Low Molecular Weight Antigens Research Articles

Antigenuria in visceral leishmaniasis: detection and partial characterisation of a carbohydrate antigen

The detection of antigen in the urine is increasingly being used for diagnosis of parasitic infections. A urinary antigen has recently been demonstrated in visceral leishmaniasis (VL), using a latex agglutination test. The results of our study show that the detected antigen is: heat-stable, precipitates with acetone and ethanol but not TCA, is sensitive to periodate and acid hydrolysis but not to pronase E, lipase, or neuraminidase. The antigen is a low molecular weight glycoconjugate that can be extracted by phenol–water, partitions into the aqueous phase when extracted with Triton X-114 or chloroform/methanol, and can be labelled by biotin hydrazide. Since this urinary antigen cannot be characterised by conventional SDS-PAGE and Western blotting, we used an affinity transfer blotting system in which antigens were captured onto nitro-cellulose paper previously coated with a specific antibody. Using this system a low molecular weight antigen (LMWA) spanning an area of the nitro-cellulose membrane corresponding to molecular weight of 5–20 kDa was detected in the urine of VL patients (from Nepal, Sudan, Brazil, Yemen and Spain) and of experimentally infected animals. No LMWA was detected in the urine of patients with malaria, schistosomiasis, or other nonparasitic diseases including typhoid and brucellosis. Immunoprecipitation, using antibody-coated latex, followed by immunoblotting showed that the LMWA is the target antigen in the previously described latex agglutination test (‘KATEX’). The antigen is detectable in both the promastigote and amastigote stages of the parasite. Monoclonal antibodies (mAbs) against Leishmania glycoconjugates strongly react with this molecule. These results suggest that the detected antigen is highly specific and diagnostic for VL.

Specific identification of three low molecular weight membrane-associated antigens of Helicobacter pylori.

A large number of Helicobacter pylori proteins are antigenic, but antibodies to these proteins persist in spite of the eradication of the infection. The analysis of sera from H. pylori-infected and non-infected patients, before and 3 and 5 months after eradication, showed that the antibody response against unknown H. pylori antigens at 32, 30, 22 and 14 kDa in sodium dodecylsulphate polyacrylamide gel electrophoresis decreased by > or = 60% at 3 months and > or = 70% at 5 months after treatment. Two-dimensional gel electrophoresis and mass spectrometry allowed the identification of eight proteins at these positions: neuraminyl-lactose-binding haemagglutinin precursor, 3-oxoadipate CoA-transferase subunit A, elongation factor P, peptidoglycan-associated lipoprotein precursor, hypothetical protein HP0596, adhesin-thiol peroxidase, 50S ribosomal protein L7/L12 and subunit b' of the F(0) ATP synthase. Three of these eight, expressed as recombinant proteins (32 kDa neuraminyl-lactose-binding haemagglutinin precursor, 30 kDa peptidoglycan-associated lipoprotein precursor and 22 kDa hypothetical protein HP0596), reacted specifically with sera from infected patients, while the 14 kDa 50S ribosomal protein L7/L12 cross-reacted with one out of five sera from H. pylori-negative patients. The other recombinant proteins did not show significant immunoreactivity. Four low molecular weight antigens were identified by these methods, three of which were specific. Immunoreaction with these three proteins (neuraminyl-lactose-binding haemagglutinin precursor, peptidoglycan-associated lipoprotein precursor and hypothetical protein HP0596) could provide a serological assessment not only of H. pylori infection, but also of eradication.

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, Mr∼6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml−1 with a linear response for insulin from 10 μg ml−1 to 10 ng ml−1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.

Partial characterization of a non-proteinaceous, low molecular weight antigen of Eimeria tenella.

A low molecular weight (LMW) antigen of Eimeria tenella, initially identified using a murine monoclonal antibody (mAb C(3)4F(1)) raised against E. tenella sporozoites, was partially characterized using enzymatic degradation. solvent extraction, and immunization into various inbred lines of mice. The LMW antigen could be isolated using Folch extraction (methanol/chloroform/ water) and the epitope recognized by mAb C(3)4F(1) was resistant to degradation by alpha-amylase, pronase, and proteinase K, but was sensitive to sodium m-periodate treatment or digestion using mixed glycosidases (from Turbo cornutus). These observations suggest that the antigenic epitope recognized by mAb C(3)4F(1) is carbohydrate-dependent and, based on our ability to isolate the LMW antigen by Folch extraction, the epitope probably resides on a polar glycolipid. The inability of sporozoite-immunized nude mice to elicit a serum antibody response to this molecule indicates that it acts as a T-dependent antigen. Furthermore, sporozoite-immunized male CBA/N mice (with an X-linked immunodeficiency) also failed to elicit a serum antibody response to this molecule, which is consistent with a carbohydrate antigenic epitope. We propose that this antigenic molecule be designated ET-GL1 to reflect its origin and probable structure (E. tenella glycolipid 1).

Localization and immunogenicity of a low molecular weight antigen of Eimeria tenella.

A low molecular weight (LMW) antigen recognized by a murine monoclonal antibody (C(3)4F(1)) was localized within endogenous stages of Eimeria tenella (USDA strain 80). Using indirect fluorescent antibody assay and immunoelectron microscopy, the LMW antigen was found in: sporozoites, first, second and third generation meronts, gamonts, unsporulated oocysts, and sporocysts. The antigen was observed in the cytoplasm and pellicle of the parasite, and in the parasitophorous vacuole, sporocyst walls and cytoplasm of infected host cells. The immunogenicity of this LMW antigen was assessed by antigen-specific serum antibody responses in chickens orally inoculated with live oocysts or injected intramuscularly with dead sporozoites. LMW antigen-specific serum antibodies were detected using Western blots of E. tenella sporozoites as early as 4 days after sporozoite injection and 6 days after oocyst inoculation. Unusually, the monoclonal antibody C(3)4F(1) reduced the binding of immune chicken serum to the antigen in a competitive antibody binding assay, but not the reverse, suggesting that there is a single, immunodominant epitope on this antigen.

The relationship between circulating and intestinal Heligmosomoides polygyrus-specific IgG1 and IgA and resistance to primary infection.

Specific serum and intestinal immunoglobulin (Ig)G1 and IgA responses to Heligmosomoides polygyrus were measured in a panel of seven inbred mouse strains which exhibit 'rapid' (<6 weeks (SWRxSJL)F1), 'fast' (<8 weeks, SJL and SWR), 'intermediate' (10-20 weeks, NIH and BALB/c) or 'slow' (>25 weeks, C57BL/10 and CBA) resolution of primary infections. Mice with 'rapid', 'fast' or 'intermediate' response phenotypes produced greater serum and intestinal antibody responses than those with 'slow' phenotypes. The F1 hybrids ((SWRxSJL)F1) of two 'fast' responder strains showed the earliest antibody response with maximum titres evident within 6 weeks of infection. There was a negative correlation between the serum IgG1 responses and worm burdens in individual mice within a number of mouse strains, and also between serum IgG1 and IgA responses and worm burdens in the 'rapid' ((SWRxSJL)F1) responder strain. The presence of IgG1 in the gut was found to be due to local secretion rather than plasma leakage. Using Western immunoblotting, serum IgG1 from 'rapid' and 'fast' responder but not 'slow' responder mice was found to react with low molecular weight antigens (16-18 kDa) in adult worm excretory/secretory products.

XENOREACTIVE ANTIBODY RECOGNITION OF RELEVANT ANTIGENS IN SWINE KIDNEY AND LUNG

535 Background: The role of antibody in swine-to-baboon lung xenograft injury is different from that in hyperacute rejection of heart and kidney xenografts, which involves the binding of xenoreactive antibody to Galα(1-3)Gal epitopes on the endothelium. Removal of xenoantibody by pretransplant ex-vivo column absorption or kidney perfusion is not beneficial to subsequent pulmonary graft survival; however, the use of pretransplant lung perfusion does have a beneficial effect on subsequent pulmonary graft outcome. One possible explanation for this finding is that pretransplant lung perfusion removes additional antibodies that are specific for unique lung antigens. The purpose of this study was to determine if these relevant unique antigens exist on the pulmonary microvasculature. Methods: (1)Xenoreactive Antibody Depletion: Human plasma was perfused through porcine lungs and kidneys harvested by standard technique in order to absorb out xenoantibody. (2)Antigen Extraction: Antigens from porcine lungs and kidneys were extracted by organ perfusion with a solution of Triton-X 100 and protease inhibitors. Antigens from porcine aortic endothelial cells (PAEC) were obtained from culture for comparison. (3)Western Blot Analysis: Membrane antigens were separated by SDS page, transferred to membranes, and stained with normal human serum, human serum perfused through porcine lung, or human serum perfused through porcine kidney, and labeled with anti-IgM. Results: Perfusion of serum through both the lung and through the kidney removed higher molecular weight (HMW) bands, between 97.4kD and 220 kD, which were recognized by normal IgM in human serum; this removal was consistent across all three cell extract types (PAEC, lung microvascular, and kidney microvascular). These HMW bands were previously found to correspond to antigens that possess Galα(1-3)Gal epitopes. Normal human serum also stained numerous lower molecular weight (LMW) antigens, corresponding to bands between 30kD and 46kD, in kidney and lung extracts; these were not present in PAEC extracts. However, antibodies recognizing these LMW antigens were not significantly depleted from human serum by lung or kidney perfusion. Conclusions: Porcine lung perfusion did not remove any additional xenoreactive antibody from human serum as compared to kidney perfusion. This suggests that there are no lung-specific antigens that are recognized by human xenoreactive antibodies, and therefore that antibody removal does not explain the beneficial effects of pretransplant porcine lung perfusion on pulmonary xenograft survival.

Value of Western Blotting in the Clinical Follow-Up of Canine Leishmaniasis

Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker.

A low molecular weight antigen (33/35K antigen) and its relation to peptic ulcer disease in Japan

A low molecular weight antigen (33/35K antigen) and its relation to peptic ulcer disease in Japan

Improved diagnosis of reactivated toxoplasmosis.

AIMS: To identify antigens detected by western blotting in primary Toxoplasma gondii infection and determine their role in diagnosis of reactivated toxoplasmosis. METHODS: Twenty three immunocompromised patients were tested by...

Bacterial cell wall components as immunomodulators—I. lipopeptides as adjuvants for parenteral and oral immunization

We investigated the immunostimulatory properties of synthetically prepared lipopeptides derived from the cell wall of Gram negative bacteria. These compounds constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also immunoadjuvants in parenteral or oral immunization. By coupling the lipopeptides to haptens or low molecular weight antigens which are not immunogenic per se, highly immunogenic conjugates can be prepared. Lipopeptide antigen conjugates as synthetic vaccines give protection by enhancing the antibody-mediated immune response, and they stimulate the cellular immune response in vivo by priming of cytotoxic T-cells.

Antigenicity and specificity of very low molecular weight Onchocerca volvulus polypeptides in the range 2.2–12.5 kD

Onchocerca volvulus polypeptides in the molecular mass range of 2.2 to 12.5 kD were separated by Tricine-SDS-PAGE and the serological recognition of these very low molecular weight antigens (VLMW-OvAg) was then investigated by immuno-blotting. Sera from 21 onchocerciasis patients as well as from 53 individuals with other filariases were used to determine the sensitivity and specificity of detection of individual VLMW-OvAg. In onchocerciasis patients, up to 16 VLMW-OvAg were recognized predominantly by IgG1 and IgG4, while only few antigens were recognized by IgG2 and IgG3. The antigen recognition pattern varied individually, but 4 VLMW-OvAg of 8.6, 6.2, 5.4, and 5.1 kD, respectively, were bound by IgG4 from more than 90% of the onchocerciasis patients. Six VLMW-OvAg of 7.3, 5.8, 5.4, 4.0, 3.8, and 3.6 kD were recognized exclusively by IgG1 from onchocerciasis patients. In amicrofilaraemic filariasis patients with lymphatic pathology, a strong reactivity of IgG3 to an OvAg of 2.2 kD was observed, indicating a possible contribution of this antigen to the pathogenesis. In the molecular mass range below 13 kD, no specific carbohydrate residues or phosphorylcholine-containing (PC) determinants could be identified by lectin-blotting or PC-specific immunoblotting, respectively. Two-dimensional separation and immunoblotting distinctly resolved more than 40 antigenic polypeptides, the majority focusing at acidic isoelectric points. In O. volvulus-infected chimpanzees the IgG1- and IgG4-reactivity against OvAg below 13 kD appeared concurrently with onset of patent infection. These data suggest that some of these VLMW-OvAg might be associated with the production and release of microfilariae from gravid female worms as well as be involved in immune-mediated pathogenesis during filarial infections.

Molecular structure of the “low molecular weight antigen” of Toxoplasma gondii: a glucose α1-4 N-acetylgalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic

Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be identical with the low molecular weight antigen which elicits an early immunoglobulin M immune response in humans. A detailed study of the structures of these glycolipid antigens was performed. Radiolabelled glycolipids were extensively analysed by chemical and exoglycosidase treatments in combination with high pH anion-exchange chromatography, gel-filtration and lectin affinity chromatography. In addition, carbohydrate fragments prepared and purified from bulk preparations of unlabelled glycolipids by high performance liquid chromatography were subjected to two-dimensional 1H nuclear magnetic resonance spectroscopy, fast-atom bombardment-mass spectrometry, and methylation linkage analysis in order to elucidate the structure of T. gondii GPIs. The following structures were identified:(ethanolamine-PO 4)-Manα1-2Manα1-6(GalNAcβ1-4)Manα1-4GlcNα-inositol-PO 4-lipid and the novel structure(ethanolamine-PO 4)-Manα1-2Manα1-6(Glcα1-4GalNAcβ1-4)Manα1-4GlcNα-inositol-PO 4-lipid both with and without terminal ethanolamine phosphate. Evidence is provided, that only T. gondii GPIs bearing the unique glucose- N-acetylgalactosamine side branch are immunogenic in humans and that this structure is widely distributed among T. gondii isolates. Monoclonal antibodies have been characterized to recognize structures with different degrees of side-chain modification. We suggest that these reagents in combination with recently devised techniques for insertional mutagenisis in T. gondii should greatly facilitate the cloning of genes essential for GPI side-chain modification.

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.

Low molecular weight antigen arrays delete high affinity memory B cells without affecting specific T-cell help

An ongoing, T-cell dependent, secondary antibody response to an epitope can be suppressed in vivo by low molecular weight, soluble polymers, bearing multiple copies of the same epitope. This study illustrates that such suppressive T-cell independent antigen arrays target the epitope-specific, high affinity, memory B cells for long-term functional elimination. Splenocytes from hyperimmune unsuppressed donors, when adoptively transferred into irradiated recipients will readily reconstitute a secondary anti-hapten response after antigenic challenge. No such response was observed with splenocytes transferred from hyperimmune donors suppressed with antigen arrays. The extent of suppression depended on antigen array dose and duration of exposure in the donor animals. The suppressive antigen array carryover from the donors into the recipients was negligible and insufficient to account for the observed suppression. B cells from hyperimmune mice producing high affinity anti-fluorescein antibodies, generated by multiple fluoresceinated ovalbumin (FL-OVA) injections, were helped efficiently by T cells from hyperimmune donors, which were either unsuppressed or suppressed with antigen arrays. Accordingly, help from T cells, specific for the carrier protein remains intact after such suppression. Neither lipopolysaccharide (LPS), nor additional transferred carrier-primed T cells could reverse the unresponsiveness of adoptively transferred splenocytes from suppressed animals. Flow cytometry showed that the number of hapten-specific B cells was markedly reduced after suppression. Collectively, these data show that the long term elimination of an ongoing T-cell dependent antibody response by suppressive exogenous antigen arrays is due to the functional deletion of high affinity, antigen-specific B cells, even in the presence of adequate T-cell help. The long-term nature of such functional deletion strongly suggests physical deletion of the antigen-specific B cell population.

Suppression of the antibody response to a polymorphic peptide from the platelet alloantigen integrin beta3 with low molecular weight antigen arrays.

The allelic human platelet alloantigens PIA1/PIA2 are determined by a 33 Leu-Pro substitution in the second disulfide loop of the integrin beta3 subunit of the fibrinogen receptor, alphaIIb beta3 (GPIIb-IIIa). Alloantibodies to PIA1 cause neonatal alloimmune thrombocytopenia. We studied the suppression of specific Ab production to a disulfide-looped peptide spanning the polymorphic region of integrin beta3, 24AWCSDEALPLGSPRCD39 (LPL). Mice immunized with LPL coupled to OVA (LPL-OVA) produced Abs specific for LPL. When immunized animals were injected with low m.w. dextran heavily derivitized with LPL (Dex(low)LPL(high)), levels of Ab to LPL fell immediately and remained low 1 mo later. Both high affinity and total Abs were affected. Arrays with lower peptide density or a high m.w. backbone did not induce well sustained suppression. Abs to OVA were unaffected by the arrays. Naive mice given Dex(low)LPL(high) were tolerant to subsequent immunization with LPL-OVA. In transfer experiments, irradiated recipients of spleen cells or purified 8 cells from animals suppressed with Dex(low)LPL(high) did not respond to LPL-OVA. Spleen cells from suppressed animals did not suppress the response to LPL-OVA in recipients of immune B cells. These results demonstrate that peptide arrays, by a mechanism sensitive to molecular configuration, induce tolerance to peptide immunization and suppress an ongoing, high affinity Ab response. Peptide arrays induce the elimination or irreversible anergy of specific memory B cells and do not require a non-B spleen cell population to maintain suppression.

Durable elimination of high affinity, T cell-dependent antibodies by low molecular weight antigen arrays in vivo.

Ongoing Ab responses to a T cell-dependent Ag can be suppressed in hyperimmune animals by exogenous, multivalent Ag arrays. The pharmacologic basis for this suppression was studied by varying the molecular mass, ligand valence, and dose of Ag arrays, and then determining their efficacy, pharmacokinetics, and tissue distribution. Arrays ranging in molecular mass from 30 to 500 kDa caused initial clearance of specific serum Abs, but only the smaller arrays caused persistent suppression despite their relatively lower binding avidity and shorter retention in vivo. Suppression by the smaller arrays at lower doses was biphasic, implying two distinct modes of Ab elimination. High affinity IgG was eliminated preferentially, as shown by calibrated variable ligand-density ELISA. Suppressive arrays were localized discretely in the splenic germinal centers of hyperimmune animals. These results indicate that Ag array mass, ligand valence, and dose all play critical roles, and histologic compartmentalization may also be a pertinent parameter, in determining suppressive efficacy in vivo.

Humoral and cellular immune responses to Fasciola hepatica experimental primary and secondary infection in sheep

Blood leukocyte changes, serum hepatic enzyme levels, lymphocyte proliferation in response to Concanavalin A (ConA) and to parasitic excretory-secretory products (FhESP), and antibody (IgG and IgM) responses (ELISA and Western blot) were studied in sheep, the natural susceptible host of F. hepatica, during the first 3 months of an experimental primary or secondary infection. The proportion of flukes established was similar in once- and twice-infected groups, but the flukes originating from the secondary infection migrated more rapidly to the bile ducts. Primary infection induced a marked peripheral eosinophilia from 3 to 13 weeks post-primary infection (PPIW). FhESP-specific IgM were produced from PPIW 2 with peaks in PPIW 3 and 9–10; FhESP-specific IgG increased from PPIW 2 to 6 and became stable afterwards. Western blotting revealed 12 major antigenic fractions in FhESP from 12, 15, 20, 24, 27, 28.5, 30, 41, 51, 56, 69 and 156 kDa; some non-specific ones have been characterized. A sequential recognition of higher then lower molecular weight antigens was observed. FhESP-specific lymphocyte proliferation was marked from PPIW 2 to 5. In contrast, ConA stimulation of lymphocytes was decreased. After secondary infection in PPIW 6, immune responses were modified. The ConA-induced lymphocyte proliferation was transitorily increased. In contrast, the humoral response, in particular against the early recognized antigens, and the level and the duration of the FhESP-specific lymphocyte proliferative response, were reduced.

Low molecular weight Cooperia oncophora antigens: Characterization and humoral immune responses in calves mono-infected with 100 000 infective larvae

Characteristics of the humoral immune response of Cooperia oncophora-infected calves to low molecular weight antigens of C. oncophora were studied. Immunoblotting with sera obtained from calves 6 weeks after a single oral infection with 100 000 third-stage (L 3) C. oncophora larvae revealed several corresponding antigenic fragments between adult worms and the fourth-stage (L 4) larvae. No reactivity in the immune sera was found against the L 3 stage. A previously defined complex of low molecular weight proteins (12–15 kDa) was found on both L 4 and adult Cooperia stages, but not on the L 3 stage. C. oncophora adults differed from the L 4 larvae at the 31 32 and 37 kDa level. Several adult and L 4 proteins were bound by biotinylated Concanavalin A, as was also true for L 3 proteins. A 31 32 kDa glycoproprotein of adult worms was recognized by a monoclonal antibody with specificity for phosphorylcholine. Using monoclonal antibodies in ELISA and Western blotting, the serum antibody response of C. oncophora-infected calves to adult worm antigen was almost entirely IgG1. Binding of the IgG1 antibodies was restricted to a complex of reduced 12–15 kDa protein(s) and a 27 kDa fragment of adult worms. The data suggest that the systemic humoral immune response of calves during a primary infection with C. oncophora consists mainly of an IgG1 response, and is directed to a non-glycoslyated Cooperia protein (molecular weight estimated at 12–15 kDa under reducing conditions and 18 kDa under nonreducing conditions). This protein is probably present in both L 4 larvae and adults. Since it was not bound by immune sera from calves mono-infected with several other nematodes, the 12–15 kDa protein complex may represent a Cooperia-specific component that can be used for serodiagnosis.

Temporal recognition of sperm autoantigens by IgM and IgG autoantibodies after vasectomy and vasovasostomy

Temporal patterns of IgM and IgG autoantibodies to sperm proteins were studied by western blot analysis at intervals after bilateral vasectomy, vasectomy followed one month later by vasovasostomy, or sham operations. Responses were detected to eight major autoantigens at 21–23, 36, 41, 51, 57, 63, 68–71 and 75–83 kDa, by study of staining patterns of sequential serum samples from individual animals and by analysis of the incidence of reaction to each protein. The four lower molecular weight antigens (21–23, 36, 41 and 51 kDa) provoked mainly IgG responses. The strongly stained set of higher molecular weight antigens (57, 63, 68–71 and 75–83 kDa) tended to show more clearly defined temporal patterns of IgM followed by IgG response, including a high incidence of IgM antibody at the 2-week interval. Three of the larger peptides (57, 63 and 68–71 kDa) appeared highly immunogenic, since some reactions were detected even in sham-operated rats. The classical patterns of IgM and IgG antibody responses to the majority of the dominant sperm autoantigens are in accord with the hypothesis that vasectomy mimics immunization with spermatozoa. The high incidence of IgM antibodies in the earliest sample, taken 2 weeks after vasectomy, suggests that the initial immunizing event takes place within about a week after the operation. Vasovasostomy did not bring about a decrease in antisperm antibodies. Instead, some animals demonstrated an increased reaction to certain antigens after reversal of vasectomy, even though the vasovasostomies were anatomically successful.