Multiplexed tests for autoantibodies to extractable nuclear antigens using the Luminex 100 ™ flow cytometer

Abstract Objective: Develop a convenient multiplexed immunoassay for the detection of autoantibodies to the extractable nuclear antigens Sm, RNP, SS-A and SS-B. Compare the results obtained with this new technology to clinical diagnosis and the results found with standard ELISA methodology. Demonstrate the high performance, increased efficiency and cost savings of this technology. Methods: A no-wash multiplexed assay that detects autoantibodies to the 4 extractable nuclear antigens in a single well was developed. Each of the 4 antigens is coated on a unique bead. The beads are then mixed in a single well as a four-plex on Sm, RNP, SS-A and SS-B. This test is called the QUANTA Plex™ ENA Profile 4. Three groups of sera were tested on the Luminex 100 ™ with these multiplexed antigens as well as on the corresponding ELISAs. Discrepant samples were tested by Ouchterlony immunodiffusion. Also, a five-plex with Scl-70 and a six-plex with both Scl-70 and Jo-1 were examined. Results: 170 samples from healthy volunteers were tested on the QUANTA Plex ENA Profile 4 and the 4 corresponding ELISAs. All samples were negative on SM, RNP and SS-B, while there was 1 false positive on SS-A. The cutoff between negative and positive on the assays ranged from 5 to 13 S.D. above the average of the healthy volunteers. 232 ANA positive samples were tested, including 119 with either SLE or Sjogren's syndrome. There was extremely high correlation between the results with ELISAs and the multiplexed assay. Counting all 402 samples, the relative sensitivity of the QUANTA Plex™ tests ranged from 92% to 96% and the relative specificity was between 96% and 99%. Linear regression comparing the values of the samples on ELISA and the QUANTA Plex™ assays was between 0.73 and 0.82 for 3 of the tests. Additionally, adding beads with Scl-70 and Jo-1 to the QUANTA Plex™ ENA Profile 4 produced performance comparable to the corresponding ELISAs and did not change the reactivity of any sera to the original 4 beads. Conclusion: The QUANTA Plex ENA Profile 4 is a labor saving immunoassay system with a small footprint that yields results that are virtually identical or in some cases superior to ELISA.