Application of Monoclonal Antibodies in Veterinary Parasitology

The discovery of hybridoma technology by Kohler and Milstein in 1975, heralded a new era in antibody research. Mouse hybridomas were the first reliable source of monoclonal antibodies. The generation of monoclonal antibodies from species other than rats and mice, has developed slowly over the last 30 years. The advent of antibody engineering and realization of the advantages of non murine antibodies has increased their relevance recently. However, in the area of veterinary parasitology, monoclonal antibodies are just beginning to fulfill the promises inherent in their great specificity for recognizing and selectively binding to antigens. This review describes the recent advances in the application of monoclonal antibodies for immunodiagnosis / prophylaxis and immunotherapy of parasitic diseases.


Introduction
antibodies therefore high specific activity of labelling is possible in radioimmunoassay, enzyme linked The advent of biotechnology has invigorated immunosorbent assay and fluorescent immunoassay.research on the control of parasitic diseases.A Mabs could be advantageously used as immunothorough study of parasite antigens is a prerequisite for sorbent for antigen purification using imunosorbent control programmes based on protection by column chromatography.vaccination, accurate serodiagnosis and perhaps Large amount of Mabs (1-20 mg per ml ascites immune modulation to diminish pathological fluid) could be obtained with modest investment.sequelae.The utilization of hybridoma technology to Mabs specific for a particular target antigen can be produce antigen-specific monoclonal antibodies obtained even without prior purification of antigen.(Mabs) has resulted in great strides towards obtaining pure antigens relevant for immunodiagnostic purposes Applications in Protozoal Infections and for research on vaccines.This technology was Coccidiosis: Coccidiosis is the most important discovered in 1975 by two scientists Georges Kohler parasitic disease of poultry resulting in over $ 300 of West Germany and Caesar Milstein of Argentina million in annual losses.Recent studies on Eimeria who jointly with Neil Jerne of Denmark were awarded using monoclonal antibodies have laid the ground the noble prize for Physiology and Medicine.
work for isolating functional antigens in immuni-

Advantages of Monoclonal Antibodies
zation trials in chickens.The characterization and localization of antigens of Eimeria spp.by monoclonal Each Mab recognizes only one antigenic antibodies (MAb) has focused predominantly on determinant or epitope.Therefore, a Mab may be surface antigens of sporozoites and merozoites or selected which has specificity for an antigenic deterspecific organelles such as sporozoite refractile minant unique to a particular parasite and false bodies.The monoclonal antibodies produced against positive reactions due to cross-reactivity are mini-Eimeria tenella recognized all strains of this species mized or eliminated.
While diagnostic reagents vary from laboratory that were tested but did not react with any of six other Eimeria species.to laboratory but the parasite antigen recognized or To assess the role of specific antigen of E. tenella isolated using a Mab is invariant, thus permitting in infectivity, the effect of three distinct monoclonal reproducibility and standardization in diagnostic tests.
antibodies on parasitic establishment and development All the antibodies produced (100%) are active was studied in vitro (Danforth, 1983).All three Babesia equi respectively were successfully produced antibodies influenced the ability of parasites to enter (Bruning et al., 1997).Both Mabs reacted specifically cells by 24 hrs and nearly completely inhibited the in indirect ELISA when isolated whole merozoites invitro development of the parasites by 96 hrs.These were used as antigens.Similarly, MAb BEG3 produced studies suggest that all the three antigens recognized against a species specific 19 kDa antigen of B. equi by the different monoclonal antibodies are important significantly inhibited the invitro growth of B. equi for the intracellular establishment and development of parasite (Avarzed et al., 1998 specificity.In calves experimentally infected with B. Since the sporozoite antigens identified by bigemina, the seroconversion was detected at about 10 hybridoma antibodies, were found in such minute days post inoculation.The test can be useful for the amounts that it become necessary to utilize genetic epidemiology studies, particularly in areas where B. engineering in order to produce enough protein for bovis and B. bigemina have overlapping distributions.immunization and one such protein, designated 5401 Theileriosis: Theileria parva is an intracellular has been shown the use of genetically engineered protozoan parasite that causes a disease termed East antigen as potential vaccine candidates against Coast fever in cattle in East and Central Africa.Cattle coccidiosis.The antibodies production in birds has become infected when sporozoites of T. parva are been elicited and partial protection against coccidial secreted into the blood during feeding of the tick infection has also been produced.The progress intermediate host.obtained from these investigations makes the Monoclonal antibodies have been developed development of vaccine against coccidia by use of that identify common antigenic determinants on the birds own protective immune response within the surface of several strains of T. parva sporozoites.realm of possibility (Danforth and Augustine, 1985).These antibodies have been found to neutralize the Some work has also been done on the stage cross infectivity of sporozoites in an invitro assay reactive Mab by use of indirect immunofluroscent (Dobberlaere et al., 1984;Musoke et al., 1984) and antibodies (IFA) and western blot analysis.One such invivo when sporozoites are pretreated with Mabs cross reactive monoclonal antibody designated 1205 prior to inoculation into cattle.One of these protective was used to study redistribution, parasitophorus monoclonal antibodies was subsequently used to vacuole incorporation and insitu antigen production isolate a 68K antigen from the surface of T. parva during the intracellular parasite development of sporozoites (Dobbelaere et al., 1985).These results Eimeria acervulina and E. tenella (Danforth et al., indicate that sporozoite surface antigens common to different stocks of T. parva may have potential as a 1994).

Babesiosis:
broad spectrum vaccine; however, field testing of Babesia (causing babesiosis or Red these antigens will undoubtedly require antigen water fever) is an intraerythrocytic protozoan parasite production by gene cloning on other methods. of livestock transmitted by a tick intermediate host.
Toxoplasmosis: Toxoplasma gondii is an Monoclonal antibodies generated against the crude intracellular protozoan parasite that infects virtually parasite fractions of Babesia bovis have been used to all warm-blooded animals and is of considerable identify and isolate pure parasite antigens.One of public health importance.The antigens of T. gondii these antigens, of 44K molecular weight, has been have been the subject of considerable research aimed used to protect cattle against virulent challenge at improved diagnosis and vaccine development.(Wright et al., 1983(Wright et al., , 1985)).These results suggest that a Monoclonal antibodies have been used to identify candidate antigen has been identified with potential as antigens from the surface (Handman et al Antibodies directed the 70 kDa and 34 kDa protein of Babesia caballi and against a 30K molecular weight tachyzoite surface addition, Cryptosporidium infectivity in experimenantigen have been used to kill parasites invitro in the tally infected animals was decreased with the use of presence of complement (Kaspar et al., 1983).immune colostrum in combination with Mabs However, active immunization with the purified (Perryman et al., 1990).antigen failed to confer any protection (Kaspar et al.,

Helminth Infection
1985).Another study showed that monoclonal antibodies directed against 35K and 14K molecular Fasciolosis: Very few studies have been reported on weight tachyzoite surface antigens were able to confer the production of monoclonal antibodies to Fasciola passive protection in mice (Jhonson et al., 1983).A spp. with most work on Fasciola hepatica rather than monoclonal antibody directed against a 58K Fasciola gigantica.These include monoclonals which molecular weight tachyzoite cytoplasmic antigen was were reactive with tegumental antigens present in the found to confer passive protection and the affinity-tegument-I (TI) granules and glycocalyx of the fluke isolated antigen conferred complete protection against (Hanna and Trudgett, 1983;Hanna et al., 1988) and lethal challenge in mice (Sharma et al., 1984).Murine monoclonals against some excretory-secretory (E/S) Mabs against Neospora caninum tachyzoites were products of F. hepatica (Solano et al., 1991).produced to identify the cross-reactive antigens A monoclonal antibody-based capture ELISA between N. caninum and T. gondii and it is reported was developed for detection of a 26 to 28 kDa antigen that some of the proteins could be useful in developing of F. hepatica in the feces of infected cattle (Abdel et vaccines or drugs for controlling the diseases caused al., 1998).Mab was recognized as M2D5/D5F10.by the two parasites (Liao et al., 2005).This test is highly specific and sensitive and it can Cryptosporidiosis: Cryptosporidium parvum is an diagnose the infection as early as 6 weeks duration and obligate enteric protozoan parasite which infects the can detect as little as 300 pg of coproantigens/ml of gastrointestinal tract of animals and humans.It is fecal supernatant.The assay results correlated well transmitted by the fecal-oral route via the oocyst stage with the number of flukes suggesting that it is possible through ingestion of contaminated water or food, to estimate fluke burden.direct contact with infected humans or animals, or MM3 Mabs, produced by immunizing mice with contaminated surfaces.A number of zoite surface a 7 to 40 kDa purified and O-deglycosylated fraction glycoproteins are known to be expressed and believe of F. hepatica E/S antigen, were used for the detection to be involved in invasion and infection of host of F. hepatica E/S antigens in feces of infected hosts epithelial cells.In the absence of protective treatment (Mezo et al., 2004).This experiment indicated that for this illness, antibodies targeted against these zoite capture ELISA using Mab MM3 assay is a reliable and surface glycoproteins offers a rational approach to ultra sensitive method for detecting subnanogram therapy.Mabs against sporozoite surface antigens amounts of F. hepatica antigens in feces from sheep have proven useful in the characterization of unique and cattle, facilitating early diagnosis.The MM3 antigens and in detection of oocysts from feces for capture ELISA assay detected 100% of sheep with one diagnostic purposes (Anusz et al., 1990).The major fluke, 100% of cattle with two flukes and 2 of 7 cattle goals for developing anti-C.parvum Mab were -first with one fluke.The false negative animals (5/7) were is Mabs against sporozoite antigens can inhibit probably not detected because the F. hepatica in these parasite invasion and lessen the severity of infection in animals was immature.animals (Tilley et al., 1991) and secondly, Mabs that MM3 sero and MM3 copro ELISA test in sheep target oocyst-wall antigens have been valuable in experimentally infected with F. hepatica or F. detection of Cryptosporidium in fecal material and for gigantica showed that fasciolosis could be detected at determination of the extent of oocyst contamination in 4 weeks post infection from serum samples and copro water supplies (Smith and Rose, 1990; Sterling and antigens could be detected at 4-7 weeks in F. hepatica Arrowood, 1986).and 3-6 weeks in F. gigantica (Valero et al., 2008).The Despite the advantages of Mabs, targeting single study demonstrated that MM3 sero and MM3 copro antigenic determinants may be inadequate when ELISA could be applied in the studies on dealing with multiple antigenic determinants involved epidemiology, pathogenesis and treatment in animals in host -C.parvum interaction and result in ineffective and humans where F. hepatica and F. gigantica cotherapy.This disadvantage could be overcome by exists.using a combination of Mabs each targeting different A 28 kDa F. gigantica cysteine proteinase antigenic sites or by using polyclonal antibodies.In (FgCL-3) was purified from the E/S product of bubalian origin flukes with an average yield of (AgB), a thermostable lipoprotein that constitutes a 116.75µg/ fluke (Dixit et al., 2003).Fagbemi and considerable fraction of the cystic hydatid fluid, as a Hillyer (1992) previously isolated a 28 kDa protease suitable source for vaccination and immunodiagnosis from whole worm extract of F. gigantica.It is not clear of cystic echinococcosis due to its high sepecificity.that the 28 kDa recognized in whole worm extract is They further reported that the genetic immunization of the same as that in the E/S products.Fagbemi (1995) BALB/c mice with second subunit of antigen B (Eg also produced Mabs which were reactive with the 28 AgB8/2) for the production of monoclonal antibodies.kDa protease and a 27/28 kDa doublet of F. gigantica.Fusion products between spleen cells and myeloma The 28 kDa protease specific Mab could be a useful cells produced six MAbs.All MAbs identified the four tool for direct isolation of the protease from the whole AgB subunits with molecular weights of 8, 16, 24 and worm extract and/or E/S products of F. gigantica by 36kDa.Further work on specificity and sensitivity of antibody affinity chromatography.These proteases these MAbs as well as their use in detecting circulating possess immuno-diagnostic properties and showed parasite antigens and in antigen purification will be 100% sensitivity in detection of low and high grade assessed in future studies.experimental fasciolosis in sheep and buffaloes (Dixit Dirofilariosis: Monoclonal antibodies were prepared et al., 2002,2004).However, under field situation of for the detection of parasite antigens in the serum of natural F. gigantica infection, this sensitivity of the test Dirofilaria immitis infected dogs by counter immuno declined to 97% but specificity remained 100% (Raina electrophoresis (Weil, et al., 1985).Monoclonal et al., 2006).
antibodies were also generated against adult worms Most of E/S proteases which are 26-30 kDa in for the identification of circulating antigens associated size resemble mammalian liver Cathepsin-L proteases with immune-complex glomerulo-nephritis in dogs both in amino acid sequence and substrate specificity infected with D. immitis.Using these antibodies (Brady et al., 1999;Tort et al., 1999).Currently, these antigen-capture ELISA detected circulating antigens are referred as the Fasciola Cathepsin-L cysteine in 75% of infected dogs.Two bands of 62 and 28kDa proteinases (Dixit et al., 2008).First 14-N terminal respectively were detected on western blot by aminoacids of 28 kDa F. gigantica cathepsin L monoclonal antibody.Parasite antigen detection with cysteine protease (FgCL-3) on sequence analysis the Mab-based ELISA appears to be superior to showed an identity of 64% with N-terminal sequence previously described diagnostic methods for canine of recombinant FgCL-1(rFgCL-1) produced by dirofilariosis in terms of sensitivity, specificity and in Grams et al. (2001).The Mab 2B11-E8-E3 was relation to infection intensity (Nakagaki et al., 1993).developed against recombinant F. gigantica Cathepsin Trichinosis: Studies in rodents have shown that L-A (rFgCL-A).This Mab detected 0.8ng of rFgCL-A antigens of immunological value may be derived from and specifically reacted with 30 kDa rFgCL-A and three stages of the life cycle of T. spiralis, the tissuenative 28-29 kDa Cathepsin -L in E/S products and dwelling muscle larvae, the intestinal adult stage and crude worm extract of F. gigantica.Immunolo-the migrating newborn larvae (Wakelin and Denham, calization by Mab found Cathepsin -L in the epithelial 1983).Monoclonal antibodies against the muscle lining of gut (Inthakanok, 2007).larvae stage of T. spiralis were produced by fusing Cystic Echinococcosis: Cystic echinococcosis is an spleen cells from mice inoculated orally with infective endemic cosmopolitan zoonotic helminthic disease parasites (Gamble and Graham, 1984).Most caused by the larval stage of Echinococcus monoclonal antibodies recognized antigens shared by granulosus.Two hybridomas were produced using larval and adult stage of T. spiralis and by other swine spleen cells from mice experimentally infected with parasites, particularly Trichuris suis.However, hydatid parasite E. granulosus (Craig et al., 1981).several monoclonal antibodies were specific for T.These two hybridomas secreted antibody with anti-E.spiralis larvae or adult antigens.Because of the granulosus cyst fluid (EgCF) activity.However, these demonstrated diagnostic value of assays utilizing monoclonal antibodies produced against E. larval ES products, one hybridoma with specificity for granulosus cross react with sera from Taenia ovis, an ES antigen of muscle larvae (7C2C5) was selected Taenia hydatigena and F. hepatica infected sheep.for further study.The Mab 7C2C5 recognized a single Anyhow the partially purified E. granulosus antigen antigen epitope present on proteins of molecular wt.showed improved specificity in an indirect ELISA for 45000, 49000 and 53000 (Ts45, Ts49, Ts53).These the diagnosis of infected sheep.

M
.C. (1994), In vitro and In vivo immunolabeling of programmes.Hybridoma technology is just beginning sporozoites, schizonts and sexual stages of Eimeria to be applied in the field of parasites immunoacervulina and E. tenella by a species and stages crossreactive monoclonal antibody.Parasitol.Res., 80: 594-599.diagnostic and the recent success in research 12. Dobbelaere, D.A.E., Spooner, P. R., Barry, W. C. and lrvin, A. employing these techniques should encourage greater D. (1984), Monoclonal antibody neutralizes the sporozoite interests.Preliminary reports indicate that the stage of different Theileria parva stocks.Parasite Immunol., 6: 361.monoclonal antibodies with immunodiagnostic value 13.Dixit, A.K., Yadav, S.C. and Sharma, R.L. (2002), 28 kDa have been produced against many important parasites.Fasciola gigantica cysteine proteinase in the diagnosis pre-The use of monoclonal derived reagents have greatly patent ovine fasciolosis.Vet.Parasitol., 109: 233-247.increased the specificity of diagnosis by eliminating 14.Dixit, A.K., Yadav, S.C., Saini, M. and Sharma, R.L. (2003): Purification and characterization of 28 kDa cysteine cross reactions between closely related parasite spp., proteinase for immunodiagnosis of tropical fasciolosis.J. without suffering a significant loss of sensitivity.It is Vet.Parasitol.17: 5-9.hoped that continuing research along with the use of 15.Dixit, A.K., Yadav, S.C. and Sharma, R.L. (2004), new antibody-based strategies for immunodetection, Experimental bubalian fasciolosis: Kinetics of antibody response using 28 kDa Fasciola gigantica cysteine proteinase prophylaxis and immunotherapy will pave new means as antigen.Trop.Anim.Health Prod., 36: 49-54.to combat the notorious parasites.16.Dixit, A.K., Dixit, Pooja and Sharma, R.L. (2008), Immunodiagnostic/protective role of Cathepsin L cysteine ). E. tenella.Although the precise nature of establish-Monoclonal antibodies directed against a 58 kDa Babesia bigemina merozoite antigen reacted ment and development has not been determined, other studies indicate that one of the monoclonal antibodies, strongly with immune sera from experimentally and recognizing a 22K sporozoite antigen, binds to the naturally infected cattle in Western Blots (Molly et al., sporozoite surface and in the presence of complement, 1998).Further, competitive inhibition ELISA detected mediate cell lysis (Danforth et al.,1985; Danforth, antibodies directed against a single epitope on the 58 1986) supporting the immunotherapeutic use of kDa antigen with 95.7% sensitivity and 97% hybridoma technology in poultry coccidiosis.
considered antigen B and used in a triple-antibody indirect ELISA for the 2. Anusz, K.Z., et.al. (1990), Detection of Cryptospordetection of antibodies to T. spiralis (Gamble et al., recognized by hybridoma antibodies.Proc.Fed.Am.Soc.capable of mediating eosinophil-induced destructionExp.Biol., 44: 1334. of nematodes invitro may also be important in 9. Danforth, H.D. (1983), Use of antibodies directed against protection against infection.Eimeria tenella sporozoite to determine stage specificity and in vitro effect on parasite penetration and development.Am.Conclusion J. Vet.Res., 44: 1722-1727.10.Danforth, H.D. (1986), Use of hybridoma antibodies As failure to detect low level patent infection combined with genetic engineering in the study of protozoan incurs the risk of having a reservoir capable of parasites: A review.In: L. P. Joiner, P. L. Long and L. R.