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

✍ Scribed by J.M. Oviedo; F. Valiño; I. Plasencia; A.G. Serrano; C. Casals; J. Pérez-Gil

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 144 KB
• Volume: 293
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5111

✦ Synopsis

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 g/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.