Improvements in the clinical sensitivity of ANA ELISA testing

Abstract

Objective: Develop an anti-nuclear antibody (ANA) ELISA that has high sensitivity for all samples that are ANA positive by immunofluorescence on HEp-2 cells, with an emphasis on detecting patients with clinically defined diseases. It has been well documented that commonly used ANA ELISAs fail to detect certain clinically important antibodies. The current work aims to correct those deficiencies.Methods: Optimal antigenic extracts of HEp-2 cells and nucleoli were obtained by testing numerous biochemical preparations for reactivity by ELISA with appropriate sera. In order to discover which other antigens were clinically important and needed to be added to the extracts, sera from patients with systemic lupus erythematosus (SLE), scleroderma and Sjögren's Syndrome (SS) that were negative on the extracts were examined in detail to discover their antigenic specificity.Results: A nucleolar extract that preserves reactivity with fibrillarin, the most diagnostically important nucleolar autoantibody in scleroderma, was obtained. A HEp-2 extract that reacts with a high proportion of ANA positive sera with no known specificity was also developed. In examining clinically defined sera it was found that 5%–10% of centromere positive sera recognized only CENP-A or CENP-B, while the other 80%–90% recognized other centromere antigens. Approximately 1%–2% of patients with SLE, scleroderma and SS have anti-SS-A 52kd as their only known autoantibody. Including the optimized extracts, both CENP-A and CENP-B, and SS-A 52 kd in the ANA ELISA yields a highly sensitive screening test with specificities equal to or better than standard immunofluorescent techniques.Conclusion: By optimizing the extracts and antigens included in the ANA ELISA, we have obtained a sensitivity on clinically important samples that is equal to immunofluorescence on HEp-2 cells. These reactivities correct the deficiencies noted in other ANA ELISAs without compromising high specificity.