Development of an enzyme-linked immunoassay for sensitive detection of native and recombinant human interferon-gamma using whole IgG fraction as polyclonal tracer


Aybay C.

JOURNAL OF IMMUNOASSAY & IMMUNOCHEMISTRY, cilt.25, sa.4, ss.321-334, 2004 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 25 Konu: 4
  • Basım Tarihi: 2004
  • Doi Numarası: 10.1081/ias-200033828
  • Dergi Adı: JOURNAL OF IMMUNOASSAY & IMMUNOCHEMISTRY
  • Sayfa Sayıları: ss.321-334

Özet

Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against human interferon gamma (IFN-gamma) were produced and used for development of a sensitive enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of native and recombinant human IFN-gamma in tissue culture fluid and human sera. The human IFN-gamma ELISA was constructed using mAb CAy-IFNg111 as the capture antibody (Ab) and biotinylated polyclonal mouse immunoglobulin G (IgG) as the tracer Ab. The assay is completed within 4 hr at room temperature (RT). The human IFN-gamma ELISA worked in tissue culture medium and human serum and was capable of detecting both recombinant and native human IFN-gamma. The assay dynamic range extended from 16 to 1000 pg/mL and the sensitivity level was less than 3 pg/mL of human IFN-gamma with averaged intra- and inter-assay variation coefficients less than 8% for both. The results demonstrated that without the need of an antigen-affinity purification, biotinylation of protein G-purified pAb, obtained from 1 mL of mouse blood, was sufficient for constructing the tracer reagent for the establishment of a highly sensitive ELISA (40,000 test) for the quantitative detection of native and recombinant human IFN-gamma in culture supernatant and human sera.