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 polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate 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 immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein concentrations, neither dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, nor phosphatidylcholine or phosphatidylglycerol from egg yolk had significant effects on the binding of antibodies to SP-B up to protein-to-lipid weight ratios of 1:20. Coating of ELISA plates with SP-B concentrations higher than 1 microg/mL produced a substantial decrease in the binding of antibodies to the protein that was prevented by the presence of negatively charged but not zwitterionic phospholipids. Characterization of the secondary structure of SP-B by far-UV circular dichroism showed that phospholipids induced pronounced changes on the conformation of SP-B when the solvent was evaporated and dry lipid-protein films were formed, a necessary step to expose protein to antibodies in ELISA. Under these conditions, negatively charged lipids, but not zwitterionic ones, induced a marked decrease on the ellipticity of SP-B that would be associated with a conformation that is significantly more exposed to antibodies.
Oviedo
J M
JM
Departamento Bioquímica, Universidad Complutense, 28040 Madrid, Spain.
Valiño
F
F
Plasencia
I
I
Serrano
A G
AG
Casals
C
C
Pérez-Gil
J
J
D000818:Animals; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera / Q000276:immunology; D008051:Lipid Bilayers; D008823:Micelles; D010743:Phospholipids / Q000032:analysis* / Q000737:chemistry; D011510:Proteolipids / Q000032:analysis*; D011663:Pulmonary Surfactants / Q000032:analysis*; D013552:Swine
D000838:Anions; D000911:Antibodies, Monoclonal; D007106:Immune Sera; D008051:Lipid Bilayers; D008823:Micelles; D010743:Phospholipids; D011510:Proteolipids; D011663:Pulmonary Surfactants