Journal of Immunological Methods, 131 (1990) 41-48
Measurement of free human leukocyte elastase and human leukocyte elastase/oq proteinase inhibitor complexes by an enzyme-linked immunosorbent assay M . D . K r a m e r 1, M. Mt~ller-Bardorff 1, M . M . S i m o n z, W. T i l g e n 1, E. Schickel a a n d D. P e t z o l d t 1 I Universitiits-Hautklinik, Voss-Strasse 2, D-6900 Heidelberg, F.R.G., and 2 Max-Planck-lnstitut/~r Immunbiologie, Stiibeweg 51, D-7800 Freiburg~ F.R.G.
(Received 11 September1989, revisedreceived 5 March 1990, accepted 22 March 1990) We report on an ELISA procedure for the quantitative analysis of total human leukocyte (PMN)-elastase, i.e., the simultaneous determination of (i) free (non-complexed) PMN-elastase and of (ii) PMN-elastase that is complexed to a I proteinase inhibitor. Simultaneous detection of both forms of PMN-elastase was achieved using a monoclonal antibody that recognizes a PMN-elastase-specific epitope present on both the uncomplexed and the complexed form of the enzyme. The test system described is reliable, easy to perform and permits the determination of total PMN-elastase in complex biological fluids such as plasma or seminal fluid. It is to be expected that this test system will be useful for investigations of human PMN-elastase in biological specimens obtained from both normal and pathological conditions. Key words: Leukocyteelastase (EC: 22.214.171.124), human; al-Proteinase-inhibitor; ELISA
Introduction The role of polymorphonuclear (PMN) leukocytes in the inflammatory response has been extensively documented (for review see, Havemann and Janoff, 1978). Excessive neutrophil stimulation in response to trauma or infectious foci may be dele.terious to the organism and lead to local
Correspondence to: M.D. Kramer, Universit~its-Hautklinik, Forschungsgruppe Hautklinik, Im Neuenheimer Feld 324, D6900 Heidelberg,F.R.G. (Tel.: 49/6221/563288 or 3289). Abbreviations: A, absorbance; al-PI , a 1 proteinase inhibitor; DMSO, dimethylsulfoxide; ELISA, enzyme-linked immunosorbent assay; HSF, human seminal fluid; mAb, monoclonal antibody(ies); PBS,phosphate-bufferedsaline; PBS/Tw, PBS containing 0.05% (v/v) Tween 20; PMN, polymorphonuclear (leukocyte); PMN-elastase, human leukocyte elastase; PMSF, phenylmethylsulfonylfluoride; RT, room temperature; Tw, Tween 20.
tissue destruction or even systemic effects such as failure of shock-sensitive organs (Fritz et al., 1984). The major secretable constituents of the neutrophil secretory granules responsible for these extracellular phenomena are the neutral proteinases, such as PMN-elastase and cathepsin G, as well as oxidative compounds generated at least in part by the granule associated enzyme myeloperoxidase. Free PMN-elastase in the extracellular, particularly the intravascular, space is readily complexed by its major inhibitor aa proteinase inhibitor ( a 1PI, formerly named al-antitrypsin) and in cases when al-PI is exhausted by the broad spectrum proteinase inhibitor a2 macroglobulin (Travis and Salvesen, 1983). The test systems previously described to determine the presence of the enzyme have measured P M N elastase/al-PI complexes. In such protocols the complex is specifically bound via anti-PMN-elastase antibodies and quantified
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
42 via enzyme-labelled anti-al-PI antibodies (Neumeier et al., 1982). It is known, however, that during severe inflammation free elastase may occur locally, due to either consumption of the local inhibitor potential, to proteolytic a n d / o r oxidative inactivation of al-PI, or to release of active PMN-elastase from the enzyme/inhibitor complex (Beatty et al., 1980; Carlsson et al., 1983; Johnson et al., 1986). Such observations suggest that the determination of total PMN-elastase via analysis of enzyme/inhibitor complexes could be misleading. We have developed an assay system which permits the quantitation of total PMN-elastase, i.e., free PMN-elastase as well as PMN-elastase/al-PI complexes. This assay uses a monoclonal antibody which recognizes an epitope of PMN-elastase that is accessible in both the free enzyme and the enzyme complexed with al-PI.
Materials and methods
Materials Leukocyte elastase (PMN-elastase, EC 3.4.21. 11) purified from human purulent sputum (cat. no. SE 563) was obtained from Elastin Products Co., via E. Lotti, Geneva, Switzerland. The chromogenic peptide substrate (cat. no. L-1335) for the determination of PMN-elastase activity was obtained from Bachem, Heidelberg, F.R.G. Polyclonal sheep anti-PMN-elastase antibodies (cat. no. AHP 051) were from Serotec, via Camon Laborservice, Wiesbaden, F.R.G. Human standard plasma (cat. no. OTFI 06/07) was from Behringerke, Marburg, F.R.G. Purified avproteinase inhibitor was kindly provided by Dr. N. Heimburger, Behringwerke, Marburg, F.R.G. Control plasma samples standardized for their content of PMN-elastase/al-PI complexes were from Merck, Darmstadt, F.R.G. Chromogenic substrate assay for PMN-elastase activity A 25 mM stock solution of the chromogenic substrate MeO-Suc-Ala-Ala-Pro-Val-pNA (L1335, Bachem) was prepared in DMSO and stored at - 2 0 ° C . 10 /~1 of the diluted stock solution (1/10 in assay buffer/Tw; assay buffer/Tw = 30
mM Tris/HC1, pH 7.4, 60 mM NaC1, 0.05% NaN 3, 0.05% Tween 20) were added to 100 /~1 of the enzyme-containing sample (diluted appropriately in assay buffer/Tw) in flat bottom microtiter plates. Liberation of free nitroaniline was quantified by absorbance measurements at 405 nm directly from the plate using an automated ELISA reading apparatus.
Preparation of PMN-elastase / al-PI complexes To prepare PMN-elastase/al-PI complexes, a constant amount of al-PI (2 # g / m l in assay buffer/Tw) was mixed with graded amounts of PMN-elastase (between 5 and 0.61 ~tg/ml in assay buffer/Tw) in a flat bottom microtiter plate and reacted at room temperature for 1 h with permanent shaking. Subsequently 200/~1 of the reaction mixture were mixed with 10 /~1 of the diluted chromogenic PMN-elastase substrate and absorbance at 405 nm determined at different time points. In addition, dilutions of the different reaction mixtures were assayed for PMN-elastase/a V PI complexes and for total PMN-elastase in the ELISA assay system described below. Derivation of monoclonal antibodies recognizing PMN-elastase or al-proteinase inhibitor Immune spleen cells were obtained from mice which had been immunized with commercially obtained PMN-elastase or al-proteinase inhibitor. Cell fusion and selection and stabilization of hybridoma cell lines in culture was performed according to established protocols (Justus et al., 1988). A monoclonal antibody (secreted by hybridoma clone 'Ela 6.2.4' (isotype IgG1)) that recognizes free PMN-elastase as well as PMN-elastase/alproteinase inhibitor complexes was used to establish the enzyme immunoassay. A monoclonal antibody (secreted by hybridoma clone 'AT 1'), that recognizes free aa-proteinase inhibitor as well as PMN-elastase/al-proteinase inhibitor complexes, was used for the determination of enzyme/inhibitor complexes. Affinity purification of PMN-elastase and characterization of mAb Ela 6.2.4 5/~g of human PMN-elastase in 0.1 M Tris-HC1 buffer pH 8.0 were applied to a column of 1 ml of
43 aprotinin-agarose (no. A-2268, Sigma Chemie, Deisenhofen, F.R.G.) and eluted with 0.1 M glycine-HCl buffer (pH 4.5) containing 0.4 M NaC1. Fractions of 500 /~1 were collected. The material eluted by glycine-HC1 buffer was immediately neutralized by the addition of 1 M Tris-HC1 buffer (pH 8.0). One tenth of the resulting fractions was directly coated onto flat bottomed activated microtiter plates (F16, no. 4-69914; Nunc, Wiesbaden, F.R.G.). Afterwards non-specific binding sites were blocked by the addition of 0.2% w / v gelatine in PBS. The wells were then incubated with mAb Ela 6.2.4 (1 /~g/ml in PBS) and afterwards with peroxidase-labelled antibodies specific for the Fc region of mouse IgG (no. 315-035-046, Dianova, Hamburg, F.R.G.) diluted 1/5000 in PBS/Tw. After extensive washing bound peroxidase-labelled antibodies were quantified as described below. In parallel a 1/100 dilution of each sample was analysed in the ELISA test system for total PMN-elastase as described below whilst a 1/10 dilution of the same sample in assay buffer was analysed for enzyme activity in the chromogenic substrate assay as described above. Immunohistochemistry on frozen sections of affected skin from a female patient suffering from the skin disease called acute neutrophilic dermatosis ('Sweet's syndrome') was performed using mAb Ela 6.2.4, biotinylated species-specific secondary antibodies and preformed biotinylated streptavidin/peroxidase complexes according to a method previously described (Kramer et al., 1989).
Enzyme immunoassay for the simultaneous quantification of free PMN-elastase and PMN-elastase / a 1proteinase inhibitor complexes Activated flat bottom microtiter plates (F16, no. 4-69914; Nunc, Wiesbaden, F.R.G.) were coated with 100 /xl/well of a 1/500 dilution of polyvalent sheep anti-PMN-elastase IgG in 50 mM Na2HCO 3, 3 mM NaN 3, pH 9.6 for 18 h at 4°C. Non-specific binding sites were blocked by 200 /~l/well of PBS, 0.2% w / v gelatine (4070, Merck, Darmstadt, F.R.G.) for 1 h at RT with permanent shaking. The further steps of this assay were as follows: (i) precoated plates were washed ( x 2) with 200
/~l/well of PBS containing 0.05% Tween 20 (Sigma Chemie, Deisenhofen, F.R.G.); (ii) diluted test sample (100 /~l/well) was incubated for 1 h at RT with permanent shaking; (iii) the plate was washed ( x 5) with PBS/Tw; (iv) monoclonal Ela 6.2.4 (100/ll/well) diluted to 1/~g/ml in PBS/Tw was added and the plate incubated for 1 h at RT with permanent shaking: (v) the plate was washed ( x 5) using PBS/Tw; (vi) peroxidase-labelled antibodies specific for the Fc region of mouse IgG (no. 315-035-046; Dianova, Hamburg, F.R.G.) diluted 1/5000 in PBS/Tw were added (100 /d/well) and the plate incubated for 1 h at RT; (vii) the plate was washed ( x 5) using PBS/Tw; (viii) bound peroxidase was detected by incubation with 100 /~l/well of 1 mg/ml o-phenylenediamine (P-2903, Sigma) and 1 # l / m l H202 (7210, Merck) in 0.1 M phosphate buffer, pH 6.0; (ix) the reaction was stopped after 5 rain by addition of 100 /zl/well of 1 M HzSO 4 and the reaction product quantified by measuring absorbance at 492 nm using an automated ELISA reading apparatus. In order to exclusively measure PMNelastase/al-proteinase inhibitor complexes the mAb Ela 6.2.4 in step (iv) was substituted by mAb AT 1 (1/~g/ml in PBS/Tw). Human seminal fluid Human seminal fluid specimens were obtained from the Andrology Department of the Dermatology Clinics, University of Heidelberg. We are indebted to Drs. Biltz and Pekar, Andrology Department, Dermatology Clinics, University of Heidelberg, F.R.G. for obtaining these samples.
Characterization of the anti-PMN-elastase monoclonal antibody secreted by hybridoma clone Ela 6.2.4 The anti-PMN-elastase monoclonal antibody used in this study was characterized by immunochemical and immunohistochemical analyses. MAb Ela 6.2.4 was used to monitor affinity purification of PMN-elastase via aprotinin-agarose. As
-OS A v
-0.3 -0.2 -0.1
Fraction Number Fig. 1. Characterization of anti-PMN-elastase mAb Ela 6.2.4. 5/tg of PMN-elastase was bound to and eluted from aprotinin-agarose as described in the materials and methods section. The resulting fractions were analysed for (i) reactivity with mAb Ela 6.2.4 in a direct solid-phase ELISA (circles), (ii) for their reactivity in the novel ELISA assay for total PMN-elastase (squares), and (iii) for enzyme activity in the chromogenic substrate assay (triangles). Inset: immunohistochemical staining by mAb Ela 6.2.4 of infiltrating neutrophilic granulocytes in the skin disease acute neutrophilic dermatosis. (---) epidermal surface. Space bar: 200 #m.
shown in Fig. 1 immunoreactivity with mAb Ela 6.2.4 co-migrated with (i) PMN-elastase enzyme activity as detected by the chromogenic substrate MeO-Suc-Ala-Ala-Pro-Val-pNA, and (ii) PMNelastase immunoreactivity as detected by the novel enzyme immunoassay. The inset to Fig. 1 demonstrates the specific reactivity of mAb Ela 6.2.4 with the infiltrating neutrophilic granulocytes observed in acute neutrophilic dermatosis. Taken together these data provided strong evidence that mAb Ela 6.2.4 was reacting with PMN-elastase, the elastolytic proteinase associated with intracellular granules of neutrophilic granulocytes. Standardization of the enzyme immunoassay for PMN-elastase and PMN-elastase / a t-proteinase inhibitor complexes In order to provide reference preparations of PMN-elastase/aa-PI complexes for the standardization of the assay system aliquots of aFPI (2 t~g/ml) were used after reaction with graded amounts of PMN-elastase (between 5 and 0.61
/Lg/ml). These concentrations correspond to a molar ratio of al-PI to PMN-elastase between about 1:5 and 2:1. After 1 h at room temperature under permanent shaking PMN-elastase activity was determined on the chromogenic peptide substrate MeO-Suc-Ala-Ala-Pro-Val-pNA. In the absence of aFPI, enzyme activity increased with time for all of the concentrations tested (Fig. 2A). When, however, 2 /Lg al-PI were mixed with 1.25 or 0.61 /~g PMN-elastase (i.e., a molar excess of al-PI ) enzyme activity was completely blocked (Fig. 2B). Subsequently, the same preparations were tested for the presence of PMN-elastase/aFPI complexes, using anti-PMN-elastase antibodies as the capture reagent and anti-al-PI mAb AT 1 as the detecting antibody (as described previously by Neumeier et al. (1982)). As expected, enzyme/inhibitor complexes were only detectable in the presence (Fig. 2D) but not in the absence of ai-PI (Fig. 2C). The fact that the absorption values decreased at higher concentrations of PMN-elas-
45 tase m a y be due either to free PMN-elastase competing with the e n z y m e / i n h i b i t o r complex for the anti-PMN-elastase antibodies a n d / o r to the nonspecific degradative capacity of free PMN-elastase. In parallel experiments (data not shown) the presence of immunoreactive a l - P I was ascertained using an experimental set-up for the detection of al-PI, i.e., using polyvalent anti-al-PI antibodies
as capture reagent and m A b AT-1 as detecting antibody. W h e n the same reaction mixtures were tested in the novel assay system for total (i.e., free and complexed) PMN-elastase using anti-PMN-elastase antibodies as capture reagent and a n t i - P M N elastase m A b Ela 6.2.4 as detecting antibody, c o m p a r a b l e slopes were obtained in the absence (Fig. 2 E ) and presence (Fig. 2 F ) of al-PI.
Quantification of total PMN-elastase
10 15 20 25 30
S 10 "6 20 25 30
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T o assess the sensitivity of the assay system for total PMN-elastase, graded a m o u n t s of P M N elastase were tested. T h e resulting standard curves displayed a linear slope over a concentration range between 50 and 1.6 ng P M N - e l a s t a s e / m l , corresponding to a detection limit of 10 n M P M N elastase. The reproducibility of the assay system was assessed by calculating the standard deviation of 16 replicate determinations of samples containing different concentrations of PMN-elastase. Coefficients of variation were 4.4% at 100 n g / m l , and 3.4% at 6.25 n g / m l , respectively. To explore whether the assay system could be used to quantify PMN-elastase in complex sampies, we assayed h u m a n plasma and h u m a n seminal fluid ( H S F ) after the admixture of graded a m o u n t s of PMN-elastase. Titration curves with linear slopes and regression coefficients between 0.9766 and 0.9955 were obtained for PMN-elastase in dilution buffer, in plasma and in h u m a n
' ' ' 1
2.0 1.0 PHN-Elastase IHo./ml I
if . . . .
. . . . . . .
Elattat~ [l~/ml I
Fig. 2. Standardization of the assay system for total PMNelastase. Graded amounts of human PMN-elastase (5 (filled triangles), 2.5 (open triangles), 1.25 (filled circles), and 0.61 (open circles) ttg/ml) were preincubated in the absence (A, C, and E) or presence (B, D, or F) of a constant amount of al-PI (2 #g/ml) as detailed in the materials and methods section. Enzyme activity was determined by the addition of a PMN-elastase specific chromogenic peptide substrate (A and B). A 1/10 dilution of each reaction mixture was tested for PMN-elastase/al-PI complexes using anti-PMN-elastase antibodies as the capture reagent and an anti-arPI mAb as detecting antibody (C and D). The reaction mixtures were also tested in the ELISA system for total PMN-elastase. E: 1/320 dilution of reaction mixtures containing graded amounts of PMN-elastase only (without addition of a:-PI). F: 1/640 dilution of the reaction mixture containing graded amounts of PMN-elastase and a constant amount of al-PI.
46 seminal fluid. T h e d e t e c t i o n limit for P M N - e l a s tase was in all cases a b o u t 10 n M ( d a t a n o t shown).
o Simultaneous detection of free and complexed PMN-elastase in human seminal fluid compared to the conventional detection of PMN-elastase / a 1-PI complexes W e c o m p a r e d the two assays, i.e., d e t e r m i n a tion of either t o t a l P M N - e l a s t a s e o r P M N - e l a s tase/al-PI complexes under controlled conditions. G r a d e d a m o u n t s o f p u r i f i e d P M N - e l a s t a s e were m i x e d with a s t a n d a r d a m o u n t of s e m i n a l fluid, t h a t was f o u n d in s e p a r a t e e x p e r i m e n t s n o t to c o n t a i n significant a m o u n t s of P M N - e l a s t a s e , a n d then tested in the E L I S A system for t o t a l P M N elastase as well as in the E L I S A for P M N - e l a s t a s e / a l - P I complexes. A s s h o w n in Fig. 3 signific a n t a b s o r b a n c e values were o b t a i n e d in the novel E L I S A for total P M N - e l a s t a s e , only. H o w e v e r , w h e n a l - P I o r s e r u m (as a source of a l - P I ) were a d d e d to H S F t o g e t h e r with P M N - e l a s t a s e , to
.. ,W,: !
6O0 800 1000
Total PMN-Etastase [ng/mt] Fig. 4. Determination of total PMN-elastase and PMN-elastase/al-PI complexes in individual seminal fluid samples. 50 individual seminal fluid samples were tested at a 1/20 dilution in the assay for total PMN-elastase and in the assay for PMN-elastase/al-PI complexes. The concentrations of PMNelastase were calculated by mathematical regression analysis. Each point represents an individual HSF sample for which PMN-elastase was determined as total PMN-elastase (abscissa) or PMN-elastase/al-PI complex (ordinate). The coefficient of correlation between both variables was found to be 0.918.
allow f o r m a t i o n o f a t - P I - P M N - e l a s t a s e c o m plexes, d o s e - d e p e n d e n t a b s o r b a n c e values were o b t a i n e d in b o t h a s s a y systems ( d a t a n o t shown).