MEASUREMENT OF BACTERICIDAL PERMEABILITY-INCREASING PROTEIN IN HUMAN-BODY FLUIDS BY SANDWICH ELISA/

A sensitive sandwich ELISA has been developed to measure levels of nat ive 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 concentrati ons of rBPI spiked into pooled human plasma samples averaged 83% and r anged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI(23) av eraged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Be cause LBP is present in normal human plasma and shares sequence homolo gy with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference wit h BPI detection. At 100 mu g/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI(23). Matched serum and plasma s amples were collected from 20 healthy adults to measure endogenous lev els of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in pla sma 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) approximate to 60,000), which com igrated with native BPI extracted from human neutrophils. These data d emonstrate that low levels of holo-BPI are present in plasma, and sugg est that additional quantities of BPI were released from neutrophils d uring the process of coagulation.