Title of Invention

" A PROCESS FOR THE PREPARATION OF PLASMODIUM FALCIPARUM SPECIFIC MONOCLONAL ANTIBODY CLONE"

Abstract

A process for the preparation of Plasmodium falciparum specific monoclonal antibody which comprises: a. Culturing hybridoma clone having characteristics as herein described at temperature of 37° C in humidified atmosphere of 5-10 % CO2 / 90-95 % air in a medium such as herein described. b. Injecting the culture obtained from step (a) to pristane primed BALB/c mice for ascites production . c. Isolating monoclonal antibody from culture supernatant/ascites fluid by a manner as herein described. d. Purifying monoclonal antibody using affinity column in a manner as herein described.

Full Text

This invention relates to a process for preparation of Plasmodium falciparum specific monoclonal antibody. In particular the present invention relates a process for preparation of plasmodium falciparum specific monoclonal antibody from a new hybridoma clone PLD-C13 useful for diagnosis of malaria and screening of antimalarials useful for diagnosis of malaria and screening of antimalarials. Lactate dehydrogenase (LDH), the terminal enzyme of the glycolytic peihway, has been shown to be present in malaria parasites and is known to play a regulatory role in energy metabolism of these parasites. The LDH from malarial parasites has been found to be biochemically and immunologically distinct from the host LDH and used as a marker enzyme for biochemical characterization of rnalarial parasites Earlier we have damonstrated the potential of monospecific polyclonal antibodies against plasmodial LDH for immunodiagnosis of malaria (patent application no. 991/ DEL/ 94). An immunodiagnostic test (based on parasite LDH detection) has been developed employing the polyclonal anti-LDH antibodies. The method is simple, rapid and reliable for detecting malaria parasites in finger prick blood samples (patent application no. 992/DEL94) However, in order to refine the test system, replacement of polyclonal antibodies with monoclonal antibody is required We have generated a number of monoclonal antibodies against the plasmodial LDH (Kaushal et al. 1995. Monoclonal antibodies against lactate dhydrogenase of Plasmodium Knowlesi. Indian J. Exp. Bid. 33: 6-11). Though these menodonal antibodies showed specificity plasmodial LDH, did not show enough sensitivity for detecting the malaria parasites in blood samples. This emphasized theneed to produce a monoclonal antibody against parasite LDH for use in the immunodiagnostic test. The plasmodial LDH activity is directly related to the parasite load The killing of parasites by drugs results in loss of LDH activity, and the inhibition of PLDH activity may also leads to death of the parasites. Therefore, the monoclonal antibody inhibiting specifically the parasite LDH activity can be exploited for the screening of antimalarials. The applicants, by sustained and continuous research work were be able to identify a monoclonal antibody, produced by a new hybridoma clone, which can detect malaria parasite LDH in the blood samples from malaria infected individuals and can be used for the screening of antimalarials. Earlier we have invented a process for the preparation of a new hybridoma clone PLD-C13 which has been deposited in the Central Drug Research Institute, one of the constituent laboratories of the applicant at Lucknow, U.P., India. Tne new hybridoma clone PLD-C13 is different from the previously reported anti-LDH hybridoma clones in secreting a monoclonal antibody of different epitope specificity. The PLD-C13 hybridoma clone secretes monoclonal antibod} which is directed against the loop region of the parasite LDH. The monoclonal antibody produced by the hybridoma clone is IgGl isotype, inhibits specifically malaria parasite LDH activity and useful for detecting malaria parasites in blood samples as well as for the screening of antimalarials. produce a monoclonal antibody against parasite LDH for use in the immuriodiagnostic test. The plasmodial LDH activity is directly related to the parasite toad The killing of parasites by drugs results in loss of LDH activity, and the inhibition of PLDH activity may also leads to death of the parasites. Therefore, the monoclonal antibody inhibiting specifically the parasite LDH activity can be exploited for the screening of antimalarials The applicants, by sustained and continuous research work were be able to identify a monoclonal antibody, produced by a new hybridoma clone, which can detect malaria parasite LDH in the blood samples from malaria infected individuals and can be used for the screening of antimalarials. In our co-pending application no 292/Del/99, we have described and claimed a process for the preparation of a new hybridoma clone PLD-C13 which ahs been deposited in the Central Drug Research Institute, one of the constituent laboratories of the applicant at Lucknow, U.P., India. The main objective of the present invention is to provide a process fro the preparation of anti-plasmodial lactate dehydrogenase monoclonal antibody secreted by a new hybridoma clone having accession no. PLD-C13 given by Central Drug Research Institute, one of the constituent laboratories of the applicants. The main objective of the present invention is to provide a process fro the preparation of anti-plasmodial lactate dehydrogenase monoclonal antibody secreted by a new hybridoma clone having accession no. PLD-C13 given by Central Drug Research Institute, one of the constituent laboratories of the applicants. Accordingly, the present invention provides a process for the preparation of plasmodium falciparum specific monoclonal antibody which comprises: a. Culturing hybridoma clone having characteristics as herein described at temperature of 37° C in humidified atmosphere of 5-10 % CO2 / 90-95 % air in a medium such as herein described. b. Injecting the culture obtained from step (a) to pristane primed BALB/c mice for ascites production . c. Isolating monoclonal antibody from culture supernatant/ascites fluid by a manner as herein described. d. Purifying monoclonal antibody using affinity column in a manner as herein described. In an embodiment of the invention after wherein the monoclonal antibody specific to parasite lactate dehydrogenase is purified using protein A Sepharose column (affininty column); In an embodiment of the invention after wherein the culture supernatant/ascities fluid is applied to the affinity column in presence of 2-3 M sart at pH ranging between 8-9; In an embodiment of the invention after wherein the monoclonal antibody absorbed to the affinity column is eluted with a buffer pH ranging between 5-6; In an embodiment of the invention after wherein the hybridoma clone used secretes a monoclonal antibody useful for detection of malaria parasites in blood samples; In an embodiment of the invention after wherein the hybridoma clone used secretes a monoclonal antibody specifically binding to the loop region of plasmodial LDH; In an embodiment of the invention after wherein the hybridoma clone used secretes a monoclonal antibody useful for the screening of antimalarials; The process of the present invention utilises new hybridoma clone PLD-C13 for the preparation of monoclonal antibody useful for diagnosis of malaria. The said monoclonal antibody is produced by the maintenance of new hybridoma clone in in vitro culture/pristane primed BALB/c mice. The monoclonal antibody is purified from the culture supernatants/ascites fluid. The monoclonal antibody is specific to plasmodial LDH as it shows high reactivity with LDH from malarial parasites ( P. falciparum, P. vivax, P. cynomolgi, P. knowlesi, P. yoelii and P. berghei) but do not cross-react with normal red blood cell LDH, three known isoenzymic forms of mammalian LDH or enzyme from several other sources (bacterial, hel- minth and protozoan parasites). These findings suggest that the target epitope of the monoclonal antibody of the present invention is conserved within Plasmodium spp. The monoclonal antibody of the present invention is an IgGl isotype, inhibits the enzyme activity specifically of malaria parasite LDH and directed against an epitope located in the loop region of the plasmodial LDH. The monoclonal detects malaria parasites (based on LDH) in blood samples. The monoclonal antibody can also be used in screening of antimalarials. The details of the invention are given in the Examples provided below which are given by way of illustaration only and, therefore, should not be construed to limit the scope of the present invention. Example 1 The anti-LDH monoclonal antibody is prepared as follows: Maintenance of PLD—C13 hybridoma clone: The hybridoma clone PLD-C13 is maintained in In vitro culture at 37°C in humidified atmosphere of 5-10% CO2/ 90-95% air using DMEM/ RPMI-1640 supplemnted with 10-20% fetal bovine serum. The medium is changed regularly and the subculture is done every 3rd-5th day. The cells harvested from the culture are injected to pristane primed BALB/c mice for ascites production (Potter et al. 1972. Growth of primary plasmacytoma in mineral oil-conditioned peritoneal environment. J. Natl. Cancer Inst. 49: 305). IsoType analysis: The isotype of the secreted monoclonal antibody from culture supernatant ( concentrated using cen-tricon-10 membrane filters, Amicon, USA ) was determined by Ouchterlony analysis (Ouchterlony 1958. Diffusion in gel methods for immunological analysis. Prog. Allergy 5_: 1) using goat anti-mouse IgM, IgGl, IgG2a, IgG2b, IgG3 and IgA. Purification of monoclonal antibody: The purification of monoclonal is done by protein-A Sepharose affinity coluam. The pooled culture supernatant/ascites fluid is applied to Protein-A Sepharose column in a buffer, pH between 3-9, containing 2-3 M NaCl/MgCl2- After washing the column with the same buffer, the antibody absorbed to the column is eluted with buffer having pH between 5-6. The column fractions obtained are tested in ELISA for antibody using purified malaria parasite LDH and the fractions showing high reactivity to parasite LDH are pooled and dialysed extensively against buffered saline, pH around 8.4. The purified monoclonal antibody against P. falciparum LDH is used in -he immunodiagnostic test for examining the presence of malarial parasites based on detection of LDH. The monoclonal antibody specific to malaria parasite LDH can also be purified using Protein-G Sepharose or Diethy-lamino- ethyl cellulose/ Diethylaminoethyl sephacel or Sepha-dex G-200 / Sephacryl S-200 columns. The purified monoclonal antibody is conjugated to radish peroxidase (HRP) following the procedure described elsewhere (Nakane and Kawoi 1974. J. Histocchem. 22.: 1084-1091). The procedure for the preparation, purification and conjugation of anti-plasmodial LDH polyclonal antibody is described in our previous patent applications nos. 991/DEL/94 and 992/DEL/94. Preparation of Blood Samples: The blood samples from normal or malaria infected individuals are collected by finger prick and processed as described in previous patent application no. 992/DEL/94. Briefly, 100 ul of blood samples from normal or malaria infected individuals, collected by finger prick, are diluted about four times with lysis buffer (buffer with pH in the range of 7.2 containing 150 mM sodium chloride, 1% Triton X-100 and 5 mM ethylenediamine tetra-acetic acid) and vortexed for 5 minutes. Enzyme linked immunosorbent assay: The enzyme linked immunosorbent assay (ELISA) is done in microtitre ELISA plate using the following procedure. Briefly, the wells of microtitre plates are coated with purified malaria parasite LDH (about 20-100 ng/ well) diluted in buffer-1 ( buffer, pH 7.2-9.6) by incubation for about 2 h to overnight at around 37°C. The plates are then washed with buffer-1 and the uncoated sites are blocked by incubation with around 200 ul of milk (defatted milk powder prepared In buffer-1) at around 37° tor about 2 h. Following 3-5 washes with buffer-2 (buffer, pH 7.2-9.6, containing around 0.05% Tween-20), the plates are incubated with around 100 ul of appropriately diluted culture supernat-ants/ ascites fluid for about 2 h at around 37°C. The plates are washed 3-5 times with buffer-2 and peroxidase conjugated anti-mouse IgG (around 1:2500) is added to the wells. After incubation for about 1.5 h at around 37°C, the plates are washed and color is developed by adding the substrate solution (around 0.5-lmg/ml 0-phenylenediamine in buffer, pH around 5.0, containing around 1 ul/ml H2O2) and keeping for about 5-20 min. The reaction is stopped by adding about 5 N H2SO4, and the absorbance is read at 490 nm using microtitre plate ELISA reader. Inhibition ELISA: The monoclonal antibody is used in inhibition ELISA for the screening of antimalarials. Briefly, wells of the microtitre plates are coated with purified P. falciparum LDH (around 5 ng/well) by incubating at around 37°C for about 2 h to overnight. The blocking of the uncoated sites and the washing of the plate is done as described for ELISA. The peroxidase conjugated monoclonal antibody is preincubated with the test compound (antimalarial/peptide) for about 1 h at around 37°C, then added to the wells of microtitre plate and incubated for about 2 h at around 37°C. The plate is washed and developed as described for ELISA. Immunodiagnostic test for detection of malaria parasites: The preparation of dipsticks and their subsequent use for malaria diagnosis is done as described previously (patent application no. 992/DEL/94). Briefly, 1-2 µ1 (1-2 ug) of purified monoclonal antibody against plasmodial LDH is spotted on Nitrocellulose paper (NCP) strip and air dried for about 30 minutes at around 50° C or 1-4 h at around 37°C. After washing the dipsticks with buffer-1 (buffer, pH around 7.2-9.6) The unreacted sites of the coated strips are blocked by incubation with around 3% non-fat dry milk powder (prepared in buffer-1, pH around 7.2-9.6) for 1-2 h at around 37°C. The strips are washed 3-5 times with buffer-2 (buffer, pH around 7.2-9.6, containing around 0.05% Tween-20) air' dried and stored dessicated. The diazotized paper or nylon membrane strips ( instead of NCP ) can also be used for preparing the dipsticks. The immunodot-dipsticks are incubated with blood samples, prepared as described above, for 1.5 h at around 37°C. The dipsticks are washed 3-5 times with buffer-2 and further incubated with peroxidase conjugated ( conjugated by the method of Nakane and Kawoi, 1974, J. Histochem.22. 1084-1091 ,' or alkaline phosphatase cojugated ( cojugated by the method of Avrameas, 1969, Immunochemistry,6, 43-52) anti-plasmodial LDH monoclonal/polyclonal antibody (designated as PLD-M13) at 37° C for about 1 hour followed by incubation with substrate solution of peroxidase (buffer, pH around 7.2-9.0 containing around 0.3 mg/ml 4-chloro-l-naphthol or around 50 ug/ml diaminobenzidine and 1 ul/ml H2O2 ) or alkaline phosphatase ( around 0.33 mg/ml nitroblue tetrazolium and around 0.165 mg/ml 5-bromo-4-chloro-3- indolyl phosphate In buffer with pH around 9.5 containing around 100 mM sodium chloride and around 5 mM magnesium chloride ) for about 5-10 minutes at room temperature. EXAMPLE 2 The immunodiagnostic test can also be done using micro-titre ELISA plates. The extraction of blood samples and the preparation of antibody conjugates is done in the same way as in example 1. The preparation of the microtitre plates for use in the malaria diagnostic test is done as follows: The wells of the microtitre plates (polystyrene/polyvinyl) are coated with 1-2 ug of anti-plasmodial LDH monoclonal antibody by incuation at around 37°C for about 2 h. The blocking of the uncoated sites of the ELISA plate and the preparation of the blood sample is done as described for the dipsticks in Example 1. The plate is then incubated with about 100 ul of extracted blood samples at about 37°C for about 1.5 h. After washing 3-5 times with Buffer-2, around 100 ul of peroxidase or alkaline phosphatase conjugated anti-plasmodial LDH monoclonal or polyclonal antibodies are added to the wells of the microtitre plate and incuabation is done at around 37° C for about 1 hour. The plate is finally washed 3-5 times with buffer-2 and developed using substrate solution for peroxidase( buffer, pH around 5.0 containing around 0.4-1 mg/ml of orthophenyle-nediaxnine (0PD) or around 0.5-1 mg/ml of 2,2'-azino-bis[3- thylbenz-thiazoline-6 sulfon: ABTS) and around 1 ul/ml H2O2 or alkaline phosphatase (buffer, pH around 9.8-10.4 containing around 1 mg/ml p-nitropheny 1 phosphate; pNPP) for about 5-10 minutes at room temperature. The advantage of the present invention is that the PLDM13 monoclonal antibody is directed against a new epitope located in the loop region of the plasmodial LDH and detects malaria parasites in blood samples at a sensitivity 10-20 times more as compared to previous!}, produced anti-LDH monoclonal antibodies. The monoclonal antibody can also be used in screening of antimalarials (LDH based). We Claim: 1. A process for the preparation of plasmodium falciparum specific monoclonal antibody which comprises: a. Culturing hybridoma clone having characteristics as herein described at temperature of 37° C in humidified atmosphere of 5-10 % CO2 / 90-95 % air in a medium such as herein described. b. Injecting the culture obtained from step (a) to pristane primed BALB/c mice for ascites production . c. Isolating monoclonal antibody from culture supernatant/ascites fluid by a manner as herein described. d. Purifying monoclonal antibody using affinity column in a manner as herein described. 2. A process for the preparation a process for preparation of plasmodium falciparum specific monoclonal antibody substantially as herein described with reference to the Examples.

Documents:

277-del-1999-abstract.pdf

277-del-1999-claims(cancelled).pdf

277-del-1999-claims.pdf

277-del-1999-complete specification (granted).pdf

277-del-1999-correspondence-others.pdf

277-del-1999-correspondence-po.pdf

277-del-1999-description (complete).pdf

277-del-1999-form-1.pdf

277-DEL-1999-Form-2.pdf

277-del-1999-form-4.pdf

277-del-1999-form-9.pdf