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

Academic Article
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Overview

authors

  • Teyton, Luc
  • Lotteau, V.
  • Boyer, B.
  • Charron, D. J.

publication date

  • 1986

journal

  • Journal of Immunological Methods  Journal

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.

subject areas

  • Cell Line
  • Collodion
  • HLA-DR Antigens
  • Histocompatibility Antigens Class II
  • Humans
  • Immunosorbent Techniques
  • Membrane Proteins
  • Molecular Weight
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Identity

International Standard Serial Number (ISSN)

  • 0022-1759

Digital Object Identifier (DOI)

  • 10.1016/0022-1759(86)90033-5

PubMed ID

  • 3517174
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Additional Document Info

start page

  • 73

end page

  • 79

volume

  • 89

issue

  • 1

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