Trypanosoma cruzi: characterization of two recombinant antigens with potential application in the diagnosis of Chagas' disease.
A genomic library of Trypanosoma cruzi, constructed in the vector lambda gt11, was screened with a hyperimmune rabbit antiserum against tissue culture trypomastigotes. Two clones, B12 and B13, containing inserts of 350 and 600 bp, respectively, were isolated. Sequencing data indicated that both clones present a pattern of tandemly repeated nucleotide units of 60 bp for B12 and 36 bp for B13. Southern blot analysis suggests that both corresponding genes exist as a single copy. The inserts of both recombinants were subcloned in the vector pMSgt11, in phase with the lacZ gene. Recombinant proteins were affinity purified on pAPTG-agarose columns and employed to immunize rabbits, as well as to immunoselect human chagasic antibodies. By Western blot, antibodies to B12 reacted with bands of 230 kDa in trypomastigotes and 200 kDa in epimastigotes, while those to B13 identified bands of 140 and 116 kDa in trypomastigotes and epimastigotes. Immunoprecipitation of radioiodinated parasites revealed that the 140-kDa antigen recognized by antibodies to B13 is located on the membrane of trypomastigotes but not epimastigotes. The potential application of either recombinant antigen in the serological diagnosis of Chagas' disease was evaluated initially by RIA. It was observed that B13 presents a very good performance with sensitivity of 97%. For B12, the corresponding value was 82%. The reactivity to B13 was also evaluated by ELISA tests run in parallel with conventional serological reactions for Chagas' disease. Analysis of 209 serum samples indicates that B13 presents similar or even better performance in relation to the use of total epimastigote antigens, making it a promising candidate for the diagnosis of Chagas' disease.
Gruber
A
A
Departamento de Bioquímica, Universidade de São Paulo, Brazil.
Zingales
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood / Q000276:immunology; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D014355:Chagas Disease / Q000175:diagnosis*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D013045:Species Specificity; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000276:immunology*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins