Quantitation of Pulmonary Surfactant Protein SP-B in the Absence or Presence of Phospholipids by Enzyme-Linked Immunosorbent Assay

• Published in: Analytical biochemistry 2001-06, Vol.293 (1), p.78-87
• Main Authors: Oviedo, J.M., Valiño, F., Plasencia, I., Serrano, A.G., Casals, C., Pérez-Gil, J.
• Format: Article
• Language: English
• Subjects: Animals; Anions - chemistry; Anions - metabolism; Antibodies, Monoclonal - immunology; Blotting, Western; Circular Dichroism; Enzyme-Linked Immunosorbent Assay; epitope exposure; Immune Sera - immunology; Lipid Bilayers; Micelles; monolayers; Phospholipids - analysis; Phospholipids - chemistry; protein quantitation; Proteolipids - analysis; Pulmonary Surfactants - analysis; secondary structure; Swine; thin films
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2001.5111

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 μg/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.