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A quantitative dot immunobinding assay for human HLA class II antigens using nitrocellulose membrane filters

Authors
Journal
Journal of Immunological Methods
0022-1759
Publisher
Elsevier
Publication Date
Volume
89
Issue
1
Identifiers
DOI: 10.1016/0022-1759(86)90033-5
Keywords
  • Hla-Dr Antigens
  • Nitrocellular Membranes
  • Radioimmunoassay
Disciplines
  • Biology
  • Medicine

Abstract

Abstract 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.

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