Stage-specific Monoclonal Antibodies Research Articles

Characterization of Leishmania ( Leishmania) tropica axenic amastigotes

Optimum conditions for generating Leishmania ( Leishmania) tropica axenic amastigotes (AxA) in culture were determined, pH 5.5/36 °C, and the parasites characterized by different techniques, including light microscopy, macrophage infection, stage specific antigen expression and differential display. AxA were morphologically similar to amastigotes and 15.5-fold more infective than stationary phase promastigotes for mouse peritoneal macrophages. Western blotting with promastigote stage specific monoclonal antibodies to either lipophosphoglycan (T2) or a 60 kDa flagella antigen (F3) showed a dramatic decrease in antigen expression when AxA were compared to promastigotes. Similarly F3 gave strong immune fluorescent staining of the promastigote flagellum, but no fluorescence was detected when AxA were examined. Conversely, Western blotting with the amastigote specific monoclonal antibody (T16) showed that this antigen is more highly expressed in AxA than promastigotes. Differential display-PCR was used to identify several parasite genes showing stage specific expression. One gene selectively expressed by AxA was partially sequenced and identified as Leishmania ( L.) tropica amastin. Amastigote specific expression of this gene was further confirmed by reverse transcriptase-PCR (RT-PCR) using AxA and infected macrophages. No amastin expression was observed with promastigotes. Expression of the cysteine protease B ( cpb) and protein kinase A catalytic isoform 1 subunit ( pkac1) in promastigotes and AxA was also examined by RT-PCR. Pkac1 was strongly expressed by promastigotes, while cpb expression was only seen with AxA or infected macrophages. L. ( L.) tropica AxA will prove useful for further studies on parasite differentiation and gene regulation, as well as for drug screening.

Diagnosis of Trypanosoma cruzi chronic infections in humans: usefulness of the complement regulatory protein antigens and lytic antibodies in the control of cure.

Chagas disease, highly prevalent in Central andSouth America, is caused by the hemoflagellate,protozoan, Trypanosoma cruzi transmitted mainlyby vector contamination and blood transfusion. Theparasite is found in the vertebrate host in two stages:trypomastigotes and amastigotes. The trypo-mastigotes are first inoculated as metacyclics,which invade the host mucosa or the broken skin,through contaminated feces and urine of the tri-atoma vector. They transform and divide as intrac-ellular amastigotes, the multiplicative forms, toemerge as trypomastigotes, which circulate in theblood and re-invade various cells, promiscuously.The amastigotes are intracellular but may main-tain the cycle without trypomastigotes, as demon-strated in vitro with stage specific monoclonal an-tibodies by Andrews et al. (1987).The infection has an acute parasitemic phase,which lasts 1-2 months, often with no symptoms,and a chronic pauciparasitemic phase. Parasitemultiplication at the acute phase is followed byinflammatory reactions at the site of inoculation(delay-type hypersensitvity at the eye mucosa orskin), satelity lymphadenitis, and, other acute in-flammatory reactions (fever, splenomegaly, loss ofweight, weakness, myalgia), more severe in infec-tions induced by high inocula, including in acci-dental, non-treated laboratory infections in humans.Serology is useful for diagnosis of the acute phase,based on high levels of IgM anti- T. cruzi measuredby tests with fixed epimastigotes from axenic cul-tures or with recombinant antigens specific oftrypomastigote or amastigote.Diagnosis of chronic Chagas disease (CCD)relays chiefly on conventional serology (CS) i.e.indirect immunofluorescence (IIF), ELISA, hemag-glutination and complement fixation reaction. Al-though infections are thought to be life-long, de-tection of parasites is difficult in CCD. It requiresindirect methods of parasite amplification(hemocultures and xenodiagnosis, reviewed inChiari et al. 1989, Chiari 1992), not used in clini-cal routine. Both are rather fastidious, slow, of lowefficiency and time consuming. Such techniquesare not yet replaced by modern techniques of mo-lecular biology, presently reviewed by Chiari.Blood sub-inoculations in mice (babies or adultsimmunosuppressed) is also helpful to uncoverparasites during CCD.The xenodiagnosis is the more used parasito-logical test and gives consistent results in the lit-erature, differently from hemocultures. In severaldifferent studies of xenodiagnosis and hemocul-tures in parallel, the former was almost always moreefficient (8/11 reports) as reviewed by Junqueira(1996). Its drawn back is the invasiveness repre-sented by the need of hundreds of bites when thevector feeds on the patient, and injects saliva, re-sulting in local allergies. Although such problemis easy to overcome by artificial feeding throughmembranes an additional problem is the use of se-rial xenodiagnosis, proven to increase the test posi-tivity from 30-40% in one test (Schenone et al.1974) to about 70% in three tests (Coura et al.1991).The sensibility of hemocultures varies from 0%to 98% in the literature according to the volume ofblood, the temperature for maintaining and pro-cessing it, anticoagulants and culture medium used,time of culture, i.e. 4-6 months of culture withmonthly exams are recommended before a nega-tive test is released (Galvao et al. 1989, Luz et al.1994). We selected hemocultures to follow uptreated patients. Among them one third have para-sitological tests positives, even though about 90%remained positive by CS (Cancado 1985, Ferreira1990). However, in spite of positive CS, 30-40%of such treated patients lack functional antibodies

Infection and Stage Conversion during Murine Pulmonary Toxoplasmosis: A Study with Three Different Strains of Toxoplasma gondii

A murine model of pulmonary toxoplasmosis was examined morphologically and immunochemically using 3 strains of Toxoplasma gondii. BALB/c and NMRI mice were infected with tachyzoites of a virulent strain (RH) or with brain cysts of an avirulent (GAIL), or moderately virulent, strain (NED). Depending on the strain of T. gondii, the degree of infection, number of parasites, and replicative potential of T. gondii in lungs varied. In lungs of mice infected with the RH strain, the number of parasites increased in mice during the survival period. Only tachyzoites were found, as confirmed by stage-specific monoclonal antibodies. In lungs of mice infected with the GAIL strain or the NED strain, different stages of parasites were detected. Up to day 14 after infection, only tachyzoites were present, followed by bradyzoites between days 14 and 20 after infection. The number of cysts decreased and finally could not be detected in this organ. In general, the number of cysts that developed in the lungs was smaller than the number that developed in the brain.

Antigens responsible for eosinophil hyporesponsiveness in Brugia pahangi microfilariae injected mice

In order to identify the eosinophil hyporesponsiveness factor in the microfilaremic host, stage-specific monoclonal antibodies against microfilariae (Mf) of Brugia pahangi were produced. One of these (MfG2a) was established for the first time as a monoclonal antibody of IgG2a isotype against Mf. MfG2a recognizes the eosinophil hyporesponsiveness factor, the 42 kDa excretory/secretory antigen of Mf. Treatment of MfG2a significantly ( P < 0.05) induced eosinophil response with rapid reduction of microfilaremia in previously Mf injected mice which became amicrofilaremic within 2 weeks. Eosinophil hyporesponse was observed in the control microfilaremic mice and the microfilaremia persisted at high levels. Another monoclonal antibody, MfG1 of the IgG1 class, recognized the 64-kDa surface antigen of Mf, MfG1 was less effective in eosinophil response- or microfilaremia reduction. These data suggest that the 42-kDa microfilarial excretory/secretory antigen might be responsible for the eosinophil hyporesponsiveness in B. pahangi Mf injected mice.

Identification and enrichment of human osteoprogenitor cells by using differentiation stage-specific monoclonal antibodies

A major problem in developmental bone biology is the inability to clearly identify early progenitor cells of the osteogenic and related lineages. Identification of these cells is important for the study of their normal development and for determination of potential changes in skeletal diseases. The objective of the present study was to obtain specific markers for early progenitor cells. Monoclonal antibodies were raised against human marrow stromal fibroblastic cell cultures, known to be rich in progenitors for the stromal lineages. Antibodies were selected initially by their reactivity with these marrow cultures and their immunohistochemical localization in human fetal tissues, in progenitor cell regions adjacent to osteoblastic cells. Antibody HOP-26 was strongly reactive with cells in marrow stromal colonies at early stages of differentiation, before the induction of alkaline phosphatase activity, and decreased dramatically after the cells reached confluence. In sections of human fetal limb, binding of HOP-26 was restricted to cells in close proximity to the developing bone, in periosteum, and between the developing bone trabeculae. In adult trabecular bone tissue, HOP-26 was reactive with occasional cells present within the marrow spaces with osteoblasts, adipocytes, and fibrous tissue unreactive. No antibody binding was detected in sections of skin, muscle, appendix, brain, tonsil, or liposarcoma, or cultured SaOS II, MG63, or skin cells. In primary cell suspensions, HOP-26 was unreactive with blood cells but strongly reactive with 0.59 ± 0.27% of nucleated marrow cells. The antigen associated with these cells was detectable both intracellularly and on the cell surface, and by using immunopanning, HOP-26 selected the marrow stromal fibroblastic colony-forming units (CFU-F). HOP-26 provides the means to identify osteogenic progenitor cells directly and with high specificity. The present studies demonstrate the value of this antibody in providing enriched populations of progenitor cells for experimental studies of osteogenic differentiation and in histopathology.

Methods to Produce Stage-Specific Monoclonal Antibodies Against Mouse Trophoblast Cells by Intrasplenic and In Vitro Immunizations

Monoclonal antibodies recognizing epitopes expressed by invasive mouse trophoblast cells were obtained by immunizing male mice of the NMRI strain with mouse egg-cylinders obtained by culturing blastocysts on agarose for 4 days. Immunization was achieved by intrasplenic and in vitro methods. The procedures described outline a rational way of producing and selecting antibodies which are stage-specific for the mouse trophoblast at implantation and early placentation. Preimplantation stage-specificity was tested and adhesive blastocysts only were detected by 4 antibodies. Thus, these antibodies become markers for that developmental stage which characterizes an adhesive blastocyst ready to implant. Postimplantation stage-specificity was demonstrated by 6 antibodies, which labelled trophoblast cells of day 7 implantations only. Thus, there is a specific expression of some epitopes by the invasive trophoblast. Cell surface epitopes were detected on adhesive blastocysts by 4 antibodies and on the invasive trophoblast of the egg-cylinder by 7 antibodies. Shedded antigens from egg-cylinders in vitro were detected by 5 antibodies. Four of these antibodies detected substances released only from early implantation stages. A functional effect was demonstrated for an IgM antibody which impaired hatching and outgrowth of blastocysts in vitro.

Toxoplasma gondii : Kinetics of Bradyzoite-Tachyzoite Interconversion in vitro

Stage-specific monoclonal antibodies have been used to investigate the bradyzoite–tachyzoite interconversion of Toxoplasma gondii in vitro. The differentiation of bradyzoites isolated from brain cysts (strains 76K and BQNC2) and grown in culture proceeded through intermediate stages which expressed both the specific markers of bradyzoites (P36, P34, P21, and P18) and a specific marker of tachyzoites (SAG1—P30). Differentiation started before parasite division, but large vacuoles containing intermediate stages were also found, suggesting that these were able to multiply. Intermediate stages were also observed during the differentiation of peritoneal tachyzoites (strain Prugniaud) into bradyzoites in vitro. Triggering of bradyzoite protein synthesis is not a single event since one of the four bradyzoite-specific proteins (P21) always appeared later than the others during bradyzoite differentiation. During interconversion, heterogenous vacuoles containing parasites expressing different levels of bradyzoite or tachyzoite proteins were observed. This observation together with the fact that differentiation is not synchronous within a culture or within a host cell suggest a complex triggering process.

Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages.

By using stage-specific monoclonal antibodies, an in vitro model has been developed to analyze the kinetics of expression of stage-specific antigens during the conversion process between tachyzoites and bradyzoites of Toxoplasma gondii. Following infection of murine macrophages with bradyzoites, the expression of bradyzoite-specific antigens declined, whereas the expression of tachyzoite-specific antigens increased during the first 72 h postinfection. Conversely, in gamma interferon-treated murine macrophages infected with tachyzoites, the inhibitory effect of gamma interferon on replication of parasites was accompanied by the induction of bradyzoite-specific antigens.

Protective Immunity against Coccidiosis Elicited by Radiation-Attenuated Eimeria maxima Sporozoites That Are Incapable of Asexual Development

Eimeria maxima oocysts were exposed to various doses of gamma radiation that did not affect sporozoite invasion of intestinal epithelium but did prevent subsequent merogonic development therein. Although merogony and oocyst formation were inhibited, parasites exposed to 12 kRad radiation induced a level of immunity against E. maxima challenge equivalent to that induced by non-irradiated oocysts. Chickens immunized per os with 20 kRad-treated E. maxima oocysts were not protected against coccidial challenge. Immunization of chickens with a single low dose (five oocysts) of non-irradiated (0 kRad) or irradiated (12 kRad) E. maxima was effective in preventing weight depression after coccidial challenge. Immunofluorescence staining of intestinal tissue from chickens infected with irradiated (12 or 20 kRad) or non-irradiated (0 kRad) E. maxima oocysts with developmental stage-specific monoclonal antibodies showed that sporozoite invasion was similar in all groups. However, merogonic development was not observed at any time postinfection in chickens infected with irradiated oocysts, unlike the case with chickens infected with non-irradiated parasites. These results suggest that sporozoite-infected host cells are capable of eliciting complete protection against E. maxima challenge.

X-irradiation of Eimeria tenella oocysts provides direct evidence that sporozoite invasion and early schizont development induce a protective immune response(s)

Sporulated oocysts of the protozoan parasite Eimeria tenella were attenuated by exposure to various doses of X-radiation to inhibit intracellular replication and thus determine whether sporozoites alone can induce a protective immune response. Exposure to doses greater than 15-kilorads had a significant effect on development, as indicated by the absence of oocyst production in chickens infected with parasites treated with 20 or 30 kilorads of radiation. Infection with nonirradiated or 15-kilorad-exposed parasites led to either normal or reduced oocyst shedding. Equivalent protection was afforded chickens inoculated with a minimum immunizing dose of either nonirradiated or 20-kilorad-irradiated E. tenella oocysts. Immunofluorescence staining of cecal tissue from chickens inoculated with 10(7) nonirradiated or 20- or 30-kilorad-irradiated oocysts with stage-specific monoclonal antibodies showed no significant difference in sporozoite invasion between treatment groups. Normal merogonic development was observed at appropriate times (48, 60, 72, and 96 h) postinfection in chickens inoculated with nonirradiated oocysts. In contrast, irradiated parasites exhibited minimal merogonic development at 48 h postinfection. Furthermore, no merogonic stages were observed at times of otherwise peak merozoite development (60, 72, and 96 h) in cecal tissue from chickens inoculated with irradiated parasites. Infection of chicken cells with irradiated or nonirradiated parasites in vitro corroborated these findings and indicate that events early after sporozoite invasion induce a protective immune response against this parasite.

Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development.

Myosin isoforms contribute to the heterogeneity and adaptability of skeletal muscle fibers. Besides the well-characterized slow and fast muscle myosins, there are those isoforms that appear transiently during the course of muscle development. At a stage of development when two different myosins are coexpressed, the possibility arises for the existence of heterodimers, molecules containing two different heavy chains, or homodimers, molecules with two identical heavy chains. The question of whether neonatal and adult myosin isoforms can associate to form a stable heterodimer was addressed by using stage-specific monoclonal antibodies in conjunction with immunological and electron microscopic techniques. We find that independent of the ratio of adult to neonatal myosin, depending on the age of the animal, the myosin heavy chains form predominantly homodimeric molecules. The small amount of hybrid species present suggests that either the rod portion of the two heavy chain isoforms differs too much in sequence to form a stable alpha-helical coiled coil, or that the biosynthesis of the heavy chains precludes the formation of heterodimeric molecules.

Species- and infective stage-specific monoclonal antibodies to Leishmania major produced by an in vitro immunization method

Monoclonal antibodies specific to the infective-stage promastigotes of Leishmania major are needed for developing rapid diagnostic assays of infected sand flies. An in vitro immunization protocol was applied for the production of monoclonal antibodies using small amounts of L. major. Infective-stage promastigotes were isolated from sand flies ( Phlebotomus papatasi) 7–10 days after infection and used as antigen for immunization. Two weeks after a primary immunization, murine splenocytes were removed and immunized in vitro with antigen in murine EL-4 thymoma cell conditioned medium. Three fusions were performed using X63-Ag·653 myeloma cells as fusion partners and two fusions were performed using FOX-NY cells. Antibodies specific to promastigotes were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Initially 56 monoclonal antibodies were selected, and their species and stage specificity were determined using both an ELISA and an indirect fluorescent antibody assay (IFA). Twelve monoclonal antibodies showed species specificity to L. major when tested against four sympatric species of Leishmania. Four other monoclonal antibodies showed species and infective-stage specificity to L. major promastigotes. When tested in immunoblots, all four species- and stage-specific monoclonal antibodies bound to five protein bands that were unique to the infective-stage promastigotes.

Amastigote stage-specific monoclonal antibodies against Leishmania major.

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.

Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein.

Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.

Developmental stage-specific antigens in the nervous system of the cockroach

A specific effort was made to obtain monoclonal antibodies that bind to macromolecules that play a role in the development of the nervous system. It was considered that good candidates for such molecules were those that were only transiently present in the embryonic nervous system. Hybridomas were prepared from spleen cells taken from mice that had been immunized with nerve cords from cockroach embryos at the 43–50% stage of development. The hybridoma supernatants were screened for antibody binding to frozen sections of both embryonic and adult thoracic ganglia. Cell lines that produced monoclonal antibodies that transiently bound to the embryonic nervous system were saved and cloned. These developmental stage-specific monoclonal antibodies either did not bind to the adult nervous system or bound to it with a pattern very different from that in the embryonic nervous system. The developmental stage-specific antigens detected by these monoclonal antibodies were organized into four categories based on the part of the embryonic nervous system in which they were transiently localized. These include binding to the cell bodies of all neurons, cell bodies of subsets of neurons or neuroblasts, subsets of axons, and the neuropile. Preliminary biochemical characterization of the antigens showed that many of these antibodies were recognizing carbohydrate epitopes. Functions for these antigens, most of which are components of the cell surface, are tentatively proposed.

A tubulin-related 55 kilodalton surface antigen recognized by different Trypanosoma cruzi stage-specific monoclonal antibodies from infected mice

Thirteen monoclonal antibodies (MAbs) specific for the membrane of live Trypanosoma cruzi have been obtained from BALB/c infected mice. Most of them had greater avidity for intact than for disrupted parasites. According to the staining by indirect immunofluorescence of the different live developmental stages of the parasite the MAbs could be divided into several groups. Three of them were trypomastigote specific, one amastigote-specific and two epimastigote-specific. The rest reacted with either all stage forms or with various combinations of the different stages. However, despite the fact that they seemed to correspond to stage-specific antibodies, ten of them reacted with the same 55 50 kDa antigen by immunoblotting. Similarly, a 55 kDa protein was immunoprecipitated from these MAbs. By contrast, a single band or a dimer of about 25 kDa was the predominant antigen(s) immunoprecipitated by the same MAbs in absence of protease inhibitors. This smaller protein may arise from proteolysis of the 55 kDa band. This protein is related to tubulin since tubulin (a 55 kDa protein) but not other cytoskeleton proteins blocked the binding of these MAbs to T. cruzi, and some MAbs react with pig α-tubulin by immunoblotting.

B cell sensitivity to natural killing: correlation with target cell stage of differentiation and state of activation.

When 13 B cell lines were phenotyped with a panel of B cell, stage-specific monoclonal antibodies and ordered with respect to differentiation state; their sensitivity to natural killer (NK) cell-mediated conjugate formation and cytolysis was found to be stage dependent. Target cell lines expressing an early B cell phenotype (B1+B2-CALLA+DU-ALL1 +/-) or an intermediate phenotype (B1+B2+CALLA-DU-ALL1+) were NK resistant. Late phenotypic B cells (B1+B2-CALLA-DU-ALL1-) were highly susceptible to NK cytolysis. Individual B cell lines when induced with sodium butyrate or retinoic acid expressed altered B cell phenotype and NK susceptibility. For example, SB, an intermediate B cell line and initially NK resistant, when induced to express a later B cell phenotype became NK sensitive. BJA.B, a late B cell line and initially NK sensitive, when induced to differentiate lost most of its sensitivity to natural killing. Since loss of the B2 antigen is associated with B cell activation, we further phenotyped the B cell lines and induced B cell lines with the 4F2 and 5E9 (anti-transferrin receptor) monoclonal reagents. All cell lines tested expressed each of these antigens, but with varying intensities. While the intensity of 4F2 expression appeared to correlate well with NK sensitivity on both resting and differentiated B cell lines, the intensity of 5E9 expression did not. Peripheral blood B cells express a similar pattern of reactivity to natural killing when stimulated with pokeweed mitogen (PWM) in vitro. B cell sensitivity to NK-mediated events peaked at day 4 of incubation and correlated with the loss of the B2 antigen and acquisition of the 4F2 and 5E9 antigens; sensitivity to natural killing was diminished in the presence of the PCA-1 antigen. The expression of the NK-susceptible phenotype among B cells appears to be differentiation stage- and activation state-dependent. It is during this period of ontogeny that the NK cell may cytolytically regulate the B cell transition to a plasma cell.

Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies

Stage-specific Trypanosoma cruzi surface antigens were characterized by using monoclonal antibodies (MAb) which bind specifically to the metacyclic trypomastigotes derived from either the insect vector or acellular cultures. A protein with an apparent molecular weight of 90 000 was detected by the MAb 5E7 on the surface of cultured metacyclics of four strains of T. cruzi: G, CL, Y and Tulahuen. The MAb 1G7, which binds to an epitope of the 90 kDa antigen distinct from that recognized by the MAb 5E7, reacted with metacyclics of the G and Tulahuen strains but not of the Y or CL strain. A polypeptide of approximately 82 kDa, identified by the MAb 3F6, was found in the metacyclics of all four T. cruzi strains. The MAb 3F6 also detected a 75 kDa antigen in the G strain metacyclics. The stage-specific MAb and the polyclonal antibodies from mice protected against acute T. cruzi infection by immunization with killed G metacyclics identified the same set of major surface proteins of G metacyclic trypomastigotes.

Onchocerca gibsoni: Increase of circulating egg antigen with chemotherapy in bovines

Monoclonal antibodies directed to stage-specific surface antigens of Onchocerca gibsoni eggs were used in immunoradiometric assays to detect antigens in the sera of cattle infected with O. gibsoni. Two monoclonal antibodies detected antigens, presumably of egg origin, in sera. The target antigens appeared to be carbohydrate in nature and of variable molecular weights. Significant increases in levels of circulating egg antigens were found after treatment of infected cattle with benzimidazole compounds. These drugs cause disruption of embryogenesis and accelerated loss of worm uterine contents. In contrast, administration of either macrofilaricides or microfilaricides to infected cattle did not alter pretreatment levels of circulating egg antigens. Measurement of changes in levels of circulating antigens by immunoradiometric assays with stage-specific monoclonal antibodies provides a new means of assessing the efficacy of drugs and their site of action in onchocerciasis.

Identification of stage-specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies.

Stage-specific antigens of Toxoplasma gondii have not been reported. We developed a panel of monoclonal antibodies that identified unique surface membrane antigens on the sporozoite and oocyst wall of T. gondii. These monoclonal antibodies failed to react with T. gondii tachyzoite surface membrane antigens in several immunologic assays. A comparison of surface membrane radioiodinated tachyzoites and sporozoites by one- and two-dimensional electrophoresis showed that T. gondii sporozoites have two major membrane proteins of approximate m.w. 67,000 and 25,000, not present in the tachyzoite stage. Two of the stage-specific monoclonal antibodies (2E4 and 5A4) were directed against the sporozoite 67,000 m.w. protein. In addition, T. gondii sporozoites are deficient in the major radioiodinated tachyzoite protein P30, which is identified by both human and mouse convalescent antitoxoplasma antisera. A convalescent antitoxoplasma serum failed to recognize sporozoite antigens by both immunoprecipitation and reaction with nitrocellulose transfer blots.