GAD65 Antibody ELISA With Extended Reportable Range: Validation and Guidance for Neurological Practice

To (1) validate GAD65-ELISA detection and quantification for type 1 diabetes mellitus and autoimmune neurological diagnoses, (2) correlate ELISA results (reference range < 5 IU/mL) with established radioimmunoprecipitation assay (RIA; ≤ 0.02 nmol/L), and (3) define ELISA clinical utility and pitfalls.

Serum performance for diabetes (FDA-cleared, undiluted) was verified, and neurological laboratory-developed serum and CSF dilution protocols were validated to extend the reportable range beyond > 250 IU/mL. ELISA and RIA values were correlated, including established neurological RIA cut-offs (serum ≥ 20 nmol/L; CSF any positive).

ELISA met analytical criteria (precision, accuracy, sensitivity, specificity, reference range) in serum and CSF and was clinically equivalent to RIA for autoimmune diabetes. Neurological ELISA cut-offs were established at 10,000 IU/mL (serum) and 100 IU/mL (CSF). Precision was better below 10,000 IU/mL (CVs < 20%) than above (CVs 30.8% serum; 26.5% CSF). ELISA-RIA correlation and agreement was stronger below the neurological cut-off (R² = 0.89) than above (R² = 0.36). Positive agreement for RIA-defined neurological disease was 100% in serum and CSF; all serums were > 10,000 IU/mL. Clinical specificity was 97.5% in serum and 100% in CSF, exceeding reported RIA specificity. Screen results > 250 IU/mL spanned a wide range of dilution values; many were below the neurological cut-off. Most patients with paired serum/CSF positivity showed elevated GAD65 IgG indices.

GAD65 ELISA and RIA have equivalent sensitivity for autoimmune diabetes and neurological testing, with higher specificity for ELISA. A serum cut-off of 10,000 IU/mL is informative but requires clinical context, and dilution of screen > 250 IU/mL samples is essential for neurological interpretation.