A quantitative dot immunobinding assay for human HLA class II antigens using nitrocellulose membrane filters.
A quantitative method for the evaluation of human HLA-DR antigen expression has been developed. Cell membrane proteins were solubilized in Nonidet P-40 or deoxycholic acid detergent and diluted in a Triton X-100 containing sample buffer. The samples were subsequently spotted on a nitrocellulose membrane filter and fixed by immersion in isopropyl alcohol-acetic acid solution. The membrane was saturated in a 5% BSA blocking buffer and sequentially incubated with specific monoclonal anti-HLA-DR antibody, and 125I-labelled protein A. Each spot was then assayed for radioactivity in a gamma scintillation counter. Immunoadsorbant purified HLA-DR antigen was used to standardize the method and a reference dosage curve was established with serial dilutions of the purified HLA-DR antigen. The method permitted the detection of HLA-DR antigens with reproducibility in the ng range, in cellular extracts, physiological and pathological fluids, and in fractions eluted from affinity columns.
D002460:Cell Line; D003101:Collodion; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology; D008970:Molecular Weight
D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; D003101:Collodion