Identification of HLA antibody utilizes bead-based technology, which offers a snapshot of the antibody profile at the time of collection. However, antibody profiles can fluctuate with time, specifically a current serum profile may not detect historical sensitization. Memory B cells in circulation specific to HLA-alloantigen can be present without detection by single antigen bead assay, though may be differentiated into plasma cells and rapidly produce high affinity HLA specific antibodies. Karahan et al published a protocol for stimulation of memory B cells and characterization for the presence of HLA specific IgG. The aim of this project is to reproduce the findings of Karahan et al in the Western Australian solid organ recipient cohort.
The sample cohort was selected based on the history of sensitization. Peripheral blood and serum samples were collected from healthy donors consisting of multiparous women, and individuals who had never been exposed to alloantigens. Specifically, B cells from PBMC were incubated with agonists and growth media for 10 days at 37°C. The stimulated cells were characterised by Immunophenotyping assay using flow cytometry. The supernatant was purified for IgG using a protein G affinity purification method to be analysed via single antigen bead (SAB), flow cytometry and ELISA to detect IgG specificities and quantity.
We observed an expansion of CD19+ B cells in 10 days of stimulation and those cells carried a characteristic antibody-secreting-cell phenotype. There was a variation in the production of total IgG among tested individuals, ranging from 0.32 – 316 ng/ml. In immunized individuals, 3/7 were found to have HLA-specific IgG antibody in concentrated supernatants. Furthermore, the HLA specificities detected in the supernatants were absent in the current serum, and all detected HLA IgG antibody in the cell culture supernatants were accounted by a single mismatch eplet from the sensitizing partner. In all unsensitized individuals, the concentrated supernatant was negative for HLA specificities by SAB assay as expected. Therefore, the stimulation assay was shown to be specific to B cell memory, however, further investigation is required to examine the assay sensitivity as four of seven multiparous subjects failed to produce HLA IgG antibody.