Measurement of bactericidal/permeability-increasing protein in human body fluids by sandwich ELISA

Abstract

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI 23) in human body fluids. The linear range for the rBPI and rBPI 23 standard curves were 100–6000 pg/ml and 25–800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI 23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 μg/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI 23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9–72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet ( M r ≈ 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.