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.     

Identification of outer oocyst wall proteins of three Cryptosporidium (Apicomplexa: Cryptosporidiidae) species by 125I surface labeling.

Autoradiography of oocyst wall surface proteins of three Cryptosporidium spp. revealed common bands at 285 to 290, 145 to 148, 120, 57, and 32 kilodaltons (kDa). Cryptosporidium baileyi and C. muris share proteins at 180, 100, 80 to 81, 29, and 18 to 19 kDa; C. baileyi and C. parvum share one protein at 46 to 47 kDa; and C. muris and C. parvum share a protein at 67 to 69 kDa. Additional protein bands, each unique to one species, were also observed.     

T cell recognition of cell surface antigens. I. Specificity

Mouse fibroblast monolayers can be used as an immunoadsorbent to separate normal, unsensitized rat lymphocytes with receptors for foreign cell surface antigens. We used this approach to investigate if the specificity of thymus-dependent cellular immune reactions is based on a functional diversity of the T lymphocytes recognizing the relevant antigens. T lymphocyte diversity was demonstrated by the following findings.

• 1) Lymphocytes that are adsorbed to fibroblasts of a given phenotype are relatively restricted in their reactivity to cellular antigen. They strongly react against the adsorbing monolayer type, but their reactivity to another, third party fibroblast type is decreased.
• 2) Lymphocytes that were preabsorbed on one fibroblast type and transferred to fresh monolayer cultures decreased their reactivity to the absorbing fibroblast type, but could fully react against unrelated third party fibroblasts.
• 3) Pretreatment of the lymphocytes with relevant subcellular antigen prevents their specific adherence to fibroblast monolayers, and, thus, recognition of cellular antigen.
• 4) Using mosaic fibroblast monolayers composed of fibroblasts of two unrelated phenotypes, we were able to demonstrate two different lymphocyte populations, each characterized by the capacity to recognize either of the adsorbing fibroblast types.

     

Identification of cross-reactive antigens between Mycoplasma pulmonis and Mycoplasma arthritidis.

Serological cross-reactivity between Mycoplasma pulmonis and Mycoplasma arthritidis was investigated by enzyme-linked immunosorbent assay, immunoanalysis of electrophoretic blots, and protein A immunoprecipitation reactions. The results demonstrate that one-way cross-reactivity was present in both hyperimmunized and naturally infected rats and that the predominant cross-reactive antigens were M. pulmonis surface proteins. Distinct immunoblot patterns were demonstrated for M. pulmonis and M. arthritidis, allowing differentiation of the two species. The response to M. arthritidis antigens during natural infections differed greatly from that during hyperimmunization. Evidence suggested that nonprotein antigens were major determinants eliciting the antibody response to this mycoplasma.     

Identification and preliminary characterization of saliva-interacting surface antigens of Streptococcus mutans by immunoblotting, ligand blotting, and immunoprecipitation

The ability of surface protein antigens of Streptococcus mutans to interact with salivary components was examined by Western blot and immunoprecipitation methods. Immunoblotting of S. mutans OMZ175 wall-associated antigens revealed 10 major antigens, designated according to their estimated molecular weights. Four of them, with molecular weights of 135,000, 125,000, 120,000, and 115,000 in their denaturated form, bound salivary components. This property was further investigated by immunoprecipitation experiments: the reactivity with saliva was confirmed for antigens with molecular weights of 135,000, 125,000, and 120,000 in their native form, and their locations on the bacterial cell surface were established. These three antigens were characterized as glycoproteins; they directly bound concanavalin A, and pronase abolished their antigenicity, which was partly retained after treatment with NaIO4. Because of their distribution in several other stains of S. mutans, it will be of interest to study their possible implication in the mechanism of attachment of streptococcal strains to saliva-coated tooth surfaces.     

Identification of endothelial cell surface antigens encoded by genes other than HLA. A combined immunoprecipitation and proteomic approach for the identification of antigens recognized by antibodies against endothelial cells in transplant recipients

It has been known for some time that transplant recipients may have antibodies to endothelial cells which are not detected on lymphocytes. However, little progress has been made in the analysis of these endothelial antigens. In the present experiments we have attempted to characterize endothelial cell surface antigens to which antibodies were produced during graft rejection. We have used a panel of endothelial cells from umbilical cord veins and found that antibodies with a polymorphic pattern in the panel appeared to correlate with transplant failure of kidney allografts and with the development of transplant-related coronary artery disease (TCAD) in heart transplant recipients. Among 39 patients with kidney allografts, 21 were negative for antibodies to endothelial cells and did well and 18 were positive and had frequent transplant loss (p = 0.001). In 18 patients with TCAD and 20 patients of a comparator group without TCAD, association of coronary disease with endothelial cell antibodies was observed (p < 0.02). To characterize the endothelial antigens responsible for these serologic reactions we performed immunoprecipitation of reactive antibodies with the corresponding endothelial cell surface antigens, followed by protein identification of the target antigens. Nine proteins were identified in these experiments, 5 were non-polymorphic and appeared to represent autoantigens. Four of the isolated proteins appeared to be polymorphic. They were the Human Major Histocompatibility Complex class I chain-related gene A (MICA), already known to be associated with antibody production and graft failure, human keratin 1, a protein known to be polymorphic and expressed on the surface of endothelial cells, eukaryotic translation initiation factor (EIF) 2A and ErbB3-binding protein 1. The possible role of keratin 1 and the other antigens in allograft rejection requires further investigation.     

Association of some cell surface antigens of lymphoid cells and cell surface differentiation antigens with early rat pregnancy

Monoclonal antibodies and the immunoperoxidase technique were used to localize some cell surface antigens of rat lymphoid cells and cell surface differentiation antigens on cryostat sections of early rat pregnancies. The W3/13 leucocyte sialoglycoprotein was detected almost constantly on trophoblast. The immunoglobulins were more associated with mother's rather than with embryo-derived tissues. We were unable to detect considerable amounts of class I and class II major histocompatibility complex-derived antigens on trophoblast and adjacent decidual cells. The Ia+ cells of the lymphocyte type were occasionally detected in the sites exhibiting presence of immunoglobulins. The Thy-1 cell surface differentiation antigen was detected on the cells producing Thy-1+ material among decidual cells. Depletion of Thy-1 was followed by the regression of decidualized tissue. The OX-2 antigen, known as minor glycoprotein of rat thymocytes, was detected on trophoblast cells and endothelia of decidual vessels, the latter exhibiting also class I major histocompatibility complex-derived antigens. The non-pregnant uterine tissues, as well as the oviduct epithelium were also investigated. The possible role of some of these antigens in the maintenance of the 'immunologically privileged' stage of trophoblast, and in the control of the rearrangement of maternal tissues surrounding the embryo, is discussed.     

Identification of leishmanial antigens in the sera of patients with American visceral leishmaniasis.

Circulating immune complexes are present in the sera of patients with visceral leishmaniasis caused by Leishmania donovani chagasi. In order to determine whether these complexes contain parasite antigens, sera were collected from Brazilian patients with visceral leishmaniasis and from hospitalized control subjects with other diagnoses. High-molecular-weight complexes were precipitated from pooled sera with 2.5% polyethylene glycol. Approximately 140-fold-more protein was precipitated from patient sera than from control sera; 12% of the total patient serum protein was precipitated. Patient serum precipitates contained immunoglobulins G (525 mg/dl), M (27 mg/dl), and A (8 mg/dl). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the patient serum precipitates revealed multiple bands, including a prominent band at 70 kilodaltons, that were not seen in precipitates of control sera. The 70-kilodalton band was recognized by human and hamster sera with antileishmanial antibodies, but not by control sera. Finally, immunization of BALB/c mice with the high-molecular-weight precipitates from patients elicited antileishmanial antibodies against L. donovani chagasi antigens as detected by enzyme-linked immunosorbent assay and Western blot (immunoblot) assay. In summary, sera of patients with American visceral leishmaniasis were found to have high-molecular-weight complexes that contained one or more parasite antigens. These complexes may play a role in the immunology of the disease, and detection of circulating parasite antigens has potential diagnostic importance.     

Hapten-sandwich labeling. IV. Improved procedures and non-cross-reacting hapten reagents for double-labeling cell surface antigens.

New procedures are presented for preparation of hapten-antibody conjugates with the bifunctional amidinating reagent methyl-p-hydroxybenzimidate (HB). Conjugates with improved solubility are effective for hapten-sandwich labeling of cell surface antigens with high sensitivity and specificity. Several non-cross-reacting hapten-antihapten antibody systems are described which are well-suited for simultaneous labeling of different surface antigens.     

Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens.

An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 125I-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commerically available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. We have used the assay to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas.     

Fractionation on lymphocyte surface antigens. I. Rapid method for eliminating labeled lipid from cell surface antigens iodinated by the lactoperoxidase catalysed reaction.

Fractionation of lactoperoxidase iodinated cell surface material on miniature DEAE-cellulose columns provided a rapid method for separating labeled lipid from cell surface antigens. The procedure also removed poorly solubilized aggregates yielding a labeled preparation which demonstrated stable, reproducible immunoprecipitation results. Using these fractionated antigens components tentatively designated as human 'T' cell specific antigens have been identified.     

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis.

The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL or subclinical L. major infection is an IFN-gamma-producing Th1-like response.     

Humoral immune response in tuberculosis: initial characterization by immunoprecipitation of 125iodine labelled antigens and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

125Iodine-labelled Mycobacterium tuberculosis antigens were immunoprecipitated with tuberculosis patients' sera and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A group of four polypeptide antigens of 55, 38, 28 and 18 kD were thus identified. The 38 and 28 kD polypeptides were the major antigens. Antibody response differed from one patient to another, both with respect to the number and quantity of antigens precipitated. Untreated patients and those undergoing treatment with antimycobacterial drugs also showed marked differences in their antibody response. Generally, immunoprecipitates from treated patients showed a larger number of antigen bands and the relative intensities of the bands was also greater. No correlation was observed between the immunoprecipitation profile and antibody titres determined by enzyme linked immunosorbent assay.     

Identification of atypical mycobacteria by thin-layer chromatography of their surface antigens.

The knowledge that the surface (Schaefer) antigens of certain smooth-colony atypical mycobacteria are multiglycosylated C-mycosidic peptidoglycolipids was used to devise a sensitive thin-layer chromatographic (TLC) procedure for the identification of Mycobacterium avium/M. intracellulare/M. scrofulaceum serotypes. TLC maps of the type-specific peptidoglycolipids from 17 of the 31 serotypes are presented. The primary use of the technique is to corroborate results obtained by seroagglutination. Without the aid of seroagglutination, the TLC procedure almost invariably requires the availability of reference strains or the specific peptidoglycolipids derived therefrom.     

Identification of larval cross-reactive and egg-specific antigens involved in granuloma formation in murine schistosomiasis mansoni.

Cross-reactive humoral immune responses between antigens of different developmental stages of the worm Schistosoma mansoni have previously been demonstrated. In contrast, information on antigenic cross-reactivity at the T-cell level is still very sparse. The present study examined the cross-reactive T-cell responses to eggs and crude and fractionated soluble egg antigens (SEA) in infected mice prior to (from 0 to 4 weeks of infection) and after (5 weeks and onwards) egg deposition. Splenic lymphocyte proliferation to unfractionated SEA was detected as early as 2 weeks postinfection and increased rapidly by 4 weeks postinfection. Injections of live eggs into the lungs of infected mice at 4 weeks postinfection demonstrated enhanced granuloma formation, indicating the presence of primed T cells that respond to egg antigens. Further experiments with the artificial granuloma model and polyacrylamide gel electrophoresis-separated SEA fractions demonstrated that in mice infected for 4 weeks the 60- to 66-, 93- to 125-, and greater than 200-kDa SEA fraction-coated beads elicited significant pulmonary granulomas. By 6 weeks postinfection, when eggs are deposited in the livers, in addition to the cross-reactive fractions (60 to 66, 93 to 125, and greater than 200 kDa), beads coated with fractions of 25 to 30, 32 to 38, and 70 to 90 kDa also elicited significant granulomatous reactions. These antigenic fractions are considered to have elicited egg stage-specific T-cell responsiveness. In addition hepatic granuloma T cells from the 6th week of infection demonstrated the strongest blastogenic response to the 60- to 66-kDa cross-reactive fraction. Thus, in vitro and in vivo experiments demonstrated T-cell cross-reactivity between the larval and egg stages of the worm. On the basis of these observations, the appearance of the primary circumovum granulomatous response in infected mice is considered to represent the sum of larval cross-reactive and egg-specific T-cell responsiveness.     

Metabolically dependent and independent cell surface antigens.

In contrast to metabolically dependent tissue-specific cell surface antigens (MDA) which are available for reactions with antibodies only on surfaces of metabolically active cells, the availability of the universally distributed blood group A or Forssman-type antigens on cell surfaces was found to be independent of the metabolic activity of cultured cells. In case of the MDA, cytotoxic reactions were induced by antibodies alone, resulting in a disorganization of the cellular sheet without a significant release of radioactive label. Radioactive release in this cytotoxic reaction was increased by the addition of complement without additionally affecting the degree of cell sheet disorganization. In case of Forssman and blood group A antigens, such morphologically demonstrable cytotoxicity required complement, and the resulting pathology was always accompanied by extensive release of cellular contents. The ability of anti-MDA antibodies to induce cytotoxic reactions in the absence of complement may be related to a vital role of MDA in cellular function.     

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.     

Identification and biochemical characterization of antigens of tachyzoites and bradyzoites ofToxoplasma gondii with cross-reactive epitopes

The aim of the present work was the identification and biochemical characterization of antigens from the tachyzoite and bradyzoite stages ofToxoplasma gondii that share cross-reactive epitopes. Our previous work has demonstrated the induction by tachyzoite excreted-secreted antigens of both a humoral and a cell-mediated protective response. We investigated the question as to whether some bradyzoite and tachyzoite (excreted-secreted, soluble or membrane) antigens share cross-reactive epitopes. Using immunoprecipitation techniques, we identified four tachyzoite antigens with molecular weights of 63, 43, 39, and 28.5 kDa, which were recognized by sera raised against bradyzoite extracts. We also detected a 43-kDa bradyzoite antigen that was recognized both by sera raised against tachyzoite antigens and by chronic-phase human sera with residual IgG antibodies. In an attempt to define the biochemical nature of these antigens, we show that the 43- and 28.5-kDa antigens seem to be glycosylated since they bind to concanavalin A, as does a 37-kDa tachyzoite antigen.