LAUSR

Aim This study is focused to determine the practicality of using the internal positive control bead as a qualitative measure in One Lambda’s Single Antigen Bead assay. Methods In the present study, forty samples (n = 40) were tested using One Lambda (Canoga Park, CA) Single Antigen Bead (SAB) Assay for HLA Class I and fifty-one samples (n = 51) were tested for HLA Class II antibody specificities using manufacturer’s suggested protocol. To treat serum with DTT, 5μL of 50 mM DTT was added to 195 μL of patient serum and incubated at 37 °C for 15 min. Results Forty samples were assayed to determine the identification of anti-HLA class I antibodies and 52 samples were tested to determine the identification of anti-HLA class II antibodies. For this study, 1000 mean fluorescence intensity (MFI) was considered positive. Data listed as in Table. Of the + to = beads, half were borderline, demonstrating low reactivity in an untreated sample but being 200–300 MFI from the positive threshold following treatment. A similar proportion followed the opposite path. Of the = to + group, half demonstrated borderline reactivity, just under the positive threshold. Following DTT treatment, the average MFI rose by only a few hundred (200–300) MFI. Conclusions Dithiothreitol treatment is a commonly used reducing reagent used to denature disulphide bonds in IgM molecules to remove background or prevent steric hindrance. This treatment, in regards to SAB testing, is performed to increase the positive control MFI. DTT is efficient at cleaving exposed disulfide bonds, thus eliminating IgM-based interference. DTT is also able to obviate C1 interference but does so poorly. EDTA prevents complement interference far more efficiently through Ca2+ chelation. Based on our results, we question the utility of using DTT to increase the positive control bead MFI. Due to lot-to-lot variability in the density of the IgG molecule on the surface of the positive control bead, One Lambda’s published positive control bead cutoff is sufficient to determine if the assay is valid. Download : Download high-res image (47KB) Download : Download full-size image