Determination of the frequency of HLA antibody secreting B-lymphocytes in alloantigen sensitized individuals

Sera from prospective transplant patients are usually screened for HLA antibodies prior to transplantation, but presently available tests do not permit quantification of the humoral alloantigen directed response. We adapted a culture system for isolated human B-lymphocytes to assay the secretion of HLA-antibodies on a single cell basis. B-cell supernatants were screened for HLA antibodies by complement dependent cytotoxicity. The assay assigns precursor frequencies for HLA-alloantibody secreting B-lymphocytes (BCPFs), and simultaneously allows for dissection of the humoral alloantigen directed response into its monoclonal components. The lymphocytes of 15 HLA-seropositive multiparous women that were used to validate the assay, were found to contain HLA-BCPFs ranging from 0 to 123 per 106 B-lymphocytes (mean: 43 ± 45 per 106 B-lymphocytes). The HLA-specificities of antibodies in the B-cell supernatants were in agreement with serum specificities. Genuine HLA reactivity of B-cell supernatants was confirmed using an ELISA with purified HLA class I antigens. When applied to lymphocytes of patients on transplant waiting lists, the present assay may enable the unraveling of serum specificities in their components, thus supplementing HLA antibody serum screening data