DELISA - SENSITIVE NONISOTOPIC ASSAY FOR GAD(65) AUTOANTIBODIES, A KEY RISK-ASSESSMENT MARKER FOR INSULIN-DEPENDENT DIABETES-MELLITUS

Nonisotopic assays for the measurement of autoantibodies to 65-kDa glu tamic acid decarboxylase (GAD(65)) have not previously achieved perfor mance equivalent to radiobinding assays (RBA). We have developed a mod ified ELISA protocol, DELISA, for measuring autoantibodies to GAD(65) in serum. The method overcomes the problems of poor sensitivity and sp ecificity associated with conventional ELISAs. Serum containing GAD(65 ) autoantibodies is incubated with biotinylated GAD(65) (bGAD(65)). Su fficient soluble Protein A-dextran conjugate is added to bind the immu noglobulins in the sample, including GAD(65) autoantibodies to which G AD(65) is bound. After incubation, the mixture is transferred to a str eptavidin-coated microtiter well, which binds free bGAD(65) but not bG AD(65) bound to autoantibodies, Streptavidin-bound bGAD(65) is detecte d by means of a peroxidase-GAD(65)MAP conjugate. The method appears to have comparable sensitivity and specificity to those of RBAs. Reactio n of the antibodies with soluble antigen to increase the binding rate and the use of high serum concentrations and very low antigen concentr ations to increase sensitivity are critical elements of the method.