PROCESSING PROIL-1-BETA DECREASES DETECTION BY A PROIL-1-BETA SPECIFIC ELISA BUT INCREASES DETECTION BY A CONVENTIONAL ELISA

Recent investigations have noted that conventional IL-1beta enzyme lin ked immunoassays (ELISA) may underestimate proIL-1beta concentrations. In an attempt to circumvent this problem, we have devised a proIL-1be ta specific ELISA which sandwiches proIL-1beta between a carboxy-termi nus specific, capture antibody, and an amino-terminus specific, detect ion antibody. The amino-terminus specific antibody was generated again st amino acids 3-21 of the intact proIL-1beta molecule. This sandwich ELISA does not recognize mature 17 kDa IL-1beta and is not inhibited b y the coexistence of mature, 17 kDA IL-1beta. We compared this proIL-1 beta specific ELISA (amino-terminus ELISA) to the conventional IL-1bet a ELISA (carboxy-terminus ELISA) on test samples that contained either proIL-1beta or mature IL-1beta. When purified proIL-1beta is cleaved by increasing concentrations of IL-1beta converting enzyme or porcine pancreatic elastase, there is a dose dependent increase in the signal from the conventional IL-1beta ELISA and a concurrent decrease in sign al from the proIL-1beta specific ELISA. Furthermore, when the proIL-1b eta specific ELISA is used to quantify cellular sources of IL-1beta, t here is no detectable release of proIL-1beta into the supernatants fro m either fresh blood monocytes or alveolar macrophages, despite detect able mature IL-1beta. The cell associated compartment of IL-1beta is l argely proIL-1beta and the relative amounts of IL-1beta that remain in tracellularly are quite large. Specifically, when assayed at 18 h, the proIL-1beta 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) monocyte s and 1.6 ng/ml/10(6) macrophages by conventional IL-1beta ELISA. Thus , the 5-10-fold deficit in the detection of intracellular IL-1beta by a conventional IL-1beta ELISA, combined with the enhanced detection af ter enzymatic processing of proIL-1beta, confirms that a proIL-1beta s pecific ELISA is needed to accurately quantify proIL-1beta.