Quantitative liquid-phase chemiluminescence ELISA: detection of subtle epitope differences in HuIFN-beta. J Interferon Cytokine Res 1999 Sep;19(9):995-8
Date
10/03/1999Pubmed ID
10505740DOI
10.1089/107999099313190Scopus ID
2-s2.0-0032855198 (requires institutional sign-in at Scopus site) 2 CitationsAbstract
We have developed a new liquid-phase, chemiluminescence-enhanced, inhibition ELISA (LP-CEI-ELISA) to explore the binding sites recognized by two neutralizing monoclonal antibodies (mAb) against recombinant human IFN-(beta)ser (rHuIFN-(beta)ser). In this assay, the initial antigen-antibody reaction occurs in solution under more physiologic conditions than in a standard solid-phase ELISA. Subsequently, the reaction mixture is applied to a membrane that is exposed to a second, peroxidase-labeled mAb, chemiluminescent reagents are added, and the membrane is photographically recorded. Competitive inhibition of binding of a second, labeled mAb by the first mAb decreases the signal detected. Two well-characterized mAb A1 and A7, have been shown to recognize distinct epitopes on rHuIFN-(beta)ser and to neutralize its antiviral and antiproliferative activity (Proc. Natl. Acad. Sci. USA 88, 4040-4044, 1991). In conventional solid-phase ELISA, mAb A1 does not inhibit the binding of A7 to rHuIFN-(beta)ser, but we observed partial inhibition in the new liquid-phase assay. In contrast, A7 did not inhibit the binding of A1, consistent with the solid-phase ELISA results. This observation suggests that in the LP-CEI-ELISA, A1 and A7 may recognize epitopes differently than in solid-phase assays. Thus, the LP-CEI-ELISA, which is simple, sensitive, and quantifiable, appears also to be able to detect subtle, conformational differences of epitopes not evident in a standard solid-phase ELISA.
Author List
Golgher RR, Redlich PN, Totti DO, Grossberg SEAuthor
Philip N. Redlich MD, PhD Professor in the Surgery department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Binding, CompetitiveEnzyme-Linked Immunosorbent Assay
Epitopes
Humans
Interferon beta-1a
Interferon beta-1b
Interferon-beta
Luminescent Measurements
Recombinant Proteins
