Purification and partial characterization of immobilization antigens from Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elicited immobilizing antibodies (Ab) when injected into rabbits. An effort was made to purify and characterize these Ag (referred to as immobilization Ag) because of their potential role in protective immunity in fishes. Mice immunized with theront cilia were used for production of immobilizing monoclonal antibodies (MAb). Hybridomas were screened by indirect immunofluorescent light microscopy and immobilization of live parasites. Six hybridomas producing immobilizing MAb were cloned. Immobilizing MAb were used to affinity purify Ag solubilized with Triton X-114 and Na deoxycholate. Two membrane protein Ag of approximately 48 and 60 kDa were identified. Immobilizing MAb failed to react with these Ag on Western blots and, conversely, MAb that reacted with the Ag on Western blots did not immobilize live organisms. These results suggest that immobilization required native conformational epitopes which were altered by Western blotting procedures. Individual MAb reactive on Western blots recognized both the 48- and 60-kDa proteins indicating the presence of common epitopes. Affinity purified Ag elicited immobilizing antisera when injected into rabbits, mice, and channel catfish.     

Identification of antigens of Sarcocystis cruzi sporozoites, merozoites, and bradyzoites with monoclonal antibodies

Sporozoites and culture-derived merozoites of Sarcocystis cruzi were used to elicit monoclonal antibodies (MAb's) in mice. Some of these antibodies reacted with the surface of live sporozoites and merozoites as determined by immunofluorescence. An array of similar antigens was identified in Western blots of sporozoites by both anti-merozoite MAb's and an anti-sporozoite MAb. At least 1 antigen in blots of bradyzoites was identified by anti-merozoite MAb's and a cluster of antigens was identified by an anti-sporozoite antibody. These results indicate that several surface epitopes of sporozoites and merozoites are shared with molecules of bradyzoites and that antigen patterns of molecules bearing these epitopes in 3 stages of Sarcocystis may be either distinct or similar.     

Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous

Epitope mapping (identification of an antigenic site recognized by an antibody) is an important component of vaccine development and immunological assays. It is widely accepted that in Western blots, antibodies react exclusively with continuous epitopes: discontinuous epitopes are assumed to be irreversibly destroyed by electrophoresis under the denaturing conditions used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Here, we demonstrate that the epitopes recognized by four different monoclonal antibodies were identified as discontinuous epitopes when characterized by radioimmunoprecipitation assays and enzyme-linked immunosorbent assays, yet each of these antibodies reacted with the corresponding antigen on Western blots. Reaction on Western blots may be due to epitope renaturation during or after the transfer of the protein to a membrane. Therefore, positive reactions on Western blots do not necessarily indicate that epitopes are continuous and this caveat should be kept in mind while characterizing them.     

Target antigens of transmission blocking immunity of Plasmodium vivax malaria. Characterization and polymorphism in natural parasite isolates.

A panel of 20 anti-Plasmodium vivax female gamete mAb has been established and was characterized with respect to their transmission-blocking properties in membrane-feeding experiments and their target Ag identified. Seven mAb suppressed the infectivity of P. vivax parasites to Anopheles tesselatus mosquitoes. The m.w. of the Ag recognized by these mAb were ascertained by SDS-PAGE and Western blots. Three sets of polypeptides of low Mr--20, 24, and a doublet of 37/42 kDa--have been defined as target Ag of transmission-blocking antibodies of P. vivax. All epitopes of these target Ag were found to be dependent on the tertiary conformational structure of the Ag. Polymorphism of target Ag of transmission-blocking immunity was investigated in over 30 natural isolates of P. vivax in Sri Lanka based on the reactivity of a mAb with an isolate as assessed by the indirect immunofluorescent test with the use of live extracellular female gametes, and in Western blots with the use of extracted gametes. The functional consequences of antigenic polymorphism on immunity was investigated in transmission-blocking assays by using membrane-feeding experiments. A majority of target Ag of transmission-blocking immunity were found to be polymorphic, exhibiting size as well as epitope polymorphism. Results indicate that failure of a mAb to affect the infectivity of a parasite isolate of P. vivax to mosquitoes can be caused by polymorphism of the target Ag among isolates.     

Acanthamoeba castellanii: characterization of an adhesin molecule.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

Sulfated oviductal glycoproteins in the rabbit: quantitation by competitive enzyme-linked immunosorbent assay

The rabbit oviductal epithelium synthesizes and secretes a family of antigenically related, sulfated oviductal glycoproteins (SOG). Anti-SOG monoclonal antibodies (Mabs) were produced and two (Mab 1 and Mab 2) were selected for further characterization. Periodate oxidation of Western blots of oviductal fluid did not affect the binding of Mab 1 or Mab 2, thus suggesting that these antibodies recognized protein rather than carbohydrate epitopes on SOG. The specificity of Mab 1 was determined by Western blot analysis of tissues obtained from estrous rabbits and from the male rabbit reproductive tract. SOG was identified in tissue extracts of both the oviductal ampulla and isthmus. Cervix was the only non-oviductal tissue with which Mab 1 cross-reacted. Mab 1 was used to isolated SOG from whole oviductal fluid by immuno-affinity chromatography. Affinity-purified SOG and Mab 1 were used to develop a quantitative, SOG-specific, competitive enzyme-linked immunosorbent assay. This assay was used to quantify SOG in rabbit oviductal fluid collected during estrus and pseudopregnancy. SOG secretion during pseudopregnancy was resolved into two transient episodes of increased secretion. Maximum SOG secretion (X = 1039 +/- 199 micrograms/day) occurred within 48 h of the induction of pseudopregnancy. A second period of enhanced SOG secretion (X = 308 +/- 46 micrograms/day) occurred during the fifth and sixth days of pseudopregnancy. Baseline SOG secretion occurred during estrus at approximately 60% of maximum postovulatory secretion.     

卵形鲳鲹对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫（Cryptocaryon irritans）的幼虫对卵形鲳鲹（Trachinotus ovatus）进行腹腔注射和体表感染,然后每隔1周用阻动试验（Immobilization assay）检测免疫鱼的抗血清和皮肤培养液对激刺隐核虫幼虫的阻动效价,在第14周,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示：两种免疫方法都能让卵形鲳鲹的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异：两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤粘膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是粘膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。     

Use of recombinant P-glycoprotein fragments to produce antibodies to the multidrug transporter

Multidrug-resistance of human cancer cells may result from expression of a 170,000 dalton multidrug efflux pump called P-glycoprotein. To identify this multidrug transporter, and to study its structure and function, we have generated polyclonal rabbit antibodies against the amino-terminal and carboxy-terminal halves of the molecule using recombinant protein fragments produced in Escherichia coli. Two recombinant P-glycoprotein fragments, representing amino acids 140-228 and 919-1280, were overproduced in Escherichia coli by an inducible T7 expression system, gel-purified and injected into rabbits. Both antisera specifically immunoprecipitate 3H-azidopine and 35S-methionine labeled P-glycoprotein from multidrug-resistant cells and detect P-glycoprotein on Western blots with high sensitivity. Because these antisera were raised against epitopes in the amino- and carboxy-terminal halves of P-glycoprotein, they should be useful as research tools to define the function of these two halves of the molecule.     

Anti-idotype and recombinant antigen in immunotherapy of colorectal cancer

The CO17-1A/GA733 antigen (Ag), bound by monoclonal antibodies (MAb) CO17-1A and GA733 that define two different epitopes on the Ag, has proven a useful target in passive and active immunotherapy of colorectal carcinoma (CRC). Previous studies suggest that the antitumor effects demonstrated in MAb-treated patients may be mediated by idiotypic cascades. In approaches to active immunotherapy against the Ag, polyclonal goat and monoclonal rat anti-idiotypic antibodies (Ab2) directed against MAb CO17-1A or GA733 (Ab1) were administered as alum precipitates to 54 patients with CRC (stage Dukes' B, C, and D). The majority of the patients treated with the various Ab2 preparations developed anti-anti-idiotypic antibodies (Ab3) that specifically bound to the CO17-1A or GA733 epitope and shared idiotopes with the corresponding Ab1. Approximately 30% of the patients tested developed specific cellular immunity, i.e., Ag-specific T-cells mediating delayed-type hypersensitivity (DTH) reaction in vivo or proliferating on stimulation with the Ag in vitro. The humoral and cellular immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 Ag has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant Ag preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T-cells), similar to the native Ag. Antibody titers elicited in experimental animals by recombinant Ag were significantly higher than those elicited by Ab2, presumably because Ag expresses numerous epitopes, whereas Ab2 mimics a single epitope. Recombinant CO17-1A/GA733 Ag has potential as a vaccine for CRC patients.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

Field immobilization of raccoons (Procyon lotor) with Telazol and xylazine

The effectiveness of tiletamine plus zolazepam (Telazol) and xylazine was evaluated as an immobilizing combination for raccoons (Procyon lotor). Fifteen raccoons were injected intramuscularly with a 3:2 mixture of Telazol (3.2+/-0.6 mg/kg [mean+/-SD]) and xylazine (2.1+/-0.4 mg/kg) at Pictured Rocks National Lakeshore, Michigan, USA, during May-October, 2001-03. Mean induction time was 4.8+/-3.8 min; mean recovery time was 128.5+/-48.4 min. No linear relationships were found between the amount (mg/kg) of Telazol-xylazine injected and induction (r2 = 0.06, P = 0.40) or recovery times (r2 = 0.01, P = 0.78). Mean heart rate, respiratory rate, and body temperature declined through 20 min after induction (P< 0.05). No mortality occurred and no short-term adverse effects were observed in recaptured individuals. I conclude that a 3:2 mixture of Telazol-xylazine is a safe and effective immobilizing agent for raccoons when conducting nonsurgical field procedures. Immobilizing raccoons with Telazol at 3 mg/kg and xylazine at 2 mg/kg should provide up to 60 min of handling time and usually allow full recovery in about 120 min.     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.     

Identification of a novel GPI-anchored CRISP glycoprotein,MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm

To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100% amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.     

A strategy for production of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Serum antibody responses to sheep hydatid cyst fluid (SHCF) and a purified Antigen 5 (Ag5) were examined in ELISA, immunoelectrophoresis (IEP) and immunoprecipitation (IP) to facilitate production of monoclonal antibodies (MAb) to E. granulosus Ag5 and Antigen B (AgB). Although sera from mice immunized with SHCF contained antibodies of various classes, the fusions using these donor mice resulted in mainly anti-AgB MAb, possibly due to the preferential selection of MAb to AgB by the SHCF-based ELISA screening system. Donor mice immunized with Ag5 also produced several classes of antibodies, and the resultant fusions enabled selection of IgG MAb to Ag5.     

Inhibition of human tumor growth by IgG2A monoclonal antibodies correlates with antibody density on tumor cells

Eight monoclonal antibodies (MAb) of IgG2a isotype that were produced against human melanomas were tested for tumor growth-inhibiting properties in nude mice injected with human melanoma cells of various origins. Four of the eight MAb inhibited growth of these tumors, and all four of these antibodies reacted in antibody-dependent macrophage-mediated cytotoxicity (ADMC) assays in vitro. The MAb that were inactive in vivo also did not react in these assays in vitro. The number of antibody-binding sites per cell on the tumor cell surface was significantly higher for tumoricidal MAb as compared to unreactive MAb. On the other hand, the percentage of tumor cells binding the MAb and the binding affinity to these cells were the same for the two groups of MAb. Also, tumoricidal and nontumoricidal MAb bound with similar affinity and antibody density to Fc receptors on macrophages. The importance of the number of antibody sites on the tumor cell surface for tumor destruction by MAb was confirmed by the demonstration of tumoricidal effects of mixtures of MAb that were by themselves not tumoricidal. MAb binding to different molecules on melanoma cells were complementary in ADMC, whereas MAb directed to the same molecule but to different epitopes were not.     

Role of Non-Surface Antigens in Controlling Paramecium Surface Antigen Synthesis*

SYNOPSIS. Paramecium aurelia exposed to antisera prepared against cells of a different surface antigenic type are often induced to transform to a new serotype. One possible explanation is that paramecia that are so affected have antigens related to the ciliary antigens, but not accessible to immobilizing antibodies. It is these secondary antigens that are bound by the antibodies, thereby forcing the cells to alter their pattern of antigen synthesis. Examination of affected paramecia has disclosed that secondary antigens are often present but the specificity of these antigens cannot account for the activity of the antisera. It is therefore proposed that antibodies directed against substances other than the immobilization antigens may induce transformation. Two kinds of antiserum, neither of which contains immobilizing antibodies of any sort, are able to markedly alter the expression of the serotypes. One was obtained by immunizing rabbits with culture fluid in which paramecia had been growing. The 2nd was made by injecting rabbits with normal sera from other rabbits.     

Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies.

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.     

Identification of Immunodominant Neutralizing Epitopes on the Hemagglutinin Protein of Rinderpest Virus

The immunodominant epitopes on the hemagglutinin protein of rinderpest virus (RPV-H) were determined by analyzing selected monoclonal antibody (MAb)-resistant mutants and estimating the level of antibody against each epitope in five RPV-infected rabbits with the competitive enzyme-linked immunosorbent assay (c-ELISA). Six neutralizing epitopes were identified, at residues 474 (epitope A), 243 (B), 548 to 551 (D), 587 to 592 (E), 310 to 313 (G), and 383 to 387 (H), from the data on the amino acid substitutions of hemagglutinin protein of MAb-resistant mutants and the reactivities of MAbs against RPV-H to the other morbilliviruses. The epitopes identified in this study are all positioned on the loop of the propeller-like structure in a hypothetical three-dimensional model of RPV-H (J. P. M. Langedijk et al., J. Virol. 71:6155-6167, 1997). Polyclonal sera obtained from five rabbits infected experimentally with RPV were examined by c-ELISA using a biotinylated MAb against each epitope as a competitor. Although these rabbit sera hardly blocked binding of each MAb to epitopes A and B, they moderately blocked binding of each MAb to epitopes G and D and strongly blocked binding of each MAb to epitopes E and H. These results suggest that epitopes at residues 383 to 387 and 587 to 592 may be immunodominant in humoral immunity to RPV infection.