Development of an Enzyme‐Linked Immunoassay for Sensitive Detection of Native and Recombinant Human Interferon‐γ Using Whole IgG Fraction as Polyclonal Tracer

Abstract

Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against human interferon gamma (IFN‐γ) 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‐γ in tissue culture fluid and human sera. The human IFN‐γ 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‐γ ELISA worked in tissue culture medium and human serum and was capable of detecting both recombinant and native human IFN‐γ. The assay dynamic range extended from 16 to 1000 pg/mL and the sensitivity level was less than 3 pg/mL of human IFN‐γ 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‐γ in culture supernatant and human sera.

Keywords:

• Human interferon gamma
• Monoclonal antibody
• Polyclonal antibody ELISA