title: Removal of false-positive reactions from plasma in an enzyme immunoassay for bovine interferon-gamma.
abstract: A monoclonal antibody-based sandwich enzyme immunoassay (EIA) for bovine interferon-gamma (IFN-gamma) has been developed and can be used in conjunction with a whole blood culture system to diagnose tuberculosis in cattle. During its development, normal bovine plasma samples were tested to establish background levels of circulatory IFN-gamma. Of 191 samples tested, 81 (42.4%) were positive (OD > 0.1) when tested undiluted in intact monoclonal antibody (IgG1)-coated wells compared to only 8 (4.2%) in F(ab')2-coated wells, which suggested non-specific interference in the EIA rather than circulatory IFN-gamma. Reactivity of all remaining samples was removed by diluting plasmas 1/2 with 1% casein-PBS-0.05% Tween 20 supplemented with an optimum amount (5%) of normal mouse serum (NMS). Serum pools derived from BALB/c, DBA/2, C3H/HeJ, CBA/CaH and Swiss, but not C57BL/6J, mice were found to inhibit equally the reactions of five strong false-positive bovine plasma samples but had no effect on the titre of IFN-gamma in the sample. Sera from other species tested were less effective. This suggests that the interfering factors possess a high degree of specificity, since the immunoglobulin heavy chain of IgG1 produced by all these five strains of mice are allotypically identical and different to IgG1 produced by C57BL/6J mice. The use of F(ab')2 antibody fragments to coat plate wells and sample diluent containing 5% NMS has resulted in an EIA for bovine IFN-gamma that is virtually free from false-positive reactions, has a high degree of reproducibility and a sample detection limit equivalent to approximately 80 pg/ml recombinant bovine IFN-gamma.
authors.affiliation: Research and Development Division, CSL Limited, Parkville, Australia.
authors.forename: S L
authors.initials: SL
authors.lastname: Jones
authors.forename: J C
authors.initials: JC
authors.lastname: Cox
authors.forename: J M
authors.initials: JM
authors.lastname: Shepherd
authors.forename: J S
authors.initials: JS
authors.lastname: Rothel
authors.forename: P R
authors.initials: PR
authors.lastname: Wood
authors.forename: A J
authors.initials: AJ
authors.lastname: Radford
mesh_terms: D000818:Animals; D002417:Cattle; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007371:Interferon-gamma / Q000097:blood*; D013045:Species Specificity
chemical_list: D007140:Immunoglobulin Fab Fragments; D007371:Interferon-gamma