Analysis and potential of excretory-secretory antigens of Setaria cervi for immunodiagnosis of human filariasis.

The excretory-secretory (E-LS) products released by the adult Setaria cervi, a bovine filarial parasite, were used to raise polyvalent hyperimmune serum in rabbits. Analysis of E-S products, using anti-E-S serum showed the presence of 10-14 immunogenic proteins, the rabbit anti-E-S serum showed reciprocal antibody titres in the range of 100,000-250,000 by enzyme linked immunosorbent assay. The anti-E-S antibodies could detect circulating antigen in filarial patients sera by Counter immunoelectrophoresis.

Monoclonal antibodies against lactate dehydrogenase of Plasmodium knowlesi.

Lactate dehydrogenase (LDH) of malarial parasites has been demonstrated to be biochemically and immunochemically distinct from the equivalent host enzyme. The polyclonal antibodies raised against the purified plasmodial LDH showed specificity to Plasmodium spp. Six hybridoma cell lines secreting monoclonal antibodies specific to Plasmodium knowlesi LDH have been obtained. The two monoclonal antibodies (2A3B7 and 4A6A7) showed high reactivity with LDH from simian (P. knowlesi. P. cynomolgi), human (P. falciparum, P. vivax) and rodent (P. berghei, P. yoelii) malarial parasites and did not cross-react with red cell LDH as well as with isoenzymic forms of mammalian LDH (A4, B4 and C4). One monoclonal antibody (4A6A7) strongly inhibited the enzyme activity specifically of plasmodial LDH and did not have any effect on the activity of red cell LDH. The other monoclonal (2A3B7) did not show inhibitory effect on parasite LDH. These findings as well as competitive immunoassay studies suggest the presence of at least two parasite specific epitopes on plasmodial LDH.

Monoclonal antibodies against antigenic epitopes common between Setaria cervi and Brugia malayi.

Several common antigens between the bovine (Setaria cervi) and human (Brugia malayi) filarial parasites have been demonstrated [Immunol Investig, 16 (1987) 139]. Hybridoma cell lines producing monoclonal antibodies against such common antigenic epitopes were obtained by immunizing the BALB/c mice with S. cervi antigen, fusing the spleen cells with Sp2/0 myeloma cells and screening the culture supernatants for antibody against both S. cervi and B. malayi antigens by ELISA. Nine monoclonal antibodies directed against antigenic epitopes common between the bovine and human filarial parasites were identified. Two monoclonal antibodies (I3B4 and I5D6) showed reactivity with the antigen(s) present in filariasis patients serum and thus may have potential for detecting circulating antigen in filaria infected individuals.

Enzymes of PEP-succinate pathway in Setaria cervi and effect of anthelmintic drugs.

Localization of different enzymes of PEP-succinate pathway has been done in Setaria cervi, a bovine filarial worm. Succinate dehydrogenase and fumarate reductase were localized in mitochondria rich particulate fraction while all other enzymes were cytosolic. The in vitro effect of certain antifilarial/anthelmintic agents on these enzymes was also investigated. Sumarmin, at low concentration, could cause a marked inhibition of most of the enzymes of this pathway. Centperazine, an antifilarial drug being developed by CDRI showed significant inhibitory action on pyruvate kinase, lactate dehydrogenase, fumarase and succinate dehydrogenase while CDRI compound 72/70 showed significant inhibition of PEP-carboxykinase activity. Diethylcarbamazine and levamisole, however, were found to be more or less ineffective at lower concentrations against all the enzymes of this pathway.

Comparative analysis of surface components of adult, micro-filariae and infective larvae of Brugia malayi.

A comparative analysis of surface proteins of adult, microfilariae and infective larvae of Brugia malayi, the human filarial parasite, has been carried out using IODOGEN (1,3,4,6-tetrachloro-3,alpha 6 alpha-diphenyl-glycoluril) and lactoperoxidase methods. SDS-polyacrylamide gel electrophoretic and autoradiographic analyses revealed the presence of 9 proteins (15-200 kDa) in adults, while microfilariae and infective larvae showed 8 and 6 proteins (15-120 kDa), respectively. The pattern of proteins radiolabelled by IODOGEN method was very similar to that of proteins labelled by the lactoperoxidase method. Since these proteins are released by the protease treatment of whole parasites, they are likely to be present on the surface of the parasite.

Antibodies to lactate dehydrogenase of Plasmodium knowlesi are specific to Plasmodium species.

Polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to lactate dehydrogenase (LDH) of P. knowlesi by immunodot enzyme staining method. The anti-LDH antibodies are most probably directed towards an epitope distinct from the catalytic site as shown by the specific enzyme staining of LDH after binding with antibody on nitrocellulose paper. These antibodies showed reactivity with LDH from different strains (H, P and W1 strains of P. knowlesi) and species (P. cynomolgi B, P. berghei, P. yoelii, P. falciparum and P. vivax) of malarial parasites but did not cross-react with three isoenzymic forms of mammalian LDH (A4, B4 and C4) as well as with LDH from some protozoan and helminth parasites. These findings suggest that the anti-LDH antibodies have defined specificity to Plasmodium spp.

Ancylostoma ceylanicum: I. Protein and antigenic composition of adult and larval stages.

The protein and antigenic composition of adult and larval stages of Ancylostoma ceylanicum, a human hookworm maintained in golden hamsters (Mesocricetus auratus), was studied employing immunochemical techniques. SDS-polyacrylamide gel electrophoresis revealed the presence of 47 and 43 protein bands in adult worms and infective larvae respectively in the molecular weight range of 10-170 kD. Crossed immunoelectrophoretic analysis, using immune rabbit sera, showed the presence of 32 antigenic peaks in adults and 19 in infective larval stage. Most of the antigens were common between adult and larval stage as evidenced by cross-line immunoelectrophoresis, although some stage specific antigens were also identified. These studies also demonstrate the complex nature of adult worms as compared to larvae.

Antigenic characterization of excretory-secretory products of Setaria cervi.

Excretory-secretory products (ES) of Setaria cervi, a bovine filarial parasite, were prepared by in vitro maintenance of the worms in a protein free defined medium at 37 degrees C. RPMI-1640 medium proved to be the best as the worms remained motile in it for the maximum period of time without change of medium, and approximately 12-15 mg protein per 100 adult worms could be obtained. Crossed immunoelectrophoretic analysis of ES products revealed 11-15 antigens and most of them were common to somatic antigenic preparations from both adult worms and microfilariae. The ES products were also found to contain 3-4 host proteins, including serum albumin.

Identification of antigenic proteins of setaria cervi by immunoblotting technique.

Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites.

Protein and antigenic composition of adult and microfilarial stages of Setaria cervi.

The protein and antigenic pattern of adult (female/male) and microfilarial stages of Setaria cervi, a bovine filarial parasite, was studied using certain immunochemical techniques. SDS-polyacrylamide gel electrophoretic analysis showed the presence of 35-40 protein bands in adults and 25-29 protein bands in microfilariae in molecular weight range of 10,000-200,000. Immunoelectrophoresis revealed the presence of 9-10 precipitin lines in adult and only 4 precipitin lines in microfilarial antigenic preparations. Crossed immunoelectrophoresis resolved these antigenic preparations further, and revealed the presence of 22-24 antigens in adults and 12-14 in microfilariae. These results demonstrate complex nature of somatic extracts of adult stage as compared to microfilariae and also reveal some qualitative and quantitative differences between these stages.

Comparison of immunoblot and immunoprecipitation methods for analyzing cross-reactive antibodies to filarial antigens.

Qualitative analysis of antibody responses in helminth infections is essential not only for developing better immunodiagnostic antigens but also for understanding immune recognition and its relevance to immunopathogenesis and protective immunity. In this study 2 qualitative analytic methods (immunoprecipitation and immunoblotting) were compared for the ability to define the extent of cross-reactivity in the serum antibodies from patients with various forms of filariasis (caused by Brugia malayi, Wuchereria bancrofti, Loa loa and Tetrapetalonema perstans) or other non filarial helminth infections (ascariasis, strongyloidiasis, trichinosis, echinococcosis and schistosomiasis). Our results demonstrated that the spectrum of cross-reactive antibodies identified by immunoprecipitation was limited because of the selective radiolabeling of particular filarial antigens, while immunoblotting was able to detect a much wider range of cross-reactive antibodies in both filarial and non-filarial serum pools. In addition, this latter procedure was easily adapted for simultaneous analysis of different antibody isotopes (e.g., IgE and IgG) to the same antigens in individual sera. Immunoblotting thus provides an excellent tool for studying the spectrum of antibodies of different isotypes evoked during helminth infections and for discriminating between those responses that are species-specific and those that are cross-reactive.

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae.

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.

Excretory-secretory and somatic antigens in the diagnosis of human filariasis.

In order to compare the immunodiagnostic value of excretory-secretory (E-S) antigens derived from adult Brugia malayi worms with somatic antigens derived from adults, microfilariae (Mf) and infective larvae (L3) of these parasites, well defined serum pools from patients with filarial (brugia, bancrofti, loa and perstans) and non-filarial (ascaris, stronglyoides, toxocara, echinococcus, cysticercus and schistosoma) helminth infections were tested against antigens derived from these different life cycle stages of B. malayi in a Staphylococcus aureus radioimmunoprecipitation assay (S. aureus RIA). The adult brugia antigens proved significantly more discriminatory than those of the other parasite stages, with the homologous brugia serum pool also showing greater reactivity to adult than to L3 and Mf antigens. Similar results were obtained when individual sera from patients (rather than serum pools) were tested in the same assay. The most surprising finding was the minimal reactivity seen between the adult filarial antigens and the non-filarial serum pools despite the presence in these pools of strong antibody reactivity with their homologous antigens. The reasons underlying the unexpected specificity of this S. aureus RIA for discriminating among sera from filarial and non-filarial infections were analysed qualitatively by immunoprecipitation techniques. It was found that use of the chloramine-T method for radioiodination resulted in preferential labelling of the low molecular weight (mol. wt) proteins (10-70,000 daltons) in the B. malayi adult somatic antigen and that these antigens were bound primarily by the filarial and not the non-filarial serum pools. These findings suggest that lower mol. wt helminth antigens may show greater species specificity than those with higher mol. wt, and those with higher mol. wt, greater cross-reactivity. If substantiated by further analysis, such results would have important implications for the subsequent isolation of diagnostically important filarial parasite antigens.

Identification and characterization of excretory-secretory products of Brugia malayi, adult filarial parasites.

Although E-S antigens may be particularly important for both the pathogenesis and immunodiagnosis of helminth infections, little is known about the immunochemistry or functional roles in human filarial infections. In the present paper, we have done some initial identification and characterization of E-S products of adult Brugia malayi by employing a combination of sensitive biochemical and immunochemical techniques. E-S products, collected by incubating B. malayi adults in vitro in a defined protein-free medium, were radiolabeled with 125I. SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography of labeled E-S products revealed 11 protein bands in the m.w. range of 10,000 to 70,000. Comparison of radiolabeled E-S products and adult somatic antigen (B.m.A) in SDS-PAGE indicated many common bands, and crossed immunoelectrophoresis and competitive Staph-A RIA confirmed the presence of most E-S antigens in B.m.A. Of the 11 E-S bands, two appeared to be derived from the surface of the adult worms and microfilariae as shown by SDS-PAGE and autoradiography of lodogen surface-labeled parasites; the presence of two host proteins in E-S was detected by crossed-line immunoelectrophoresis. The E-S antigens were highly immunogenic when tested both with rabbit antiserum raised against B.m.A and with a serum pool of patients with natural filarial infection.

Antigenic analysis of Giardia lamblia from Afghanistan, Puerto Rico, Ecuador, and Oregon.

The trophozoite antigens of Giardia lamblia to which host humoral and cellular immune responses are directed have not been identified. Therefore, we initiated studies to characterize these antigens in strains of G. lamblia from Afghanistan, Oregon, Ecuador, and Puerto Rico. By polyacrylamide gel electrophoresis, the electrophoretic mobility patterns of proteins of the four strains were similar; molecular weights of protein bands ranged between 12,000 and 140,000. The antigens which reacted with rabbit and anti-G. lamblia antisera by immunoelectrophoresis were also similar for the four strains. However, comparison by crossed immunoelectrophoresis showed the Oregon strain, which has been the longest in culture, lacked a set of anodic antigens and the single neutral antigen which were present in the other three strains. In addition, other minor antigen differences between the strains were detected by this technique. When we employed trophozoites from each strain as antigen in an enzyme-linked immunosorbent assay against 10 human antisera of various liters, we also detected some differences between the strains. Although polyacrylamide gel electrophoresis and immunoelectrophoresis revealed gross similarity among G. lamblia from widely differing geographic locations, subtle differences detected by crossed electrophoresis and enzyme-linked immunosorbent assay suggest the existence of potentially important antigenic differences among these strains.