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PubMed · 8228277

Processing proIL-1 beta decreases detection by a proIL-1 beta specific ELISA but increases detection by a conventional ELISA.

M D Wewers·H A Pope·D K Miller
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Abstract

Recent investigations have noted that conventional IL-1 beta enzyme linked immunoassays (ELISA) may underestimate proIL-1 beta concentrations. In an attempt to circumvent this problem, we have devised a proIL-1 beta specific ELISA which sandwiches proIL-1 beta between a carboxy-terminus specific, capture antibody, and an amino-terminus specific, detection antibody. The amino-terminus specific antibody was generated against amino acids 3-21 of the intact proIL-1 beta molecule. This sandwich ELISA does not recognize mature 17 kDa IL-1 beta and is not inhibited by the coexistence of mature, 17 kDa IL-1 beta. We compared this proIL-1 beta specific ELISA (amino-terminus ELISA) to the conventional IL-1 beta ELISA (carboxy-terminus ELISA) on test samples that contained either proIL-1 beta or mature IL-1 beta. When purified proIL-1 beta is cleaved by increasing concentrations of IL-1 beta converting enzyme or porcine pancreatic elastase, there is a dose dependent increase in the signal from the conventional IL-1 beta ELISA and a concurrent decrease in signal from the proIL-1 beta specific ELISA. Furthermore, when the proIL-1 beta specific ELISA is used to quantify cellular sources of IL-1 beta, there is no detectable release of proIL-1 beta into the supernatants from either fresh blood monocytes or alveolar macrophages, despite detectable mature IL-1 beta. The cell associated compartment of IL-1 beta is largely proIL-1 beta and the relative amounts of IL-1 beta that remain intracellularly are quite large. Specifically, when assayed at 18 h, the proIL-1 beta ELISA detected 4.6 +/- 1.4 ng/ml per 10(6) monocytes and 13.8 ng/ml per 10(6) macrophages vs. 0.8 +/- 0.3 ng/ml/10(6) monocytes and 1.6 ng/ml/10(6) macrophages by conventional IL-1 beta ELISA. Thus, the 5-10-fold deficit in the detection of intracellular IL-1 beta by a conventional IL-1 beta ELISA, combined with the enhanced detection after enzymatic processing of proIL-1 beta, confirms that a proIL-1 beta specific ELISA is needed to accurately quantify proIL-1 beta.

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Enzyme-Linked Immunosorbent AssayHumansInterleukin-1IsomerismMacrophagesMonocytesPeptide HydrolasesProtein PrecursorsSensitivity and SpecificityComparative StudyJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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M D Wewers, H A Pope, D K Miller. 1993-10-15. Processing proIL-1 beta decreases detection by a proIL-1 beta specific ELISA but increases detection by a conventional ELISA.. https://doi.org/10.1016/0022-1759(93)90353-9

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