Enzyme-Linked Immunosorbent Assay Screening Then Indirect Immunofluorescence Confirmation of Antinuclear Antibodies

Abstract

The purpose of this study was to analyze antinuclear antibody (ANA) screening by enzyme-linked immunosorbent assay (ELISA) followed by indirect fluorescent antibody (IFA) testing to confirm and characterize the pattern and titer of the antibody. We evaluated 4 ANA ELISAs and 1 HEp-2 IFA substrate in 224 clinically defined serum samples consisting of 30 from systemic lupus erythematosus (SLE) cases, 94 from rheumatoid arthritis cases, and 100 from healthy donors plus 495 serum samples submitted for routine ANA testing and 12 reference serum samples distributed by the Centers for Disease Control and Prevention. IFA tests were read independently by 2 certified medical technologists. ELISA sensitivities ranged from 90% to 97% compared with 80% by IFA in the SLE serum samples. The ELISAs had specificities of 36% to 94%, whereas the IFA had 99% specificity. Overall, ELISAs for ANA assays demonstrated better sensitivity and good specificity, suggesting ELISA is a more cost-effective alternative to IFA testing for initial ANA screening. Samples positive by ANA ELISA should be tested on HEp-2 to determine the titer and pattern.

Clinical significance of anti-dense fine speckled 70 antibody in patients with fibromyalgia

“…Nevertheless, several challenges attending the HEp-2 IIF methodology persist [8,9] and other technologies for ANA detection continue to evolve [10,11].…”

Recent Approaches To Optimize Laboratory Assessment of Antinuclear Antibodies

“…Since the adoption of ANA assessment as a routine clinical laboratory test, the techniques for ANA detection and/or measurement have evolved and now encompass a variety of immunological methods (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The IFA method using HEp-2 cells is considered the standard method for detecting ANAs, with a number of pros and cons (1-4, 21, 22, 24-30).…”

“…Central to the analytical challenges associated with ANA IFA testing are its labor-intensiveness (with significant amounts of training being required for competence), subjectivity in titer and pattern recognition, poor standardization of reagents, and a declining workforce in clinical laboratories (1-4, 15, 30, 32). Thus, in some laboratories, the ANA IFA technique has been replaced by high-throughput, less subjective methods, including enzyme-linked immunosorbent assays (ELISAs) and multiplex assays such as line immunoassays (LIAs) and multiplexed bead assays (MBAs) (21)(22)(23)(24)(25)(26)(27)(28)(29). The non-IFA assays differ in the source, purity, concentration, and binding capacity of the antigens, the reference materials or standards used in assay development, the secondary antibodies (conjugates), and the detection signals.…”

“…A variety of manual or automated single or multiplex immunoassays (examples of the most commonly used methods include ELISAs, chemiluminescence immunoassays [CLIAs], LIAs, and MBAs), with different performance characteristics, have been developed and reported in the literature (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The advantages of these alternative approaches to ANA IFA testing include the ease of use, the possibility of performing quantitative high-throughput analyses, the lack of subjectivity, and the consolidation of ANA-related tests in a single platform.…”

“…Commercially available ANA ELISAs have various configurations of antigens (substrates) and secondary antibodies (23,24,28,30). While most ELISAs for the detection of ANAs use HEp-2 cell extracts spiked with a combination of purified, recombinant, or native proteins associated with common SARDs, quite a few do not include the HEp-2 cell extract.…”