DNA solution was determined quantitatively in
accordance with E5, Spectrophotometry determination
of the amount of DNA or RNA, "Molecular cloning: A
laboratory manual," 1989, Eos. Sambrook, J., Fritsch,
E.F., and Maniatis, T., Cold Spring Harbor Laboratory
Press.
PCR was performed using 10 ng of this genomic
DNA. PCR was performed using Tag polymerase (Takara
Co., Ltd., code R001A). Then, 5 ml of a buffer
attached to enzyme, 4 ml of a dNTP mixture attached
to enzyme, and 200 pmol each of synthetic
oligonucleotide E shown in Sequence No. 15 of the
Sequence Table and synthetic oligonucleotide F shown
in Sequence No. 16 of the Sequence Table, which were
designed based on the ribosomal protein L7/L12 DNA
sequence of Neisseria gonorrhoeae acquired from
Internet Information (Oklahoma University, N.
Gonorrhoeae Genome Project, disclosed genomic DNA
data) because of the similarity with ribosomal
protein L7/L12 DNA sequence of other bacteria, were
added to the enzyme to bring the final volume to 50
ml.
This mixture was cycled 5 times with a TaKaRa PCR
Thermal Cycler 480 for 1 minute at 95 °C, 2 minutes
at 50 °C, and 3 minutes at 72 °C and was then cycled
25 times for 1 minute at 95 °C, 2 minutes at 60 °C,
and 3 minutes at 72 °C. Electrophoresis was performed
in 1.5 % agarose gel using some of this PCR product.
This product was then stained with ethidium bromide
(Nihon Gene Co., ltd.) and observed under ultraviolet
rays Ud confirm amplification of approximately 400 bp
cDNA. After digestion treatment with restriction
endonucleases BamHI and Xhol, electrophoresis was
performed in 1.5 % agarose gel and staining with
ethidium bromide was carried out. An approximately
370 bp band was cut out from the gel. This band was
purified with SuprecOl (Takara Co., Ltd.) and then
inserted into pGEX-4T-l (Pharmacia), which is a
commercial vector. Actually, vector pGEX-4T-l and the
previous DNA were mixed together at a molar ratio of
1:3 and DNA was inserted into the vector with T4 DNA
ligase (Invitrogen Co.). Vector pGEX-4T-l into which
DNA had been inserted was genetically introduced to
Escherichia coli One-Shot Competent Cells (Invitrogen
Co., Ltd.) and then inoculated in a plate of L-Broth
(Takara Co., Ltd.) semi-sold culture plate containing
50 mg/ml ampicillin (Sigma). The plate was then set
aside at 37 °C for 12 hours and the colonies that
grew were selected at random and inoculated into 2ml
L-Broth liquid culture medium containing the same
concentration of ampicillin. Shake cultivation was
performed at 37°C for 8 hours and the bacteria was
recovered and the plasmid was separated using Wizard
Miniprep in accordance with the attached literature.
The plasmid was cleaved with restriction endonuclease
BamHI/XhoI. Insertion of said PCR product was
confirmed by cutting out approximately 370 bp DNA.
The base sequence of the DNA that had been inserted
was determined using said clone.
Determination of the base sequence of the
inserted DNA fragment was performed using the
Fluorescence Sequencer of Applied Biosystems. The
sequence sample was prepared using PRISM, Ready
Reaction Dye Terminator Cycle Sequencing Kit (Applied
Biosystems). First, 9.5 ml reaction stock solution,
4.0 ml T7 promoter primer at 0.8 pmol/ml (Gibco BRL)
and 6.5 ml template DNA for sequencing at 0.16 mg/ml
were added to a microtube with a capacity of 0.5 ml
and mixed. After layering with 100 ml mineral oil,
PCR amplification was performed for 25 cycles, where
one cycle consisted of 30 seconds at 96 °C, 15
seconds at 55 °C, and 4 minutes at 60 °C. The product
was then kept at 4 °C for 5 minutes. After the
reaction was completed, 80 ml sterilized pure water
was added and stirred. The product was centrifuged
and the aqueous layer was extracted 3 times with
phenol-chloroform. Ten microliters 3 M sodium
acetate (pH 5.2) and 300 ml ethanol were added to 100
ml aqueous layer and stirred. The product was then
centrifuged for 15 minutes at room temperature and
14000 rpm and the sediment was recovered. Once the
sediment was washed with 75 % ethanol, it was dried
under a vacuum for 2 minutes to obtain the sequencing
sajnple. The sequencing sample was dissolved in
formamide containing 4 pi 10 mM EDTA and denatured
for 2 minutes at 90 °C. This was then cooled in ice
and submitted to sequencing. One of the 5 clones
obtained had homology of the sequence with the probe
used for PCR. In addition, DNA sequences extremely
similar to the gene sequence of ribosomal protein
L7/L12 gene of the other microorganisms, for example,
Haemophilus influenzae, were discovered. The entire
base sequence and the corresponding amino acid
sequence of the structural gene moiety are as shown
in Sequence No. 21 and No. 22 of the Sequence Table.
This gene fragment clearly codes for ribosomal
protein L7/L12 gene of Neisseria gonorrhoeae.
Neisseria gonorrhoeae fusion GST ribosome protein
L7/L12 prepared by the same method as in Example 2
was obtained using the Neisseria gonorrhoeae fusion
GST ribosomal protein L7/L12 expression vector
constructed in this way.
Furthermore, hybridoma strain GCRB-3, which
produces monoclonal antibody to ribosomal protein
L7/L12 of Neisseria gonorrhoeae, was obtained in
accordance with the method in Example 3.
Example 8
Reaction of monoclonal antibody that reacts with
ribosomal protein L7/L12 of. Neisseria gonorrhoeae,
with Neisseria gonorrhoeae and other microorganisms
Monoclonal antibody was produced and recovered in
accordance with standard me^ttrods using the positive
hybridoma cells GCRB-3 obtained as previously
described.
Basically, 5 x 106 cells (in PBS) that had been
subcultured using RPMI 1640 culture medium
(containing 10 % FCS) were intraperitoneally injected
into Balb/C mice that had been intraperitoneally
injected with 0.5 ml Pristane 2 weeks earlier.
Ascites was recovered 3 weeks later and the
centrifugation supernatant was obtained.
The solution containing antibody that was
obtained was adsorbed in a Protein A column (5 ml
bed, Pharmacia) and washed with 3-bed volume of PBS.
Then eluted with citrate buffer at pH 3, the antibody
fraction was recovered and the monoclonal antibody
that was produced by each hybridoma was obtained.
The monoclonal antibody derived from the GCRB-3
hybridoma was used in ELISA.
The sandwich assay method was used to assess the
monoclonal antibody. The monoclonal antibody that
was prepared was used as an enzyme-labeled antibody
by being chemically bound to peroxidase. That is,
enzyme labeling was performed in accordance with the
method in "Analytical Biochemistry" 132 (1983), 68-73
with the reagent S-acetylthioacetic acid N-
hydroxysuccinimide for binding using horseradish
peroxidase (Sigma Grade VI). By means of the ELISA
reaction, a solution of commercial antri Neisseria
gonorrhoeae polyclonal antibody in PBS containing
0.05% sodium azide(Virostat, rabbit) was diluted to a
concentration of 10 mg/ml and poured, 100 ml at a
time, into a separate 96-well plate and adsorbed
overnight at 4 °C.
After removal of the supernatant, 200 ml 1 %
bovine serum albumin solution (in PBS) were added and
reacted for 1 hour and blocked at room temperature.
The supernatant was removed and the product was
washed with washing solution (0.02 % Tween 20, PBS).
One-hundred microliters of antigen solution, which
had been obtained by adding Triton X-100 to culture
solutions of each species of microorganisms to a
concentration of 0.3 % and then extracting the
solution for 5 minutes at room temperature, were
added to this and reacted for 2 hours at room
temperature. The supernatant was removed and the
product was further washed with washing solution.
Then 100 ul peroxidase-labeled anti-ribosomal protein
L7/L12 antibody solution at 5 ug/ml were added and
reacted for 1 hour at room temperature. The
supernatant was removed and the product was washed
with washing solution. TMB (KPL) solution was added,
100 ul at a time, and reacted for 20 minutes at room
temperature. After coloration, 100 ul 1 N sulfuric
acid were added to stop the reaction. Absorbance at
450 nm was determined.
As a result, as shown in Table 3 it is clear that
when monoclonal antibody derived from hybridoma GCRB-
3 was used as the enzyme-labeled antibody, all
strains of Neisseria gonorrhoeae tested were detected
at a sensitivity of 106 cells/ml, while reactivity of
other species belonging to the genus Neisseria and
other microorganisms could not be detected, even at
high concentrations of 108 cells/ml and therefore,
antibody with specific reactivity tc Neisseria
gonorrhoeae can be obtained by using monoclonal
5 antibody to ribosomal protein L7/L12.
Table 3
10 (+: Positive; -: Negative)
Example 9
Acquisition of ant-ribosomal protein L7/L12
monoclonal antibody specific to genus Neisseria
After inoculating an appropriate amount of
Neisseria gonorrhoeae strain IID821 (obtained from
Tokyo University School of Medicine Laboratories) in
a chocolate agar culture medium, the strain was
cultivated for 24 hours in a CO2 incubator under
conditions of 37 °C and 50% CO2. The colonies
that grew were suspended in a TE buffer to a final
concentration of approximately 5 x 109 CFU/ml.
Approximately 1.5 ml of this suspension was
transferred to a microcentrifugation tube and
centrifuged for 2 minutes at 10,000 rpm. The
supernatant was discarded. The sediment was
resuspended in 567 pi TE buffer. Then 30 pi 10 % SDS
and 3 pi 20 mg/ml Proteinase K solution were added
and thoroughly mixed. The suspension was incubated
for another hour at 37 °C. Next, after adding 80 pi
10 % cetyl trimexhyl ammonium bromide/0.7 M NaCl
solution and thoroughly mixing the product, it was
incubated for 10 minutes at 65 °C. Next, 700 pi
chloroform-isoamyl alcohol solution at a volume ratio
of 24:1 was raddled and stirred well.
The solution was centrifuged for 5 minutes (while
being kept at 4 °C) at 12,000 rpm using a
microcentrifugation device and the aqueous fraction
was transferred to a new microtube. Isopropanol was
added to the fraction at 0.6-times its volume and the
tube was vigorously shaken to form sediment of the
DNA. The white DNA sediment was scooped with a glass
rod and transferred to a different
microcentrifugation tube containing 1 ml 70 % ethanol
(cooled to -20 °C) .
Next, the product was centrifuged for 5 minutes
at 10,000 rpm and the supernatant was gently removed.
Then another 1 ml 70 % ethanol was added and the
product was centrifuged for 5 more minutes. Once the
supernatant had been removed, the sediment was
dissolved in 100 ml TE buffer to obtain the DNA
solution. The concentration of the genomic DNA
solution was determined quantitatively in accordance
with E5, Spectrophotometry determination of the
amount of DNA or RNA, "Molecular cloning: A
laboratory manual," 1989, Eds. Sambrook, J., Fritsch,
E.F., and Manxatis., T. , Co It* o-gnrrng Harbor Laboratory
Press.
PCR was performed using 10 ng of this genomic DNA.
Taq polymerase (Takara Co., Ltd., code R001A) was
employed for PCR. Then, 5 ml buffer attached to
enzyme, 4 ml. dNTP mixture attached to enzyme, and 200
pmol each of synthetic oligonucleotide E shown in
Sequence No. 15 of the Sequence Table and synthetic
oligonucleotide F shown in Sequence No. 16 of the
Sequence Table, which were designed based on the
ribosomal protein L7/L12 DNA sequence of Neisseria
gonorrhoeae acquired from Internet information
(Oklahoma University, N. Gonorrhoeae Genome Project,
disclosed genome DNA data) because of the similarity
with ribosomal protein L7/L12 DNA sequence of other
bacteria, were added to the enzyme to bring the final
volume to 50 ml .
This mixture was cycled 5 times with a TaKaRa PCR
Thermal Cycler 480 for 1 minute at 95 °C, 2 minutes
at 50 °C, and 3 minutes at 72 °C and was then cycled
25 times for 1 minute at 95 °C, 2 minutes at 60 °C,
and 3 minutes at 72 °C. Electrophoresis was performed
in 1.5 % agarose gel using some of this PCR product.
This product was then stained with ethidium bromide
(Nihon Gene Co., ltd.) and observed under ultraviolet
rays to confirm amplification of approximately 400 bp
cDNA. After digestion treatment with restriction
endonucleases BamHT and Xhcl, electrophoresis was
performed in 1.5 % agarose ^e-L and staining with
ethidium bromide was carried out. An approximately
370 bp band was cut out from the gel. This band was
purified with SuprecOl (Takara Co., Ltd.) and then
inserted into pGEX-6P-l (Pharmacia) , which is a
commercial vector. This same vector can function as
an expression vector for the desired molecule, which
can express fused protein with GST protein, by
insertion of the desired gene fragment into the
appropriate restriction endonuclease site. Actually,
vector pGEX-€P-l and the previous DNA were mixed
together at a molar ratio of 1:5 and DNA was inserted
into the vector with T4 DNA ligase (Invitrogen Co.).
Vector pGEX-6P-l into which DNA had been inserted was
genetically introduced tx> Escherichia coli One-Shot
Competent Cells (Invitrogen Co., Ltd.) and then
inoculated in a plate,of L-Broth (Takara Co., ltd.)
semi-sold culture plate containing 50 ug/ml
ampicillin (Sigma) . The plate was then set aside at
37 °C for 12 hours and the colonies that grew were
selected at random and inoculated into 2 ml L-Broth
liquid culture medium containing the same
concentration of ampicillin. Shake cultivation was
performed at 37 °C for 8 hours and the bacteria was
xecovered and the plasmid was separated using Wizard
Miniprep in accordance with the attached literature.
The plasmid was cleaved with restriction endomiclease
Bamfll/Xhol. Insertion of said *CR product »a&
confirmed by cutting out approximately 370 bp DNA.
The base sequence of the DNA that had been inserted
was determined using said clone.
Determination of the base sequence of the
inserted DNA fragment was performed using the
Fluorescence Sequencer of Applied Biosystems. The
sequence sample was prepared using PRISM, Ready
Reaction Dye Terminator Cycle Sequencing Kit (Applied
Biosystems) . First, 9.5 pi reaction stock solution,
4.0 ml T7 promoter primer at 0.8 putol/pl (Gibco BRL)
and 6.5 ml template DNA for sequencing at 0.16 pg/pl
were added to a microtube with a capacity of 0.5 ml
and mixed. After layering with 100 pi mineral oil,
PCR amplification was performed for 25 cycles, where
one cycle consisted of 30 seconds at 96 °C, 15
seconds at 55 °C, and 4 minutes at 60 °C. The product
was then kept at 4 °C for 5 minutes. After the
reaction was completed, 80 pi sterilized pure watex
was added and stirred. The product was centrifuged
and the aqueous layer was extracted 3 times with
phenol-chloroform. Ten microliters 3 M sodium
acetate (pH 5.2) and 300 pi ethanol were added to 100
ml the aqueous layer and stirred. The product was
then centrifuged for 15 minutes at room temperature
and 14000 rpm and the sediment was recovered. Once
the sediment was washed with 75 % ethanol, it was
dried under a vacuum for 2 minutes to obtain the
sequencing sample. The sequencing sample was
dissolved In formamide containing 4 pi 10 mM EDTA and
denatured for 2 minutes at 90 °C. This was then
cooled in ice and submitted to sequencing
One of the 4 clones obtained had homology of the
sequence with the probe used for PCR. In addition,
DNA sequences extremely similar to the gene sequence
of ribosomal protein L7/L12 gene of the other
microorganisms, for example, Haemophilus influenzae ,
were discovered. The entire base sequence and the
corresponding amino acid sequence of the structural
gene moiety are as shown in Sequence ID No. 21 and No.
22 of the Sequence Table. This gene fragment clearly
codes for Neisseria gonorrhoeae ribosomal protein
L7/L12.
50 ml Escherichia coli into which expression
vector had been inserted was cultivated overnight in
a two-fold concentration YT medium at 37 °C. Then,
450ml of the two-fold concentration YT medium was
heated at 37 °C for 1 hour. 50 ml of the Escherichia
coli solution that had been cultivated overnight was
introduced to 450ml of the aforementioned medium.
After cultivation for one hour at 37 °C, 100 ul 500
mM IPTG was introduced and cultivated for 4 hours at
25 °C. The product was then recovered and introduced
2 50 ml each to a centrifugation tube and centrifuged
tor 20 minutes at 5000 rpm. The supernatant was
discarded and dissolved in 25 ml each 50 mM Tris-HCl
at a pH of 7.4 and- Lysis buffer containing 25 %
sucrose.
Furthermore, 1.25 ml 10% NP-40 and 123 ml 1 M
MgCla were added and transferred to a plastic tube.
Sonication was performed 1 minute x 5 times while ice
cold. The product was centrifuged for 1.5 minutes at
12,000 rpm and the supernatant was recovered.
Next, the aforementioned supernatant was adsorbed
on a glutathione sepharose column {manufactured by
Pharmacia) conditioned with PBS. Then, the column was
washed with PBS three times the bed volume. Elution
was performed with 50 mM Tris-HCl at a pH of 8.0
containing 10 mM glutathione. The protein content in
the fraction was determined by the pigment bonding
method (Bradford method; BioRad Co.) and the main
fraction was acquired. The main fraction was
dialyzed three times against 3L PBS.
1 ml of a cleavage buffer containing 500 mM Tris-
HCl (pH 7.0), 1.5 M NaCl, 10 mM EDTA, and 10 mM DTT
was added to 10 ml of 1 mg/ml solution of the.
resulting GST fusion ribosomal protein L7/L12. 100
ml of 2 u/ml PreScission Protease (manufactured by
Pharmacia company) was further added and reacted at 4
°C to separate the GST moiety from ribosomal protein
L7/LI2.
The reaction solution was passed through a
glutathione sepharose column which had been
conditioned with PBS. The solution coming out from
the column was recovered. One bed volume of PBS was
passed through and also recovered. Purity of the
purified ribosomal protein L7/L12 that was obtained
was confirmed by electrophoresis to be approximately
90 %, showing that a purity satisfactory for an
immunogen could be guaranteed.
First, 100 mg protein antigen of ribasomal
protein L7/L12 of Neisseria gonorrhoeae was dissolved
in 200 ml PBS and then 200 ul Freund's complete
adjuvant was added and mixed and emulsification was
performed. 200 ml was intraperitoneally injected to
immunize mice. Then, the same emulsion antigen was
intraperitoneally injected after 2 weeks, after 4
weeks, and after 6 weeks. A two-fold concentration
antigen emulsion was further injected
intraperitoneally after 10 weeks and after 14 weeks.
The spleen was excised 3 days after the final
immunization and submitted to cell fusion.
After thoroughly mixing 2 x 107 myeloma cells per
108 spleen cells from mice, which had been recovered
aseptically, in a glass tube, the mixture was
centrifuged for 5 minutes at 1500 rpm and the
supernatant was discarded. The cells were thoroughly
mixed.
The myeloma cells used for cell fus-ion were
obtained by cultivation of cell strain NS-1 with an
RPMI 1640 culture medium containing 10 %"FCS,
cultivating this product beginning 2 weeks before
cell -frrsion using an RPMI 1640 medium containing 0.13
raM azaguanine, 0.5 ug/ml MC-210, and 10 % FCS for 1
weeks, and then further cultivating the cell strain
for 1 week with an RPMI 1640 medium containing 10 %
FCS. 30 ml of RPMI 1640 culture medium 50 ml that had
been kept at 37°C was added to the mixed cell sample
and centrifuged at 1,500 rpm. After removal of the
supernatant, 1 ml 50 % polyethylene glycol that had
been kept at 37 °C was added and stirred for 2 minute.
10 ml RPMI 1640 medium kept at 37 °C was added and
the -solution was vigorously mixed for approximately 5
minutes as it was suctioned and evacuated from a
sterile pipette.
After centrifugation for. 5 minutes at 1,000 rpm
and removal of the supernatant, 30 ml HAT culture
medium were added to bring the cell concentration to
5 x 106 cells/ml. This mixture was stirred till
uniform and then poured, 0.1 ml at a time, into a 96-
well culture plate and cultivated at 37 °C under 7 %
C02. HAT medium was added,. 0.1 ml at a time, on Day 1
and at Week 1 and Week 2.
Then the cells that had produced the desired
antibody were assessed by ELISA. Solutions o-f
xifeesomaJl protein L7/L12 of Neisseria gonorrhoeae
dissolved in PBS containing 0.05 % sodium azide were
diluted to 10 ug/ml and were poured, 100 ul at a time,
into separate 96-well plates and adsorbed overnight
St. 4 °C^ After removal of the supernatant, 200 ul 1 %
bovine serum albumin solution (in PBS) were added and
reacted and blocked for 1 hour at room temperature.
The supernatant was removed and the product was
washed with a washing solution (0.02 % Tween 20, PBS).
100 ul cf a culture solution of fus^ron cells was
added and reacted for two hours ^at room temperature.
The supernatant was removed and the product was
further washed with a washing solution. Then, 100 ul
of a peroxidase-labeled goat anti-mouse antibody
solution at 50 ng/ml was added and reacted for one
hour at room temperature. The supernatant was
removed and the product was washed with a washing
solution. TMB (KPL) solution was added, 100 ul at a
time, and reacted for 20 minutes at room temperature.
After coloration, 100 pi 1 N sulfuric acid were added
to stop the reaction. Absorbance at 450 nm was
determined.
As a result, positive wells that reacted with
ribosomal protein L7/L12 were detected, confirming
presence of the antibody to ribosomal protein L7/L12.
Therefore, the cells in the positive wells were
recovered and cultivated with HAT medium in a 24-well
pla-&td;c plate. The fused -medium that had been
cultivated was diluted with an HT medium to a cell
count of approximately 20 cells/ml. Then 50 ul the
medium was mixed with 106 six-week-old mouse thyroid
©ells suspended in the HT medium in a 9 6-well culture
plate. The cells were cultivated for 2 weeks at 37 °C
under the conditions of 7 % C02. The antibody titer in
the culture supernatant was determined by the
aforementioned ELISA method and, the cells those
showed a positive reactian with ribosomal protein
L7/L12 were recovered. Furthermore, the same dilution
and cloning procedure was repeated to obtain a total
of 4 clones of hybridoma AMGC-5 to AMGC-8.
Monoclonal antibody was produced and recovered
according to standard methods, using the positive
hybridoma cells obtained as previously described.
Specifically, cells subcultured in RPMI 1640
medium (containing 10 % FCS) was diluted in serum-
free medium to about 2 x 105 cells/ml, 3.3 x 105
cells/ml, and 5 x 10s cells/ml in 25 cm2 culture flask,
and the total was made 5 ml. After cells were grown
for 3 to 5 days in 7 % C02 at 37 °C, flask that
contains the least number of original cells was
selected among flasks in which cells were grown. The
same procedure was repeated until the cells diluted
to 2 x 105 cells/ml grow to 2 x 106 cells/ml in 3 to 4
days, iifaereby -abri-ftfrt tag the cells with serum-free
medium, Sext, cioaincf *as performed in a 96-well
plate for bacterial culture to select cells
exhibiting fastest growth and a highest antibody
titer. The selected cells were grown in a 24-well
plate and diluted with a serum free medium in a 25
cm2 culture flask to a concentration of about 2 x 105
cells/ml and the total volume was made to 10 ml.
After incubation for 3 to 4 days in 7 % CO2 at 37°C
to a concentration of 1 x 106 cells/ml, the culture
broth 100 ml, 1 x 106 cells/ml was transferred to a
bottle for mass cultivation which were grown in the
same manner in a 75 cm2 flask. 100 ml of a serum-free
medium was added to the mixture and was incubated at
37 °C for two days while stirring. 200 ml of the
serum-free medium was added again and the mixture was
incubated for a further two days. The culture broth
was divided into four aliquots, one volume serum-free
medium was added to each portion, followed by
incubation for two days. After further addition of
400 ml of the serum-free medium, the culture broth
was incubated for 6 days. The culture broth was
collected and centrifuged at 10,000 rpm for 15
minutes to obtain culture supernatant containing the
target antibody. After adding sodium azide to final
concentration 0.1 %, the culture supernatant was
stored at 4 °C. 100 ml of the supernatant containing
the antibody was dri luted 5--xjaict with PBS and adsorbed
in a protein G colasm- {5 ml toed, Pharmacia) and
washed with 3-bed volume of PBS. Then eluted with
citrate buffer at pH 3, the antibody fraction was
recovered and monoclonal antibody produced by each
hybridoma was obtained.
The monoclonal antibodies originating from the
four hybridoma clones were evaluated according to the
OIA method described in International Patent
Application Japanese Laid-open No. 509565/1995.
Specifically, the CIA method comprises preparing
a reactive substrate by reacting an antibody for
capture on a silicon wafer having a thin film layer
of silicon nitride, causing this substrate to react
with an antigen which is an extract of microorganisms
for a prescribed period of time, causing the captured
antigen to react with an antibody (an amplification
reagent) which is an enzyme-labeled antibody, and
finally adding a substrate solution to produce a
thin-film precipitate. The antigen-antibody reaction
can be judged visually by a degree of light,
interference color produced in the precipitate.
The monoclonal antibody preparation was used as a
capture antibody to be immobilized on a silicon wafer
having a silicon nitride thin film layer in the OIA
method. Moreover, peroxidase-labeled AMGC-i
monoclonal antibody which can ntm-specifically react
with ribosomal proteins L7/L.12 protein of a variety
of" microorganisms described in 8efer«ace Eac-ample was
used as the detect antibody. That is, enzyme
labeling was performed in accordance with the method
in "Analytical Biochemistry" 132 (1983), 68-73 with
trhe reagent S-acetylthioacetic acid N-
hydroxysuccinimide for binding using horseradish
peroxidase (Sigma Grade VI).
In the OIA reaction, monoclonal antibody in a PBS
containing 0.05 % sodium azide was diluted with 0.1 M
HEPES (pH.8.0) to a concentration of 10 jig/ml and
added onto a silicone wafer which has a thin film
layer of silicon nitride, 50 ml at a time, to react
for 30 minutes at room temperature, followed by
washing with distilled water and use. 15 ml of
antigen solution, which had been obtained by adding
0.5 % Triton X-100 to culture suspension of various
species of microorganisms and then extracting the
suspension for 5 minutes at room temperature, was
added to the specimen obtained in the above-described
procedure and reacted for 10 minutes at room
temperature. Then, 15 ml of 20 mg/ml peroxidase-
labeled AMGC1 was added and reacted for 10 minutes.
After washing with distilled water, a substrate
solution (KPL Co.) was added, 15 ml at a time, and
reacted for 5 minutes at room temperature. The
product was washed with distilled water to judge the
concentration of detection signals as an intensity of
light interference by naked eyes.
As a result, as shown in Table 4 it is clear that
when monoclonal antibody derived from hybridoma AMGC-
8 was used as the capture antibody, all strains of
Neisseria genus tested were detected at a sensitivity
of 108 cells/ml, while reactivity of other
microorganisms could not be detected. Thus, the
antibody with specific reactivity to Neisseria genus
was confirmed to have been obtained by using the
monoclonal antibody to ribosomal protein L7/L12 .
5 Table 4
(+: Positive; -: Negative)
10 Example 10
Acquisition of a polyclonal antibody which
specifically reacts with ribosomal protein L7/L12 of
Haemophilus influenzae using a ribosomal protein
L7/L12 protein-immobilized affinity column
A centrifuqal supernatant of Haemophilus
influenzae cell extract, which had been treated with
0.5% Triton X-100 was used as an antigen. About 1.2
ml of a physiological saline solution containing 100
mg of antigen was emulsified with the addition of 1.5
ml of Freund's adjuvant. The emulsion was
subcutameously injected into four SPF Japanese White
Rabbits to immunize the animals. The rabbits were
immunized 5 to 6 times, once every two weeks, and the
antibody titer was confirmed.
The antibody titer was confirmed by the ELISA
method. Solutions of ribosomal protein L7/L12 of
Haemophilus influenzae dissolved in PBS containing
0.05 % sodium azide diluted to 10 ug/ml were poured,
100 ul at a time, into 96-well plates and adsorbed
overnight at 4 °C. After removal of the supernatant,
200 pi 1 % bovine serum albumin solution (in PBS)
were added and reacted and blocked for 1 hour at room
temperature. The supernatant was removed and the
product was washed with a washing solution (0.02 %
Tween 20, PBS). 100 ul of a solution obtained by
diluting normal rabbit serum and immunized rabbit
antiserum was added and reacted for two hours at room
temperature. The supernatant was removed and the
product was further washed with a washing solution.
Then, 100 ul of a peroxidase-labeled goat anti-rabbit
IgG antibody solution at 50 ng/ml was added and
reacted for one hour at room temperature. The
supernatant was removed and the product was washed
with a washing solution. OPD solution (Sigma Co.)
was added, 100 ul at a time, and reacted for 20
minutes at room temperature. After coloration, 100
ml of 1 N sulfuric acid was added to stop the
reaction. Absoriance at 492 nm was determined.
After confirming that the antibody titer had
increased, a large quantity of blood was collected.
Blood was collected in a centrifugal tube made of
glass from the ear artery, allowed to stand for one
hour at 37 °C, then overnight at 4 °C. The mixture
was centrifuged at 3000 rpm for 5 minutes and the
supernatant was recovered. The resulting 4 lots of
anti-serum were stored at 4 °C.
Next, an affinity column with immobilized
ribcLSoraal protein L7/L12 of Haemophilus influenzae
and Neisseria gonorrhoeae was prepared. HiTrap NHS-
activated column (1 ml, manufactured by Pharmacia)
was used. Immediately after replacing the column
with 6 ml of 1 mM HC1, 1 ml of a PBS solution of
ribosomal protein L7/L12 adjusted to 1 mg/ml was
charged. The column was allowed to stand for 15
minutes -at 25 °C. This procedure was repeated 5
times, thereby feeding the total 5 ml of the PBS
solution of ribosomal protein L7/L12. Then, 6 ml of
Buffer A (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3), 6
ml of Buffer B (0.1 M acetic acid, 0.5 M NaCl, pH 4),
and 6 xrl of Buffer A were charged as blocking
reagents. After allowing to stand for 15 minutes at
25 °C, 6 ml of Buffer B, 6 ml of Buffer A, and 6 ml
of Buffer B were further added. The mixture was then
equilibrated with 6 ml of PBS.
Using tiis. affinity column with immobilized
riirosomal protein L7/L12 of Baaemophilus influenzae,
the polyclonal antibody in the resulting anti-serum
was purified using the supernatant of Triton X-100
treated bacteria of Haemophilus influenzae as an
antigen. This antiserum was first diluted with PBS to
a volume of 5 times, passed through a 0.45 urn filter,
then caused to be adsorbed in the column immobilized
with ribosomal protein L7/L12 of Haemophilus
Influenzae at a flow rate of 0.5 ml/min. After
elntion from the column- with 0.1 M glycine (pH 2.1)
and immediately neutralizing with 1 M Tris-HCl (pH
9.0), eluted fractions of the target antibody were
recovered by the ELISA method, the same as anti-titer
measuring method. These fractions were passed
through the affinity column immobilized with the
ribosomal protein L7/L12 of Neisseria gonorrhoeae,
which was equilibrated with PBS whereby the antibody
that reacts with the ribosomal protein L7/L12 of
Neisseria gonorrhoeae was adsorbed and fraction which
passed through without adsorption was recovered.
The polyclonal antibody purified in this manner
«as evaluated -by the same OIA method as in Example 6.
The purified antibody was used as a capture
antibody for the OIA method. Moreover, peroxidase-
labeled AMGC-1 monoclonal antibody described in
Reference Example was used as the detect antibody.
That is, enzyme labeling was performed in accordance
with the method in "Analytical Biochemistry" 132
(1983), 68-73 with the reagent S-acetylthioacetic
acid N-hydroxysuccinimide for binding using
horseradish peroxidase (Sigma Grade VI) .
In the OIA reaction, the purified polyclonal
antibody in a PBS containing 0.05 % sodium azide was
diluted with 0.1 M HEPES (pH 8.0) to a concentration
of 10 ml/ml and added onto a silicone wafer, 50 ml at
a time, to react for 30 minutes at room temperature,
followed by washing with distilled water and use.
15 ml of antigen solution, which had been
obtained by adding 0.5 % Triton X-100 to culture
suspension of various species of microorganisms and
then extracting the suspension for 5 minutes at room
temperature, was added to the specimen obtained in
•the above-described- procedure and reacted for 10
minutes at room temperature. Then, 15 ml of 20 mg/ml
peroxidase—laiyeled AMGC1 was added and reacted for 10
minutes. After washing with distilled water, a
substrate solution (KPL) was added, 15 ml at a time,
and reacted for 5 minutes at room temperature. The
product was washed with distilled water to observe
the blue color concentration by the naked eyes.
As a result, as shown in Table 5 it is clear that
when the purified polyclonal antibody of APHI2-2 was
used as the capture antibody, Haemophilus influenzae
tested at a sensitivity of 10% bacteria/ml was
detected, while reactivity ctf other microorganisms
could not be detected. Thus, the antibody with
specific reactivity to Haemophilus influenzae was
confirmed to have been obtained by using the
polyclonal antibody purified by an affinity column
immobilized with the ribosomal protein L7/L12 of
Haemophilus influenzae.
Table 5
15 (+: Positive; -: Negative)
Reference Example 1
Acquisition of monoclonal antibody that reacts non-
specificaHy with ribosomal protein L7/L12 of various
bacteria
So called sandwich assay in which an antigen is
sandwiched between a capture antibody and a labeled
antibody for detection is useful to detect
microorganisms by optical immunoassay and the ELISA
method because of its high detection sensitivity. In
this instance, not only an antibody which
specifically reacts with antigens originating from
the subject microorganism, but also another antibody
which recognizes an antigen epitope different from
that of the specific antibody are required.
Antibodies which react non-specifically with
ribosomal protein L7/L12 originating various
microorganisms are very useful as an antibody which
can constitute a sandwich assay with an antibody
which reacts specifically with ribosomal protein
L7/L12.
Fortunately, ribosomal proteins L7/L12 protein of
a variety of aler-ooxejanisms fearste * region wherein the
amino acid sequence is homologous. Here, the
inventors have been successful in acquiring a
monoclonal antibody which exhibits a cross reaction
with ribosomal proteins L7/L12 protein of various
species of microorganisms from Neisseria gonorrhoeae.
It has been discovered that an antibody to anti-
ribosomal protein L7/L12 having no specificity, which
was acquired from one species of microorganisms can
be used for sandwich assay cf several kinds of
microoxganisms .
Cloning of ribosomal protein L7/L12 genes from
Neisseria gonorrhoeae, mass expression in Escherichia
coli and purification of the same protein, and
preparation of monoclonal antibody to the same
protein were performed.
After inoculating an appropriate amount of
Neisseria gonorrhoeae strain IID821 (obtained from
Tokyo University School of Medicine Laboratoxies) in
a chocolate agar culture medium, tke strain, w-as
cultivated for 24 hours in a C02 incubator under
conditions of 37 °C ancjf &*8xxj&u©cx % C02 • The colonies
that grew were suspended in a TE buffer to a final
concentration of approximately 5 x 109 CFU/ml.
Approximately 1.5 ml of this suspension was
transferred to a microcentrifugation tube and
centrifuged for 2 minutes ^X -10,£-&fl rpnu Jtixe:
supernatant was discarded. The sedas&ent «&
resuspended in 567 ul TE buffer. Then 30 ul 10 % SDS
and 3 ul 20 mg/ml Proteinase K solution were added
and thoroughly mixed. The suspension was incubated
for another hour at 37oC. Next, after addiag 80 ml
10 % cetyl trimethyl ammonium bromide/0.7 M NaCl
solution and thoroughly mixing the product, it was
incubated for 10 minutes at 65 °C. Next, 700 ml
chloroform-isoamyl alcohol solution at a volume ratio
of 24:1 was added and stirred well. The solution was
centrifuged for 5 minutes (while being kept at 4 °C)
at 12,000 rpm using a microcentrifugation device and
the aqueous fraction was transferred to a new
microtube. Isopropanol was added to the fraction at
0 .6-times its volume and the tube was vioporously
shaken to form sediment of the DNA. The white DNA
sediment was scooped with a glass rod and transferred
to a different microcentrifugation tube containing 1
ml 70 % ethanol (cooled to -20 °C) .
Next, the product was centrifuged fox 5 ttrinutes
at 10,000 rpm and the supernatant was gently removed.
Then another 1 ml 70 % ethanol was added and the
product was centrifuged for 5 more minutes. Once the
supernatant had been removed, the sediment was
dissolved in 100 ul TE buffer to obtain the DNA
solution. The concentration of the genomic DNA
solution was determined quantitatively is ^acronisDoe
with E5, Spectrophotometry determination «itfte
amount of DNA or RNA, "Molecular cloning: A
laboratory manual," 1989, Eds. Sambrook, J., Fritsch,
E.F., and Maniatis, T Cold Spring Harbor Laboratory
Press.
PCR was performed using 10 ng of this genomic
DNA. Taq polymerase (Takara Co., Ltd., code R001A)
was employed for PCR. Then, 5 ml of a buffer
attached to enzyme, 4 ml of dNTP mixture attached to
enzyme, and 2 00 pmol of each of synthetic
oligonucleotide E shown in Sequence No. 15 of the
Sequence Table and synthetic oligonucleotide F shown
in Sequence No. 16 of the Sequence Table, which were
designed based on the ribosomal protein L7/L12 DNA
sequence of Neisseria gonorrhoeae acquired from
Internet information (Oklahoma University, N.
Gonorrhoeae Genome Project, disclosed genomic DNA
data) because of the similarity with ribosomal
protein L7/L12 DNA sequence of other bacteria, were
added to the enzyme to bring the final volume to 50
ml.
This mixture was cycled 5 times with a TaKaRa PCR
Thermal Cycler 480 for 1 minute at 95 °C, 2 minutes
at 50 °C, and 3 minutes at 72 °C and was then cycled
25 times for 1 minute at 95 °C, 2 minutes at 60 °C,
and 3 minutes at 72 °C. Electrophoresis was performed
in 1.5 % agarose gel using some of this PCR product.
This product was then stained with ethidium bromide
(Nihon Gene Co., ltd.) and observed under ultraviolet
rays to confirm amplification of approximately 400 bp
cDNA. After digestion treatment with restriction
endonucleases BamHI and Xhol, electrophoresis was
performed in 1.5 % agarose gel and staining with
ethidium bromide was carried out. An approximately
370 bp band was cut out from the gel. This band was
purified with SuprecOl (Takara Co., Ltd.) and then
inserted into pGEX-4T-l (Eharmacia) , which is a
commercial vector. Actually, vector pGEX-4T-l and
the previous DNA were mixed together at a molar ratio
of 1:3 and DNA was inserted into the vector with T4
DNA ligase (Invitrogen Co.). Vector pGEX-4T-l into
which DNA had been inserted was genetically
introduced to Escherichia coli One-Shot Competent
Cells (Invitrogen Co., Ltd.) and then inoculated in a
plate of L-Broth (Takara Co., Ltd.) semi-sold culture
plate containing 50 mg/ml ampicillin (Sigma). The
plate was then set aside at 37 °C for 12 hours and
the colonies that grew were selected at random and
inoculated into 2 ml L-Broth liquid culture medium
containing the same concentration of ampicillin.
Shake cultivation was performed at 37 °C for 8 hours
and the bacteria was recovered and the plasmid was
separated using Wizard Miniprep in accordance with
that attached literature. The plasmid was cleaved with
restriction endonuclease BamHI/XhoI. Insertion of
said PCR product was confirmed by cutting out
approximately 370 bp DNA. The base sequence of the
DNA that had been inserted was determined using said
clone.
Determination of the base sequence of the
inserted DNA fragment was performed using the
Fluorescence Sequencer of Applied Biosystems. The
sequence sample was prepared using PRISM, Ready
Reaction Dye Terminator Cycle Sequencing Kit (Applied
Biosystems). First-, 9.5 ptl reaction stock solution,
4.0 ul T7 promoter primer at 0.8 pmol/ul (Gibco BRL)
and 6.5 ul template DNA for sequencing at 0.16 ug/ul
were added to a microtube with a capacity of 0.5 ml
-and mixed. After superposition with 100 ul mineral
oil, PCR amplification was performed for 25 cycles,
where one cycle consisted of 30 seconds at 96 °C, 15
seconds at 55 °C, and 5 minutes at 60 °C. The product
was then kept at 4 °C for 4 minutes. After the
reaction was completed, 80 pi sterilized pure water
was added and stirred. The product was centrifuged
and the aqueous layer was extracted 3 times with
phenol-chloroform. Ten microliters 3 M sodium
acetate (pH 5.2) and 300 ul ethanol were added to 100
ul aqueous layer and stirred. The product was then
centrifuged for 15 minutes at room temperature and
t4:,0frG -rpm and the sediment was recovered. Once the
sse^imeai was washed warth 75 % ethanol, it was dried
under a vacuum for 2 minutes to obtain the sequencing
sample. The sequencing sample was dissolved in
formamide containing 4 ml 10 mM EDTA and denatured
for 2 minutes at 90 °C. This was then cooled in ice
and submitted to sequencing. One of the 5 clones
obtained had homology of the sequence with the probe
used for PCR. In addition, DNA sequences extremely
similar to the gene sequence of ribosomal protein
L7/L12 gene of the other microorganisms, for example,
Haemoophilus influenzae, were discovered. The entire
base sequence and the corresponding amino acid
sequence of the structural gene moiety are as shown
in Sequence No. 21 and No. 22 of the Sequence Table.
This gene fragment clearly codes for Neisseria
gonorrhoeae ribosomal protein L7/L12.
Neisseria gonorrhoeae GST fused ribosome protein
L7/L12 prepared by the same method as in Example 2
was obtained using the Neisseria gonorrhoeae GST
fusion ribosomal protein L7/L12 expression vector
constructed in this way. Furthermore, hybridoma
strain AMGC1, which produces monoclonal antibody to
ribosomal protein L7/L12 of Neisseria gonorrhoeae,
was obtained in accordance with the method similar to
the method of Example 3. Monoclonal antibody was
produced and recovered in accordance with standard
methods using the positive hybridoma ceils of AMGC1
strain obtained as previously described.
Specifically, cells subcultured in RPMI 1640
medium (containing 10 % FCS) was diluted with a
serum-free medium to about 2 x 105 cells/ml, 3.3 x 105
cells/ml, and 5 X 105 cells/ml in 25 cm2 culture
flasks, and the total was made 5 ml. After cell were
grown for 3 to 5 days in 7 % CO2 at 37 °C, a flask
which contains the least number of original cells was
selected among flasks in which cells were grown. The
same procedure was repeated until the cells diluted
to 2 x 105 cells/ml grow to 2 x 106 cells/ml in 3 to 4
days, thereby adapting the cells with the serum-free
medium. Next, cloning was performed in a 96-well
plate for bacteria cultivation to select cells
exhibiting fastest growth and a highest antibody
titer. The selected cells were grown in a 24-well
plate and diluted with a serum-free medium in a 25
cm2 culture flask to a concentration of about 2 x 10
cells/ml and the total volume was made 10 ml. After
incubation for 3 to A days in 7 % CO2 at 37 °C to a
concentration of 1 x 106 cells/ml, the culture broth
100 ml, 1 x 106 cells/ml was transferred to a bottle
for mass cultivation which were grown in the same
manner in a 75 cm2 flask. 100 ml of a serum-free
medium was added to the mixture, which was incubated
at 37 °C for two days while stirring. 2.00 ml of the
serum-free medium J*as- added ajgain and the mixture was
incubated for a. furtfrer two days. -Hie culture broth
was divided into four aliquots, the serum-free medium
was added to each portion, followed by incubation for
two days. After further addition of 400 ml of the
serum-free medium the culture broth was incubated
for 6 days. The culture broth was collected and
centrifuged at 10,000 rpm for 15 minutes to obtain a
culture supernatant including the target antibody.
After the addition of sodium azide to final
concentration 0 .1 %, the culture supernatant was
stored at 4 °C. 100 ml of the solution containing the
antibody that was obtained was diluted 5-fold with
PBS and adsorbed in a Protein A column (5 ml bed
volume, Pharmacia) and washed with 3-bed volume of'
PBS. Then eluted with citrate buffer at pH 3, the
antibody fraction was recovered and monoclonal
antibody produced by each hybridoma was obtained.
The monoclonal antibody derived from the hybridoma
was used in ELISA.
To evaluate the antibo-dy, 9-6-well plates
sensitized with ribosornal proteins L7/L12 of various
microorganisms were used as an antigen. The
monoclonal antibody prepared was reacted, followed by
the reaction of horseradish peroxidase-labeled anti-
mouse IgG (manufactured by MBL, Code 330) as a
secondary antigen, and finally the antibody was
detected using an eivz^sm x«actioT* cvl In the ELISA reaction, soU&tioms *>t r^csmtbiuan-t
ribosomal protein L7/L12 of Neisseria gonorrhoeae,
Haemophilus influenzae, and Streptococcus pneumoniae
dissolved in PBS containing 0.05 % sodium azide
diluted to 1 mg/ml were poured, 100 ml at a time,
into separate 96-well plates and adsorbed overnight
at 4 °C. After removal of the supernatant, 200 ml 1%
bovine serum albumin solution (in PBS) were added and
reacted and blocked for 1 hour at room temperature.
The supernatant was removed and the product was
washed worth a washiirg solution (0.02 % Tween 20,
PBS). Solutions of AMGC1 antibody, at concentrations
of 0.1-1 mg/ml, in an amount of 100 ul each, were
added and reacted for two hours at room temperature.
The supernatant was removed and the product was
further washed with a washing solution. Then, 100 ul
of a solution of 5 mg/ml horse radish peroxidase-
labeled anti-mouse IgG (manufactured by MBL, Code
330) was added and reacted for one hour at room
temperature. The supernatant waa removed and the
product was washed with a washing solution. TMB (KPL)
solution was added, 100 pi at a time, and reacted for
20 minutes at room temperature. After coloration,
100 ul 1 N sulfuric acid were added to stop the
reaction. Absorbance at 450 nm was determined.
As a result, as shown in Table 6 it was confirmed
that when the monoclonal antibody orig±natiiig_fxom;
hybridoma AMGC1 was used, this antibody -eaa r^sct
with ribosomal proteins L7/L12 of all bacteria such
as Neisseria gonorrhoeae, Haemophilus influenzae, and
Streptococcus pneumoniae.
(+: Positive)
The AMGC1 antibody obtained here is very useful
as an antibody used for the detection of
microorganisms in the so-called sandwich assay by
optical immunoassay and ELISA method, in combination
with an anti-ribosomal protein L7/L12 antibody which
is specific to each microorganism.
Industrial Applicability
According to the present invention, not only
microorganisms can be detected specifically according
to species, but also microorganisms of all serotypes
in the same species can be"detected at a high
precision, by using antibodies to intracellular
molecules of the same function.
By using antibodies to ribosomal proteins L7/L12
of various microorganisms as such antibodies,
Haemophilus influenzae and Neisseria gonorrhoeae can
be detected precisely.
Moreover, specific antibodies used for the
detection of various kinds of microorganisms can be
prepared by using intracellular molecules exhibiting
same functions in various microorganisms as an
antigen.
REMARKS TO DEPOSITED MICROORGANISMS
The organization in which the microorganisms have
been deposited:
National Institute of Bioscience and Human
Technology, the Agency of Industrial Science
and Technology
Address: 1-1-3, Higashi, Yatabe-cho, Tsukuba-shi,
Ibaraki-ken, Japan (Postal Code: 305).
Date of deposition: July 28, 1999
Number of deposition given by the deposition
organization: FERM BP-6807
WE CLAIM;
1. Antibodies for detecting bacteria, directed to ribosomal protein
L7/L12 of bacteria which react specifically with ribosomal
protein L7/L12 of bacteria from which the ribosomal protein
L7/L12 is isolated and which can distinguish said bacteria
genus or species from other bacteria genus or species,
respectively.
2. The antibodies as claimed in claim 1, where said bacteria are
bacteria which cause a sexually transmitted disease (STD).
3. The antibodies as claimed in claim 1, where said bacteria which
cause respiratory tract infection.
4. The antibody as claimed in claim 3, where the causative
bacteria of respiratory tract infection are bacteria of Hae-
mophilus influenzae.
5. The antibody as claimed in claim 3, where the causative
bacteria of respiratory tract infection are bacteria of Strep-
tococcus pneumoniae.
6. The antibody as claimed in claim 2, where the causative
bacteria of sexually transmitted diseases (STD) are bacteria of
Neisseria gonorrhoeae.
7. The antibody as claimed in claim 6, which is the antibody to
ribosomal protein L7/L12 Neisseria gonorrthoeae and which
recognized a continuous amino acid sequence moiety from 5 to
30 amino acids including the 115th alanine in the amino acid
sequence of Sequence ID No. 22 of the Sequence Table.
8. A reagent kit for detecting bacteria which is characterized by
comprising an antibody as claimed in any of claims 1 to 7.
9. A method of preparing any antibody described in any one of
claims 1 to 7, characterized by the fact that ribosomal protein
L7/L12 of bacteria obtained by a gene manipulation procedure
or by isolation from bacteria, or a peptide moiety thereof, or a
synthesized peptide corresponding to the peptide moiety is used
as an immunogen.

An antibody for detection of microorganisms, a
method of detection of microorganisms, and a reagent kit for detection of microorganisms, which is species specific for every species of microorganisms and with which all serotypes within the same species can be detected, are provided. Antibody to intracellular molecules with the same function in each type of microorganism, particularly antibody to ribosomal protein, that is, ribosomal protein L7/L12, is made and antibody that reacts specifically with the microorganism in question is selected.

Documents:

IN-PCT-2001-147-KOL-FORM-27.pdf

in-pct-2001-147-kol-granted-abstract.pdf

in-pct-2001-147-kol-granted-claims.pdf

in-pct-2001-147-kol-granted-correspondence.pdf

in-pct-2001-147-kol-granted-description (complete).pdf

in-pct-2001-147-kol-granted-form 1.pdf

in-pct-2001-147-kol-granted-form 13.pdf

in-pct-2001-147-kol-granted-form 18.pdf

in-pct-2001-147-kol-granted-form 2.pdf

in-pct-2001-147-kol-granted-form 26.pdf

in-pct-2001-147-kol-granted-form 3.pdf

in-pct-2001-147-kol-granted-form 5.pdf

in-pct-2001-147-kol-granted-priority document.pdf

in-pct-2001-147-kol-granted-reply to examination report.pdf

in-pct-2001-147-kol-granted-sequence listing.pdf

in-pct-2001-147-kol-granted-specification.pdf

in-pct-2001-147-kol-granted-translated copy of priority document.pdf


Patent Number 233799
Indian Patent Application Number IN/PCT/2001/147/KOL
PG Journal Number 15/2099
Publication Date 10-Apr-2009
Grant Date 08-Apr-2009
Date of Filing 06-Feb-2001
Name of Patentee ASAHI KASEI KOGYO KABUSHIKI KAISHA
Applicant Address 2-6, DOJIMAHAMA 1-CHOME, KITA-KU, OSAKA-SHI, OSAKA 530-8205
Inventors:
# Inventor's Name Inventor's Address
1 ETOH TAKASHI 168-30, OBUCHI, FUJI-SHI, SHIZUOKA 417-0801
2 MATSUYAMA KENJI 5036 BUCKINGHAMROAD, BOULDER, CO 80301
3 SHIRAI TAKASHI 541-36 SHIMOTOGARI, NAGAIZUMICHO, SUNTO-GUN, SHIZUOKA 416-0943
PCT International Classification Number C07K 16/12
PCT International Application Number PCT/JP99/04122
PCT International Filing date 1999-07-30
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/230204 1998-07-31 Japan