ELISA technique for quantification of surfactant protein B (SP-B) in bronchoalveolar lavage fluid.

Abstract

Surfactant protein B (SP-B) is one of the essential constituents of the alveolar surfactant system, presenting mainly in the form of SP-B dimer. The measurement of this molecule in biologic samples has been hampered by its extreme hydrophobicity and intimate association with surfactant lipids. We developed a solid-phase, adsorption-based enzyme-linked immunosorbent assay (ELISA) technique for the quantification of SP-B in aqueous solutions. The ELISA employs the hydrophobicity of SP-B for direct binding of this compound to polystyrol immunosorbent plates. Samples are mixed with propanol (1:1 vol/vol) to achieve a homogeneous dispersion of their lipophilic constituents prior to adsorption to the wells. After fluid removal by evaporation, trifluoroethanol is added to optimize SP-B-polystyrol binding, and is then removed, again by evaporation. Subsequent washing procedures (diisopropyl-ether/butanol; Tween 20 in phosphate buffered saline [PBS]) selectively remove phospholipids. Solid phase-bound SP-B is detected by a monoclonal mouse antibody against porcine SP-B, cross-reacting with the apoprotein of human origin. For amplification, a biotinylated anti-mouse antibody and the avidin/biotin-peroxidase technique are used. Steep calibration curves with an excellent reproducibility are obtained for SP-B dimer (range: 0.3125 to 40 ng/well), either introduced directly into the immunosorbent-plate wells or previously admixed with synthetic phospholipid mixtures. SP-B monomer is detected with approximately 10% efficiency as compared with the dimer (wt/wt). Cross-reactivities with human SP-A and SP-C or albumin are negligible. Experiments with spiking of human bronchoalveolar lavage fluid (BAL) samples with different quantities of SP-B dimer revealed virtually complete apoprotein recovery.(ABSTRACT TRUNCATED AT 250 WORDS)