GAD65Ab and ICA512Ab are strongly associated with insulin-dependent (Type 1) diabetes mellitus. A novel, simple radio-antigen binding assay with microSepharose conjugated with monoclonal antibodies specific for human immunoglobulin light chains was developed to provide diagnostic sensitivity and specificity of GAD65Ab and ICA512Ab for Type 1 diabetes. The Receiver Operating Characteristic (ROC) curve was used to determine the upper level of Normal in 583 new onset Type 1 diabetic patients and in 829 matched controls. The sensitivity of GAD65Ab and ICA512Ab was 66% (384/583) and 41% (211/520), respectively, and the diagnostic specificity was 96% for both autoantibodies. Levels, but not frequency, of GAD65Ab were higher among female Type 1 diabetes patients, whereas ICA512Ab levels did not differ between males and females. Positivity for GAD65Ab. ICA512Ab or both showed a sensitivity of 74% and a specificity of 92% for Type 1 diabetes. This simple, one-step centrifugation, high-capacity radio-antigen binding assay has a high precision and reproducibility to accurately detect both GAD65Ab and ICA512Ab. This assay should also prove useful in other autoantibody assays against conformation-sensitive autoantigens.