title: An ELISA technique for quantification of surfactant apoprotein (SP)-C in bronchoalveolar lavage fluid.
abstract: Pulmonary surfactant apoprotein C (SP-C) is a small, unique peptide that contributes to the reduction of alveolar surface tension. Due to the extreme hydrophobic nature of this peptide it was hitherto not possible to quantify SP-C in biological samples by immunological techniques. Using a newly developed polyclonal antibody raised against recombinant human SP-C in rabbits, we now describe an enzyme-linked immunosorbent assay (ELISA) to quantitate SP-C in bronchoalveolar lavage fluid (BALF). Solid phase binding of the hydrophobic SP-C was achieved by transfer of the standard or BALF samples (diluted in 80% isopropanol, pH 3.5) to polystyrene microtiter plates. Sequential treatment with trifluoroethanol and methanol (2x) was employed to improve antigen presentation and to minimize the influence of phospholipids. With this assay, SP-C from human, rabbit, porcine, and bovine surfactant was detectable. No cross-reactivity of the antibody to human SP-A and monomeric and dimeric SP-B was encountered. Total serum proteins did not affect ELISA signals, as evident from spiking experiments. The detection limit of the ELISA ranged below 3 ng/ml, and intra- and interassay coefficients of variation were 3.5% (n = 16) and 5.3% (n = 6), respectively. Serial dilutions of BALF showed good linearity, and excellent recovery rates were obtained upon spiking of human BALF. A mean value of 579.5 +/- 45.9 ng/ml (mean +/- SEM) SP-C was found in BALF samples of human healthy volunteers (n = 22), corresponding to 26.61 +/- 1.91 microg SP-C/mg total phospholipids (PL). SP-C levels were significantly lower in BALF of patients with acute respiratory distress syndrome (ARDS) (286.9 +/- 19.8 ng/ml [p < 0.001]; 13.92 +/- 1.93 microg SP-C/mg PL [p < 0.001], n = 48). We conclude that SP-C may be quantified with high specificity, reproducibility, and sensitivity in bronchoalveolar lavage samples by the presently described ELISA technique and that SP-C levels are significantly decreased in ARDS.
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany.
authors.forename: Reinhold
authors.initials: R
authors.lastname: Schmidt
authors.forename: Wolfram
authors.initials: W
authors.lastname: Steinhilber
authors.forename: Clemens
authors.initials: C
authors.lastname: Ruppert
authors.forename: Christina
authors.initials: C
authors.lastname: Daum
authors.forename: Friedrich
authors.initials: F
authors.lastname: Grimminger
authors.forename: Werner
authors.initials: W
authors.lastname: Seeger
authors.forename: Andreas
authors.initials: A
authors.lastname: Günther
mesh_terms: D001059:Apoproteins / Q000032:analysis*; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000032:analysis*; D015203:Reproducibility of Results; D012128:Respiratory Distress Syndrome / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D001059:Apoproteins; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C046616:pulmonary surfactant apoprotein