Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM),and lower results with egg antigen, 77.8% (IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r=0.552 (p=0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

Candidin: comparison of two antigens for cutaneous delayed hypersensitivity testing

A candidin, which is a suspension of killed yeast cells, is commonly used for intradermal tests of delayed hypersensitivity, to evaluate the immunological cellular competence of the patient, when the test is applied along with other similar tests. When working with a cellular antigen, the histopathology of positive skin tests reveals a cellular infiltrate which not only presents a characteristic hypersensitivity reaction but also a neutrophilic abscess in the central part. This research presents the results of a comparison between the yeast cell suspension and the polysaccharide antigens, both obtained from the same strains of Candida albicans. The results obtained by skin tests in one hundred individuals were 61.0% with the polysaccharide antigen and 69.0% with the yeast cell suspension antigen. Concordant results concerning the two antigens were observed in 82.0% of the individuals. The discussion section presents an assumption to explain the differences of positivity obtained with the two antigens. We conclude that the polysaccharide antigen can be utilized in the intradermal test of delayed hypersensitivity to Candida albicans.

HEMAGGLUTINATION TEST FOR THE DIAGNOSIS OF HUMAN NEUROCYSTICERCOSIS: DEVELOPMENT OF A STABLE REAGENT USING HOMOLOGOUS AND HETEROLOGOUS ANTIGENS

A hemagglutination (HA) test was standardized using formalin- and tannin-treated gander red blood cells sensitized with a total salt extract of C. cellulosae (HA-Cc) and an antigenic extract of Cysticercus longicollis (HA-Cl) vesicular fluid. A total of 61 cerebrospinal fluid (CSF) samples were assayed, 41 from patients with neurocysticercosis and 20 from a control group, which were, respectively, reactive and non-reactive to ELISA using C. cellulosae. The CSF samples from the control group did not react and 35 (85.4%) and 34 (82.9%) CSF samples from patients were reactive to the HA-Cc and HA-Cl tests, respectively. The reagents ready for use were stable up to 6 months when stored at 4°C in 50% glycerol. The present results confirm that the reagent using Cysticercus longicollis stabilized with glycerol can be used as an alternative in the immunological diagnosis of neurocysticercosis

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

DETECTION OF IgM ANTIBODIES TO Schistosoma mansoni GUT-ASSOCIATED ANTIGENS FOR THE STUDY OF THE DYNAMICS OF SCHISTOSOMIASIS TRANSMISSION IN AN ENDEMIC AREA WITH LOW WORM BURDEN

For a period of 2 years, five follow-up measures of prevalence and incidence rates were estimated in a prospective study of S. mansoni infection in a group of schoolchildren who were living in a rural area of the Municipality of Itariri (São Paulo, Brazil), where schistosomiasis is transmitted by Biomphalaria tenagophila. Infection was determined by the examination of three Kato-Katz stool slides, and the parasitological findings were analyzed in comparison to serological data. In the five surveys, carried out at 6-month intervals (March-April and September-October), the prevalences were, respectively, 8.6, 6.8, 9.9, 5.8 and 17.2% by the Kato-Katz, and 56.5, 52.6, 60.8, 53.5 and 70.1% by the immunofluorescence test (IFT). Geometric mean egg counts were low: 57.8, 33.0, 35.6, 47.3 and 40.9 eggs per gram of feces, respectively. Of the total of 299 schoolchildren, who submitted five blood samples at 6-month intervals, one for each survey, 40% were IFT-positive throughout the study, and 22% were IFT-negative in all five surveys. Seroconversion from IFT negative to positive, indicating newly acquired S. mansoni infection, was observed more frequently in surveys carried out during March-April (after Summer holidays), than during September-October. Seasonal trends were not statistically significant for detection of S. mansoni eggs in stool. The results indicate that the use of IgM-IFT is superior to parasitological methods for detection of incidence of S. mansoni infection in areas with low worm burden.

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.

ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.

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.

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

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%.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.