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, 333 (1-2), 38-50

Interleukin-32 Monoclonal Antibodies for Immunohistochemistry, Western Blotting, and ELISA

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Interleukin-32 Monoclonal Antibodies for Immunohistochemistry, Western Blotting, and ELISA

Ki-Hong Kim et al. J Immunol Methods.

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-alpha 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-32alpha, beta, gamma, and delta). In order to optimize the sandwich ELISA for IL-32, recombinant IL-32alpha 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-32alpha and KU32-52 was bound to all isoforms, whereas KU32-56 were reactive to IL-32 alpha, beta, delta isoforms but not gamma 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-32beta, IL-32gamma, IL-32delta, hIL-1alpha , IL-1beta , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. 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-32alpha and IL-32beta isoforms but not gamma and delta isoforms and had no cross-reaction with other cytokines such as hIL-1alpha , IL-1beta , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. 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.

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