HLA class II antigens positively and negatively associated with hepatosplenic schistosomiasis in a Chinese population

To identify possible associations between host genetic factors and the onset of liver fibrosis following Schistosoma japonicum infection, the major histocompatibility class II alleles of 84 individuals living on an island (Jishan) endemic for schistosomiasis japonica in the Poyang Lake Region of Southern China were determined. Forty patients exhibiting advanced schistosomiasis, characterised by extensive liver fibrosis, and 44 age and sex-matched control subjects were assessed for the class II haplotypes HLA-DRBI and HLA-DQB1. Two HLA-DRB1 alleles, HLA-DRB1*0901 (P = 0.012) and *1302 (P = 0.039), and two HLA-DQB1 alleles, HLA-DQB1*0303 (P = 0.012) and *0609 (P = 0.037), were found to be significantly associated with susceptibility to fibrosis. These associated DRB1 and DQB1 alleles are in very strong linkage disequilibrium, with DRB1*0901-DQB1*0303 and DRB1*1302-DQB1*0609 found as: common haplotypes in this population. In contrast, the alleles HLA-DRB1*1501 (P = 0.025) and HLA-DQB 1*0601 (P = 0.022) were found to be associated with resistance to hepatosplenic disease. Moreover, the alleles DQB1*0303 and DRB1*0901 did not increase susceptibility in the presence of DQB1*0601, indicating that DQB1*0601 is dominant over DQB1*0303 and DRB1*0901. The study has thus identified both positive and negative associations between HLA class II alleles and the risk of individuals developing moderate to severe liver fibrosis following schistosome infection. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

An ELISA method using serum derived HDAg for the sorological detection of HDV antigens and antibodies

One of the main difficulties related to the detection of the Hepatitis Delta Virus (HDV) antigen and antibody has been the source of the needed HD antigen since HDV containing human and animal livers are very difficult to obtain and since yield is low. This fact prompted us to try to use the serum of patients in the acute phase of HDV infection as a source of HDAg and turn to enzyme immunoassays (EIA) instead of RIA for the sake of easiness and economy in the amount of HDAg needed. The antigen for EIA was obtained from patients during the acute phase of HDV infection and the antibody from patients who have been carriers for many years. For the detection of the antigen, a sandwich type method was employed, whereas for the antibody a competition assay was developed. In order to assess the relative specificity and sensibility of the test, the antibody assay was compared to a commercial RIA (C. RIA, Abbott) and to a non-commercial RIA (NC RIA). Forty-two sera were tested by the two methods and only in two cases discrepant results were obtained. Its is concluded that: 1) sera from patients in the acute and chronic phases of HDV infection can be used as source of both antigen and antibody, for immunoassays; 2) EIA and RIA have comparable relative specificity and sensibility and 3) EIA is easier to perform, cheaper, non-hazardous, has a longer shelf-life and saves scarce HDAg.

Isolation and characterization of partially purified leptospiral antigens

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28%; hexoses),4% protein, 20% lipid and 2,7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.

Immunoelectrophoretic study on common antigens of São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and Biomphalaria snails

Immunoelectrophoretic studies on common antigens were carried out by using rabbits sera immunized against São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and antigens of Biomphalaria glabrata pigmented (Jaboatão - PE); B. glabrata albino (Belo Horizonte - MG) and B. straminea (São Lourenço da Mata, PE). Furthermore, the reverse approach was proceeded, namely, sera anti Biomphalaria snails produced in rabbits were tested against both strains of Schistosoma adult worm antigens. The analysis of the common antigens between the SLM strains of S. mansoni adult worm and B. glabrata pigmented showed 8 to 9 precipitin bands, 3 bands with B. glabrata albino and only 1 band with B. straminea crude extracts. On the other hand, the BH strain of S. mansoni adult worm antisera produced 6 to 7 bands with B. glabrata pigmented, 5 bands with B. glabrata albino and 1 band with B. straminea antigenic extract. Biomphalaria snails crude extracts were fractionated by Sephadex G-100 column and three fractions were collected from each snail strain. The fractions were tested with anti SLM and BH strains of S. mansoni adult worm sera by immunoelectrophoresis. The common antigens fractionated from Biomphalaria snails crude extracts and those found for both strains of S. mansoni adult worm mostly existed in the first fraction and they were estimated to have molecular weight over 158,000 daltons. In our laboratory, it was found a relationship between the antigenic similarities and experimental infection rates of S. mansoni towards Biomphalaria snails so that more bands were seen with increasing infection rates of S. mansoni.

Serological, electrophoretic and biological properties of Fasciola hepatica antigens

Fasciola hepatica somatic antigen, its partially purified fractions and excretion-secretion products were investigated as to serological, electrophoretic and biological properties. In a Sephadex G-100 column (SG-100), Fasciola hepatica total antigen (FhTA) gave 5 fractions, and SDS-PAGE analysis showed they were glycoproteins ranging from 14 to 94 kDa molecular weight (MW). When these fractions were analyzed by enzyme linked immunotransfer blot (EITB) and immunodiffusion in gel (ID) with serum from immunized rats with FhTA, the presence of different antigenic components was revealed. In the SDS-PAGE of excretor-secretor antigen (ESA), it was possible to observe peptides from 12 to 22 kDa, which were also present in FhTA. When the FhTA, its fractions and the ESA were analyzed by EITB with the immune rat serum (IRS), it was observed that only some fractions of the SG-100 shared antigens with the FhTA and ESA. Moreover, DTH and ITH responses were studied in FhTA immunized rats challenged with these different antigen components, revealing that the protein/carbohydrate ratio is important for inducing DTH response. The ESA was the most active component in the DTH and ITH response.

Immunodiagnosis of hydatid disease: evaluation of antigens from hydatid cyst fluid and the vesicularfluid of Taenia crassiceps metacestode

The specificity and sensitivity of the enzyme immunoassay (EIA), presently used in South America areas where hydatidosis caused by Echinococcus granulosus is endemic, was compared to two alternative EIA. One of these uses an hydatid antigen of different prepraration and the other vesicular fluid of Taenia crassiceps cisticerci (VFCC). The effect of previous neutralization in the serum sample of antibodies anti-normal ovine or murine sera and anti-phosphorylcholine on the diagnostic efficiency of these EIA was studied. The frequency of distribution of the titers obtained with normal sera, hydatid sera positive to DD5 test and hydatid sera negative to DD5 test in three EIA systems was analyzed. Results showed a significant decrease of sensitivity of the EIA using VFCC when compared to these EIA using hydatid antigens. This makes inconvenient the use of VFCC for the immunodiagnosis of hydatid disease. No significant differences between the two EIA using hydatid antigens were observed. SDS-PAGE analysis showed remarkable differences between the VFCC and the hydatid antigens composition and some differences among these latters probably due to manufacturing procedures.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1 M EDTA pH 7.5 at 90°C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Identification of Toxoplasma gondii antigens involved in the IgM AND IgG indirect hemagglutination tests for the diagnosis of toxoplasmosis

Crude Toxoplasma gondii antigens represent raw material used to prepare reagents to be employed in different serologic tests for the diagnosis of toxoplasmosis, including the IgM and IgG indirect hemagglutination (IgG-HA and IgM-HA) tests. So far, the actual antigenic molecules of the parasite involved in the interaction with agglutinating anti-T. gondii antibodies in these tests are unknown. The absorption process of serum samples from toxoplasmosis patients with the IgG-HA reagent (G-toxo-HA) demonstrated that red cells from this reagent were coated with T. gondii antigens with Mr of 39, 35, 30, 27, 22 and 14 kDa. The immune-absorption process with the IgM-HA reagent (M-toxo-HA), in turn, provided antibody eluates which recognized antigenic bands of the parasite corresponding to Mr of 54, 35 and 30 kDa, implying that these antigens are coating red cells from this reagent. The identification of most relevant antigens for each type of HA reagent seems to be useful for the inspection of the raw antigenic material, as well as of reagent batches routinely produced. Moreover the present findings can be used to modify these reagents in order to improve the performance of HA tests for the diagnosis of toxoplasmosis

REPRODUCIBILITY OF ALKALINE ANTIGENS OF Leishmania major-LIKE AND Leishmania (V.) braziliensis EVALUATED BY IgG-ELISA. COMPARISON OF ANTIGENS ADDED OF A PROTEIN INHIBITOR (PMSF) OR NOT

This paper deals with the analysis of 10 batches of L.major-like and L.(V.) braziliensis antigens added or not of a proteases inhibitor evaluated by means of an IgG-ELISA on three consecutive days using positive standard sera from patients with diagnosis of American Leishmaniasis previously tested for the presence of IgG antibodies by means of ELISA. The statistical analysis showed that for L. (V.) braziliensis the PMSF-containing antigen did not show any difference among batches or days of testing; the L.(V.) braziliensis antigen without PMSF showed statistical significance for differences among batches and a two-way ANOVA showed significant differences between antigens. L.major-like antigen prepared with or without PMSF showed differences among batches; all 3 days of testing displayed differences for the PMSF antigen but only for days 1 and 2 for the antigen without inhibitor. A two-way ANOVA showed differences among batches of the antigens but not for antigens with and without the protein inhibitor. According to the statistical analysis the L.major-like antigen added or not of PMSF has shown that it is the choice antigen for mucocutaneous leishmaniasis serology.

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens

Considering the impact of cysticercosis on public health, especially the neurologic form of the disease, neurocysticercosis (NC), we studied the frequency of positivity of anti-Taenia solium cysticercus antibodies in serum samples from 1,863 inhabitants of Cássia dos Coqueiros, SP, a municipal district located 80 km from Ribeirão Preto, an area considered endemic for cysticercosis. The 1,863 samples were tested by enzyme linked immunosorbent assay (ELISA) using an antigenic extract from Taenia crassiceps vesicular fluid (Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Of the 459 samples submitted to immunoblotting, 40 were strongly immunoreactive to the immunodominant 18 and 14 kD peptides. Considering the use of immunoblotting as confirmatory due to its high specificity, the anti-cysticercus serum prevalence in this population was 2.1%.