Development of an enzyme-linked immunoassay for sensitive detection of native and recombinant human interferon-gamma using whole IgG fraction as polyclonal tracer.
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.
Aybay
Cemalettin
C
Department of Immunology, Faculty of Medicine, Gazi University, Besevler, Ankara, Turkey. aybayc@tr.net
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry; D019921:Biotinylation; D004768:Enterotoxins / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D007371:Interferon-gamma / Q000032:analysis* / Q000097:blood / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins; D015203:Reproducibility of Results
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004768:Enterotoxins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C031818:enterotoxin B, staphylococcal; D007371:Interferon-gamma