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

J M Oviedo, F Valiño, I Plasencia, A G Serrano, C Casals, J Pérez-Gil

Publikation: Bidrag til tidsskriftTidsskriftartikelForskning

Abstrakt

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.
Udgivelsesdato: 2001-Jun-1
OriginalsprogEngelsk
TidsskriftAnalytical Biochemistry
Vol/bind293
Udgave nummer1
Sider (fra-til)78-87
Antal sider9
ISSN0003-2697
DOI
StatusUdgivet - 1. jun. 2001

Bibliografisk note

Copyright 2001 Academic Press.

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