Interleukin-32 monoclonal antibodies for Immunohistochemistry, Western blotting, and ELISA

Abstract

The members of the IL-1 family play important roles in the development and pathogenesis of autoimmune and inflammatory diseases. Especially, IL-1 and IL-18 belong to the IL-1 family because they share structural similarity and require caspase-1 for processing. Currently, IL-18 has been studied for its biological effects in the broad spectrum of Th1- or Th2- related autoimmune diseases. IL-18 also uses a similar signaling pathway as that of IL-1 family members. Taken together these results, IL-18-inducible genes might also contribute to autoimmune and inflammatory diseases. It has recently been reported that an inducer of TNF-α was identified as one of IL-18 inducible genes in IL-18 responsible cells and named as a new cytokine IL-32. We have produced novel monoclonal anti IL-32 antibodies in order to help study IL-32 function and to develop improved diagnosis of IL-32-expressing tumors. Several mAbs reactive to IL-32 isoforms were prepared and characterized by the epitope analysis and Western blotting performed using various deletion mutants and IL-32 isoforms (IL-32α, β, γ, and δ). In order to optimize the sandwich ELISA for IL-32, recombinant IL-32α was added, followed by the addition of a biotinylated mAb KU32-52 into the microtiter plate wells pre-coated with a mAb KU32-07 or mAb KU32-56. The bound mAb was probed with a streptavidin conjugated to HRP. The epitope analysis and Western blot analysis revealed that mAb KU32-07 could detect only IL-32α and KU32-52 was bound to all isoforms, whereas KU32-56 were reactive to IL-32 α, β, δ isoforms but not γ isoform. These sandwich ELISAs were highly specific and had a minimal detection limit of 80 pg/ml (mean + 3 SD of zero calibrator) and measuring range of up to 3000 pg/ml. An ELISA using a coating mAb KU32-07 and a capturing biotinylated mAb KU32-52 had no cross-reaction with other cytokines such as IL-32β, IL-32γ, IL-32δ, hIL-1α , IL-1β , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-α. Intra-assay coefficients of variation were 11 to 6% ( n = 16) and inter-assay coefficients were 10 to 5% ( n = 9). Another ELISA using a coating mAb KU32-56 and a capturing biotinylated mAb KU32-52 detected both IL-32α and IL-32β isoforms but not γ and δ isoforms and had no cross-reaction with other cytokines such as hIL-1α , IL-1β , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-α. One mAb KU32-09 was shown to react strongly on immunohistochemistry. Our newly established mAbs, KU32-07, KU32-09, KU32-52, KU32-56, have different and useful properties for the detection of IL-32 by immunohistochemistry, ELISA, and Western blotting.