title: Quantification of HLA class II-specific memory B cells in HLA-sensitized individuals.
abstract: For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood. By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy-immunized females, we found memory B cell frequencies ranging from 25 to 756 spots per 10(6) B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no significant spot formation was detected. Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients.
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Gonca E
authors: GE
authors: Karahan
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Yvonne J H
authors: YJ
authors: de Vaal
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Dave L
authors: DL
authors: Roelen
authors: R&D, Pure Protein LLC, Oklahoma City, OK, USA.
authors: Rico
authors: R
authors: Buchli
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Frans H J
authors: FH
authors: Claas
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: S.Heidt@lumc.nl.
authors: Sebastiaan
authors: S
authors: Heidt
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology*; D058501:Enzyme-Linked Immunospot Assay; D005240:Feasibility Studies; D005260:Female; D006084:Graft Rejection / Q000175:diagnosis* / Q000517:prevention & control; D006683:HLA-DQ Antigens / Q000276:immunology; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007114:Immunization; D007156:Immunologic Memory; D007519:Isoantigens / Q000276:immunology; D008297:Male; D016377:Organ Transplantation; D011247:Pregnancy
chemical_list: D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; D007519:Isoantigens
keywords: Chronic rejection; HLA antibodies; Kidney transplantation; Luminex; Pregnancy-immunized women