Neoepitope immunoassay: an assay for human interleukin 1 beta based on an antibody induced conformational change

Journal of Immunoassay
H TowbinC Rordorf

Abstract

A secondary monoclonal antibody (mAb2) was generated by immunization with immune complexes of human IL-1 beta and a primary monoclonal (mAb1). mAb2 bound to a neoepitope on the IL-1 beta/mAb1-complex with a dissociation constant (Kd) of 26 pM but not to uncomplexed IL-1 beta. As assessed by the binding of labeled IL-1 beta and neutralization of bioactivity, mAb2 enhanced the affinity of IL-1 beta to mAb1; Kd-values were 108 pM in absence and 5.4 pM in presence of mAb2. By analyzing a series of mutants of IL-1 where surface loops had been exchanged with the corresponding loops of human IL-1 receptor antagonist protein, a critical region responsible for mAb2 binding was localized to the C-terminal region. In addition to mAb1/IL-1 beta-complexes, mAb2 bound pro-IL-1 beta/mAb1 complexes as well as pro-IL-1 beta suggesting that mAb2 recognized a conformation of IL-1 beta resembling that of pro-IL-1 beta. Using this pair of mAbs, chemiluminescent and enzyme linked assays with detection limits of 2 pg/ml hIL-1 beta have been established.

References

Jul 1, 1989·Journal of Bioluminescence and Chemiluminescence·S J LawJ Unger
Jan 1, 1987·Journal of Immunoassay·D Henning, K Nielsen
Dec 1, 1994·Current Opinion in Structural Biology·I A Wilson, R L Stanfield

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Citations

Oct 29, 2003·Journal of Immunological Methods·Chatchai TayapiwatanaWatchara Kasinrerk

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