Title of Invention

"A NOVEL MONOLONAL ANTIBODIES FOR DETECTING CHLAMYDIA TRACHOMATIS"

Abstract The present invention relates to novel Chlamydia trachomatis serovar specific monoclonal antibodies. The present invention further provides a kit for detecting the specific scrovars of Chlamydia trachomatis. The present invention further more provides a method for detecting specific scrovars of Chlamydia trachomatis.
Full Text NOVEL CHLAMYDIA TRACHOMATIS SPECIFIC MONOCLONAL ANTIBODIES, A PROCESS OF PREPARINF THE SAME, A DIGNOSTIC KIT FRO DETECTING CHLAMYDIA TRACHOMATIC, A METHOD OFPRODUCING THE KIT AND A METHOD FOR DETECTING CHLAMYDIA TRACHOMATISVSMG THE KIT
Field of the Invention
The present invention relates to novel Chlamydia trachomatis serovar specific monoclonal antibodies, a process of preparing said serovar specific monoclonal antibodies. The present invention further provides a kit for detecting the specific serovars of Chlamydia trachomatis and a process for preparing said process for kit for detecting specific serovars of Chlamydia trachomatis using said serovar specific monoclonal antibodies. The present invention furthermore provides a method for detecting specific serovars of Chlamydia trachomatis.
Background and Prior Art Description
Chlamydiae are obligate intracellularly growing bacteria. They are cosmopolitan in distribution infecting both humans and animals. Currently, four species, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia pecorum, belonging to the genus Chlamydia of the family Chlamydiaceae, within the order Chlamydiales, are recognized (Meijer et al 1999). C. psittaci and C. pecorum are primarily animal pathogens while C. pneumoniae and C. trachomatis are human pathogens.
C. pneumoniae has been recognized as a major cause of respiratory infections and has also been associated with asthma, exacerbation of chronic asthma, atherosclerotic disease and recently, Alzheimer's dementia (Balin et al 1998).
C.irachomatis is a human mucosal pathogen which causes three forms of disease, trachoma, genital infection and lymphogranuloma venereum. C. trachomatis is a major cause of sexually transmitted diseases and trachoma in humans (Schachter and Caldwell 980, Tories 1995, Pannekoek 2000). C.trachomatis infections
may lead to pelvic inflammatory disease (PID), resulting in tubal infertility, ectopic pregnancy and chronic pain (PearlmariMcNerley1992).
In addition, there are reports, which suggest that chlamydial infection of uterine cervix can cause cervical dysplasia, which may lead to cervical cancer/in women (Schachter 1999). Athough chlamydial infection is a non-ulcerative STD, it has been reported to facilitate the transmission of HIV, but HIV infection augments expression and progression of Human Papilloma Virus (HPV) disease leading to increased incidence of clinical cancer in women (Wasserheit 1992, Gopalkrisnan al 1999). Control of genital chlamydial infection could potentially have a major impact on HIV transmission and targeted screening for genital chlamydial infections in high-risk population should therefore be a priority for STD/HIV control programme.
C. trachomatis is divided into 15 serovars A, B, Ba, C, D, E, F, G, H, I, J, K, l_i, L2 and l_3. C. trachomatis serovars D to K are common causes of genital infections throughout the world.
Symptoms: Symptoms of C. trachomatis infection in women vary with the disease state for e.g. chlamydial mucopurulent cervicitis is characterized by mucopurulent cervical discharge, cervical ectopy and edema, spontaneous or easily induced cervical bleeding. Incubation period is uncertain chlamydial PID is characterized by lower abdominal pain, adexnal tenderness on pelvic examination and there is often an evidence of mucoprulent cervicitis. Incubation period for chlamydial salpingitis is 2-3 weeks after cervical infection.
In males, chlamydial non-gonococcal urethrhis (NGU) is characterized by dysuria and uretheral discharge; chlamydial epidedymitis is characterized by fever, epididymal or testicular pain, evidence of NGU and epididymal tenderness and mass.
Prevalence of disease (endemic/epidemic) world-wide
Europe: According to the studies done in West, prevalence of endocervicitis due to C. trachomatis has been estimated to be in the range of 3%-5% in asymptomatic women to over 20% in women seen in STD clinics (Judson 1985). Since infection with C. trachomatis is often asymptomatic, many infected individuals are not readily identified and go untreated (Smith et al 1987). Therefore, it is important to identify patients infected with C. trachomatis not only to reduce transmission but also to minimize the risk of more serious infections and their sequelae (Scholes et al 1996). It is therefore desirable to identify chlamydial infections in infected individuals and administer appropriate treatment before further complications arise. There is a strong need to develop new diagnostic assays prognostic markers for early identification of the microorganism.
Asia: In 1999, the estimated new cases of chlamydial infection (in million) among female adults were.
 Central Asia and Eastern Europe = 3.25
 South and South East Asia = 23.96
 East Asia and Pacific = 2.74
India: A high prevalence of Chlamydia lower genital tract infection has been reported in our country. Published prevalence ranges of C trachomatis in women in India are:
 Community based married women = 0.5 to 28%
 Gynaecological OPD patients = 0.2 to 31.3%
 Gynaecological patients with "Vaginitis" = 2.6 to 12.2%
 Gynaecological patients with Cervical Erosion = 3.0%
 Infertility and PID patients = 0.5 to 24.2%
In male patients attending STD Clinic, the prevalence ranges from 20 to 30%. There a few kits available in the market for the detection of C trachomatis. The kits thus available for the detection of the said bacteria are mainly imported from abroad and are very costly. Also, these kits involve only the identification of the
bacteria perse. There are no means of detection of the various serovars bacteria through the known diagnostic kits.
Thus, there is a need to develop a novel kit which is not only able to detect the bacteria per se., but also is able to identify the specific serovar. The detection of the specific serovar makes the process of treatment very easy. At present, the samples collected from the patient is first tested for the presence of the bacteria and then genotyping is done using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) to detect the specific serovar. The process for detecting the bacteria and then identification of the serovar takes very long time, something about 2-3 days. There is no kit available at present which is able to detect the presence of the bacteria and also identify the specific serovar of C trachomatis. The Inventors have for the first time attempted to develop a kit that is able to not only detect the bacteria but is also able to identify some of the specific serovars.
Antibodies are proteins that help the white blood cells to fight against viruses and bacteria by binding to foreign invaders and signaling the immune cells to attack. Monoclonal antibodies that are made in laboratory to bind only one single type of molecule. They have being currently used for wide variety of purposes like cancer treatment, detection of disease causing bacteria and viruses and other related molecules. Laboratory provided identical antibodies can target specific antigens. Monoclonal antibodies are being used in clinical trials to determine their effectiveness in diagnosis, treatment, detections of organisms causing diseases. Monoclonal antibodies are highly purified antibodies and produced artificially. The monoclonal antibodies of the present invention are highly purified antibodies (References: 1.rex.nci.nih.gov/PATIENTS/INFO_TEACHER/bookshelf/NIHjmmune/html/imm35.html.
2. yourmedicalsource.com/library/lymphoma/NHL_glossary.html
3. www.cdad.com/tricare/glossarv/glossary.html
4. www.ilar.org/glossarv/glossary m.htm )
OBJECTS OF THE INVENTION
The main object of the present invention provides a novel Chlamydia trachomatis
serovar specific monoclonal antibodies,
Another object of the present invention provides a process of preparing said
serovar specific monoclonal antibodies.
Yet another object of the present invention provides a a kit for detecting the
specific serovars of Chlamydia trachomatis
One more object of the present invention provides a process for preparing said
process for kit for detecting specific serovars of Chlamydia trachomatis using
said serovar specific monoclonal antibodies.
Still another object of the present invention provides a method for detecting
specific serovars of Chlamydia trachomatis.
SUMMARY OF THE INVENTION
The C.trachomatis is a human mucosal pathogen which causes three forms of disease, rachoma, genital infection and lymphogranuloma venereum. C. trachomatis is divided into 15 serovars namely A, B, Ba, C, D, E, F, G, H, I, J, K, LI, L2 and l_3. C. trachomatis serovars D to K are common causes of genital infections throughout the world.
To know the type of serovar of C. trachomatis and also to determine the prevalent serovar in female genital tract in patients coming to the Applicant's hospital, genotyping was done using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Predominance of serovar D was found in female genital infections.
To develop diagnostic assay for C. trachomatis and in particular serovars D, the Applicants developed a serovar D specific monoclonal antibody using hybridoma technique from an Indian patient's isolate of C trachomatis. This developed antichlamydial hybrid clone can be used for detection of C. trachomatis infection.
The Applicants have for the first time isolated various clones which are serovars specific and also developed novel diagnostic kits using these novel clones which has an advantage to detect the bacteria and serovars D, which is major cause of this disease in Asian population particularly India. The identified monoclonal antibody clone for serovars D is very specific and has identified specific epitopic or the target site. The present work has advantage of being serovars specific thereby being cost effective and consumes less time.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
Fig 1a &b: Sequencing results for monoclonal antibodies of the serovar D of C.trachomatis. The sequence comparison was found to be matching with the serovar D against which the antibody has been raised.
Fig. 2a and 2b: Nucleotide sequence for monoclonal antibodies of the serovar D of C.trachomatis. The sequence comparison was found to be matching with the serovar D against which the antibody has been raised.
Fig. 3: A micrograph showing inclusion bodies of Chlamydia trachomatis stained by using antichlamydial monoclonal antibody producing hybrid clone (D serovar specific) as the first antibody and anti-mouse IgG FITC labeled as second antibody.
Fig.4. A graph showing diagnostic kit Identification of C.trachomatis serovar D in the clinical samples.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the main object described above the present invention provides novel monoclonal antibodies namely H 5.6 and E4.2 having epitope sequence DVKTGAE for detection of Chlamydia trachomatis.
Another embodiment of the present invention provides monoclonal antibodies wherein said antibodies are specific towards serovar D of Chlamydia trachomatis.
Another embodiment of the present invention relates to monoclonal antibodies, wherein said antibodies have 100 % sensitivity for detecting Chlamydia trachomatis.
One more embodiment of the present invention relates to monoclonal antibodies, wherein antibodies have 100 % sensitivity for detecting Chlamydia trachomatis serovar D.
Yet another embodiment of the present invention relates to monoclonal antibodies, wherein it takes about 2-3 hrs to detect Chlamydia trachomatis serovar D.
Another embodiment of the present invention provides a method for detecting Chlamydia trachomatis causing genital infections by novel monoclonal antibodies namely H 5.6 and E4.2 having epitope sequence ID No.1, said method comprising steps:
(a) applying suspensions of Chlamydia antigens to a nitrocellulose
(NC)grid,
(b) incubating NC grids in Bovine serum albumin in Phosphate borate
buffer solution (PBS) for 2 hrs at room temperature,
(c) adding monoclonal antibodies namely H 5.6 and E4.2 having
epiotpe SEQ ID No.1 to the NC grids,
(d) allowing the mixture of step (c) to be incubated at room
temperature for 1 hr,
(e) washing the NC grids of step (d) with PBS and Tween-20,
(f) adding and reacting the NC grids of step (e) with conjugate of
horseradish peroxidase goat antimouse immunoglobulin (IgG) for
for 1 hr,
(g) washing again the NC grids to remove excess of IgG,
(h) detecting the enzyme tagged immunoglobulin complexed to C.trachomatis by colorimetric reaction.
Another embodiment of the present invention provides a process for preparing a diagnostic kit for detecting Chlamydia trachomatis said kit comprising steps of:
(a) preparing monoclonal antibodies H 5.6 and E4.2 having epitope
sequence DVKTGAE against Chlamydia trachomatis by a process such
as herein described,
(b) providing a predetermined amount of PBS-T buffer, 2% BSA , rabbit anti-
mouse IgG HRPO conjugate as second antibody and 5M sulfuric acid,
(c) providing a structure fro carrying out the test, and
(d) providing an instruction manual which describes the procedure for
carrying out the tests.
Yet another embodiment of the present invention provides a diagnostic kit for detecting Chlamydia trachomatis said kit comprising of Novel monoclonal antibodies namely H 5.6 and E 4.2 having epitope sequence DVKTGAE against Chlamydia trachomatis said kit comprising steps of:
(a) a structure for carrying out the diagnostic procedure,
(b) monoclonal antibodies namely H 5.6 and E4.2 having epitope
sequence DVKTGAE
(c) chemical reagents selected group contain PBS-T buffer, 2% BSA ,
rabbit anti-mouse IgG HRPO conjugate as second antibody and 5M
sulfuric acid adding 2% BSA, and
(d) an instructing manual which describes the procedure for carrying
out the tests.

Another embodiment of the present invention provides a use of Novel monoclonal antibodies namely H 5.6 and E4.2 having epitope having sequence as DVKTGAE for detecting Chlamydia trachomatis.
Yet another embodiment of the present invention relates a structure wherein the structure is selected from group comprising of 96 well plates, test tubes, slides and other specimen observing structures.
Yet another embodiment of the present invention relates to NC girds, wherein NC grid has size of 2*2 inches.
Still another embodiment of the present invention relates to concentration of antigen wherein 0.25ul of antigen is added in step (a).
One more embodiment of the present invention relates to concentrations of BSA wherein BSA in step (b) is about 2%.
Another embodiment of the present invention relates to concentration of PBS wherein PBS in step (c) is bout 0.05%.
Still another embodiment of the present invention relates to the ratio of PBS and horseradish peroxidase conjugated goat antimouse immunoglobulin IgG wherein ratio of PBS and horseradish peroxidase conjugated goat antimouse immunoglobulin IgG is 1:1000.
Yet another embodiment of the present invention relates to type of method performed wherein the method performed is DOT ELISA for detecting serovars of Chlamydia trachomatis.
One more embodiment of the present invention relates to the size of monoclonal antriboideswherein monoclonal antibodies of step (c) are 40 kDa.
Specificity and sensitivity of the kit and method vis-a vis the existing Indian/foreign kits.
Throughout the world including India, the diagnostic kits developed so far only detect the presence of bacteria C.trachomatis, however there are no means available for detecting the specific serovars from the kits. The only method available for detecting the serovar is genotyping using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Therefore, the detection of specific serovars is under in vitro culture conditions which is time consuming and expensive. The present invention however, expands the scope for the detection by identifying C. trachomatis and its specific serovars, thereby improving the method of treatment. Till date it takes more time in treating a patient. If the bacteria becomes or is resistant towards the prescribed antibiotics, then a method of treating is also difficult. Further, this augments to change the treatment method. However, with the invented diagnostic methodology it not only saves time in detection of bacteria C. trachomatis but also its serovars, particularly serovars D which is predominant in Asian population particularly India. The sensitivity and specificity of the developed serovar specific antichlamydial clone, is 91.6% and 100% respectively as compared to known foreign kit. The Figure 2 shows identification of inclusion bodies of C. trachomatis specifically serovar D by using antichlamydial monoclonal antibody against the said serovar.
EXAMPLES
Example 1
Process of making the monoclonal antibodies Indigenous Raw Materials
used and other sources:
Cell growth containers (Tissue culture flasks, 96 well plates, 24 well plates, Pipetting tips, Media storage bottles, Petri discs etc.).
Cell growth media (EME medium, DME medium, RPMI-1640, Antibiotics, PCS
etc.).
Anti mouse HRPO conjugate Cell lines- Myleoma, McCoy
Balb/c mice
Imported Chemicals Used:
Hypoxanthine, Aminopterine, Thymidine, polyethylene glycol 1500, etc.
Indigenous Instruments Used:
Laminar air flow cabinet
Pipetting devices
Auto clave
Oven
Centrifuge
Imported Instruments Used:
Incubator
Microscope
Freezer
Scale of the technology developed:
Laboratory scale: Yes
Pilot scale: No
Commercial scale: No
Edge of the technology over existing commercially available technology in
terms of accuracy:
Specificity: 100%
Sensitivity: 91.6%
Cost effectiveness: A microphotograph showing inclusion bodies of Chlamydia trachomatis stained
by suing antichlamydial monoclonal antibody producing hybrid clone (D serovars
specific) as the first antibody and anti-mouse IgG FITC labeled as second
antibody.
Example 2
A kit for detecting Chlamydia trachomatis
A kit for detecting Chlamydia trachomatis said kit comprising:
A structure, wherein the structure is a 96 well plate or a 20 pre- grided nitrocellulose (NC) of 2X2 inches, commonly known as ELISA plate. The kit contains monoclonal antibodies namely H5.6 and E4.2 having epitope sequence DVKTGAE. Further, the kit contains predetermined quantity of chemical reagents which consist of Phosphate buffer saline buffer-T(PBS-T), bovine serum albumin (BSA), a second antibody of rabbit anti-mouse IgG HRPO conjugate and stop buffer which is sulfuric acid 5M provided in separate containers. The monoclonal antibody can be precoated on the structure or can be given separately on the structures. The kit also contains an instructing manual which describes the procedure for carrying out the tests. All the above described elements of the kit are preferably packaged together in a container. However, it is also possible that these elements may be made available separately. The procedure of the as described in the instructing manual is described in the Examples 4 and 6.
EXAMPLE 3
Method of preparing the kit for detecting Chlamydia trachomatis
Immunization process for making the antibodies against the Chlamydia
trachomatis:
The Balb/C mice (4-6 weeks old female) were injected intravenously with whole cell sonicated antigen of C.trachomatis isolates. The dosage included the first intravenous injection (10 days), followed by a booster dose after three days. Before the fusion check the antibody titer serum of immunized.
Preparation of Spleenocytes:
The immunized mice were killed and spleen taken out and stored in RPMI 1640 medium. The isolated spleen after mincing was centrifuged at 160 g for 5 min.
The obtained pellet was checked for its viability using trypan blue and
resuspended in RPMI 1640.
Preparation of mouse myeloma cell (SP2/0) for fusion:
Split the myeloma cultures 1:2 daily 3-days preceding the fusion.
Wash the myeloma cells three times with RPMJ-1640 medium before mixing the
spleen cells
Check the viability before fusion with trypan Blue.
Fusion Procedure for preparing the monoclonal antibodies:
• Combine the cells in a ratio of 4:1 immunized spleen cells to myeloma
cells (i.e. 2xl07 SP2/0 and 8xl07 immunized spleen cells).
• Centrifuge the mixed cells suspension using glass round bottom 50 ml
tube at 160xg for 5 min.
• Aspirate as much medium as possible, and stand the tube upside down
position for draining of medium completely.
• Add 1.0 ml pre-warmed 50% Polyethylene Glycol 1500 and gently
resuspend the cells at 37°C water bath for 2 mins.
• Slowly dilute out the PEG-4, 000 with pre-warmed base medium (about 40
ml), drop by drop, over the next 3-5 mins. (Dilute out slowly in the first part
and quickly in the later part) Centrifuge cells at 1,100 rpm for 7 mins.
• Resuspend cells in 40 ml HAT as hybridization medium (RPMI-1640,
(Gibco, USA) with 10"4 M hypoxanthine, 10~7 M aminopterine, 10"5 M
thymidine containing 20% fetal calf serum and gentamycin & amphotericin
B) at a concentration plate into 96 well feeder cell plates, 100 ul/well.
• Plate 100 ul per well of a % well flat bottom plate, which has feeder cells.
• Incubate at 37°C with 5% CO2 and 100% humidified incubator.
• The cultures were observed under the microscope and after 72 hours
fusion efficiency was determined.
HAT medium selection for hybrid cell:
On day 1-2: Observed cell on inverted microscope for myeloma cell death and contamination. Cells in HAT control should not be alive (myeloma cell only die). Day 7: Observe cell to see whether they have clusters of proliferating cells in the bottom and marked them.
Day 8-11: Change half volume of medium in the well with fresh HAT medium and this half volume of medium was used for screening of hybrid clones for secrete antibody by ELISA test
Screening of hybrid clones by ELISA:
88% fusion efficiency was achieved after 72 hours of fusion, 70 hybrid clones
could be observed microscopically. Supernatants from these hybrid clones were
collected and primary screening was done for antichlamydial antibody bv ELISA
test using antimouse immunoglobulins conjugated with HRPO.
Selection of desired clones in HT medium:
Select the clones whose supernatant gave a strong and /or desired reaction for
further hybrid cloning for a single cell clone in HT medium
Determination of Immunoglobulin class of monoclonal antibody:
The immunoglobulin classes of the monoclonal antibodies were determined by
indirect immunofluoresence assay using class specific antimouse
immunoglobulins conjugated with FITC (Dakopat, Denmark) as the second
antibody.
EXAMPLE 4
Process of detecting the Chlamydia trachomatis
Specificity of monoclonal antibody by DOT ELISA:
Suspensions of chlamydial antigens (C, J, A, I, L3, H, F, G, K, B, Ba, D, E, L1 and L2) were applied on each of 20 pre- grided nitrocellulose (NC) 2X2 inches mounted on used X Ray film for rigidity. Approximately 0.25 jJ of each antigen preparation was applied to each grid position and the NC squares were allowed

to dry at room temperature (20-25 °C) for 6-24 hrs before stereotyping was performed The NC squares were incubated in 2% bovine serum albumin in PBS for 2 h at room temperature in separate chamber of polystyrene slide box. To each chamber was added the monoclonal antibody supernatant (neat) to be tested The mixture was allowed to react and incubated for 1 h, at room temperature. The NC squares were then washed four times with PBS -0.05% Tween20. 1:1000 dilution in PBS of horseradish peroxidase conjugated goat antimouse immunoglobulins IgG was then used to cover each NC Square. The conjugate reaction was allowed to proceed at room temperature for 1 h with gentle rotation. The conjugate solution was then removed and NC squares were then washed three times with PBS-tween20 and then PBS. Enzyme tagged immunoglobulin complexed to C trachomatis monoclonal antibodies were detected by a colorimetric reaction using a solution of 4-chloro-lnaphthol (0.5mg/ml) and hydrogen peroxide (0.025%) in phosphate buffered saline. Standard antigens included in each assay were observed closely to determine optical color development (usually 5-10 min) when rinsing with water stopped reaction.
After being rinsed, the NC squares were dried overnight at room temperature. Each antigen dot was scored for color production (- designating no color change: + dark grey color (code -202B according to RHS colors monitor) Specificity of the monoclonal antibody was then determined by comparing the reactivity profile pattern with chlamydial antigens. The specificities of the monoclonal antibodies in the DOT ELISA assay were determined. We tested 70 hybrid clones (Table 1). There were 68 clones, which were found to be specific for various serovars including (Table 2). However, two hybrid clones were found to be very type specific for D antigen in DOT ELISA (Fig. 3). These two clones were not specific for other antigens (Table 3).
TABLE 1: STANDARD SEROVAR OF CHLAMYDIA TRACHOMATIS
(Table Removed)
Table 2: Identified clones representing different serovars
Reactivity Clones
B-Complex
(B,Ba,D,E,LI and L2)- E4.2, E4.3, B2.2, C10.2, B3.3, D5.1, D9 2, D10.4, D11.2,
F4.2, G1.5and H5.6. C-Complex (A,C,H,l,J,Kand L3) - D10.4, D11.2, F4.2 and G1.5.
Intermediate Group
(F and G) - B2.2, B3.3, C10.2, D5.1, D9 2, D10.4, D11.2, E4.3,
F4.2andG1.5.
TABLE: 3 the absorbance diagnostic values for identified C. trachomatis serovar D in comparison to the other species of Chlamydia and the control samples.
(Table Removed) EXAMPLE 5
Characterization of monoclonal antibody of hybrid clone:
The secretary monoclonal antibody of serovar D specific hybrid clone was characterized for antigen specificity using purified MOMP antigen (40 KDa) of I Chlamydia trachomatis by ELISA test (Table 4; Fig 1 and 2).
Table 4: A DOT ELSIA to confirm monoclonal antibody clones H5.6 and E4.2 for detection of serovar D of C. trachomatis
(Table Removed)
(-) = Negative, (+) = Positive
NOTE: Color matching done with RHS Colors monitor and found positive result with code 202B (Dark grey) color
Example 6
A kit for detecting Chlamydia trachomatis Propagation of selected hybrid
clones:
Selected serovar D specific hybrid clones were propagated in RPMI1640 medium containing 20% fetal calf serum and gentamycin & amphotericin B in the 45 cm tissue culture flask, subsequent culture and stored in liquid nitrogen. The available kits for diagnosis of C.trachomatis are genus specific and species. Specific monoclonal antibody based on MOMP present in all 15 known Human serovars. However the monoclonal antibody developed by us is D serovar specific and has no cross reactivity with other species of Chlamydia and for a specific sequence of the MOMP gene (Copy enclosed) and has no cross reactivity with other species of Chlamydia.
Detection of C.trachomatis in clinical specimens in the present diagnostic kit by present invention takes 2-3 hours which is almost similar to the existing kit (Microtrak.Trinity Biotech, Ireland) for antigen detection by DFA and 4-5 hours by ELISA. However for detection of C.trachomatis in culture system it takes 48- 72 hours (Fig.4).
Further, we provide a comparative chart in form of Tables 5, 6 and 7, which shows that the present kit is better in detecting the Chlamydia trachomatis as compared to the existing kits.
Stepwise representation of the detection process is given below: (a) Detection of C. trachomatis in clinical samples by DFA (A kit for detecting Chlamydia trachomatis)
(a) :
Clinical specimen (Fix with methanol for 10 minutes)

Block with 2% BSA for 60 minutes

Wash with PBS-T

Add culture supernatant (monoclonal antibody) of hybrid clone (invention) and
incubate for 60 minutes
Wash with PBS-T

Add Rabbit anti-mouse IgG FITC conjugate as 2 antibody and incubate for 30
minutes

Wash with PBS-T

Mount the slide and observed under the Flouresence microscope(b) Detection of C. trachomatis in clinical samples by ELISA (A kit for detecting Chlamydia trachomatis)
Coat the clinical specimen in 96 well plates and incubate for 60 minutes

Wash with PBS-T

2% BS A use as blocking agent for 60 minutes

Wash with PBS-T

↓ Add culture supernatant (monoclonal antibody) of hybrid clone (invention) and
↓ incubate for 60 minutes

Wash with PBS-T

Add Rabbit anti-mouse IgG HRPO conjugate as 2 antibody and incubate for 60
minutes

↓ Stop the reaction with 5 M sulfuric acid and take OD by ELISA reader at 450 nm.

Tables 5 and 6 showing comparision between present method and the earlier known methods for detecting Chlamydia trachomatis the
(Table Removed) References:
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We claim
1. Novel monoclonal antibodies namely H5.6 and E4.2 having a sequence complementary to an epitope with sequence DVKTGAE for detecting Chlamydia trachomatis.
2. The monoclonal antibodies as claimed in claim 1, wherein said antibodies are specific to serovar D of Chlamydia trachomatis.
3. The monoclonal antibodies as claimed in claim 1, wherein Chlamydia trachomatis causes genital infection.
4. The monoclonal antibodies as claimed in claim 1, wherein antibodies have 100 % specificity for detecting Chlamydia trachomatis serovars.
5. The monoclonal antibodies as claimed in claim 4, wherein antibodies have 100 % specificity for detecting Chlamydia trachomatis serovar D.
6. A method for detecting the Chlamydia trachomatis with the novel monoclonal antibodies
of claim 1, comprising the steps as follows;
(a) applying suspensions of Chlamydia antigens to a nitrocellulose (NC) grid,
(b) incubating NC grids in Bovine serum albumin (BSA) in Phosphate borate buffer solution (PBS) for 2 hrs at room temperature,

(c) adding monoclonal antibodies namely H 5.6 and E4.2 having sequence complementary to an epitope with sequence DVKTGAE to the NC grids,
(d) allowing the mixture of step (c) to be incubated at room temperature for 1 hr,
(e) washing the NC grids of step (d) with PBS and Tween-20,
(f) adding and reacting the NC grids of step (e) with conjugate of horseradish
peroxidase goat antimouse immunoglobulin (IgG) for 1 hr,
(g) washing again the NC grids to remove excess of IgG, and
(h) detecting the enzyme tagged immunoglobulin complexed to C.trachomatis by colorimetric reaction.
7. A method as claimed in claim 6, wherein NC grid has size of 2X2 inches.
8. A method as claimed in claim 6, wherein 0.25ul of antigen is added in step (a).
9. A method as claimed in claim 6, wherein BSA in step (b) is 2%.
10. A method as claimed in claim 6, wherein PBS in step (b) is 0.05%.
11. A method as claimed in claim 6, wherein ratio of PBS and horseradish peroxidase conjugated goat antimouse immunoglobulin IgG is 1:1000.
12. A method as claimed in claim 6, wherein monoclonal antibodies of step (c) are 40 kDa.
13. Novel monoclonal antibodies, method for detecting Chlamydia trachomatis, substantially as herein described with reference to the accompanying drawings and figures.

Documents:

792-DEL-2003-Abstract-(19-01-2011).pdf

792-DEL-2003-Abstract-(23-06-2010).pdf

792-del-2003-abstract.pdf

792-DEL-2003-Claims-(19-01-2011).pdf

792-DEL-2003-Claims-(23-06-2010).pdf

792-del-2003-claims.pdf

792-DEL-2003-Correspondence Others-(06-03-2012).pdf

792-del-2003-Correspondence Others-(11-04-2012).pdf

792-DEL-2003-Correspondence-Others-(05-07-2010).pdf

792-DEL-2003-Correspondence-Others-(19-01-2011).pdf

792-DEL-2003-Correspondence-Others-(23-06-2010).pdf

792-del-2003-correspondence-others.pdf

792-del-2003-correspondence-po.pdf

792-del-2003-description (complete).pdf

792-del-2003-description (provisional).pdf

792-DEL-2003-Drawings-(05-07-2010).pdf

792-DEL-2003-Drawings-(23-06-2010).pdf

792-del-2003-drawings.pdf

792-DEL-2003-Form-1-(19-01-2011).pdf

792-DEL-2003-Form-1-(23-06-2010).pdf

792-del-2003-form-1.pdf

792-del-2003-form-18.pdf

792-DEL-2003-Form-2-(19-01-2011).pdf

792-DEL-2003-Form-2-(23-06-2010).pdf

792-del-2003-form-2.pdf

792-del-2003-form-26.pdf

792-del-2003-form-3.pdf

792-del-2003-form-4.pdf

792-del-2003-form-5.pdf

792-del-2003-GPA-(11-04-2012).pdf

792-DEL-2003-GPA-(23-06-2010).pdf

792-DEL-2003-Petition 137-(23-06-2010).pdf


Patent Number 246263
Indian Patent Application Number 792/DEL/2003
PG Journal Number 08/2011
Publication Date 25-Feb-2011
Grant Date 23-Feb-2011
Date of Filing 10-Jun-2003
Name of Patentee INDIAN COUNCIL OF MEDICAL RESEARCH
Applicant Address V. RAMALINGASWAMI BHAWAN, ANSARI NAGAR, POST BOX 4911, NEW DELHI-110 029, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 ARUN LATA MITTAL C/O. INDIAN COUNCIL OF MEDICAL RESEARCH V. RAMALINGASWAMI BHAWAN ANSARI NAGAR,POST BOX 4911 NEW DELHI-110 029,INDIA.
2 AMIT KUMAR C/O. INDIAN COUNCIL OF MEDICAL RESEARCH V. RAMALINGASWAMI BHAWAN ANSARI NAGAR,POST BOX 4911 NEW DELHI-110 029,INDIA.
PCT International Classification Number G01N 33/53
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA