Detection of Schistosoma mansoni membrane antigens by immunoblot analysis of sera of patients from low-transmission areas

Schistosoma mansoni surface membrane components play a relevant role in the host-parasite interaction, and some are released in vivo as circulating antigens. n-Butanol extraction favors the release of membrane antigens like alkaline phosphatase, which has been shown to be specifically recognized by antibodies from S. mansoni-infected humans and animals. In the present study, components in the n-butanol extract (BE) of the adult S. mansoni worm membrane fraction were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE [15%]) and further analyzed by immunoblotting (immunoglobulin G) using defined sera. S. mansoni-infected patient sera, but not sera of uninfected patients or sera obtained from patients infected with other parasite species, specifically and variably recognized up to 20 polypeptides in the molecular mass range of approximately 8 to >80 kDa. There were some differences in the number, intensity, and frequency of recognition of the BE antigens among sera from Venezuelan sites of endemicity with a different status of schistosomiasis transmission. Antigens in the 28- to 24-kDa molecular mass range appeared as immunodominants and were recognized by S. mansoni-positive sera from all the sites, with recognition frequencies varying between 57.5 and 97.5%. Immunoblotting with BE membrane antigens resulted in a highly sensitive (98.1%), specific (96.1.0%), and confirmatory test for the immunodiagnosis of schistosomiasis in low-transmission areas.     

Purification of two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis

Two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis were purified using species-specific monoclonal antibodies and characterized. The molecular weights of the proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were approximately 70,000 and approximately 72,000, respectively. The 70 kDa protein, which appears as a diffuse band on silver staining, was resolved into a doublet by Western blotting with monoclonal antibody. Though of similar molecular weight and amino acid composition, the two proteins were shown to be distinct by peptide mapping and Western blotting of the purified material. The two proteins are recognized specifically by human visceral leishmaniasis serum and not by serum from cutaneous leishmaniasis or Chagas' disease. These proteins will be useful in developing a direct serodiagnostic assay for visceral leishmaniasis.     

Western immunoblot analysis of the antigens of Haemobartonella felis with sera from experimentally infected cats

Cats were experimentally infected with a Florida isolate of Haemobartonella felis in order to collect organisms and evaluate the immune response to H. felis. Cryopreserved organisms were thawed and injected intravenously into nonsplenectomized and splenectomized cats. Splenectomized animals were given 10 mg of methylprednisolone per ml at the time of inoculation. Blood films were evaluated daily for 1 week prior to infection and for up to 60 days postinfection (p. i.). Blood for H. felis purification was repeatedly collected from splenectomized animals at periods of peak parasitemias. Organisms were purified from infected blood by differential centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes for immunoblot analysis. Serum was collected from nonsplenectomized animals prior to and for up to 60 days p.i. and was used on immunoblots to identify antigens. The combination of splenectomy and corticosteroid treatment resulted in marked, cyclic parasitemias without concurrent severe anemia, providing an opportunity to harvest organisms in a manner that was not lethal to the animals. Several antigens (150, 52, 47, 45, and 14 kDa) were identified. An antigen with a molecular mass of approximately 14 kDa appeared to be one of the most immunodominant and was consistently recognized by immune sera collected at various times during the course of infection. These data suggest that one or more of these antigens might be useful for the serologic diagnosis of H. felis infections in cats.     

Novel nuclear antigens recognized by human sera in lymphocytes latently infected by Epstein-Barr virus

We have used three latently infected cell lines, X50-7, JC-5, and Raji, to identify two new nuclear antigen complexes by Western immunoblotting with human anti-EBNA (Epstein-Barr Virus Nuclear Antigen) sera. One antigen complex, termed EBNA III, is composed of a group of high molecular weight proteins between 130 and 160 kDa and the other antigen complex, termed EBNA IV, is a size-related group of polypeptides between 28 and 62 kDa. Both the EBNA III and EBNA IV groups of proteins display variation in size among the different strains of EBV. Cell fractionation of X50-7, JC-5, Raji, and C16, a cell clone of P3HR1, showed that both new antigen complexes were completely recovered from the nuclei of latently infected lymphocytes as were previously described EBNA I and II. Because these new antigens are only detected by anti-EBNA sera in EBV infected cells, it seems likely that they may be encoded by the viral genome and play some role in the immortalization of lymphocytes by the virus.     

Borrelia burgdorferi B31 Erp proteins that are dominant immunoblot antigens of animals infected with isolate B31 are recognized by only a subset of human lyme disease patient sera

Sera from animals infected with Borrelia burgdorferi isolates yield intense immunoblot signals from the B31 ErpA/I/N and ErpB/J/O proteins, which have apparent molecular masses of 19 and 60 kDa, respectively. Since B. burgdorferi proteins with those molecular masses are of immunodiagnostic importance, Lyme disease patient sera were used in studies of B31 lysates and recombinant B31 ErpA/I/N and ErpB/J/O proteins. Immunoblot analyses indicated that only a minority of the patients produced antibodies that recognized the tested B31 Erp proteins. Southern blot analyses of Lyme disease spirochetes cultured from 16 of the patients indicated that all these bacteria contain genes related to the B31 erpA/I/N and erpB/J/O genes, although signal strengths indicated only weak similarities in many cases, suggestive of genetic variability of erp genes among these bacteria. These data indicate that Erp proteins are generally not the 19- and 60-kDa antigens observed on serodiagnostic immunoblots.     

Semiquantitative Species-Specific Detection of Bartonella henselae and Bartonella quintana by PCR-Enzyme Immunoassay

Bartonella henselae is the main causative agent of cat-scratch disease, and both B. henselae and Bartonella quintana cause angioproliferative disorders such as bacillary angiomatosis. To increase the sensitivity of Bartonella detection by PCR and to improve the species differentiation, we developed a semiquantitative, species-specific PCR-based enzyme immunoassay (EIA). The 16S rRNA gene was selected as the target sequence. Internal nucleotide sequences derived from the amplified 16S rRNA region were used to develop species-specific oligonucleotide probes for B. henselae and B. quintana. Biotin-labeled PCR products were immobilized on streptavidin-coated microtiter plates, hybridized to a digoxigenin-labeled probe, and detected with antidigoxigenin peroxidase conjugate. No cross-hybridization with other Bartonella or non-Bartonella species was observed. This EIA was as sensitive as dot blot hybridization and was 10 times more sensitive than visualization of PCR products on agarose gels. Serial dilutions of B. henselae and B. quintana suspensions demonstrated that an optical density (OD) of approximately 0.200 was equivalent to 5 CFU in the reaction mixture. By comparing the OD of the bacterial dilutions with that obtained from clinical specimens we could determine that the number of CFU in clinical samples ranged from 10(3) to 10(6) CFU/ml. The PCR-EIA developed in the present study is a rapid, sensitive, and simple method for the diagnosis of B. henselae and B. quintana infections.     

Electrophoretic and immunoblot analyses of Rhizopus arrhizus antigens.

Four antigen preparations from Rhizopus arrhizus were made and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and column chromatography. Electrophoretic analyses of these antigens indicated that there are 18 to 28 component bands with a molecular mass range of approximately 10,500 to 83,000 daltons. Seven of these bands appear to be components common to three antigen preparations. Several of the bands identified by SDS-PAGE were composed of glycoproteins or carbohydrates as determined by their affinity for concanavalin A. Western blots, using sera from five patients with mucormycosis, consistently identified five different determinants in the R. arrhizus antigens separated by SDS-PAGE. This suggests that several of the Rhizopus antigens are present during mucormycosis. Four of the antigenic determinants recognized by patient sera reacted with the concanavalin A-peroxidase stain, indicating that they are composed of glycoproteins or carbohydrate. Enzyme-linked immunosorbent assays of sera from five patients with mucormycosis and with rabbit antisera resulted in antibody titers ranging from 1:64 to 1:32,000 for the R. arrhizus antigens.     

Use of sera from humans and dolphins with lacaziosis and sera from experimentally infected mice for Western Blot analyses of Lacazia loboi antigens

Antibodies in the sera of patients with lacaziosis recognized an ~193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.     

Antibodies against membrane antigens of cytomegalovirus infected cells in sera of patients with a cytomegalovirus infection

The appearance of a new virus specific antigen was demonstrated by an indirect immunofluorescence technique on cell surfaces of CMV infected human fibroblasts, 48–72 hr after inoculation. The development of antibodies to these membrane antigens was followed in thirty-nine serial sera from twelve patients with a virologically and/or serologically confirmed CMV infection. In all patients except one, membrane antibodies could be detected. Sera from four patients collected before infection were negative, as were sera taken 1–13 days after onset of symptoms. From the end of the second week of illness CMV-membrane antibodies as well as CMV macroglobulin antibodies to intracellular antigens were detectable. The membrane antibodies persisted until 335 days after onset of illness. They were mainly of the IgM class. Most positive sera also reacted with non-infected fibroblasts but to a lesser degree. This reaction became negative after prior absorption of the sera with non-infected fibroblasts, whereas the reaction with CMV-infected cells remained positive.Controls consisted of forty-five sera from healthy persons and nine sera from patients suffering from other herpes virus infections with antibodies to herpes simplex, zoster/varicella and Epstein-Barr virus. All but two sera were negative in the membrane immunofluorescence test.     

Outer membrane proteins of nontypable Haemophilus influenzae and reactivity of paired sera from infected patients with their homologous isolates.

Plasmid DNAs obtained from 18 Escherichia coli isolates that hybridized with the heat-stable b (ST-b) enterotoxin gene probe were examined by Southern blot analysis for genes coding for heat-labile, ST-a, and ST-b enterotoxins with specific radiolabeled DNA probes. Four E. coli isolates contained plasmids coding for both heat-labile and ST-b enterotoxins, and one isolate contained a plasmid coding for ST-a and ST-b. Five of 11 isolates of antibiotic-resistant enterotoxigenic E. coli isolates containing ST-b-coding DNA transferred a plasmid coding for both antibiotic resistance and ST-b to E. coli K-12, suggesting that the widespread use of antibiotics could increase the distribution of genes coding for ST-b.     

Identification and partial characterization of Trypanosoma cruzi antigens recognized by T cells and immune sera from patients with Chagas'' disease.

Trypanosoma cruzi antigenic specificities involved in human T-cell and antibody responses were compared in chronic chagasic patients affected with cardiomyopathy (C) or with the indeterminate form (I), the asymptomatic form of chronic Chagas' disease. T-cell Western blotting (immunoblotting) was performed to identify the most active antigens in epimastigote extracts (EPI-Ag). The patterns of peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses induced by fractions blotted to nitrocellulose were heterogeneous, but the computation of their frequency distribution disclosed some important antigen specificities. Molecules ranging from 100 to 150 kilodaltons (kDa) were frequently stimulatory to PBMC from I patients (5 of 8 cases) and were less so when confronted with C patient (1 of 7 cases) lymphocytes. In contrast, both groups of patients actively responded to fractions ranging from 48 to 57 and 28 to 32 kilodaltons (kDa). The Western immunoblotting patterns of antibody reactivity displayed by 17 C and 15 I patients were also similar, yielding outstanding staining in the molecular mass ranges of 70 to 80 and 43 to 57 kDa. The latter antigen complex was recognized by 100% of the 32 chronic Chagas' disease serum specimens tested and closely corresponded to the migratory position recognized by T cells of most patients tested. The identification of the active molecules contained in the 43- to 57-kDa region was sought, with a focus on GP57/51, an antigen with well-established serodiagnostic properties. Immunoblotting analysis of EPI-Ag with a monoclonal antibody to GP57/51 confirmed its presence within the predicted molecular weight region. Highly purified GP51 was then used to demonstrate directly its capacity to promote specific PBMC proliferative responses in vitro. Data computed from a survey with 12 patients have shown a linear correlation (r = 0.93) between PBMC responses to EPI-Ag and to purified GP51, suggesting that the immune response to this particular glycoprotein may be an important component of human immune responses against T. cruzi.     

Excretory-secretory antigenic components of Paragonimus heterotremus recognized by infected human sera.

Antigenic components of Paragonimus heterotremus metabolic products were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis of sera from patients with P. heterotremus infection, from patients with other illnesses, and from healthy adults. By SDS-PAGE, it was found that the metabolic products comprised more than eight major polypeptides. Immunoblot analysis revealed 11 components which were strongly recognized by paragonimiasis antisera. These antigenic components had molecular masses ranging from less than 12.3 kDa to 144 kDa. One antigenic band of 31.5 kDa was found to give a consistent reaction with paragonimiasis antisera (97% sensitivity). Of the other patient sera, only sera from patients with Fasciola sp. infection reacted with antigenic bands of 56, 38, and 18.5 kDa. The present findings suggest that the 31.5-kDa component is sensitive and specific for the diagnosis of human P. heterotremus paragonimiasis.     

Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera.

Antigenic components in the cytoplasmic extract of Candida albicans were examined after fractionation by concanavalin A-Sepharose and DEAE-Sephacel ion-exchange chromatography. Fractions from the DEAE column were tested by fused rocket immunoelectrophoresis for their reactivity with antibodies in the sera of 20 patients with disseminated candidiasis. Three groups of fractions (regions A, B, and C) from the DEAE column were defined by their reactivity with these sera. Immunoblot analysis with 20 human sera identified 18 antigenic components in regions A, B, and C. Region A contained nine antigens, region B contained four antigens, and region C contained five antigens. Region A contained an antigen with an apparent molecular weight of 48,000 that was recognized by 7 of 10 sera from patients with disseminated candidiasis. Immunoprecipitation experiments with labeled proteins from region A and 51 human sera also demonstrated the presence of a major antigen whose apparent molecular weight is 48,000 to 52,000. The 48- to 52-kilodalton protein is an abundant protein in region A and is the most frequently recognized protein by antibodies in the sera of patients with disseminated candidiasis. Patients with disseminated candidiasis had significantly higher levels of antibody (immunoglobulin G) (P less than 0.001) directed against the 48- to 52-kilodalton protein than did patients with noninvasive forms of candidiasis, patients with other fungal infections, or normal, healthy persons.     

Novel antigens of Helicobacter pylori correspond to ulcer-related antibody pattern of sera from infected patients

Recently, we reported that the patterns of antibodies to Helicobacter pylori protein antigens in serum may be useful for screening patients at high risk for ulcers (P. Aucher et al., J. Clin. Microbiol. 36:931-936, 1998). Here we report the identification, by a combination of electrophoretic, immunochemical, and protein sequencing methods, of five antigens that correspond to this antibody pattern: groEL, catalase A, flagellin A, beta-ketoacyl-acyl carrier protein synthase I (beta-ketoacyl-ACP S), and peptidyl prolyl cis-trans isomerase (PPiase). Beta-Ketoacyl-ACP S and PPiase are reported for the first time as antigens of diagnostic interest in infections by H. pylori. The antigenicity of the five antigens, together with those of CagA and VacA, was tested in an immunoblot assay with water-soluble protein extracts from two H. pylori pathogenic strains (HP 141 and ATCC 43579) and panels of sera from H. pylori-positive patients with gastroduodenal ulcers (GDU), nonulcer dyspepsia (NUD), as well as sera from H. pylori-negative healthy volunteers. For catalase A, groEL, and flagellin A antigens, no overall statistically important values were found making it possible to discriminate between patients with GDU and NUD. For both H. pylori strains, the mean performance indices (MPI) presenting percentages of correctly classified patients with GDU and NUD showed that the most significant antibody patterns were as follows: anti-VacA + anti-beta-ketoacyl-ACP S (MPI = 76.1), anti-VacA + anti-PPiase (MPI = 71.8), and anti-CagA + anti-VacA + anti-beta-ketoacyl-ACP S (MPI = 70.5). Antibody patterns detected with these antigen profiles may therefore be useful in developing a diagnostic test designed to predict the clinical severity of the H. pylori infection within the adult population of France.     

Toxoplasma gondii antigens recognized by sequential samples of serum obtained from congenitally infected infants.

The Sabin-Feldman dye test, the immunoglobulin M (IgM) immunosorbent agglutination assay, and the immunoblot technique were used to study the evolution of the antibody response to Toxoplasma gondii and to examine antigens of the organism recognized by antibodies in the sera of 12 congenitally infected infants and 7 mothers. In the sera of eight infants, a significant rise was noted in the dye test titers, while the serum of only one infant demonstrated a late increase in the IgM immunosorbent agglutination assay titer. In each infant and mother, antigens with approximate masses of 35,000 and 115,000 daltons were strongly recognized by IgG antibodies. An antigen(s) with an approximate mass of 4,000 daltons was recognized by IgM antibodies in the sera of each of the mothers but was recognized by antibodies in the sera of only two of the infants.     

Monoclonal antibodies against human erythrocyte membrane antigens and the antigens recognized by these antibodies

Six monoclonal antibodies (KOR-E1-E6) were raised against human erythrocyte membrane antigens. Aggregating reactions of normal human erythrocytes with or without enzyme treatment and specific antigen deficient (null type) erythrocytes were used for detection of the antigens. SDS-polyacrylamide gel electrophoresis with Western blotting and immunoperoxidase methods were also used to confirm the results. The antigen recognized by KOR-E1 and KOR-E5, which was sensitive to protease, trypsin, and neuraminidase, and only expressed on human erythrocytes, was identified as Pr1h. The antigen recognized by KOR-E2 and KOR-E6 was identified as the EnaTS portion of glycophorin A, because the antigen was sensitive to protease and trypsin, but resistant to neuraminidase, and was not present on En(a-) erythrocytes. The antigen recognized by KOR-E3 that was protease-, trypsin-, and neuraminidase resistant, and absent on En (a-) erythrocytes, was identified as Wrb antigen. As KOR-E4 reacted with all erythrocytes examined, the antigen it recognizes could not be determined.     

Leishmania donovani molecules recognized by sera of filaria infected host facilitate filarial infection

Parasitology Research - We earlier found that F6 fraction of human filaria Brugia malayi cross-reacted with sera of Leishmania donovani infected hamsters and immunization with F6 inhibited both...     

Reactivity of dog sera to whole-cell or recombinant antigens of Borrelia burgdorferi by ELISA and immunoblot analysis

Enzyme-linked immunosorbent assays (ELISAs) with separate preparations of 10 purified recombinant antigens of Borrelia burgdorferi sensu stricto were used to test sera from 36 dogs not vaccinated with whole cells of this agent and from five dogs vaccinated with whole-cell B. burgdorferi bacteria. All dogs lived in tick-infested areas of Connecticut and south-eastern New York state, USA. The non-vaccinated dogs had limb or joint disorder, lameness and fever during the period 1984-1991 and had antibodies to B. burgdorferi, as determined by a polyvalent ELISA with whole-cell antigen. In re-analyses of sera for total immunoglobulins in ELISAs with recombinant antigens, reactions were most frequently recorded when outer-surface protein (Osp) F, protein (p)35, p37, p39 and p-41G (a flagellin component) were tested separately. Western immunoblots of a subset of 16 sera, positive by ELISA with whole-cell antigen and representing a range of antibody titres (640-40960), verified immune responses to these or other lysed whole-cell antigens. Sera from vaccinated dogs contained antibodies to OspA, OspB, p22, p37 and p41-G. Therefore, serological reactions to OspF, p35 and p39 were the most important indicators of natural exposure to B. burgdorferi. Serum reactivities to these recombinant antigens in ELISAs can be used to help identify possible natural infections of canine borreliosis in dogs not vaccinated with whole-cell B. burgdorferi and to provide information on the geographic distribution of this bacterium.     

Recognition of mycobacterial antigens by sera from patients with leprosy.

Mycobacterium leprae sonic extracts prepared from armadillo-derived bacteria were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) procedures and probed with serum or plasma samples from 20 patients with lepromatous leprosy and 14 healthy endemic controls. Five proteins of 33, 25, 18, 15, and 12 kilodaltons (kDa) were frequently recognized; the 33- and 15-kDa proteins were, respectively, recognized with high intensity by 16 and 13 of the 20 samples from patients with leprosy, whereas only one healthy donor had antibodies that recognized the 15-kDa protein. By the use of M. leprae-specific murine monoclonal antibodies it was demonstrated that the 33-, 25-, and 15-kDa antigens were different from those bound by the available murine monoclonal antibodies. The 18- and 12-kDa proteins detected had molecular masses similar to those detected by the corresponding murine monoclonal antibodies. The serum and plasma samples from patients with leprosy were also used to probe Western blots of a soluble extract of M. tuberculosis. They recognized, among others, antigens with molecular weights similar to those detected in the M. leprae antigenic preparations, although with less intensity and at a lower frequency.     

Selection and characterization of recombinant clones that produce Mycobacterium leprae antigens recognized by antibodies in sera from household contacts of leprosy patients.

A Mycobacterium leprae expression library was constructed in the vectors EX1, pEX2, and pEX3 and screened with a pool of 19 well-absorbed sera from household contacts of leprosy patients. Twelve selected recombinants that were further characterized differed clearly from recombinants selected with murine monoclonal antibodies. Whereas the monoclonal antibodies recognized mainly six recombinant antigens, the human sera from contacts reacted with a range of different recombinant antigens. None of the contact recombinant antigens was identical or related to well-characterized antigens from M. leprae or other mycobacteria selected with monoclonal antibodies, including proteins of the heat shock families. Two groups of recombinant antigens could be distinguished: one that was recognized by all sera used in the pool and one that was recognized by only a limited number of sera. These antigens, selected with sera from household contacts of previously untreated lepromatous leprosy patients, may be relevant to the immune responses during the early phase of infection with M. leprae.