1. Performance Characteristics of the FARRZYME™ High Avidity Anti-dsDNA IgG Antibody ELISA Brenda B. Suh-Lailam 1, Tyson R. Chiaro 1, K. Wayne Davis 1, Andrew R. Wilson 1, Anne E. Tebo 1, 2 Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology 1 and the Department of Pathology 2, University of Utah School Of Medicine, Salt lake City, UT ABSTRACT INTRODUCTION BACKGROUND: The FARRZYME™ High Avidity Anti-dsDNA IgG Antibody ELISA, measures specific high avidity IgG autoantibodies directed against double stranded deoxyribonucleic acid (dsDNA) found in human serum. When used in combination with other serological tests and clinical findings, the FARRZYME assay aids in the diagnosis of systemic lupus erythematosus (SLE). OBJECTIVES: The analytical principle of an anti- dsDNA IgG antibody assay determines both its diagnostic and predictive capabilities in systemic lupus erythematosus (SLE). The main objective of this study was to evaluate the analytical concordance and characteristics of the FARRZYME™ high avidity anti-dsDNA IgG ELISA with other assays for screening and confirming the presence of these antibodies. METHOD: We investigated the FARRZYME™ dsDNA IgG antibody ELISA for agreement with six different commercial ELISAs for screening and the confirmatory Crithidia luciliae indirect immunofluorescence test (CLIFT) using 100 anti-nuclear antibody (ANA) positive sera with a homogeneous pattern and titers ≥1:160 by indirect immunofluorescence assay (IFA) and 100 adult healthy control samples. The assay was also assessed for imprecision and interfering substances using commercial quality control materials for hemoglobin, icterus and lipemia. RESULTS: The positivity rates for screening assays in the ANA positive group ranged from 55.0-88.0% and were 68% and 18% for the CLIFT and FARRZYME assays respectively. The per cent positive agreement between the FARRZYME and all anti-dsDNA IgG assays was 100%. All FARRZYME antibody-positive samples displayed high level anti-dsDNA IgG antibodies in all screening assays as well as the CLIFT. Specificities for the anti-dsDNA IgG ELISAs ranged from 84-100% and were 84.0% and 100.0% for the CLIFT and FARRZYME assays respectively. The intra- and inter-assay imprecision ranged from 13.9 -16.5% however, 8% of the results could not be reproduced between lots. Except for hemoglobin, interference was observed with the performance of this assay with both bilirubin and lipemia. CONCLUSION: Our data suggest that the FARRZYME assay although less sensitive than the CLIFT, is specific and predicts the presence of high avidity anti-dsDNA IgG antibodies irrespective of screening assay employed. More prospective studies are needed to determine its clinical relevance in monitoring response to treatment in SLE patients. RESULTS REFERENCES ACKNOWLEDGEMENTS Anti-dsDNA antibodies are highly specific for the diagnosis of systemic lupus erythematosus (SLE) but are heterogeneous with respect to avidity, class and cross- reactivity. These antibodies can be detected by an enzyme-linked immunosorbent assay (ELISA), Crithidia luciliae indirect immunofluorescence test (CLIFT) and the Farr assay, a radioimmunological method based on the ammonium sulfate precipitation of immune complexes. The method of detection largely defines its clinical relevance. High avidity (HA) anti-dsDNA antibodies as detected by CLIFT and/or Farr assays have been reported to have good positive predictive values for SLE. The FARRZYME™ ELISA is a newly developed assay that is designed to detect HA anti- dsDNA IgG antibodies. The assay principle is thought to be similar to that of the Farr assay which is of limited use as it employs a radioactive substance. This study was designed to evaluate the analytical concordance of the new FARRZYME assay with six commercially available ELISAs for detecting anti-dsDNA IgG antibodies as well as the CLIFT confirmatory test. To determine it analytical performance, we performed imprecision studies following CLIS guidelines as well as interference studies using an in-house protocol. For these studies, 100 ANA positive samples with titers ≥1:160 (homogeneous pattern) and 100 healthy blood donors were employed. STUDY PROTOCOL AND METHOD Table 1. Agreement between anti-dsDNA IgG Assays Table 2. Characteristics of FARRZYME-positive Samples Good correlation between FARRZYME-positive samples with ANA- and CLIFT-positive titers ≥160. Figure 1. A Scatter-plot Showing Correlation between Assays Investigated All kits for this study were provided free-of-charge by AESKU, Dr. Fooke, Euroimmun, Bio-Rad and INOVA Diagnostics. This work was supported by the ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories and Department of Pathology, University of Utah, Salt Lake City. 1. Isenberg D., Smeenk R. Lupus 2002; 11:797-800 2. Isenberg D.A. et al. Rheumatol 2007; 0 kem112v:1-5. 3. Eilertsen G.O. et al. J Rheumatol. 2009; 36(3):552-9. 4. Clinical and Laboratory Standards Institute. EP5–A2 2004 CLSI CONCLUSION The FARRZYME assay is highly specific and predictive of the presence of highly elevated levels of anti-dsDNA antibodies. Due to its predictive value, the FARRZYME assay and/or the CLIFT can be used to confirm the presence of anti-dsDNA antibodies. Our imprecision studies suggest that further fine tuning of the FARRZYME assay is necessary to improve inter-assay or lot-to-lot precision. Based on the interference studies, lipemic and icteric serum samples should be avoided as their presence significantly affects the accuracy of results. Although high avidity anti-dsDNA antibodies may be used to predict lupus nephritis, our study has not addressed this aspect of laboratory testing. Table 4. Effect of Interfering Substances on Assay Performance Table 3. Summary of Imprecision studies RESULTS Except for hemoglobin which had interference <5%, bilirubin and lipemia showed deviations greater than 10% at concentrations evaluated. Assay Manufacturer % Positive Agreement % Negative Agreement % Positive Agreement % Negative Agreement % Overall Agreement FARRZYME Confirmatory Test FARRZYME New Test AESKU10052.817.310057.0 The Binding Site10068.724.010071.5 Bio-Rad10075.829.010078.0 Euroimmun10077.530.510079.5 Dr. Fooke10068.123.710071.0 INOVA10076.93010079.0 CLIFT10021.463.710067.0 Interfering Substance Conc. Manufacturer’s Claims of no interference Specimen Type Conc. Unspiked (IU/mL) Conc. Spiked (IU/mL) Deviation observed (%) Hemoglobin 1250 mg/dL486 mg/dL Negative10.8 10.4 3.7 Positive602.6625.93.9 Bilirubin 7.25 mg/dL20.2 mg/dL Negative10.8 11.1 2.8 Positive602.6483.919.7 Lipemia 2000 mg/dL1460 Units Negative10.8 9.2 14.8 Positive602.6353.541.3 Overall, there is excellent correlation between all assays for high level anti-dsDNA IgG antibodies. A positive FARRZYME test is associated with a significantly elevated level of dsDNA IgG antibody in all six anti-dsDNA IgG ELISAs in this study. Good intra-assay with marginal inter-assay precision. 8% of results not replicated between two lots evaluated (data not shown). SampleMean Conc. (IU/mL) Intra-assay CV (%)Total CV (%) Negative10.59.716.5 Low Positive37.82.013.9 High Positive749.50.914.2 *TBS: The Binding Site (now INOVA) Sample ID Sex/AgeANA IFA >1:40 AESK U >15 TBS >30 Bio-Rad >25 Euroimmu n >1.0 Dr. Fooke >1.0 INOVA >201 CLIFT >1:10 FARRZYME >30 1F/583205036360913.5744>1:4037 2F/426401455567346.53.88391:32038 3F/16128021139477527.77.315701:5120355 4F/121601712225443.73.45041:8066 5F/73320783584894.835111:320133 6F/78512020943047617.85.81451>1:40311 7F/701601164066763.62.86371:16043 8F/1564019118907895.86.213351:1280168 9F/4232021037067785.7715911:2560291 10F/61024020541047848.67.115371:2560286 11M/291280199755075675.916551:2560417 12F/436401403915216.648891:8041 13F/6232019813237414.65.711311:1280185 14F/916020927077555.36.815171:128093 15F/3012801508427584.33.812591:640101 16F/221602029657564.15.911521:2077 17F/461601523545302.33.94321:16064 18F/5916010962261134.510551:16030 Specimens: We used 100 anti-nuclear antibody (ANA) positive sera with a homogeneous pattern and titers ≥1:160 by indirect immunofluorescence assay (IFA) on HEp-2 cells and 100 adult “healthy” control samples from the ARUP Decades of Life study. Detection of Anti-dsDNA IgG Antibodies: All samples were screened for the presence of anti-dsDNA IgG antibodies using six commercial ELISAs from AESKU Diagnostics, The Binding Site (TBS), Bio-Rad Laboratories, Euroimmun, Dr. Fooke Laboratories, and INOVA Diagnostics. The presence of anti-dsDNA IgG antibodies were confirmed by the CLIFT (INOVA) and the high avidity FARRZYME ELISA. Analytical Performance: Imprecision (CLIS guidelines), lot-to-lot variation and interferences studies were performed using in-house protocols. Statistical Analyses: Positivity rates, specificities and correlations between assays were calculated as indicated using SAS® software, Version 9.1 of the SAS system for Windows. Copyright© 2002-2003 SAS institute Inc., Cary, NC, USA.