Title of Invention

A DEVICE AND KIT FOR ANALYZING THE PRESSENCE OF LEPTOSPIRA LGM ANTIBODIES IN HUMAN SERUM OR PLASMA

Abstract The present invention relates to a microlisa Leptospira device for analyzing the presence of Leptospira IgM antibodies present in human serum or plasma. It is a process for the preparation of the microlisa Leptospira device for the detection of IgM antibodies present in human serum or plasma. More specifically it is a kit for analyzing the presence of Leptospira IgM antibodies in human serum or plasma.
Full Text Field of the invention
The present invention relates to the Lepto IgM Enzyme Linked ImmunoSorbent Assay test device for analyzing the presence of Lepto IgM antibodies in human serum and plasma. More specifically the present invention relates to the process for making the Lepto IgM microlisa device for the detection of IgM antibodies present in human serum or plasma.
The present invention also relates to a kit for analyzing the presence of Lepto IgM antibodies in human serum or plasma.
The Enzyme Linked Immuno Assay ( ELISA) is recommended as the screening method for the detection of Lepto IgM antibodies.
Lepto IgM Microlisa is developed to detect IgM antibodies to Leptospira. The Lepto IgM antibodies are detected in acute phase.
It is an in vitro- qualitative enzyme immunoassay for the detection of IgM antibodies to Leptospira in human serum or plasma and is used as a screening test for the testing of collected blood samples suspected for Leptospirosis.
Background
Leptospira is a genus of spirochaete bacteria, including a small number of pathogenic and saprophytic species. It is an important global human and veterinary health problems. It is a wide spread zoonotic disease caused by pathogenic strains of Leptospira which are capable of infecting most
mammalian species. It was first observed in 1907 from a post mortem renal tissue slice. Leptospira genus consists of a genetically diverse group of 12 species, eight of which are pathogenic, and four of which are non-pathogenic, and saprophytic. See Faine, et al., Leptospira and Leptospirosis (2.sup.nd edition, 1999, Melbourne, Australia, MediSci); Fair, Clin. Infect. Dis 21:1 8 (1995). Levett, Clin. Microbiol Rev. 14:296 326 (2001), incorporated by reference. Infection occurs either through the direct contact with the infected animal or indirect contact with the contaminated soil or water. The organism enters the human body through the cuts or abrasions on the skin or through intact mucosa of the mouth, nose or conjunctiva. Leptospirosis is caused by spirochaete bacterium called Leptospira. Leptospira is transmitted by urine of infected animals and is contagious as long as it is still moist. It is also transmitted by the semen of the infected animals.
In human, leptospiral infection causes a wide range of symptoms and some infected persons may have no symptoms at all. It is a biphasic disease that begins with the flu like symptoms [ fever, chills, myalgias, intense headache]. Because of the wide range of symptoms, the infection is often wrongly diagnosed.
In the past decade, leptospirosis has emerged as a globally important infectious disease. It occurs in urban environment of industrialized and developing countries as well as in rural regions worldwide.
Prior Arts
WO/1995/032220 : (EN) An antigenic preparation is provided which contains a 63 Kd outer membrane protein from Leptospira which can be used immunologically as a vaccine for leptospirosis caused by this organism. Also provided in the invention are polynucleotides encoding the protein and antibodies which bind the protein which are useful in the diagnosis of leptospirosis.
WO/1996/036355 : (EN) The present invention presents novel leptospiral membrane lipoproteins, LipLl and LipL2, associated with pathogenic strains of Leptospira. LipLl is of about 35 kDa, and LipL2 is of about 41 kDa. Also disclosed are the method for purifying these proteins from Leptospira, their nucleotide and amino acid sequences, the cloning of the genes encoding the proteins and their recombinant proteins, methods for producing antibodies to these proteins, the resulting antibodies. These proteins, their immunogenic fragments, and antibodies against them, are useful for inducing an immune response to pathogenic Leptospira as well as providing a diagnostic target for leptospirosis.
US Patent No. 6,800,734 : Four antigenic preparations are provided, each of which contains a different protein from Leptospira which can be used immunologically in vaccines for leptospirosis caused by this organism. Also provided in the invention are polynucleotides encoding these four proteins and antibodies which bind the proteins for use in the diagnosis of leptospirosis.
US Patent No. 6699482: The present invention presents novel leptospiral membrane lipoproteins, LipLl and LipL2, associated with pathogenic strains
of Leptospira. LipLl is of about 35 kDa, and LipL2 is of about 41 kDa. Also disclosed are the method for purifying these proteins from Leptospira, their nucleotide and amino acid sequences, the cloning of the genes encoding the proteins and their recombinant proteins, methods for producing antibodies to these proteins, the resulting antibodies. These proteins, their immunogenic fragments, and antibodies against them, are useful for inducing an immune response to pathogenic Leptospira as well as providing a diagnostic target for leptospirosis.
US Patent No. 6685945: An antigenic preparation is provided which contains a 31 Kd outer membrane protein from Leptospira which can be used immunologically as a vaccine for leptospirosis caused by this organism. US Patent No.6482924: Four antigenic preparations are provided, each of which contains a different protein from Leptospira which can be used immunologically in vaccines for leptospirosis caused by this organism. Also provided in the invention are polynucleotides encoding these four proteins and antibodies which bind the proteins for use in the diagnosis of leptospirosis.
Application 197/CHE/2004: Leptospirosis is a zoonosis of worldwide significance, caused by infection with pathogenic Leptospira species. There is an urgent need for the development of new diagnostic strategies for leptospirosis. Clinical recognition is difficult because leptospires can affect many different organ systems, resulting in a wide variety of clinical presentations. Laboratory confirmation is of utmost importance and the standard serologic test, the microscopic agglutination test (MAT) requires the detailed knowledge of the locally occurring strains as the predominant serovars have to be selected for use as antigens. MAT is inadequate for rapid case identification as it requires analysis of paired sera to achieve sufficient
densitivity. Hence, a rapid and simple recombinant LipL32 antigen -based Latex agglutination test was developed for the first time for canine, bovine and human serodiagnosis in the developing countries. The expression of the antigen is also restricted only to the pathogenic leptospires. The recombinant antigen-coated latex beads could detect the anti-leptospiral antibodies in the acute and convalescent phase of illness and useful in the diagnosis of Leptospirosis.
US Patent No: 7108853: The invention relates to Leptospiral surface
proteins, and the nucleic acid molecules which encode them. Various uses
are described, including immunoprophylactic, diagnostic and therapeutic
methods.
These methods have suboptimal sensitivity and specificity and abundance of
false positive in low prevalence population. These patents suffer from
disadvantages like not user friendly, time consuming laborious and costly.
The object of the present invention is to overcome the above said disadvantages and hence the present invention came.
Description of the invention
The present invention is a ELISA test for the qualitative and differential detection of IgM antibodies to Leptospira in human serum and plasma.
The object of the present invention is to provide a device and a test kit for the differential detection of IgM antibodies to leptospira present in human serum or plasma.
Yet another object of the present invention is to provide a microtiter device and a test kit to a man of ordinary prudence, which will take a short time to perform the test.
Brief description with figures Figure 1 shows the microtiter wells.
Detailed description:
Principle
The test is performed in a 8 X 12 wells which contains an 8 X 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter. Lepto IgM Microlisa test is an enzyme linked immunoassay based on "Indirect ELISA". Recombinant proteins; recombinant leptospira antigens or synthetic peptides or native bacterial lysate of Leptospira biflexa or leptospira interrogans or a combination of two or more antigens of various molecular weight representing immunodominant epitopes are coated on to microtiter wells.
Specimens and controls are added to the microtiter wells and incubated. IgM Antibodies to leptospira if present in the sample, will bind to leptospira antigens adsorbed on to the surface of the wells. The plates are then washed to remove unbounded material. Horseradish Peroxidase (HRPO) conjugated to anti human IgM is added to each well. This anti human IgM conjugate will bind to leptospira specific IgM antibodies, which will complex with leptospira antigen. Finally substrate solution containing chromogen and hydrogenperoxide is added to the wells and incubated. A blue colour will develop in proportion to the amount of leptospira antibodies present in the specimen. The colour reaction is stopped by the stop solution. The enzyme substrate reaction is read by EIA reader for absorbance at a wavelength of 450 nm. If the sample does not contain leptospira IgM antibodies, then the

enzyme conjugate will not bind and the solution in the wells will be either colourless or only a faint background colour develops.
Interpretation of result
After adding the substrate, if the sample is blue in colour, then the patient's sample is reactive for Leptospira IgM antibodies. If there is no colour to the solution, then the sample is not reactive to the leptospira IgM antibodies. Stop solution such as acids are added to stop the reaction. After adding the stop solution, if the solution in the micro titer well is yellow in colour, the sample is reactive for lepospira IgM antibodies and if there is no yellow colour, then the sample is non reactive for Leptospira IgM antibodies. Specimen collection and handling
Human serum or plasma samples should be used for the test. While preparing the serum samples, remove the serum from the clot as soon as possible to avoid hemolysis. Fresh serum or plasma samples are preferred. Specimen should be free of microbial contamination and may be stored at 2 to 8°C for one week or frozen at -20°C or lower. Repeated freezing and thawing should be avoided.
Frozen sample
Lepto IgM Microlisa test is best used with fresh samples that have not been
frozen and thawed.
Allow the frozen samples to thaw in a vertical position in a rack. Don't
shake the sample. This allows the particles to settle in the bottom. The
sample should be centrifuged.
Preparation of Reagents
Prepare the reagents during the assay procedure. Reagents and the samples
should be at room temperature before beginning the assay and can remain at
room temperature during testing. Return the reagents to room temperature
after testing.
Antigen coated Microwells
Bring the pack to room temperature before opening to prevent condensation
on the microwells.
Break off the required number of strips needed for the assay and place in the
well holder. Take the strip holder with the required number of strips, taking
in to account that two each of the negative , positive controls and calibrator
should be included in the run while opening the fresh kit. However for one
or two strips each of negative and positive controls and calibrator and for
more strips each of negative and positive controls and calibrator should be
included in each subsequent runs.
Sample Preparation:
a. Tube dilution
Mark the tubes carefully for the proper identification of the samples. Dilute the serum samples to be tested, with sample diluent 1:100 in separate tubes. (1ml diluent + lOul serum samples).
b. Microwell dilution
i. Pipette lOOul of sample diluent in to the microwell. ii.Add lul of serum sample to be tested.
iii Ensure thorough mixing of the sample to be tested.
Preparation of working wash buffer
Prepare at least 25 ml of buffer for each strip by adding 1 ml concentrated
buffer with 24 ml of water. Mix well before use.
Preparation of working conjugate
Dilute conjugate concentrate 1:10 in conjugate diluent.
Preparation of wash Buffer
Take 800- 900ml of distilled water and add lOmM Phosphate buffer and 0.15M normal saline (NaCl). Stir the solution in a magnetic stirrer to obtain a homogeneous solution. Add 0.03 to 0.06 % of TritonX-100 to the above solution to make the volume up to 1000ml. Adjust the pH of the solution to 7.0 to 7.4 by adding Sodium Hydroxide.
Preparation of the working chromogenic substrate solution/ TMB)
Dilute TMB concentrate is in the ratio 1:10 in substrate solution, i.e. TMB
Diluent. The 10X solution may crystallize during storage.
TMB Concentrate (IPX)
TMB means 3,3,5,5, Tetra Methyl Benzedene, which is dissolved in
Dimethyl sulphoxide which is used as an indicator.
TMB Diluent
Buffer solution containing the substrate is the TMB Diluent.
Stopsolution
Acids are the stop solution.
According to the present invention, the process of the detection of Lepto IgM antibodies in human serum or plasma are as follows:
1. Leave A-l well as blank which is to test the quality of the substrate.
2. Add 100(4,1 negative control (ready to use) in each well No. B-l & C-l
respectively.
3. Add 100|al Positive Control in D-l and E-l wells.
4. Add 100 pi calibrator in F-l and G-l wells.
5. Add lOOjjl of sample diluent in each sample well starting from HI
followed by the addition of lul sample.
6. Apply cover seal and incubate in an incubator at 37°C+ 2°C for 30
minutes + 1 minute.
7. Take out the plate from the incubator and wash the wells 5 minutes
with the working wash solution.
8. Add lOOul of working conjugate solution in each well excluding A-l
9. Apply cover seal and incubate at 37°C+ 20°C for 30 minutes + 2
minutes.
10.Aspirate and wash the well.
11. Add lOO^il of the working substrate solution in each well including
the A-l well.
12.1ncubate at room temperature for 30 minutes in dark. 13.Add 50 ul of stop solution and read the result within 30 minutes.
According to the present invention, Leptospira IgM microlisa device for the detection of Leptospira IgM antibodies in human serum or plasma comprises of 8 X12 matrix of 96 wells about one centimeter high and 0.7cm in diameter characterized in that the leptospira antigen is added to the
microtiter wells where in the specimens and the control are added to the microtiter wells followed by the addition of enzyme conjugate.
In an embodiment of the present invention, a process for making the Lepto IgM microlisa device for the analysis of the presence of the Leptospira IgM antibodies in human serum or plasma comprises the following steps.
1. Dissolving dilute Leptospira antigen in the ratio of 200 to 800
nanograms in the buffer solution and followed by mixing it
thoroughly.
2. Stirring the solution at 50 to 100 rpm in a magnetic stirrer to obtain a
homogeneous solution.
3. Dispensing the solution in the wells in the range of 50- 200 ul.
4. Incubating the wells overnight at 2 to 10°C.
5. Washing the wells with the buffer solution of pH 7.2 to 7.6 at least 4
to 5 times.
6. Blocking the washed wells with protein solution made in 10% BSA
solution.
7. Removing the unutilized solution by inverting the wells and drying
the wells at room temperature thus obtaining the device.
In another embodiment of the present invention, the enzyme conjugate is antihuman IgM linked with an enzyme such as HRPO prepared by the following steps.
a. A 20 mg of the enzyme such as HRPO dissolved in 1ml of
distilled water.
b. Taking 0.08 molar Sodium per iodate and mixing in the ratio 1:
10 with HRPO.
c. Shaking and leaving the mixture at room temperature for 60
minutes.
d. Mixing with the activated HRPO solution obtained with 10 mg
antihuman IgM antibodies.
e. Allowing the solution to incubate for 2 hours at ambient
temperature.
f. Adding 1:20 ratio of borohydride solution with the above
solution and leaving it to incubate for 30 minutes at ambient
temperature; allowing the reaction between anti human IgM
antibodies with HRPO.
g. Adding 1:20 ratio Ethanolamine
h. Allowing the solution to incubate for 30 minutes followed by
dialyzing i. Adding buffer solution i.e. BSA in PBS at the percentage 5% to
10% thus obtaining the enzyme conjugate.
In still another embodiment of the present invention, the said negative control (-) is normal human sera diluted in saline solution which is negative for Leptospira, HIV, HCV and HBV and the positive control (+) is human sera diluted in saline solution which is positive to IgM antibodies of Leptospira and negative for HCV, HBV and HIV.
Example 1:
20 mg of enzyme such as HRPO dissolved in 1 ml of distilled water followed by taking 0.08 molar per iodate and mixing in the ratio 1:10 with HRPO; shaking and leaving the mixture at room
temperature for 60 minutes; mixing with activated HRPO solution obtained with 10 mg of antihuman IgM antibodies; allowing the solution to maturize for 2 hours at ambient temperature; adding 1:20 ratio of Sodium borohydride solution with the above solution and leaving the same to maturize for 30 minutes at ambient temperature; allowing the reaction of anti human IgM antibodies with HRPO; adding 1:20 ratio Ethanolamine; allowing the solution to maturize for 30 minutes followed by dialyzing; i.e. BSA in PBS at the percentage 9% to obtain the enzyme conjugate.


I Claim:
1. A microlisa device and a kit for detection of IgM antibodies to Leptospira present in human serum or plasma comprising 96 wells microtiter plate, one centimeter high and 0.7 cm in diameter; each wells are coated with recombinant leptospira antigens or synthetic peptide or native bacterial lysate of Leptospira biflexa or Leptospira interrogans or a combination of two or more antigens as here in described; and the kit further comprises of enzyme conjugate, substrate, wash buffer and calibrator.
2. A device as claimed in claim 1, wherein the microtiter plate is coated with recombinant leptospira antigen or synthetic peptides or native bacterial lysate of Leptospira biflexa or leptospira interrogans or a combination of two or more antigens,
by dissolving 200 to 800 nanogram of above said Leptospira antigens in a buffer
solution, stirring the solution at 50 to 100rpm to get a homogeneous solution;
dispensing 50 ul to 200 ul solution in the well and incubating the solution
overnight at 2-10°C;
washing the wells with buffer solution of pH 7.2 to 7.6;
blocking the wells with 10% BSA solution.
3. A kit as claimed in claim 1, wherein the enzyme conjugate is antihuman IgM antibodies conjugated to HRPO.
4. A kit as claimed in claim 3 where in the conjugate is prepared by dissolving 20gm of enzyme HRPO in 1ml of distilled water; mix 0.08molar Sodium per Iodate in the ratio 1:10 with HRPO;
shake the mixture well and leave it at room temperature for 60 minutes;


mix the activated HRPO solution thus obtained with 10mg anti human IgM
antibodies;
allow the solution to maturize for 2 hrs at ambient temperature;
add 1:20 ratio of Borohydride solution with the above solution and leave it to
maturize for 30 minutes at ambient temperature and allow the reaction of IgM
antibodies with HRPO;
add 1:20 ratio Ethanol amine to the above solution and allow the solution to
maturize for 30 minutes followed by dialyzing;
add BSA in PBS at the percentage 5% to 10 % to the above solution.
5. A device as claimed in claims 1 and 2 wherein the amount of antigens coated on the wells range from 50µl to 200µl.
6. A microlisa device for detection of IgM antibodies to Leptospira in human serum or plasma substantially described herein with reference to and as illustrated in accompanying drawing.

Documents:

2483-DEL-2007-Abstract-(15-04-2009).pdf

2483-del-2007-abstract.pdf

2483-DEL-2007-Claims-(15-04-2009).pdf

2483-del-2007-claims.pdf

2483-DEL-2007-Correspondence-Others-(15-04-2009).pdf

2483-DEL-2007-Correspondence-Others-1.pdf

2483-DEL-2007-Description (Complete)-(15-04-2009).pdf

2483-del-2007-description (complete).pdf

2483-del-2007-drawings.pdf

2483-DEL-2007-Form-1-(15-04-2009).pdf

2483-del-2007-form-1.pdf

2483-del-2007-form-18.pdf

2483-DEL-2007-Form-2-(15-04-2009).pdf

2483-del-2007-form-2.pdf

2483-del-2007-form-3.pdf

2483-del-2007-form-5.pdf

2483-del-2007-Post-Grant-Opposition-(01-11-2010).pdf

2483-DEL-2007-Post-Grant-Opposition-(20-09-2010).pdf


Patent Number 235937
Indian Patent Application Number 2483/DEL/2007
PG Journal Number 38/2009
Publication Date 18-Sep-2009
Grant Date 09-Sep-2009
Date of Filing 28-Nov-2007
Name of Patentee MAHAJAN; LALIT
Applicant Address N-118, GREATER KAILASH, PART-1, NEW DELHI, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 MAHAJAN; LALIT N-118, GREATER KAILASH, PART-1, NEW DELHI, INDIA.
PCT International Classification Number A61K39/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA