Am. J. Respir. Crit. Care Med., Volume 165, Number 4, February 2002, 470-474

An ELISA Technique for Quantification of Surfactant Apoprotein (SP)-C in Bronchoalveolar Lavage Fluid

REINHOLD SCHMIDT, WOLFRAM STEINHILBER, CLEMENS RUPPERT, CHRISTINA DAUM, FRIEDRICH GRIMMINGER, WERNER SEEGER, and ANDREAS GÜNTHER

Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany; and Byk Gulden, Konstanz, Germany

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 (2×) 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 µg 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 µg SP-C/mg PL [p < 0.001], n = 48). We conclude that SP-C may be quantified with high specifity, reproducibility, and sensitivity in bronchoalveolar lavage samples by the presently described ELISA technique and that SP-C levels are significantly decreased in ARDS.

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