Direct double antibody sandwich immunoassay for Pseudomonas aeruginosa elastase

Journal oflmmunological Methods, 164 (1993) 27-32 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

27

JIM06766

Direct double antibody sandwich immunoassay for Pseudomonas aeruginosa elastase M.-C. Jaffar-Bandjee a, j. Carrere a, A. Lazdunski b, O. Guy-Crotte c and C. Galabert a a CERM, H~pital Ren~e Sabran, Giens, 83406 Hy~res Cedex, France, b Laboratoire de Chimie Bacterienne, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 9, France, and c Groupe de Recherches sur les Glandes Exocrines, 27 boulevard Lei'Roure, 13009 Marseille, France

(Received 25 January 1993, revised received 5 April 1993, accepted 19 April 1993)

A direct sandwich enzyme immunoassay was developed in order to quantify Pseudomonas aeruginosa elastase. As a solid phase the wells of a microtitre plate were coated with specific IgG and horseradish peroxidase labelled IgG was used as the second antibody. The detection limit of the assay was 0.26 n g / m l and a good agreement was found with elastolytic activity determined using elastin-Congo red. This assay was simple, specific, sensitive and reproducible, and permits the determination of low levels of elastase. Key words: Elastase; Pseudomonas aeruginosa; ELISA

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen responsible for several severe and often fatal infections, especially in immunodeficient hosts such as trauma and burns victims, and in patients with chronic lung disease such as cystic fibrosis (Woods et al., 1976; Baltch and Griffin, 1977; McManus et al., 1985). As with a number of bacterial pathogens, the virulence of P. aeruginosa in cystic fibrosis is multifactorial and is the product of many interacting conditions (Liu, 1973; D6ring et al., 1987; Miller et al., 1989). P. aeruginosa secretes several extracellular products which have been shown to play a role in the pathogene-

Correspondence to: C. Galabert, CERM, H6pital Ren6e Sabran, Giens, 83406 Hy~res Cedex, France. Tel.: (33) 94 38 16 80; Fax: (33) 94 58 28 48.

sis of the disease caused by this microorganism (Liu, 1974; Nicas and Iglewski, 1985). Among these factors, elastase has been considered to be of important significance (Blackwood et al., 1983). However, its specific contribution to pathogenesis has not been described. More than 90% of P. aeruginosa strains produce elastase, although this production can be very different from one strain to another (Morihara and Tzuzuki, 1977; Obernesser and D6ring, 1982). Enzymatic activity measurements have been used to quantify elastase production, (Morihara and Tzuzuki, 1977; Woods et al,, 1986; Saulnier and Wallach, 1991), but these methods lack sensitivity. In order to determine low levels of enzyme a radioimmunoassay was developed by Obernesser and D6ring (1982), and enzyme immunoassays by Elsheikh et al. (1986) and Saulnier et al. (1992). These latter methods overcome the difficulties due to radiolabelled products, and have the same degree of sensitivity. Both are

28 indirect ELISA procedures in which the antigen is first bound to an unlabelled specific antibody and then detected indirectly by a second labelled antibody. The procedure described by Elsheikh et al. is an indirect double antibody sandwich whereas Saulnier et al. determined the elastase concentration by coating the latter on the solid phase (procedure simplified by coating the solid phase with the antigen = antigen-coated indirect assay). We describe here a direct double antibody sandwich assay for elastase in which the antigen is detected directly with labelled specific antibody.

Material and methods

Materials P. aeruginosa strains were isolated from 11 sputa of patients suffering from cystic fibrosis, hospitalized in the Pediatric Department of H6pital Ren6e Sabran (Giens, France). Strains PAO1 and PAl03 were a gift from A. Lazdunski (CNRS, Marseille). PAO1, a well characterized strain, produces most of the recognized P. aeruginosa virulence factors. PAl03 is an elastase and alkaline protease deficient mutant. The following chemicals and materials were obtained from commercial sources: P. aeruginosa elastase, specificity activity 53 m P U / m g protein, was from Nagase, Osaka, Japan; Pseudomonas isolation agar and trypticase soy broth were obtained from Difco Laboratories, Detroit, MI, USA; bovine serum albumin, o-phenylene-diamine and elastin-Congo red were from Sigma, Saint Louis, MO, USA; Freund's complete and incomplete adjuvant were obtained from Institut M6rieux, France; horseradish peroxidase, grade I, 250 U / m g was from Boerhinger Mannheim, Germany; nitrocellulose was from Schleicher and Schiill; enhanced chemiluminescence Western blotting detection reagents were obtained from Amersham, UK; the Trans-Blot cell was from Bio-Rad Laboratories, Richmont, CA, USA; horseradish peroxidase linked immunoglobulin was from Dakopatts, Denmark and microtiter plates were from Dynatech Laboratories, Chantilly, VA, USA.

Culture conditions Cultures of the P. aeruginosa strains were grown on Pseudomonas isolation agar to determine elastase production. One or two colonies of the culture were transferred to a 3 ml portion of trypticase soy broth, enriched with 0.5% yeast extract and 0.5% glucose (D6ring et al., 1982); the culture was shaken for 6 h at 30°C with maximal aeration. In order to standardize the bacterial growth conditions, an aliquot of this culture was adjusted by diluting in culture medium to a final density of 5 × 108 bacteria/ml, corresponding to 2 U McFarland (API system densitometer). Then, a 1 ml standardized inoculum was added to a 9 ml portion of TSB medium and shaken again as described above for 18 h. Cultures were centrifuged (10,000 ×g/4°C/lO min) and frozen supernatants were stored at -80°C.

Preparation of anti-elastase IgG Antiserum against P. aeruginosa elastase was produced in rabbits. Animals were immunized with three successive injections of 1 mg of pure enzyme. The first injection, diluted in 0.5 ml Freund's complete adjuvant, was subcutaneous. 3 weeks later, the second injection, diluted in 0.5 ml Freund's incomplete adjuvant, was both subcutaneous and intramuscular; 10 and 11 days later, the last injections, diluted in PBS (10 mM PO 4, 0.015 M NaC1, pH 7.4), were intramuscular and intravenous, respectively. The rabbit was bled 10 days after the last injection. The IgG fraction of the antiserum was purified by caprylic acid precipitation according to the method of Steinbuch et al. (1970). 14 mg of purified IgG were obtained from 6 ml of antiserum. The concentration of the antibody was 2.37 mg/ml.

Preparation of the anti-elastase peroxidase conjugate An immunoglobulin G fraction was used to prepare the enzyme conjugate. 7 mg of peroxidase were activated by sodium m-periodate and coupled to 14 mg of specific rabbit IgG following the procedure of Wilson and Nakane (1978). Immediately after coupling and sodium borohydride reduction, the conjugate was precipitated by adding an equal volume of cold neutral saturated ammonium sulfate solution. The pellet was wash-

29 ed twice with 50 % saturated neutral ammonium sulphate solution and finally dissolved in 8 ml PBS containing 25% (v/v) of normal rabbit serum. Aliquots were stored at -80°C.

Determination of the specificity of the polyclonal antibody The standard elastase (10 ng) and supernatants of strain cultures (10-20 /zl) were anAlysed by Western blot, using SDS-PAGE. Discontinuous SDS gel electrophoresis was performed in a modified Laemmli (1970) gel system, with a 4.5% stacking gel and a 12.5% separating gel. Transfer to nitrocellulose sheet was performed in a Trans-Blot cell overnight at 50 V and 200 mA, using transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol), as described by Burnette (1981). Immobilized proteins were then characterized by immunodetection using antibody diluted 1 in 2000 in 0.005 M sodium phosphate and 0.12 M NaC1, pH 8.0, containing 10% calf serum and 0.3% Tween 20. Antibody binding was detected using horseradish-peroxidase-conjugated mouse IgG against rabbit immunoglobulins; peroxidase activity was characterized using the chemiluminescent detection procedure according to the manufacturer's recommendations.

Assay procedure Aliquots (100 /zl) of the non-conjugated antielastase antibody at a concentration of 4.74 /zg/ml were coated on microtiter plates overnight at room temperature in 0.1 M phosphate buffer, pH 7.2. The plates were washed five times with PBS 0.01 M, pH 7.2 with 0.05% Tween 20. Coated plates could be stored for up to 3 weeks in PBS 0.05% with Tween 20 at 4°C. Culture supernatants were centrifuged for 10 min at 10,000 × g, and diluted in PBS with 0.1% bovine serum albumin. Standard dilutions ranged from 0.3 to 25 ng/ml. ELISA tests were performed in duplicate. Aliquots (100/xl) of diluted supematant or standard were added to the plate. The plates were incubated for 90 min at room temperature and then washed five times with PBS with 0.05% Tween 20. Aliquots (100 /zl) of the conjugated anti-elastase antibody at a concentration of 0.25 ~ g / m l

were added and then the plates were incubated 90 min at room temperature. The plates were washed five times with PBS with 0.05% Tween 20 and 100 /~1 of substrate solution (0.1 M phosphate-citrate buffer, pH 5.5, o-phenylene-diamine dihydrochloride 3 g/l; HzO 2 3.5 raM) were added. The enzyme reaction was allowed to proceed for 30 rain at room temperature in darkness. After stopping the reaction with 100 ~1 of 1 M HCI, absorbances were read at 490 /xm on a MR7000 Dynatech spectrophotometer.

Determination of elastase proteolytic activity Elastase activity was measured using elastinCongo red as substrate. Briefly, 0.5 ml of culture supernatant was incubated with 10 mg of elastincongo red in Tris-HCl 0.1 M pH 7, CaCl 2 1 mM at 37°C overnight. The reaction was stopped on ice by the addition of phosphate buffer Nail 2PO4, 0.7 M, pH 6. The absorbance was read at 495 nm. 1 U of enzyme was defined as the amount producing a 1.000 absorbance deviation at 495 n m / m i n per ml.

Results

Specificity of the anti-elastase polyclonal antibody Culture supernatant fractions were analysed by immunoblotting, using SDS-PAGE. As expected, a major protein band of 33 kDa, corresponding to the standard purified elastase was detected (Fig. 1). This band was present in the PAO1 strain supernatant and in the two strain supernatants from cystic fibrosis patients but absent in the PAl03 supernatant. This 33 kDa band was the only one present in purified elastase. In strain supernatants, an additional minor band in the low molecular weight region (< 14 kDa) was also observed. The treatment of samples with /3-mercaptoethanol led to a slight increase in the molecular weights, probably due to denaturation.

Optimization of the ELISA assay Optimal dilutions of the rabbit anti-elastase used to coat microtiter wells and the horseradish peroxidase anti-elastase conjugate were chosen on the basis of experiments performed at various

30

concentrations. The anti-elastase antibody optimal dilution for coating was 1 in 500, corresponding to a concentration of 4.74 /xg/ml or 0.47 p~g/well. The conjugate was diluted until it gave an absorbance of 1.6 for the highest standard elastase value (25 ng/ml). This was obtained with a 1 in 4000 dilution, corresponding to a concentration of 0.25 /~g/ml. Lower dilutions of antielastase coating failed to increase sensitivity, whereas larger concentrations of conjugate increased the background. Tests samples which had an optical density greater than 1.6 were diluted in PBS, and re-assayed. The optimal incubation time of the immunoreaction step was also investigated, and a 90 min duration was selected.

Range and detection limit An ELISA titration curve of pure elastase is shown in Fig. 2. A working range of 0.3-25 ng/ml was chosen for subsequent assays. The detection limit of the assay, defined as the concentration of elastase corresponding to the

kDa I 43

-

30

-

)D 492 =

I

nm •

=

=

|

~1~. . . . . .

1

r-I supernatant slrain

. . . . . . .

I0

ng/ml

Fig. 2. Parallel standard curves obtained with pure elastase (o) and with diluted strain supernatant (©), showing the absence of interfering reactions in the supernatant.

zero standard plus 2 SD of the zero standard absorbance (n = 12), was 0.26 ng/ml using the optimized conditions. The control strain PAO1 secreted 4.75/zg/ml of elastase and PA 103 which has been described as a non proteolytic strain (Liu, 1973), did not secrete elastase.

Linearity The relationship between log optical density and log elastase concentration (doubling dilutions of enzyme 1/2, 1/4, 1/8, 1/16) was linear over a concentration range of 0.6-25 ng/ml (Fig. 2). The curve obtained by doubling the dilution of a sample (9.22 ng/ml) was exactly parallel to the standard curve obtained with pure P. aeruginosa elastase.

20

14 iiii!iiii!i:i!<~ ¸

1

2

3

4

5

6

7

8

Fig. 1. Immunoreactivity of anti-elastase with P. aeruginosa strain supernatants. Supernatants from CF strains were submitted to S D S / P A G E before Western blotting. Lane 1: elastase standard (10 ng). Lane 2:PAO1 culture supernatant (10 /zl). Lane 3: strain supernatant from patient A (20/zl). Lane 4: strain supernatant from patients A (10/zl). Lane 5: strain supernatant from patient B (20 /xl). Lane 6: strain supernatant from patient B (10/zl). Lane 7: pure elastase standard treated by /3-mercaptoethanol (10 ng). Lane 8: strain supernatant from patient A treated by/3-mercaptoethanol (10 /xl).

Precision The intra-assay (within-series) coefficients of variation obtained from repeat analyses of two culture supernatants with elastase concentrations of 2 and 6 n g / m l were 1.8 and 2.16% respectively (Table I). Inter-assay (day-to-day) precision was estimated by assaying two culture supernatants with concentration of 0.6 ng/ml and 2 ng/ml on 12 and nine different occasions respectively. The coefficients of variation were 15.54 and 6.73% respectively (Table I).

31 TABLE I

y=2,250x÷22,68,

PRECISION DATA FOR ELASTASE ASSAYS IN CULTURE SUPERNATANTS n

Mean ng/ml

SD

CV%

10 10

6 2

0.1 0.04

1.8 2.2

9 12

2 0.6

0.137 0.09

6.73 15.54

Within-series

120

,

'

,

'

r .

.

.2 .=,7.13

.

.

.

.

.

, 0

0

100 8O

60.

40.

Day-to-day 20.~0, 0

-

0

Recovery Known amounts of pure elastase were added to a culture supernatant and the levels measured were compared with those expected. A recovery of around 100% was obtained (Table II). Correlation with elastase activity 13 culture supernatants were assayed by both the enzyme-linked immunoassay and the elastase enzyme assay and the correlation between the two methods is shown in Fig. 3. A correlation coefficient of 0.71 was obtained.

Discussion

The assay described here appears to be specific, highly sensitive and easily applicable to the quantification of elastase in strain supernatants and biological fluids. TABLE II RECOVERY OF P. AERUGINOSA ELASTASE ADDED TO CULTURE SUPERNATANTS The percentage recovery was calculated as follows: % recovery = 100 × (measured/calculated values). Elastase was added to a culture supernatant of known elastase concentration, vortexed, and then assayed as described Sample elastase (ng/ml)

Added elastase (ng/ml)

Measured elastase ng/ml)

Recovery %

2.5 2.5 2.5 2.5 2.5

0.75 1.25 2.5 5 10

3.14 3.93 5.03 7.45 11.67

100 104 100 99 93

.

5

-

.

10

-

.

15

-

.

.

20

25

-

.

30

-

.

35

-

,

40

-

45

ELISA

Fig. 3. Correlation between the sandwich enzyme immunoassay and elastase enzyme activity.

The specificity of the antibody was demonstrated by the presence of a major 33 kDa band on immunoblotting. The minor band also present in strain supernatants in the low molecular weight region was probably a degradation product of the protein. The detection limit of the assay was 0.26 n g / m l which is comparable to that of the radioimmunoassay described by Obernesser and DSring (1982) (< 1 ng/ml) and lower than that of the ELISA assay described by Elsheikh et al. (1986) (5 ng/ml). The linearity of the assay was good over a concentration range of 0.6-25 ng/ml as shown by the linear relationship between optical density and two-fold dilutions of elastase in culture supernatants (Fig. 2). Moreover, the parallelism of the standard and the assay dilution curves indicated the absence of non-specific interfering reactions. The precision of the assay was consistent with most ELISA procedures giving a within-run CV% of about 2% and a day-to-day CV% of about 10% (Table I). A good analytical recovery was obtained with the proposed assay, being around 100% for added concentrations of 0.75-10 n g / m l elastase. As is generally observed with direct ELISA procedures (Clark et al., 1986), the methodology reported here appears very specific. As expected, the PAO1 strain expressing the gene for elastase

32

produced a large amount of enzyme, whereas the deficient mutant strain PAl03 did not secrete the enzyme. The described method also has the advantage over indirect double antibody sandwich methods reported elsewhere of requiring only one species specific antibody and involving one less step in the procedure. This direct ELISA test should be of interest to those investigating elastase production in a complex biological medium. It could, for example, be used to explore secretions infected with P. aeruginosa, such as cystic fibrosis bronchial secretions. In such a complex biological medium, very specific assays are required to avoid interactions. In such cases, the measure of elastase activity is not feasible, since the detection limit is too high. With a detection limit of 0.26 ng/ml, the present immunoassay appears to be sufficiently sensitive to measure elastase production in the sputum. The application of this assay to the determination of elastase production in the sputum of cystic fibrosis patients will be undertaken in an attempt to improve our understanding of the pathogenesis of this disease.

Acknowledgements This work was supported by a grant from l'Association Fran~aise de Lutte contre la Mucoviscidose.

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