title: Development of an immunoenzymatic assay for the detection of human antibodies against Trypanosoma cruzi calreticulin, an immunodominant antigen.
abstract: We have developed an indirect immunoenzymatic assay (ELISA) for the detection of human antibodies against calreticulin (formerly known as Tc45), a dimorphic Trypanosoma cruzi antigen, described in our laboratory. PVC microtitration plates were sensitized with the monoclonal anti-calreticulin antibody (MoAb) and reacted with calreticulin present in a partially purified preparation. The presence of anti-T. cruzi calreticulin IgG in sera from infected individuals was tested. The data generated with this assay were validated by correlation, in a regression analysis, with those obtained by an indirect immunoradiometric assay (IRMA). From the 12 seropositive sera (as defined by a commercial test), eight came out positive and four negative in both assays. The 12 human sera were also analyzed in direct immunometric assays (ELISA and IRMA), where the solid phase was sensitized with a whole parasite extract. The direct ELISA and IRMA correlated positively (P<0.01). Further validation of this ELISA was achieved with an indirect immunofluorescense assay. The high degree of significance obtained when the indirect IRMA and ELISA systems were compared, indicated that the relatively small sample number used (12) was statistically satisfactory for the purposes of this investigation. Thus, the IRMA can be replaced by the ELISA, with advantages mainly derived from the cumbersome manipulation of radioactive wastes. The MoAb used as an antigen capture agent in the ELISA proposed here, recognizes a homologous protein in Trypanosoma rangeli, suggesting that individuals infected with this parasite might have crossreactive antibodies. However, the system retains its diagnostic interest, given the facts that the MoAb does not recognize a homologous protein in Leishmania mexicana, Leishmania donovani, or Crithidia fasciculata.
authors.affiliation: Programa de Inmunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 13898, Correo 21, Santiago, Chile.
authors.forename: K
authors.initials: K
authors.lastname: Marcelain
authors.forename: A
authors.initials: A
authors.lastname: Colombo
authors.forename: M C
authors.initials: MC
authors.lastname: Molina
authors.forename: L
authors.initials: L
authors.lastname: Ferreira
authors.forename: M
authors.initials: M
authors.lastname: Lorca
authors.forename: J C
authors.initials: JC
authors.lastname: Aguillón
authors.forename: A
authors.initials: A
authors.lastname: Ferreira
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000276:immunology; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification; D037282:Calreticulin; D014355:Chagas Disease / Q000097:blood*; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D012261:Ribonucleoproteins / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D007074:Immunoglobulin G; D012261:Ribonucleoproteins