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An alternative ELISA for T4 determination based on idiotype anti-idiotype interaction and a latex method for
M I Becker1, J E Aguayo, A Jamett
1Laboratorio de Inmunología, Bios Chile Ingeniería Genética S.A., Santiago.
This paper is the first report on the use of an idiotype-anti-idiotype monoclonal antibody reaction to develop an enzyme immunoassay for thyroxine (T4). We have developed a monoclonal antibody against T4, named 1F10 of IgG1 subclass and KA 5.21 x 10(8) M-1 which was used to obtain anti-idiotypic monoclonal antibodies. Anti-idiotypic antibodies were selected by a novel method, a passive agglutination assay with the idiotype monoclonal 1F10 absorbed on latex particles and subsequently characterized by RIA. One of these anti-idiotype antibodies, named 5B3--type beta antibody--of IgG1 subclass, was used to develop an enzyme-linked T4 idiotype-anti-idiotype immunosorbent assay. The T4 calibration curve, using the 1F10 idiotypic antibody adsorbed to solid phase and the 5B3 anti-idiotypic antibody conjugated to alkaline phosphatase (ALP), shows adequate performance in the range between 0.7-25 micrograms% of the analyte. The reliability of the proposed method is demonstrated by the correlation coefficient r = 0.74, found between T4 measured by RIA and our assay, with a panel of sera from euthyroid, hypothyroid and hyperthyroid individuals. The correlation coefficient was r = 0.93 within assays and r = 0.88 between assays. These results provide the basis for a new non isotopic assay for the study and diagnosis of T4-related human disease and provides a model to develop immunoassays for other haptens and small molecules of clinical interest.
This paper is the first report on the use of an idiotype-anti-idiotype monoclonal antibody reaction to develop an enzyme immunoassay for thyroxine (T4). We have developed a monoclonal antibody against T4, named 1F10 of IgG1 subclass and KA 5.21 x 10(8) M-1 which was used to obtain anti-idiotypic monoclonal antibodies. Anti-idiotypic antibodies were selected by a novel method, a passive agglutination assay with the idiotype monoclonal 1F10 absorbed on latex particles and subsequently characterized by RIA. One of these anti-idiotype antibodies, named 5B3--type beta antibody--of IgG1 subclass, was used to develop an enzyme-linked T4 idiotype-anti-idiotype immunosorbent assay. The T4 calibration curve, using the 1F10 idiotypic antibody adsorbed to solid phase and the 5B3 anti-idiotypic antibody conjugated to alkaline phosphatase (ALP), shows adequate performance in the range between 0.7-25 micrograms% of the analyte. The reliability of the proposed method is demonstrated by the correlation coefficient r = 0.74, found between T4 measured by RIA and our assay, with a panel of sera from euthyroid, hypothyroid and hyperthyroid individuals. The correlation coefficient was r = 0.93 within assays and r = 0.88 between assays. These results provide the basis for a new non isotopic assay for the study and diagnosis of T4-related human disease and provides a model to develop immunoassays for other haptens and small molecules of clinical interest.