Anti‐SSA/Ro Antibody Determination by Enzyme‐Linked Immunosorbent Assay as a Supplement to Standard Immunofluorescence in Antinuclear Antibody Screening

Abstract

The aim of this study was to investigate the frequency and possible clinical relevance of SSA/Ro antibodies, as determined by enzyme-linked immunosorbent assay (ELISA), in patient sera not exhibiting a concomitant positive reaction by the standard immunofluorescence (IF) test using HEP-2 cells as substrate. SSA/Ro reactivity, as shown by ELISA, was found in 285 (7%) of 4025 serum samples consecutively remitted for antinuclear antibody (ANA) screening. Seventy-five of these serum samples (26%), derived from 64 patients, were negative by the IF-ANA screening test. Serum samples from all 64 patients exhibiting SSA/Ro reactivity by ELISA without concomitant positivity by IF-ANA were further investigated by IF using transfected HEP-2 cells hyperexpressing the 60,000 MW SSA/Ro antigen (HEP-2000(R)) and by immunodiffusion (ID) and Western blot. In 55 of these 64 patients, SSA/Ro reactivity could be verified by one or more of the other techniques investigated. Twelve of these patients fulfilled four or more American College of Rheumatology (ACR) criteria for systemic lupus erythematosus (SLE) and another five patients exhibited a histologically confirmed cutaneous lupus erythematosus (LE). In four of the 12 IF-ANA-negative patients with a diagnosis of SLE, the SSA/Ro reactivity was only detectable by ELISA and Western blot. In conclusion, the use of a sensitive ELISA assay could provide a clinically important supplement to the routine ANA screening by IF, which does not detect certain anti-SSA/Ro-containing sera among patients with relevant autoimmune diagnoses. Detection of anti-SSA/Ro antibodies, however, does not alone signify cutaneous LE or SLE but adds weight to these diagnoses that should rely heavily on other clinical information.

SS-56, a novel cellular target of autoantibody responses in Sjögren syndrome and systemic lupus erythematosus

“…Routine laboratory diagnosis in SS and SLE includes the use of immunofluorescence or ID to evaluate the presence of autoAb's against SSA and SSB antigens (3). Several reports have suggested that these classical techniques are less sensitive than Western blot and ELISA for the detection of anti-SSA-52 antibodies (38)(39)(40). Our data confirm these findings and show that among 13 SS and 12 SLE patients who were reported to have no detectable anti-SSA antibodies by ID, six (four SS and two SLE) were found by ELISA to possess measurable antibodies against SSA-52.…”

Utility of Antinuclear Antibody Screening by Various Methods in a Clinical Laboratory Patient Cohort

“…In many studies, IFA has been used to define cohorts of CTD patients, potentially excluding patients with a CTD who are ANA negative by IFA (21,22). Our data suggest that there may be some antibodies that react with antigens not well represented or "hidden" in the HEp-2 cells, such as SS-A, or that may bind to specific epitopes with increased exposure in the solid-phase EIA (23)(24)(25)(26). To further complicate the comparison, approximately 5% of samples negative by both IFA (≤1:40) and EIA (≤1.0 U) were found to be positive on the MIA method.…”

Autoantibodies to Extractable Nuclear Antigens: Making Detection and Interpretation More Meaningful

“…Conversely, the ANA test is not sufficiently specific to establish a diagnosis of a CTD, and a more specific confirmatory test, such as an anti-doublestranded DNA (dsDNA) or an anti-ENA antibody test, is indicated. With the exception of antibodies to cytoplasmic antigens such as Jo-1 (histidyl-tRNA synthetase), it is rare to have a positive anti-ENA antibody test in the absence of a positive ANA test when acetone-fixed HEp-2 cells are used as the substrate for IIF (3,14,31). Thus, the finding of ANA-negative SLE due to the use of SS-A antigen-poor substrates such as mouse liver is no longer a valid consideration.…”