P012 : EVALUATION OF FOUR METHODS TO REDUCE BACKGROUND REACTIVITY AND INTERFERENCE IN SINGLE ANTIGEN BEAD ANTIBODY DETECTION

Abstract

Aim High negative control (NC) bead reactivity and test method interferences complicate the interpretation of single antigen bead (SAB) assays; we compared 4 methods of reducing NC values and increasing antibody (Ab) detection. Methods Samples were selected due to high NC reactivity, low positive control (PC) bead reactivity, and/or DTT enhanced reactivity. Sera were treated with: – Adsorb Out™ (One Lambda Inc.). – DTT: 5 μl 100 mM DTT per 95 μl serum. – EDTA: 5 μl of 0.114 M EDTA per 95 μl serum. – NanoSep® spin columns (SC) (100 K, Pall, Life Sciences). Untreated and treated samples were tested with class I ( n = 12) or II ( n = 9) SAB (LabScreen®). Results were exported and analysed in Microsoft Excel. Results PC bead reactivity increased with all treatments. NC bead reactivity decreased with Adsorb and SC and often decreased with EDTA and DTT; however 4/12 class I and 3/9 class II results had increased NC bead values following DTT/EDTA. Increased MFI’s were seen in 4/12 class I and 4/9 class II SAB results following DTT and EDTA treatment. In 6/8 sera tested this increase was from weak to strong positive. SC treatment resulted in decreased or undetectable Ab in most sera. Conclusion While none of these treatments simultaneously decreased background and eliminated interferences, DTT and EDTA treatment were the most successful. The use of the spin columns is discouraged due to the unexpected removal of HLA antibodies. There was no evidence of decreased MFI values with DTT or EDTA. Other than some examples of increased MFI of the NC bead, there does not appear to be any disadvantage to routinely treating sera with either method. Both EDTA and DTT reagents are less expensive than Adsorb or SC. EDTA treatment is more efficient to perform than DTT and it is more stable. We will further study the use of EDTA treatment with additional samples.