Measurement of membrane bound IgD and IgM on B lymphocytes.
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.
Zhang
M
M
Department of Clinical Immunology and Asthma OPD, Free University Berlin, Germany.
Brunnée
T
T
Kleine-Tebbe
J
J
Graf
K
K
O'Connor
A
A
Kunkel
G
G
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
D007072:Immunoglobulin D; D007075:Immunoglobulin M; D011947:Receptors, Antigen, B-Cell