P015 Removal of selected non-specific reactivity in single antigen bead assays via FBS pre-treatment

Abstract

Aim Non-specific reactivity is a known complication of single antigen bead (SAB), solid phase assays. Several approaches have been tried to reduce these reactions but are mostly related to Class I (eg: iBeads, acid treatment). No such treatments exist for Class II. Presently, the current lot of Class II SABs (LS2A01, Lot#: 011) has shown strong non-specific reactivity with some DRB1∗04 and DRB1∗16 bearing beads in patients who are otherwise non-sensitized; MFI values up to 4000. Previously, we reported that pre-treatment of patient serum with fetal bovine serum (FBS) effectively reduced non-specific binding to HLA Class I and Class II FlowPRA beads. In this study we sought to determine if FBS treatment would be effective in reducing/eliminating this non-specific class II reactivity. Methods Sera from 20 non-sensitized patients, each exhibiting the DR4/DR16 pattern, were pre-treated with FBS. Briefly, 5ul of FBS was added to 95ul of patient serum; incubated for 30 min at 37 °C; centrifuged and then used in the SAB assay. As controls, we treated sera from patients with well documented HLA antibody including DR4/DR16 (N = 10) and patients without HLA antibody (N = 10). Results Our results showed that in 100% (20/20) of the effected patients the DR4/DR16 reactivity was eliminated or significantly reduced. (Fig.). Importantly, patients with bona fide HLA antibody had no change in MFI values and additional reactivity was not observed in patients without HLA antibody following FBS treatment. Download : Download high-res image (339KB) Download : Download full-size image Conclusions In this study, we have demonstrated that the non-specific DR4/DR16 reactivity pattern was eliminated by pre-treatment with FBS. We speculate that this pattern is a result of reactivity against bovine peptides (possibly derived from culture media) present in the grove of the DR4/DR16 alleles. Pre-treating sera with FBS provides an exogenous source of soluble target proteins that interact with the antibodies thereby preventing their subsequent interaction with the beads.