Evaluation of Recombinant Antigens for Serodiagnosis of Chagas’ Disease in South and Central America

Abstract

The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions ofTrypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzirecombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.

Evaluation of recombinant antigens of Trypanosoma cruzi to diagnose infection in naturally infected dogs from Chaco region, Argentina

“…The use of recombinant antigens in the serological diagnosis of T. cruzi infections provides advantages against the use of parasite protein extracts, such as specificity, facility of production process and reproducibility (26,28). The most reactive recombinant antigen studied here could not reach by itself the sensitivity of the protein homogenate in dog sera.…”

“…However, the antigen mixture SAPA/TSSA VI had a significant reactivity (95%) that could be as useful as the protein homogenate of T. cruzi with the advantage of being specific. The specificity of these antigens, compared to other parasite infections, was analysed in humans previously (11,12,17,25,26,28,29,30).…”

Epitope mapping of a single repetitive unit of the B13 Trypanosoma cruzi antigen as fusions to Escherichia coli LamB protein

“…The insertions were sequenced using a 20-mer primer complementary to nucleotides located 69 bp upstream from the BamHI insertion site in the lamB gene. A glutathione S-transferase (GST) fusion protein carrying 16.6 repeats of the 12-amino-acid motif of B13 (B13-GST) was also produced in E. coli (GST fusion system, Pharmacia) [5]. The recombinant protein was purified from IPTG-induced E. coli lysates by affinity chromatography.…”

“…The B13-LamB fusion proteins were tested by ELISA with a panel of 80 human chronic chagasic sera, which had been previously shown to react with a B13-GST fusion protein [5]. A panel of 52 normal human sera was used as negative control.…”

“…The T. cruzi B13 antigen, located on the surface of infective trypomastigotes, is composed of arrays of a tandemly repeated 12-amino-acid motif -P(L)P(S,A)-P(L)FGQAAA(E)G(A)D(G)K, where residues within parentheses can be found replacing the preceding residue in different repeat copies [4]. B13, purified as recombinant protein from Escherichia coli, has been shown to present high sensitivity and specificity in the serological diagnosis of Chagas disease [4,5]. The characterization of B13 immunodominant epitopes has been based on the use of synthetic 9-mer peptides, derived from the B13 sequence, in competitive ELISA with B13 recombinant protein and sera from chagasic patients [6].…”