title: Measurement of membrane bound IgD and IgM on B lymphocytes.
abstract: An assay system for the determination of the membrane bound IgD (mIgD) and IgM (mIgM) on B lymphocytes was developed by the combination of two new ELISA methods with the results of flow cytometry after labeling with specific antibodies. The mIgD and mIgM of B lymphocytes were prepared by incubating mononuclear cells (MNCs) in Tween 20 containing buffer and repeated freeze/thaw cycles. Optimal results were achieved with 0.2-0.4% Tween 20 and two freezing cycles. With biotin-streptavidin amplification the sensitivity of the ELISA was 30 microU/ml for IgD and 0.5 ng/ml for IgM. In healthy persons 3.5 +/- 0.5 mU mIgD were detected on 10(6) IgD+ cells and 57.1 +/- 5.9 ng mIgM on 10(6) IgM+ cells. The mIgD/mIgM ratio was 0.065 +/- 0.005 mU/ng. The developed ELISA systems utilize only commercially available reagents and therefore provide a convenient reproducible tool for determining membrane bound IgD and IgM on B lymphocytes.
authors.affiliation: Department of Clinical Immunology and Asthma OPD, Free University Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.forename: T
authors.initials: T
authors.lastname: Brunnée
authors.forename: J
authors.initials: J
authors.lastname: Kleine-Tebbe
authors.forename: K
authors.initials: K
authors.lastname: Graf
authors.forename: A
authors.initials: A
authors.lastname: O'Connor
authors.forename: G
authors.initials: G
authors.lastname: Kunkel
mesh_terms: D000328:Adult; D001402:B-Lymphocytes / Q000378:metabolism*; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007072:Immunoglobulin D / Q000097:blood*; D007075:Immunoglobulin M / Q000097:blood*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D011947:Receptors, Antigen, B-Cell / Q000097:blood*; D012680:Sensitivity and Specificity
chemical_list: D007072:Immunoglobulin D; D007075:Immunoglobulin M; D011947:Receptors, Antigen, B-Cell