BACKGROUND: Antibodies to GAD65 (GADA) are considered highly predictive humoral markers of the type 1 diabetes mellitus and also of the insulin requirement in adult-onset patients presumptively classified as type 2 diabetics or LADA. METHODS: We present 2 methods for GADA assessment. The first one (fluid phase, ELISA Protocol A) is carried out in a 2-step procedure in which serum GADA are first allowed to react with a fixed dose of GAD65-biotin in solution and the residual free antigen is later assayed by a conventional ELISA. In the second test (solid phase, ELISA Protocol B) GADA are measured in an ELISA that depends on the ability of divalent autoantibodies to form a bridge between immobilized TrxGAD65 and liquid-phase biotinylated TrxGAD65. RESULTS: All normal control samples scored negative in both variants of ELISA and RBA, hence specificity was 100% for all methods; the relative sensitivity of ELISA Protocol A respect of the RBA was 94% and that of ELISA Protocol B was 76%. CONCLUSIONS: Although ELISA Protocol A exhibited a better performance in terms of relative sensitivity than ELISA Protocol B, the simplicity of execution and the intended use of the assay must also be taken in consideration for the final choice.
BACKGROUND: Antibodies to GAD65 (GADA) are considered highly predictive humoral markers of the type 1 diabetes mellitus and also of the insulin requirement in adult-onset patients presumptively classified as type 2 diabetics or LADA. METHODS: We present 2 methods for GADA assessment. The first one (fluid phase, ELISA Protocol A) is carried out in a 2-step procedure in which serum GADA are first allowed to react with a fixed dose of GAD65-biotin in solution and the residual free antigen is later assayed by a conventional ELISA. In the second test (solid phase, ELISA Protocol B) GADA are measured in an ELISA that depends on the ability of divalent autoantibodies to form a bridge between immobilized TrxGAD65 and liquid-phase biotinylated TrxGAD65. RESULTS: All normal control samples scored negative in both variants of ELISA and RBA, hence specificity was 100% for all methods; the relative sensitivity of ELISA Protocol A respect of the RBA was 94% and that of ELISA Protocol B was 76%. CONCLUSIONS: Although ELISA Protocol A exhibited a better performance in terms of relative sensitivity than ELISA Protocol B, the simplicity of execution and the intended use of the assay must also be taken in consideration for the final choice.
Authors: Natalia I Faccinetti; Luciano L Guerra; Adriana V Sabljic; Silvina S Bombicino; Bruno D Rovitto; Ruben F Iacono; Edgardo Poskus; Aldana Trabucchi; Silvina N Valdez Journal: Microb Cell Fact Date: 2017-11-13 Impact factor: 5.328
Authors: Aldana Trabucchi; Silvina S Bombicino; Alexandra M Targovnik; Juan I Marfía; Adriana V Sabljic; Natalia I Faccinetti; Luciano L Guerra; Ruben F Iacono; María V Miranda; Silvina N Valdez Journal: Sci Rep Date: 2019-01-29 Impact factor: 4.379