P35 and P22 Toxoplasma gondii antigens abbreviate regions to diagnose acquired toxoplasmosis during pregnancy: toward single-sample assays.

BACKGROUND: P35 and P22 Toxoplasma gondii proteins are recognized by specific IgG at the early infection stage, making them ideal for acute toxoplasmosis pregnancy control. Both proteins have been studied to discriminate between acute and chronic toxoplasmosis. However, results were hardly comparable because different protein obtainment procedures led to different antigens, the reference panels used were not optimally typified, and avidity tests were either not performed or narrowly examined. METHODS: We bioinformatically predicted P35 and P22 regions with the highest density of epitopes, and expressed them in pET32/BL21DE3 alternative expression system, obtaining the soluble proteins rP35a and rP22a. We assessed their diagnostic performance using pregnant woman serum samples typified as: not infected, NI (IgG-, IgM-), typical-chronic, TC (IgM-, IgG+), presumably acute, A (IgG+, IgM+, low-avidity IgG), and recently chronic, RC (IgG+, IgM+, high-avidity IgG). RESULTS: rP35a performed better than rP22a to differentiate A from RC, the areas under the curve (AUC) being 0.911 and 0.818, respectively. They, however, performed similarly to differentiate A from TC+RC (AUC: 0.915 and 0.907, respectively). rP35a and rP22a evaluation by avidity ELISA to discriminate A from RC rendered AUC values of 0.974 and 0.921, respectively. The indirect ELISA and avidity ELISA results analyzed in tandem were consistent with those obtained using commercial kits. CONCLUSIONS: rP35a and rP22a features suggest that, with complementary use, they could replace parasite lysate for toxoplasmosis infection screening and for acute toxoplasmosis diagnosis. Our proposal should be validated by a longitudinal study and may lead to a reliable toxoplasmosis pregnancy control, performing tests in only one serum sample.

Evaluation and comparison of the ability of online available prediction programs to predict true linear B-cell epitopes.

This work deals with the use of predictors to identify useful B-cell linear epitopes to develop immunoassays. Experimental techniques to meet this goal are quite expensive and time consuming. Therefore, we tested 5 free, online prediction methods (AAPPred, ABCpred, BcePred, BepiPred and Antigenic) widely used for predicting linear epitopes, using the primary structure of the protein as the only input. We chose a set of 65 experimentally well documented epitopes obtained by the most reliable experimental techniques as our true positive set. To compare the quality of the predictor methods we used their positive predictive value (PPV), i.e. the proportion of the predicted epitopes that are true, experimentally confirmed epitopes, in relation to all the epitopes predicted. We conclude that AAPPred and ABCpred yield the best results as compared with the other programs and with a random prediction procedure. Our results also indicate that considering the consensual epitopes predicted by several programs does not improve the PPV.

Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.

The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

Design, construction, and evaluation of a specific chimeric antigen to diagnose chagasic infection.

Chagas' disease is routinely diagnosed by detecting specific antibodies (Abs) using serological methods. The methodology has the drawback of potential cross-reactions with Abs raised during other infectious and autoimmune diseases (AID). Fusion of DNA sequences encoding antigenic proteins is a versatile tool to engineer proteins to be used as sensitizing elements in serological tests. A synthetic gene encoding a chimeric protein containing the C-terminal region of C29 and the N-terminal region of TcP2beta was constructed. A 236-serum panel, composed of 104 reactive and 132 nonreactive sera to Chagas' disease, was used to evaluate the performance of the chimera. Among the nonreactive sera, 65 were from patients with AID (systemic lupus erythematosus and rheumatoid arthritis) or patients infected with Leishmania brasiliensis, Brucella abortus, Streptococcus pyogenes, or Toxoplasma gondii. The diagnostic performances of the complete TcP2beta (TcP2betaFL) and its N-terminal region (TcP2betaN) were evaluated. TcP2betaFL showed unspecific recognition toward leishmaniasis (40%) and AID Abs (58%), while TcP2betaN showed no unspecific recognition. The diagnostic utility of the chimera was evaluated by analyzing reactivity and comparing the results with those obtained with TcP2betaN. The chimera reactivity was higher than that of the peptide fractions (0.874 versus 0.564 optical density, P = 0.0017). The detectability and specificity were both 100% for the whole serum panel tested. We conclude that the obtained chimera shows an improved selectivity and sensitivity compared with other ones previously reported, therefore displaying an optimized performance for Trypanosoma cruzi infection diagnosis.

Amperometric bioelectrode for specific human immunoglobulin G determination: optimization of the method to diagnose American trypanosomiasis.

Bioelectrodes to detect immunoglobulin G (IgG) antibodies occurring in sera of patients suffering from American trypanosomiasis were assembled. The device consisted of a gold electrode modified with a thiol sensitized with parasite proteins. The assemblage was accomplished by adsorbing IgG antibodies from confirmed infected patients followed by adsorption of anti-human IgG labeled with a redox enzyme. The appliance was used as a working electrode in a three-electrode cell containing a soluble charge-transfer mediator, also behaving as enzyme cosubstrate. The method is based on the measurement of the catalytic current after addition of the enzyme substrate, occurring when a positive serum is used to build up the biosensor. The discrimination efficiency between positive and negative sera was 100% for the samples studied. A 0.9525 correlation coefficient was obtained for results acquired by using this approach and one commercial diagnostic kit. The reproducibility, evaluated by the percentage coefficient of variation, varied between 7 and 20%. The sensitivity was 12.4 ng mL(-1) IgG, which is in the same order as that obtained with the commercial kit. Stability of the device was studied for a 7-day period and the results showed no significant change (p = 0.218). Leishmaniasic sera showed cross-reactivity when total parasite homogenate was used as antigen.