Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) that uses po lyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitat e purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been used to explore the effect of the presence of different phospholipids on the im munoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with pr otein concentrations in the range of 20-1000 ng/mL. At these protein concen trations, neither dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylgl ycerol, nor phosphatidylcholine or phosphatidylglycerol from egg yolk had s ignificant effects on the binding of antibodies to SP-B up to protein-to-li pid weight ratios of 1:20. Coating of ELISA plates with SP-B concentrations higher than 1 mug/mL produced a substantial decrease in the binding of ant ibodies to the protein that was prevented by the presence of negatively cha rged but not zwitterionic phospholipids, Characterization of the secondary structure of SP-B by far-UV circular dichroism showed that phospholipids in duced pronounced changes on the conformation of SP-B when the solvent was e vaporated and dry lipid-protein films were formed, a necessary step to expo se protein to antibodies in ELISA. Under these conditions, negatively charg ed lipids, but not zwitterionic ones, induced a marked decrease on the elli pticity of SP-B that would be associated with a conformation that is signif icantly more exposed to antibodies, (C) 2001 Academic Press.