INTRODUCTION

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Keywords: surfactant; ELISA; bronchoalveolar lavage fluid; ARDS

Pulmonary surfactant covers the alveolar surface area. By reducing the surface tension at this air-water interface, the lipoprotein complex stabilizes the alveoli, in particular at low lung volumes, and enables gas exchange at normal transthoracal pressures. It is mainly composed of phospholipids (PL, ~ 80%), neutral lipids (~ 10%), and proteins (~ 10%). Dipalmitoylated phosphatidylcholine (DPPC), in association with the hydrophobic surfactant specific apoproteins SP-B and SP-C, is primarily responsible for the extreme high surface activity. Furthermore, although present in only small amounts (1-4%), the surfactant apoproteins fulfill important functions in surfactant metabolism and host defense (1, 2).

Surfactant protein C is a small (3.5-kD) protein with an exceptionally high content of hydrophobic amino acids (with 22 of the 35 amino acids being valine, leucine, or isoleucine). In addition, SP-C is palmitoylated at the two amino-terminal-located cysteine residues. It thus represents one of the most hydrophobic proteins known in nature (3, 4). Throughout all mammalian species studied, a marked conservation of the primary structure has been noted (3, 5).

In concert with SP-B, SP-C facilitates the adsorption of surfactant phospholipids to an air-water interface and stabilizes the interfacial lipid layer during film compression (6, 7). By this, the alveolar surface tension is kept below 5 mN/m, especially at end expiration, where the alveoli would otherwise tend to collapse.

In several studies addressing the surfactant composition and function in patients with acute inflammatory lung diseases, including the acute respiratory distress syndrome (ARDS), a marked loss of surface activity was consistently noted. This was found to be due to altered composition of the surfactant material (8), inhibition of surface activity by plasma proteins and inflammatory mediators (11), incorporation into a growing fibrin network (12), and altered metabolism resulting in the predominance of the less surface active small surfactant aggregates at the expense of the large surfactant aggregates (13). Among the biochemical alterations, decreased relative concentrations of the essential phospholipids phosphatidylcholine and phosphatidylglycerol (8, 10) and of the apoproteins SP-A (14) and SP-B (9, 10) were observed and paralleled the loss of surface activity. Unfortunately, only few data as to the relative or absolute concentration of SP-C under different conditions of lung injury are currently available, although a variety of attempts were undertaken by a number of groups, leading to the development of several analytical techniques such as hydrophobic gel filtration chromatography (15), reversed-phase high-performance liquid chromatography (HPLC) (16, 17), a radiochemical method described by Qanbar and Possmayer (18), and semiquantitative approaches such as electrophoretic analysis (19). However, most of these techniques are characterized by limitations in sensitivity, reliability, and accuracy, and the development of immunological techniques was hitherto not possible due to the lack of antibodies against mature SP-C, as a consequence of the extreme hydrophobicity of the antigen (3).

In the present work, we describe a solid-phase, enzyme-linked immunoassay for quantification of surfactant apoprotein C. The strong hydrophobicity of this peptide was employed for direct binding to a hydrophobic matrix, interfering surfactant lipids were removed by selective washing steps, and a newly developed polyclonal antibody against recombinant human SP-C was engaged. The method was designed for aqueous samples with very low SP-C contents (below 15 ng/ml) and allows fast and reliable quantification of SP-C in bronchoalveolar lavage fluids. The assay can be performed simultaneously with a recently developed ELISA for quantification of the hydrophobic surfactant protein SP-B (20).

	METHODS

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Materials

2-Propanol, 1,1,1-trifluoroethanol (TFE), dithiothreitol (DTT), Tris (Merck, Darmstadt, Germany), Tween 20 (Sigma, Deisenhofen, Germany), fraction 5 bovine serum albumin (BSA) (Paesel and Lorei, Frankfurt/Main, Germany), peroxidase-conjugated anti-rabbit antibody from sheep (Calbiochem, Bad Soden, Germany), 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate [6]) (ABTS; Boehringer, Mannheim, Germany), and Sephadex LH 60 (Amersham-Pharmacia, Freiburg, Germany) were purchased from the referenced manufacturers.

The washing buffer contained 50 mM Tris-HCl and 0.5% Tween 20, pH 7.6. Blocking buffer contained 50 mM Tris-HCl + 1% (wt/vol) BSA, pH 7.6. The substrate buffer was composed of 60 mM sodium acetate and 50 mM NaH₂PO₄, pH 4.2.

The antibody against SP-C (anti-rhSP-C) was raised in rabbits by immunization with recombinant human SP-C, as described in the international patent application WO 00/05585. The immunization was carried out by standard procedures using complete and incomplete Freund's adjuvants as immune stimulants (21, 22).

The hydrophobic surfactant proteins SP-B (dimer and monomer) and SP-C were isolated from bronchoalveolar lavage fluids (BALF) using organic extraction (23) and were prepared according to Warr and coworkers (24). Dipalmitoylated recombinant human SP-C was produced and purified as described in patent application WO 95/ 32992 for the above described variant. The purity of the hydrophobic apoproteins was determined using SDS-PAGE and ranged above 95%. The concentration of the stock solutions was determined using quantitative amino acid analysis.

ELISA Procedure

All standard solutions and aqueous samples of SP-C were mixed with 2-propanol/water (80/20, vol/vol, pH 3.5). Using this solvent mixture, SP-C standard (recombinant human dipalmitoylated SP-C) was diluted to final concentrations of 3.125-400 ng/ml. Similarly, BALF was mixed 1:5 (vol/vol) with this solvent mixture. The final concentration of 2-propanol in samples and standards was 64% (vol/vol). All dilution steps were performed in 2-ml polypropylene vials (Eppendorf, Hamburg, Germany). Afterward, a volume of 100 µl of diluted sample or standard was transferred to microtiter plates (Polysorp F96; Nunc, Wiesbaden, Germany). Fluid removal was achieved by overnight evaporation at 37° C. Subsequently, 100 µl/well TFE was added and evaporated at 37° C for 3 h. Afterward, 200 µl/well methanol was applied and the plates were incubated for 20 min at ambient temperature while being gently shaken. Following removal of the organic solvent, this step was repeated without additional incubation. Afterward, the plates were washed three times with washing buffer. Nonspecific protein binding to wells was blocked with 200 µl/well blocking buffer for 2 h at ambient temperature. After triple washing, the anti-SP-C antiserum (anti-rhSP-C; diluted 1:10,000 [vol/vol] in blocking buffer) was added at a volume of 200 µl and incubated for 12-15 h at ambient temperature. Following triple washing, wells were incubated for 2 h with 200 µl of peroxidase-conjugated anti-rabbit antibody (1:1000 in blocking buffer). Following triple washing, dye conversion was initiated by charging the wells with 200 µl of ABTS solution (20 mg in 30 ml substrate buffer + 10 µl 30% H₂O₂). After overnight incubation at 4° C, absorbance was measured at 450 nm.

Electrophoresis and Western Blot Analysis

SDS-PAGE was carried out using the discontinuous procedure of Schägger and von Jagow (25) with 16.5% or 8% (wt/vol) separating gels. BALF (400 µl) and SP-C standards were freeze-dried and introduced directly to the gel. Following Coomassie staining and destaining with 50% methanol/10% acetic acid, gels were treated with 5 µg/ml dithiothreitol for 30 min and silver stained according to Morrissey (26).

Western blot analysis was performed on Immobilone P^SQ membranes (Millipore, Bad Homburg, Germany) by semidry electroblotting for 1.5 h at 2 mA/cm². Proteins were visualized using chemiluminescence detection (ECL plus; Amersham Pharmacia, Freiburg, Germany).

Patient Characteristics

Diagnosis of acute respiratory distress syndrome (ARDS) was established according to the American-European Consensus Conference criteria for ARDS, as previously stated in detail (27, 28). Flexible fiberoptic bronchoscopy was performed in a standardized manner as previously described (10). All patients received the BAL within 4 d of onset of mechanical ventilation. Twenty-two healthy, never smoking individuals without a history of cardiac and pulmonary disease served as control subjects.

Other Methods

Phospholipid content of BALF was assessed using a colorimetric phosphorus assay (28). Total serum proteins were measured by means of a commercial assay (BCA assay; Pierce, Bonn, Germany).

	RESULTS

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Serial subcutaneous applications of recombinant, nonpalmitoylated human SP-C (rSP-C), together with Freund's adjuvants into rabbits resulted in the generation of anti-SP-C antibodies. As evident from Figure 1, depicting immunostained Western blots of human SP-C standards and proteins derived from human BALF, this antiserum recognized both the isolated mature human SP-C (lane 2) and the recombinant human dipalmitoylated SP-C (lane 1). No cross-reactivity with SP-B dimer or monomer was encountered. To further determine possible cross-reactions with unknown BALF components, unprocessed BALF samples were subjected directly to SDS-PAGE and Western blot analysis (Figure 1, lane 3). Signals were found only at 4.5 kD, representing the mature SP-C, and, in trace amounts, at 21 kD, representing possibly the SP-C preprotein (pro-SP-C). Hydrophilic protein components isolated from human BALF using butanol extraction (29) were assessed in the same way and again showed weak signals at 4.5 and 21 kD (data not shown). Repeated freezing/thawing cycles of BALF samples and standards led to the appearance of several high molecular lanes between 30 and 65 kD (data not shown). Most likely, these components represent aggregated SP-C (30).

View larger version (22K): [in this window] [in a new window]   Figure 1.   Western blot analysis of recombinant human dipalmitoylated SP-C (lane 1, 250 ng), mature human SP-C (lane 2, 500 ng), and human bronchoalveolar lavage fluid (lane 3, 400 µl). Proteins were separated using an 8% SDS-PAGE gel.

When using this antiserum in the final ELISA procedure (see Table 1), steep calibration curves with excellent reproducibility were obtained with both the recombinant human dipalmitoylated SP-C as well as the isolated human mature SP-C (Figure 2). As also evident from Figure 2, there was cross-reactivity with mature SP-C from other species, but binding was weaker as compared with the human protein, resulting in flattened calibration curves, in particular in the case of bovine or porcine SP-C. Rabbit SP-C was detected with intermediate sensitivity, with the calibration curve being shifted toward the right by a factor of ~ 5. When addressing the cross-reactivity of the antiserum with other human surfactant apoproteins, no antigen signal was encountered for mono- and dimeric SP-B and recombinant human SP-A (Figure 3). The same was true for serum albumin of human source (Figure 3) and for a synthetic phospholipid mixture consisting of DPPC/phosphatidylglycerol (egg yolk)/ palmitic acid (68.5/22.5/9; wt/wt/wt; data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2.   Cross-reactivities of SP-C derived from several mammalian species and recombinant human SP-C (rhSP-C), undergoing the final ELISA protocol. The mean ± SD of four experiments is given. , rhSP-C human; , SP-C human; , SP-C rabbit; , SP-C bovine; , SP-C porcine.

View larger version (13K): [in this window] [in a new window]   Figure 3.   Cross-reactivities of major surfactant constituents when subjected to the final ELISA protocol. The mean ± SD of four experiments is given. , rhSP-C human; , SP-B dimer human; , SP-B monomer human; , rhSP-A human; , serum albumin.

To rule out possible interferences with total serum proteins, spiking experiments were performed with BALF and serum of healthy control subjects and patients with ARDS. Up to 10 µg total ARDS and control serum proteins per well showed no influence on ELISA signals in control and ARDS BALF (Figure 4).

View larger version (10K): [in this window] [in a new window]   Figure 4.   Effect of total serum proteins in the final ELISA procedure. Total serum proteins of healthy controls (n = 3) and patients with ARDS (n = 3) were diluted in 0.9% saline and added to control BALF (squares) and ARDS BALF (circles) in defined amounts (0.6-10 µg total protein/well). Depicted is the mean ± SD.

When developing an ELISA method for quantification of SP-B in BALF (20), it was found that the variation of adsorption signals was reduced upon incubation with TFE and that the interfering influence of phospholipids might be overcome by washing steps employing organic solvents. Accordingly, in the current investigation addressing quantification of SP-C, omitting the TFE incubation and the washing steps indeed resulted in a large variability of adsorption signals and an increasing influence of the phospholipids on antigen detection in independence of their relative concentration to SP-C (Figure 5A). Accordingly, TFE incubation alone was shown to improve overall antigen signal but did not reduce the interfering effects of the phospholipids (data not shown). For that reason, a variety of organic solvents was tested for their ability to selectively remove the phospholipids without interfering with the SP-C signal. Of these, methanol (MeOH) turned out to be the most effective, and sequential treatment with TFE and MeOH resulted in superimposable adsorption curves independent of the SP-C/phospholipid ratios (Figure 5B). The selective removal of phospholipids was further verified by analyzing the phospholipid content of the washing fraction using a colorimetric phosphorus assay. Approximately 95.5 ± 1.8% (mean ± SEM, n = 3) of the total phospholipids transferred to the microtiter plates were found in the MeOH fraction, thus documenting the efficacy of this washing procedure.

View larger version (11K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Figure 5.   Effect of phospholipid content on calibration curves obtained with serial dilutions of recombinant human SP-C. (A) Omitting homogenization (TFE) and washing steps (methanol). (B) Employing TFE treatment and methanol washing steps according to the final ELISA protocol. Depicted is the mean ± SD of four separate experiments. (A and B) , SP-C; , PL/SP-C = 10/1; , PL/SP-C = 50/1; , PL/SP-C = 100/1; , PL/SP-C = 250/1.

To further test the impact of matrix compounds on SP-C detection employing the final assay procedure, spiking experiments were performed with isolated human SP-C. For this purpose, 5, 10, 15, and 20 ng/well SP-C were added to the BALF of healthy volunteers (n = 10) and of patients with ARDS (n = 10). When corrected for the endogeneous SP-C content, an excellent linear recovery curve was obtained, passing the y-axis near the origin (Figure 6). Furthermore, serial dilutions of seven human BALF samples were analyzed and almost identical SP-C values were obtained when corrected for dilution (Figure 7).

View larger version (9K): [in this window] [in a new window]   Figure 6.   Recovery of the ELISA procedure. Human SP-C in defined amounts (5, 10, 15, and 20 ng/well) was added to BALF from healthy control subjects (n = 10) and from patients with ARDS (n = 10). All measured SP-C values were corrected for the baseline SP-C content of each individual lavage sample. The mean ± SD is given. y = 0.02 + 1.03* x; r = 0.99978; n = 20; p = 3.97E-6.

View larger version (13K): [in this window] [in a new window]   Figure 7.   Dilution curves of bronchoalveolar lavage fluids (BALF; n = 7) undergoing the ELISA protocol. For graphic presentation, values were corrected for dilution.

The reproducibility of the present ELISA method was investigated by analyzing several aliquots of human BALF. The coefficients of variance for intra- and interassay series were 3.5% (n = 16) and 5.3% (n = 6), respectively.

Application of this assay to BALF from healthy human volunteers revealed a mean SP-C concentration of 579.5 ± 45.9 ng/ml (mean ± SEM; n = 22), corresponding to an SP-C/ phospholipid ratio of 1:37.6 (wt/wt). SP-C levels were reduced to ~ 50% of control levels in BALF from patients with ARDS (286.9 ± 19.8 ng/ml [p < 0.001]; SP-C/phospholipid ratio = 1:71.8 [p < 0.001], n = 48, Figure 8).

View larger version (11K): [in this window] [in a new window]   Figure 8.   Concentration of SP-C in human bronchoalveolar lavage. All single values (circles) as well as mean (triangles) and median (squares) values are displayed. ***p < 0.001.

	DISCUSSION

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Quantification of the pulmonary surfactant apoprotein SP-C in biological sources is prone to several pitfalls. One currently available technique, chromatographic separation (e.g., by LH-60 chromatography), is characterized by a limited sensitivity (15) and a limited recovery of SP-C. In addition, a preceding purification of the sample, for example, by extraction with organic solvents, is required. Similarly, electrophoretic analysis with or without subsequent immunological detection via Western blot analysis usually works best in the absence of the large quantities of associated phospholipids, making a prepurification of the sample mandatory. Therefore, these methods are at best semiquantitative procedures, with limited suitability for determination of SP-C in biological samples mostly due to their poor sensitivity.

The objective of the present study was to develop a sensitive, reliable method for quantification of SP-C in aqueous solutions, with the bronchoalveolar lavage fluid most relevant. This assay uses the extreme hydrophobic nature of this apoprotein for direct binding to polystyrene surfaces and thus minimizes loss of SP-C due to extensive preceding purification of the sample. The antigen is detected by an antiserum generated in rabbits upon challenge with recombinant, nonpalmitoylated human SP-C.

Two major difficulties in measuring SP-C by ELISA techniques had to be overcome. First, to develop an ELISA assay, the availability of a suitable antibody is an absolute requirement. Until recently, an antibody that recognizes mature SP-C was not available, although many people have tried. This is due to the small size (34 amino acids) and the extreme hydrophobic nature of this protein, resulting in almost complete integration into the phospholipid matrix, and thereby probably escaping the immune system. In line with such reasoning, antibodies obtained after immunization with the preprotein of SP-C did not react with mature SP-C (31).

Second, the large excess of phospholipids, cointroduced into the wells together with SP-C and intimately interacting with this hydrophobic molecule, represents a major burden for antigen detection by the antiserum. For this reason, it was necessary to develop dilution and washing steps resulting in the dissolution of the surfactant ultrastructure and the selective removal of the phospholipids without interrupting SP-C binding to the polystyrene matrix.

This was achieved by the following procedures: (1) The sample was diluted in acidified isopropanol before application to the wells, and this provided excellent solubility of SP-C and disintegration of aggregated surfactant fractions. (2) Treatment of the dried wells with TFE yielded a homogeneous distribution of the surfactant compounds on the microtiter wells and optimized antigen presentation. (3) Incubation and washing of the wells with methanol, which was shown to be the most effective organic solvent among a variety of solvents tested, guaranteed selective removal of the interfering phospholipids. As documented by a colorimetric phosphorus assay, ~ 96% of overall applied phospholipids were retrieved in the methanol fraction.

When using the final assay protocol, superimposable absorption curves were obtained for both recombinant and natural human SP-C. Natural SP-C from other sources was also detectable, however, with lower efficacy. This may be surprising in view of the relatively conserved primary structure of SP-C. As detailed in a recent review article (5), considerable variations exist in the N-terminal region of SP-C between species, with six and five amino acids replaced in porcine and bovine SP-C, respectively, and two amino acids replaced in rabbit SP-C compared with human SP-C. Although the epitope specificity of the antibody is presently unknown, this may account for the considerable variation in cross-reactivity observed here. Among the other surfactant apoproteins, no cross-reactivity was encountered with SP-A or mono- and dimeric SP-B. Furthermore, with the exception of one protein (21 kD) possibly representing the SP-C preprotein, we could not demonstrate any cross-reactivity of the antiserum with any compound other than mature SP-C in human BALF or hydrophilic extracts from human BALF, when analyzed by immunostaining of Western blots. Accordingly, spiking experiments with human serum in relative concentrations known to occur under conditions of increased endothelial and epithelial permeability did not indicate any influence on the assay result. The currently described ELISA technique is characterized by an excellent linearity and recovery along with high reproducibility and sensitivity. It thus offers a method for the quantification of SP-C in BALF or amnion fluid analysis. The sensitivity and specificity of the presently described ELISA represent major advantages over other analytical methods addressing SP-C, such as chromatographic procedures. As very low levels of SP-C in aqueous samples, as found in BALF and amniotic fluids, are easily detected without preceding extraction procedures, and many samples can be processed in parallel, the test works well for clinical routine work. Although detected with lower sensitivity, SP-C from other mammalian species (bovine, porcine, murine, canine, rabbit) may be quantified by this ELISA protocol.

In conclusion, we describe a simple and sensitive ELISA procedure for the detection and quantification of SP-C in aqueous samples. The assay is based on a recently developed polyclonal antibody preparation and on a novel technique of selective removal of interfering phospholipids, which results in high recovery and reproducibility. Specificity is demonstrated by the absence of interference with other BALF proteins. In normal healthy volunteers, the concentration of SP-C in the bronchoalveolar lavage fluid is 579.5 ± 45.9 ng/ml (mean ± SEM; n = 22), corresponding to an SP-C/phospholipid ratio of 1:37.6 (wt/wt). SP-C levels in patients with ARDS were significantly reduced to ~ 50% of control subjects.