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-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.
Wewers
M D
MD
Department of Medicine, Pulmonary and Critical Care Division, Ohio State University, Columbus 43210.
Pope
H A
HA
Miller
D K
DK
D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007375:Interleukin-1 / Q000032:analysis* / Q000378:metabolism; D007536:Isomerism; D008264:Macrophages / Q000737:chemistry; D009000:Monocytes / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D011498:Protein Precursors / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity
D007375:Interleukin-1; D011498:Protein Precursors; C043830:interleukin 1 precursor; D010447:Peptide Hydrolases