DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO SCREEN FOR AUTOANTIBODIES TO EXTRACTABLE NUCLEAR ANTIGENS

Abstract

The purpose of this study was to determine whether it is possible to coat six different autoantigens simultaneously on a polystyrene microtiter plate for use in an Enzyme-Linked ImmunoSorbent Assay (ELISA), and still maintain the sensitivity and specificity to qualify as a diagnostic test for autoimmune disease. When compared to the traditional Ouchterlony test, the Extractable Nuclear Antigen (ENA), ELISA demonstrated high sensitivity (100.0%) and specificity (97.7%). The overall relative agreement was 98.7%. Precision, or reproducibility, was determined by testing 5 positive sera and 2 negative sera ten times per plate on three replicates. The mean intra-assay coefficient of variation for the positive sera was 5.04%, and the mean inter-assay coefficient of variation was 6.46%. The linearity of the assay was demonstrated by testing two-fold serial dilutions of five different sera and comparing the values to the log2 of the dilution. The mean r value was 0.980. The solid phase ENA plates were shown to have a shelf life of one year. To determine efficacy, sera were obtained from 440 Lupus patients in a cohort at Johns Hopkins and assayed on the ENA ELISA to determine the prevalence of autoantibodies detected in a Lupus population. The prevalence was 61.4% positive for autoantibodies to ENAs. Overall, the data in this thesis demonstrates that the ENA ELISA is equivalent to traditional methods of screening for autoantibodies in sera of patients with autoimmune disease. In addition ELISA tests have the advantages of an objective reading method, automation, time efficiency, and a high degree of sensitivity and specificity.