An assessment of the immunoprecipitation of blood-group antigens using biotinylated red cells.
This study presents a simple protocol for labelling red cell membrane proteins and glycoproteins using biotin. The advantages include an increased capacity to label and process a large number of cell samples of different phenotypes without using large quantities of radionuclides, resulting in no hazardous waste by-products as in radiolabelling methods, and a fast exposure time for detection of the biotinylated component(s). Our assessment on biotinylated red cells was based on the retention of antigenic activity with 14 antibodies (13 monoclonals, one polyclonal), which have specificities for extracellular and cytoplasmic domains of various membrane components, by ELISA, haemagglutination tests and immunoprecipitation. A luminescent method was used to detect the immunoprecipitates on nitrocellulose membrane after SDS-polyacrylamide gel electrophoresis. The development time for band detection was between 5 s and 1 min. Free amino groups that constitute a part(s) of an antigenic determinant were also biotinylated. As a result antibody binding to the red cells was abrogated.
King
M J
MJ
International Blood Group Reference Laboratory, Bristol, UK.
Hemming
N
N
D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin; D001789:Blood Group Antigens / Q000276:immunology*; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004910:Erythrocyte Membrane / Q000276:immunology; D005069:Evaluation Studies as Topic; D006386:Hemagglutination Tests; D006801:Humans; D007124:Immunoenzyme Techniques; D007553:Isotope Labeling; D008163:Luminescent Measurements; D008562:Membrane Glycoproteins / Q000097:blood / Q000276:immunology; D008565:Membrane Proteins / Q000097:blood / Q000276:immunology*; D011233:Precipitin Tests / Q000379:methods*
D000911:Antibodies, Monoclonal; D001789:Blood Group Antigens; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D001710:Biotin