P131 Using DTT as a tool to remove background in HLA antibody identification

Abstract

Aim The goal of this study is to investigate the effectiveness of DTT as a tool to remove excessive IgM, in Single Antigen Bead assays, which can lead to high negative control values. Methods In the present study, nine sera (class I) and seven sera (class II), with NC MFIs ⩾1000 MFI, were tested using One Lambda SAB Assay, neat vs. DTT, Adsorb-out, Adsorb-out x 2 and DTT & Adsorb-out. Sera were tested using the manufacturer’s protocol. Sera were DTT treated by combining 5 μL 50 mM DTT to 195 μL sera. This mixture incubated at 37 °C for 15 min. All sera were pretreated with EDTA. For this study, an elevated NC is defined as an MFI ⩾1000 (two sera demonstrated borderline positivity (953 & 816)). Results With class I, all but one DTT treated serum, demonstrated a decrease in NC MFI. The outlier serum NC MFI demonstrated a decrease but was slightly above acceptable level (1790 MFI → 1083 MFI). The sera were also treated with Adsorb-out, with all resulting NC MFIs >1000. Additionally, eight NC MFIs either did not decrease or increased following Adsorb-out. These sera were treated either with Adsorb-out x2 or DTT & Adsorb-out. Four sera continued to have NC MFIs >1000. Regarding class II DTT treated sera, all but two showed a NC MFI decrease. Of those two, both demonstrated a decrease vs. neat but not 1000 and three showed an increase. The sera were treated with Adsorb-out x 2 or DTT & Adsorb-out. Of these sera, two achieved acceptable levels. Conclusions DTT is a commonly used reducing reagent which is efficient at cleaving exposed disulfide bonds, thus eliminating IgM-based interference. This treatment, in regards to SAB testing, is performed to remove interfering IgM molecules. DTT is also able to obviate C1 interference but does so poorly. EDTA prevents complement interference far more efficient through Ca2+ chelation. Based on our results, we will further investigate the use of DTT as an effective treatment on sera with a high NC MFI which are not amendable to a single treatment of Adsorb-out.