An ELISA technique for quantification of surfactant apoprotein (SP)-C in bronchoalveolar lavage fluid.
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.
Schmidt
Reinhold
R
Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany.
Steinhilber
Wolfram
W
Ruppert
Clemens
C
Daum
Christina
C
Grimminger
Friedrich
F
Seeger
Werner
W
Günther
Andreas
A
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
D001059:Apoproteins; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C046616:pulmonary surfactant apoprotein