Inhibition of in vitro fertilization by mouse anti-mouse sperm sera and preliminary antigen identification

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.     

Advantage of Dimeric Peptide Antigens in Serodiagnosis of HIV-1 Infection

The reactivity of antibodies with dimeric and monomelic peptide antigens was compared by ELISA. A panel of highly purified synthetic peptides of HIV-1 representing defined regions, 598–609 and 524 533 (fusion domain) of gp41 and 306–320 of gpl20, were used as antigens in the ELISA. These peptides were selected and synthesized taking into account the level of sequence conservation of various strains and hydrophilicity. The analysis included sera from 52 HIV-1 infected individuals and 53 HIV-1 negative controls. Both peptides from gp41 were found to be particularly immunoreactive with sera from HIV-1 infected individuals. The frequency of reactivity to the selected peptide from gp120 (V₃ loop) in infected individuals was 82%. An interesting observation was that the dimeric peptide antigens had a detection rate more than 4-fold higher than the monomeric antigens. We found that lower levels of antibodies could be detected with dimeric antigens. The peptides reacted with few sera other than HIV-1 positive sera. These results implicate the potential dimeric peptide antigens to be utilized in the serodiagnosis of HIV-1 infection.     

Characterization of Various Recombinant Antigens from <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> for Use in the Immunodiagnosis

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser²³–Glu¹⁷⁶ of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen B1 did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.     

检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

Serodiagnosis of infectious diseases with antigen microarrays

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.     

Breadth of humoral response and antigenic targets of sporozoite‐inhibitory antibodies associated with sterile protection induced by controlled human malaria infection

The development of an effective malaria vaccine has remained elusive even until today. This is because of our incomplete understanding of the immune mechanisms that confer and/or correlate with protection. Human volunteers have been protected experimentally from a subsequent challenge by immunization with Plasmodium falciparum sporozoites under drug cover. Here, we demonstrate that sera from the protected individuals contain neutralizing antibodies against the pre‐erythrocytic stage. To identify the antigen(s) recognized by these antibodies, a newly developed library of P. falciparum antigens was screened with the neutralizing sera. Antibodies from protected individuals recognized a broad antigenic repertoire of which three antigens, PfMAEBL, PfTRAP and PfSEA1 were recognized by most protected individuals. As a proof of principle, we demonstrated that anti‐PfMAEBL antibodies block liver stage development in human hepatocytes. Thus, these antigens identified are promising targets for vaccine development against malaria.     

An immunoblotting as a method for the diagnosis of typhoid fever

The patients' sera had been referred to the National Salmonella Centre for routine Widal serology. Sera were predominately from patients suspected of having been infected with Salmonella Typhi, but also included one serum from patient with typhoid fever who was culture positive for Salmonella Typhi. The immunoblotting procedure using Salmonella Typhi somatic (O=9,12 LPS) and flagellar (H=d) antigens was used for preliminary testing of selected patients sera previously evaluated by Widal agglutination assay as containing different levels of antibodies against O and/or H antigens of Salmonella Typhi. Following Chart et al., immunoblotting reactions were graded between 0 and 3, with 0 indicating an absence of antibody binding, and 3 where antibody binding was readily observed. Sera giving reaction of 2 or 3 were considered to be antibody positive for this study. Positive immunoblotting reaction to O=9,12 LPS antigen was obtained only with the serum of patient with typhoid fever. Presence of specific anti-LPS antibodies was also observed in two other patients' sera diluted 1:50, and in case of one of them also in dilution 1:200, but intensity of antigen-antibody reaction was under positive result criterion. The most other sera positive to O=9,12 antigen in law dilutions (1:50, 1:100) by Widal assay, showed the traces of non-specific reaction by immunoblotting. Presence of positive antigen-antibody reaction was indicated for five sera in dilution 1:50 when tested with the >55 kDa H=d flagellar protein subunit, including the serum of patient with typhoid fever. Only in this serum the high level of specific antibodies was detected also in dilution 1:200, what was not observed in case of the other four, which appeared negative. All the other sera were shown not to contain antibodies to flagella antigen. Although the presented results are preliminary and additional study of more sera of people infected with Salmonella Typhi is needed, it can be concluded after Chart et al., that an immunoblotting procedure incorporating O=9,12 LPS and flagellar H=d antigens is a useful method for providing serological evidence of infection with Salmonella Typhi. In our opinion it can serve as a rapid test for the diagnosis of typhoid fever.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep

and 1972. Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep. International Journal for Parasitology, 2: 449–457. An allergenic component was separated from Ostertagia circumcincta antigens and specific reaginic antibody was separated from the corresponding 7S antibodies in sheep sera. Further evidence was obtained that the immunoglobulin class defined as IgG1A contains the reaginic or homocytotropic antibodies in sheep. Both the IgG1A antibody and P.C.A. levels continued to increase after the expulsion of the parasites, whereas the levels of anti-Ostertagia IgG1 did not.     

Molecular identities of human sperm proteins reactive with antibodies in sera of immunoinfertile women

Antisperm antibodies (ASA) can cause infertility in both men and women. It is important to delineate the sperm antigens against which these ASA are directed. Sperm proteins were separated by 2D gel electrophoresis and transferred to nitrocellulose membrane and incubated with sera from fertile women or immunoinfertile women having ASA. The corresponding immunoreactive peptide spots were cored from the gel and analyzed by the two-dimensional (2D) gel electrophoresis/matrix-assisted laser desoprtion ionization-time of flight-mass spectrometry and liquid chromatography-mass spectrometry (MALDI-TOF-MS/LC-MS). A total of 68 spots belonging to 38 different proteins and their isomers were identified. Fourteen of these proteins and their isomers reacted with both the fertile and immunoinfertile sera. Twenty-four of these proteins reacted specifically only with the immunoinfertile sera and not with the fertile sera. Among them was a novel protein designated as a hypothetical protein FLJ32704 (accession # Q96MA6). An immunodominant sequence (amino acid 151-159) of this protein was identified and a nonamer peptide based upon this sequence (IQTLG1TPR) was synthesized and examined for its immunoreactivity. This synthetic peptide reacted with 90% (36/40) of immunoinfertile sera and not with any of the fertile sera (0/40) in the enzyme-linked immnosorbent assay (ELISA). In conclusion, using the 2D gel electrophoresis/MALDI-TOF-MS/LC-MS procedure, we have identified several known and at least one novel antigen against which the antibodies are present in sera of immunoinfertile but not fertile women. Some of these antigens may find applications in specific diagonsis and treatment of infertility/immunoinfertility, and in the development of new generation of contraceptive modalities including contraceptive vaccines.     

Isolation, purification and characterization of surface antigens of the bovine filarial parasite Setaria digitata for the immunodiagnosis of bancroftian filariasis

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.     

Immunodiffusion test for diagnosing basidiobolomycosis

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ° C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.     

Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients with different clinical forms of paracoccidioidomycosis

Serum antibodies against antigens of Paracoccidioides brasiliensis have been one of the major diagnostic indicators of paracoccidioidomycosis (PCM). In the present study, released antigen preparations (exoAg) obtained from P. brasiliensis isolates were characterized in terms of their protein components electrophoretically detectable and recognizable by sera (IgG) of patients. Among five different isolates (DGO, C-9, BAT, Pb-18 and B-339) the electrophoretic profiles of exoAg varied greatly. A total of 28 different components were detected, 11 of them shared by all isolates. The most representative preparation was BAT-exoAg, which presented the largest number of protein bands (23) and the highest frequency of reacting bands (19) with sera from patients with active PCM (n = 40). Six bands reacted with more than 20% of sera. Independently of clinical forms, the sera recognized the 43-kDa (97% of tested sera), 160-kDa (78% of tested sera) and 70-kDa (60% of tested sera) antigens more frequently. Sera from patients with severe forms of acute (n = 14) or chronic (n = 10) PCM recognized a greater number of antigens, with a higher frequency, than those from moderate forms. The most pronounced reduction in reactivity was provided by sera of patients that became asymptomatic at the beginning of treatment. Remnant reactivity with BAT-exoAg was detected after clinical recovery, especially with those of 43, 70 and 160 kDa. The latter presented a stable recognition frequency (60%) during the entire follow-up, allowing us to suppose that the IgG reactivity against the 160-kDa antigen constitutes a possible persistent marker of P. brasiliensis infection.     

Ostertagia ostertagi: Isolation and partial characterization of somatic and metabolic antigens

One metabolic (ES) and two somatic extracts (AS and MS) were prepared from Ostertagia ostertagi. Partial characterization of the three preparations was accomplished by sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. In immunoblots, AS showed a low number of bands that reacted weakly with sera collected from infected calves. MS reacted strongly with the homologous sera, and a sharp group of bands appeared from 12 to 14 kDa, increasing in intensity as the infection progressed. ES showed a group of strongly immunogenic bands in the range of 16–22 kDa. The three preparations were also tested for reactivity with specific antt-Ostertagia antibodies in an enzyme-linked immunosorbant assay (ELISA). The ELISA results showed that all three worm extracts contained antigen epitopes recognized by circulating antibody in sera taken from O. ostertagi-infected calves. The strongest response was seen when antibodies of the IgGl isotype were reacted with MS and ES. When sera taken from O. radiatum-infected calves were used both somatic extracts showed high levels of cross-reactivity.     

Spherules in the serology of Coccidioides immitis. II. Complement fixation tests with human sera

Soluble and particulate spherule antigens fixed complement in tests with anti—C. immitis sera. However, the soluble antigen proved more active than the particulate one. Cross reactions were noted between the spherule antigens and anti—H. capsulatum and anti—B. dermatitidis sera. Following enzymatic treatment of the particulate preparation a soluble antigen was obtained which formed two bands with anti—C. immitis serum and one band with anti-H. capsulatum serum in an agar gel double diffusion test.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.     

Analysis of stage-specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and Western blotting

e carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.     

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases

Objective. Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. Methods. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. Results. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. Conclusions. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody experssion may be induced through antigen-driven mechanisms.Abbreviations mAb monoclonal antibody - PCNA proliferating cell nuclear antigen - PCNA/AK PCNA affinity purified by antibodies from patient serum AK - PCNA/TO30 PCNA purfied by mAb TO30 - PCNA/TOB7 PCNA purified by mAb TOB7 - SLE systemic lupus erythematosus