Neoepitope immunoassay: an assay for human interleukin 1 beta based on an antibody induced conformational change.
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.
Towbin
H
H
Ciba-Geigy Ltd., Pharmaceuticals Research Laboratories, Basel, Switzerland.
Erard
F
F
van Oostrum
J
J
Schmitz
A
A
Rordorf
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000494:pharmacology*; D000915:Antibody Affinity; D018604:Epitope Mapping / Q000379:methods*; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007375:Interleukin-1 / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D007375:Interleukin-1; D011994:Recombinant Proteins