IMMUNOLOGICAL INVESTIGATIONS, 21(6), 589-596 (1992)
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THE DETECTION OF A NOVEL NEUTROPHIL-ACTIVATING PEPTIDE (ENA-78)USING A SENSITIVEELISA
R M. Strieter, S. L. Kunkel*, M. D. Burdick, P. M. Lincoln", and A. W a l P Departments of Internal Medicine and *Pathology, Division of Pulmonary and Critical Care Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0360,and *Theodor Kocher Institute, University of Bern, CH-3000 Bern 9, Switzerland.
ABSTRACT
Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides ( 4 0 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C)and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophilactivating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GROa; 52%), and IL-8(22%). In addition, ENA-78 appears to activate neutrophils through the IL-8receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.
INTRODUCTION
The accumulation of inflammatory leukocytes in tissue is a hallmark of acute inflammation. Although the polymorphonuclear leukocyte (PMN) is the predominant inflammatory leukocyte recruited in response to acute injury, this cell may also be associated with a number of more chronic inflammatory disorders. The sequence of events resulting in the elicitation of PMNs from the vascular compartment of the post-capillary venule into the tissue interstitium are essential to the pathogenesis of acute inflammation. The migration of PMNs initially occurs with
589 Copyright 0 1992 by Marcel Dekker, Inc.
STRIETER ET AL.
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/07/15 For personal use only.
590
adherence to the vascular endothelium, followed by activation, transenao;helial diapedesis, proteolytic digestion of the basement membrane extracellular matrix, and chemotaxis along established chemotactic gradients. This latter event is due to the generation of chemotactic factors followed by subsequent mobilization of leukocytes in reaction to a chemoattractant. This event is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. Previous studies have identified a supergene family (C-X-C) of chemotactic polypeptide cytokines, that include interleukin-8 (IL-8;1-4). These peptides have in common the ability to cause neutrophil activation and chemotaxis. Recently, our laboratories have described the isolation and complete primary structure of a novel neutrophil activating peptide (ENA-78) from human pulmonary type II-like epithelial cell line, A549 cells (5). ENA-78 is a 78-amino acid peptide with a molecular weight of 8.357 kD and has four cysteine amino acids positioned identically to other members of the IL-8 supergene family. ENA-78 has 22%, 52%, and 53% amino acid sequence homology with IL-8, growth regulated oncogene/melanoma growth stimulatory activity (GROa), and neutrophil activating peptide-2 (NAP-2)) respectively (5). In a similar manner as IL-8,ENA-78 stimulates neutrophil migration and activation. Moreover, it appears that ENA-78, IL-8,NAP-2, and GROa all act through the same receptor on neutrophils (5). In this manuscript, we demonstrate a specific and sensitive method for the detection of ENA-78 from cellular supernatants using a sandwich enzyme-linked immunosorbent assay (ELISA). The ENA-78 ELISA has a threshold sensitivity of 100 pg/ml. In addition, this ELISA was specific for ENA-78 and failed to detect interleukin-1 (IL-la and IL-lp), interleukin-1 receptor antagonist (IRAP),interleukin4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), IL-8,monocyte chemoattractant protein-1 (MCP-l), transforming growth factor-f3 (TGFP), tumor necrosis factora (TNFa), GROa, NAP-2, or gamma-interferon-inducible peptide-10 (YIP-10). MATERIALS A N D METHODS
Reaeents:
Human IL-la, IL-1p and IRAP were the generous gift of UpJohn Co. (Kalamazoo, MI). Human TNFa was provided by Genentech (San Francisco, CA). Human GROa, NAP-2, and YIP-10were provided by Sandoz (Vienna, Austria). Human IL-4, IL-6, and TGFp were purchased from R&D Systems (Minneapolis, MN). Human IL-7, IL-8, and MCP-1 were purchased from PeproTech, Inc. (Rocky Hill, NJ). LPS (0111:B4) was purchased from Sigma Co. (St. Louis, MO). Cell Populations: Human umbilical endothelial cells were isolated as previously described (6), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, NJ) in medium 199, supplemented with 100 pg/ml endothelial cell growth supplement, 20% fetal bovine serum, and 100 pg/ml bovine lung heparin, and used
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DETECTION OF NEUTROPHIL-ACTIVATING PEPTIDE
59 1
prior to passage 4. Human pulmonary fibroblasts were isolated from patients undergoing thoracotomy for reasons other than interstitial lung disease as previously described (7), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, NJ) in EMEM (Whitaker Biomedical Products, Walkersville, MD) containing 1mM glutamine, 25 mM HEPES, 100 units/ml penicillin 100 n g / d streptomycin, 1% non-essential amino acids (Gibco Laboratories, Grand Island, NY), and 10%fetal calf serum (FCS), and used prior to passage 6. Renal cortical epithelial cells were isolated from patients undergoing nephrectomy for hypernephroma as previously described (8), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, NJ) in EMEM (Whitaker Biomedical Products, Walkersville, MD) containing 1 mM glutamine, 25 mM HEPES, 100 units/ml penicillin 100 n g / d streptomycin, 1%nonessential amino acids (Gibco Laboratories, Grand Island, NY), and 10% fetal calf serum (FCS), and used prior to passage 6. Pulmonary epithelial cells (A549) were purchased from ATCC (Rockville, MD), cultured as previously described (9). O n day of use endothelial cells, pulmonary fibroblasts, pulmonary epithelial cells, and renal cortical epithelial cells were cultured in the presence of 0.5%, O%, O%, and 0% serum, respectively. Human monocytes (106 cells/ml) and neutrophils (5 X 106 cells/ml) were isolated from peripheral blood as previously described (7-9), cultured in 35 mm plates (Corning Glass Works, Corning, NJ) with RPMI-1640 (Whitaker Biomedical Products, Walkersville, MD), 1 mM glutamine, 25 mM HEPES, 100 units/ml penicillin, 100 ng/ml streptomycin (Hazelton Research Products, Denver, PA), and viability was greater than 95%. Generation of ENA-78 Antibodies: Rabbit Anti-human ENA-78 antibodies were produced using a modification of our previously described method (10). Briefly, ENA-78 (5) was first administered in multiple intradermal injections of 20 pg (total) emulsified with complete Freund's adjuvant. This procedure was repeated in 10 days using 20 pg of ENA-78 emulsified in incomplete Freund's adjuvant. The rabbit was bled 10 days later with antiserum isolated and heat inactivated. A direct ELISA was then performed to establish the efficacy of the rabbit antiserum to detect human ENA-78 as follows: ELISA (96 wells) plates (Nunc-Immuno Plate Maxisorb, Neptune, NJ) were coated with 50 pl of ENA-78 diluted 1 pg/ml in coating buffer [borate-buffered saline (50 mM H3BO3, 120 mM NaCl, pH 8.6)] overnight at 4OC. The plates were then washed three times with 200 pl of wash-buffer (phosphate-buffered saline [PBS] containing 0.05% v/v Tween-20), followed by blocking the plates with 200 p1 of a solution containing PBS and 2% v/v goat serum for 1 hr at 370C. The plates were washed three times with wash-buffer and 50 p1 of rabbit anti-human ENA-78 serum serially diluted (1:lO to 1:10,000,000) in wash-buffer containing 2% v/v goat serum was placed in duplicate in each of the wells, and incubated for 1hr at 37OC. The plates were washed three times with wash-buffer followed by blocking the plates with a solution containing PBS and 2% v/v goat serum for 1 hr at 37OC.
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592
STRIETER ET AL.
The plates were washed three times with wash-buffer followed by the addition of 100 pl of k400 v/v goat anti-rabbit peroxidase (Vector Laboratories Inc., Burlingame, CA) and incubated for 30 mins at 37OC. The plates were washed three times with washbuffer, followed by the addition of 100 pl of peroxidase substrate [0.67 mg/ml orthophenylenediamine dichloride (Dako, Carinteria, CA) in 25 mM citrate/ phosphate pH 5.0, and 0.0002%v/v hydrogen peroxide], color development proceeded for 6 mins at room temperature and stopped by the addition of 50 p l of 3M H2SO4. The absorbance was measured at 490 nm on a Biotek-ELISA reader, and 1:1,000,000 titer of rabbit anti-human ENA-78 serum resulted in a significant absorbance over the negative control. The polyclonal antibodies were purified from this high titer serum using a protein A-agarose column (Pierce, Rockford, a), followed by biotinylating a portion of the polyclonal antibodies with NHS-LC-biotin (Pierce, Rockford, IL)according to manufacturer's directions. Unbound biotin was separated from bound biotin by using a Centricon 30 (Amicon, Danvers, MA). ENA-78ELISA; The sandwich ENA-78 ELISA was preformed using a modification of our previously described method (10). Briefly, concentrations of both purified capture and detection antibodies were varied until optimal concentrations were determined for the detection of a fixed concentration of human ENA-78. Subsequent studies were performed with these optimal concentrations of capture and detection antibodies. ELISA plates (Nunc-Immuno Plate Maxisorb, Neptune, NJ) were coated with 50 pl/well of ENA-78 antibody diluted to 3.2 pg/ml in coating buffer and incubated overnight at 4OC. The plates were then washed three times with 200 d/well of wash-buffer, followed by blocking the plates with 200 d/well of a solution containing PBS and 2% bovine serum albumin for 1 hr at 37OC. The plates were washed three times with 200 dl/well of wash-buffer, followed by the addition of 50 p l in duplicate of either a ENA-78 standard curve (1 pg/ml to 50 ng/ml diluted in wash-buffer supplemented with 2% FCS), diluted cytokines (0.1 ng/ml to 10 ng/ml diluted in wash-buffer supplemented with 2% FCS), or cell-derived conditioned media (NEAT, 1:5, and l:lO), and incubated for 1hr at 37OC. The plates were washed three times with 200 pl/well of wash-buffer, followed by the addition of 50 pl/well of biotinylated ENA-78 diluted to 6 pg/ml in wash-buffer supplemented with 2% FCS. After incubating for 45 mins, the plates were washed three times with 200 pl/well of wash-buffer,followed by the addition of 100 pl/well of 1:5000 diluted avidin-HRP (Dako, Carinteria, CA) in wash-buffer supplemented with 2% FCS, and incubated for 30 mins. at 37OC. The plates were washed three times with 200 pl/well of washbuffer, followed by the addition of 100 p1 of peroxidase substrate [0.67 mg/ml orthophenylenediamine dichloride (Dako, Carinteria,CA) in 25 mM citrate/ phosphate pH 5.0, and 0.0002%v/v hydrogen peroxide], color development proceeded for 6 mins at room temperature and was stopped with the addition of 50 pl/well of 3M HzSO4. The absorbance of the color development was then measured
DETECTION OF NEUTROPHIL-ACTIVATING PEPTIDE 1.6
E E
0 Q)
d
v
593
-
1.2 1.0 1.4
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/07/15 For personal use only.
0)
0 E Q
0.8
u) a
0.6
! ! 0
a
-
0.4 1
- 4
- 3
- 2
ENA-78
- 1
0
1
2
(Log ng/mI)
FIGURE 1 Human ENA-78 measured by sandwich ELISA in a dose-dependent fashion. ENA78 from 0.001 ng/ml to 50 ng/ml was measured for enzymatic, colorimetric reaction, with absorbance measured at 490 nm. The lower threshold of detection of ENA-78 was 100 pg/ml. *=negative control/absence of ENA-78.
on a Biotek-ELISA reader, and the concentration of ENA-78 was determined from the standard curve of ENA-78. Data were analyzed by MacIntosh 11computer using Statview II statistical software package (Abacus Concepts, Inc.). Data are expressed as means f SEM. Data that appeared statistically significant were compared by Student's t-test (two-tailed) for comparing the means of multiple groups, and considered significant if p values were less than 0.05.
..
RESULTS AND DISCUSSION After establishing that our ENA-78 antiserum at a dilution of 1:1,000,000 detected antigenic ENA-78 by direct ELISA, the polyclonal IgG fraction of the antiserum was purified using a protein A-agarose column and an aliquot was biotinylated for use as detection antibody. To prove whether our ENA-78 ELISA was reproducible and efficacious for the detection of antigenic ENA-78, we performed 12 independent ELISA experiments. A compilation of 12 ELISAs using ENA-78 in a
STRIETER ET AL.
594
TABLE I
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/07/15 For personal use only.
-A €TRL ENA-78 (0.1 ng/ml) ENA-78 (1ng/ml) ENA-78 (10 ng/ml) IL-la (10 ng/ml) IL-la (10 ng/ml) IL-1RA (10 ng/ml) IL-4(10 ng/ml) IL-6 (10 ng/ml) IL-7 (10 ng/ml) IL-8 (10 ng/ml) MCP-l(l0 ng/ml) TGFp (10 ng/ml) TNFa (10 ng/ml) GROa (10 ng/ml) NAP-2 (10 ng/ml) YIP-10(10 ng/ml)
0.279 0.311 0.512 1.050 0.267 0.279 0.276 0.232 0.268 0.283 0.278 0.276 0.272 0.285 0.305 0.306 0.299
concentration from 0.001 ng/ml to 50 ng/ml is shown in Figure 1. The absorbance measured in the presence or absence of ENA-78 ranged from the negative control/absence of ENA-78 (0.279kO.003) to 50 ng/ml of ENA-78 (1.46(H0.100). The lower threshold limit for significant detection of ENA-78 over either background or the next preceding absorbance was 0.100 ng/ml (p=O.OOOl), suggesting that the sensitivity for the detection of ENA-78 was 100 pg/ml. Furthermore, when antigenic ENA-78 was placed in either FCS or human plasma as compared to dilution-buffer, the detection of antigenic ENA-78 by ELISA was unaltered (data not shown). This ELISA offered the advantage of greater sensitivity of measuring ENA-78 protein as compared to a neutrophil chemotactic bioassay (5). Since it was apparent that our ENA-78 ELISA was reproducible for the detection of antigenic ENA-78. We next determined whether this ELISA was specific only for ENA-78. As shown in Table I, ENA-78, IL-la, IL-lp, IRAF', IL-4, IL-6, IL-7, IL-8, MCP-1, TGFP, TNFa,GROa, NAP-2, YIp-10 were analyzed for their ability to react with either capture or detection polyclonal antibodies in the ENA-78 ELISA (experimental n=3). All of the cytokines, with the exception of ENA-78, failed to be detected, as compared to background/negative control. These data suggested that the ENA-78 ELEA was specific for the detection of antigenic human ENA-78.
DETECTION OF NEUTROPHIL-ACTIVATING PEPTIDE
595
TABLE I1 ENA-78 (pglml)
stimulus
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1L-l400 Monocytes Neutrophils
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THE DETECTION OF A NOVEL NEUTROPHIL-ACTIVATING PEPTIDE (ENA-78)USING A SENSITIVEELISA
R M. Strieter, S. L. Kunkel*, M. D. Burdick, P. M. Lincoln", and A. W a l P Departments of Internal Medicine and *Pathology, Division of Pulmonary and Critical Care Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0360,and *Theodor Kocher Institute, University of Bern, CH-3000 Bern 9, Switzerland.
ABSTRACT
Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides ( 4 0 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C)and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophilactivating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GROa; 52%), and IL-8(22%). In addition, ENA-78 appears to activate neutrophils through the IL-8receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.
INTRODUCTION
The accumulation of inflammatory leukocytes in tissue is a hallmark of acute inflammation. Although the polymorphonuclear leukocyte (PMN) is the predominant inflammatory leukocyte recruited in response to acute injury, this cell may also be associated with a number of more chronic inflammatory disorders. The sequence of events resulting in the elicitation of PMNs from the vascular compartment of the post-capillary venule into the tissue interstitium are essential to the pathogenesis of acute inflammation. The migration of PMNs initially occurs with
589 Copyright 0 1992 by Marcel Dekker, Inc.
STRIETER ET AL.
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/07/15 For personal use only.
590
adherence to the vascular endothelium, followed by activation, transenao;helial diapedesis, proteolytic digestion of the basement membrane extracellular matrix, and chemotaxis along established chemotactic gradients. This latter event is due to the generation of chemotactic factors followed by subsequent mobilization of leukocytes in reaction to a chemoattractant. This event is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. Previous studies have identified a supergene family (C-X-C) of chemotactic polypeptide cytokines, that include interleukin-8 (IL-8;1-4). These peptides have in common the ability to cause neutrophil activation and chemotaxis. Recently, our laboratories have described the isolation and complete primary structure of a novel neutrophil activating peptide (ENA-78) from human pulmonary type II-like epithelial cell line, A549 cells (5). ENA-78 is a 78-amino acid peptide with a molecular weight of 8.357 kD and has four cysteine amino acids positioned identically to other members of the IL-8 supergene family. ENA-78 has 22%, 52%, and 53% amino acid sequence homology with IL-8, growth regulated oncogene/melanoma growth stimulatory activity (GROa), and neutrophil activating peptide-2 (NAP-2)) respectively (5). In a similar manner as IL-8,ENA-78 stimulates neutrophil migration and activation. Moreover, it appears that ENA-78, IL-8,NAP-2, and GROa all act through the same receptor on neutrophils (5). In this manuscript, we demonstrate a specific and sensitive method for the detection of ENA-78 from cellular supernatants using a sandwich enzyme-linked immunosorbent assay (ELISA). The ENA-78 ELISA has a threshold sensitivity of 100 pg/ml. In addition, this ELISA was specific for ENA-78 and failed to detect interleukin-1 (IL-la and IL-lp), interleukin-1 receptor antagonist (IRAP),interleukin4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), IL-8,monocyte chemoattractant protein-1 (MCP-l), transforming growth factor-f3 (TGFP), tumor necrosis factora (TNFa), GROa, NAP-2, or gamma-interferon-inducible peptide-10 (YIP-10). MATERIALS A N D METHODS
Reaeents:
Human IL-la, IL-1p and IRAP were the generous gift of UpJohn Co. (Kalamazoo, MI). Human TNFa was provided by Genentech (San Francisco, CA). Human GROa, NAP-2, and YIP-10were provided by Sandoz (Vienna, Austria). Human IL-4, IL-6, and TGFp were purchased from R&D Systems (Minneapolis, MN). Human IL-7, IL-8, and MCP-1 were purchased from PeproTech, Inc. (Rocky Hill, NJ). LPS (0111:B4) was purchased from Sigma Co. (St. Louis, MO). Cell Populations: Human umbilical endothelial cells were isolated as previously described (6), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, NJ) in medium 199, supplemented with 100 pg/ml endothelial cell growth supplement, 20% fetal bovine serum, and 100 pg/ml bovine lung heparin, and used
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/07/15 For personal use only.
DETECTION OF NEUTROPHIL-ACTIVATING PEPTIDE
59 1
prior to passage 4. Human pulmonary fibroblasts were isolated from patients undergoing thoracotomy for reasons other than interstitial lung disease as previously described (7), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, NJ) in EMEM (Whitaker Biomedical Products, Walkersville, MD) containing 1mM glutamine, 25 mM HEPES, 100 units/ml penicillin 100 n g / d streptomycin, 1% non-essential amino acids (Gibco Laboratories, Grand Island, NY), and 10%fetal calf serum (FCS), and used prior to passage 6. Renal cortical epithelial cells were isolated from patients undergoing nephrectomy for hypernephroma as previously described (8), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, NJ) in EMEM (Whitaker Biomedical Products, Walkersville, MD) containing 1 mM glutamine, 25 mM HEPES, 100 units/ml penicillin 100 n g / d streptomycin, 1%nonessential amino acids (Gibco Laboratories, Grand Island, NY), and 10% fetal calf serum (FCS), and used prior to passage 6. Pulmonary epithelial cells (A549) were purchased from ATCC (Rockville, MD), cultured as previously described (9). O n day of use endothelial cells, pulmonary fibroblasts, pulmonary epithelial cells, and renal cortical epithelial cells were cultured in the presence of 0.5%, O%, O%, and 0% serum, respectively. Human monocytes (106 cells/ml) and neutrophils (5 X 106 cells/ml) were isolated from peripheral blood as previously described (7-9), cultured in 35 mm plates (Corning Glass Works, Corning, NJ) with RPMI-1640 (Whitaker Biomedical Products, Walkersville, MD), 1 mM glutamine, 25 mM HEPES, 100 units/ml penicillin, 100 ng/ml streptomycin (Hazelton Research Products, Denver, PA), and viability was greater than 95%. Generation of ENA-78 Antibodies: Rabbit Anti-human ENA-78 antibodies were produced using a modification of our previously described method (10). Briefly, ENA-78 (5) was first administered in multiple intradermal injections of 20 pg (total) emulsified with complete Freund's adjuvant. This procedure was repeated in 10 days using 20 pg of ENA-78 emulsified in incomplete Freund's adjuvant. The rabbit was bled 10 days later with antiserum isolated and heat inactivated. A direct ELISA was then performed to establish the efficacy of the rabbit antiserum to detect human ENA-78 as follows: ELISA (96 wells) plates (Nunc-Immuno Plate Maxisorb, Neptune, NJ) were coated with 50 pl of ENA-78 diluted 1 pg/ml in coating buffer [borate-buffered saline (50 mM H3BO3, 120 mM NaCl, pH 8.6)] overnight at 4OC. The plates were then washed three times with 200 pl of wash-buffer (phosphate-buffered saline [PBS] containing 0.05% v/v Tween-20), followed by blocking the plates with 200 p1 of a solution containing PBS and 2% v/v goat serum for 1 hr at 370C. The plates were washed three times with wash-buffer and 50 p1 of rabbit anti-human ENA-78 serum serially diluted (1:lO to 1:10,000,000) in wash-buffer containing 2% v/v goat serum was placed in duplicate in each of the wells, and incubated for 1hr at 37OC. The plates were washed three times with wash-buffer followed by blocking the plates with a solution containing PBS and 2% v/v goat serum for 1 hr at 37OC.
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592
STRIETER ET AL.
The plates were washed three times with wash-buffer followed by the addition of 100 pl of k400 v/v goat anti-rabbit peroxidase (Vector Laboratories Inc., Burlingame, CA) and incubated for 30 mins at 37OC. The plates were washed three times with washbuffer, followed by the addition of 100 pl of peroxidase substrate [0.67 mg/ml orthophenylenediamine dichloride (Dako, Carinteria, CA) in 25 mM citrate/ phosphate pH 5.0, and 0.0002%v/v hydrogen peroxide], color development proceeded for 6 mins at room temperature and stopped by the addition of 50 p l of 3M H2SO4. The absorbance was measured at 490 nm on a Biotek-ELISA reader, and 1:1,000,000 titer of rabbit anti-human ENA-78 serum resulted in a significant absorbance over the negative control. The polyclonal antibodies were purified from this high titer serum using a protein A-agarose column (Pierce, Rockford, a), followed by biotinylating a portion of the polyclonal antibodies with NHS-LC-biotin (Pierce, Rockford, IL)according to manufacturer's directions. Unbound biotin was separated from bound biotin by using a Centricon 30 (Amicon, Danvers, MA). ENA-78ELISA; The sandwich ENA-78 ELISA was preformed using a modification of our previously described method (10). Briefly, concentrations of both purified capture and detection antibodies were varied until optimal concentrations were determined for the detection of a fixed concentration of human ENA-78. Subsequent studies were performed with these optimal concentrations of capture and detection antibodies. ELISA plates (Nunc-Immuno Plate Maxisorb, Neptune, NJ) were coated with 50 pl/well of ENA-78 antibody diluted to 3.2 pg/ml in coating buffer and incubated overnight at 4OC. The plates were then washed three times with 200 d/well of wash-buffer, followed by blocking the plates with 200 d/well of a solution containing PBS and 2% bovine serum albumin for 1 hr at 37OC. The plates were washed three times with 200 dl/well of wash-buffer, followed by the addition of 50 p l in duplicate of either a ENA-78 standard curve (1 pg/ml to 50 ng/ml diluted in wash-buffer supplemented with 2% FCS), diluted cytokines (0.1 ng/ml to 10 ng/ml diluted in wash-buffer supplemented with 2% FCS), or cell-derived conditioned media (NEAT, 1:5, and l:lO), and incubated for 1hr at 37OC. The plates were washed three times with 200 pl/well of wash-buffer, followed by the addition of 50 pl/well of biotinylated ENA-78 diluted to 6 pg/ml in wash-buffer supplemented with 2% FCS. After incubating for 45 mins, the plates were washed three times with 200 pl/well of wash-buffer,followed by the addition of 100 pl/well of 1:5000 diluted avidin-HRP (Dako, Carinteria, CA) in wash-buffer supplemented with 2% FCS, and incubated for 30 mins. at 37OC. The plates were washed three times with 200 pl/well of washbuffer, followed by the addition of 100 p1 of peroxidase substrate [0.67 mg/ml orthophenylenediamine dichloride (Dako, Carinteria,CA) in 25 mM citrate/ phosphate pH 5.0, and 0.0002%v/v hydrogen peroxide], color development proceeded for 6 mins at room temperature and was stopped with the addition of 50 pl/well of 3M HzSO4. The absorbance of the color development was then measured
DETECTION OF NEUTROPHIL-ACTIVATING PEPTIDE 1.6
E E
0 Q)
d
v
593
-
1.2 1.0 1.4
Immunol Invest Downloaded from informahealthcare.com by University of Otago on 01/07/15 For personal use only.
0)
0 E Q
0.8
u) a
0.6
! ! 0
a
-
0.4 1
- 4
- 3
- 2
ENA-78
- 1
0
1
2
(Log ng/mI)
FIGURE 1 Human ENA-78 measured by sandwich ELISA in a dose-dependent fashion. ENA78 from 0.001 ng/ml to 50 ng/ml was measured for enzymatic, colorimetric reaction, with absorbance measured at 490 nm. The lower threshold of detection of ENA-78 was 100 pg/ml. *=negative control/absence of ENA-78.
on a Biotek-ELISA reader, and the concentration of ENA-78 was determined from the standard curve of ENA-78. Data were analyzed by MacIntosh 11computer using Statview II statistical software package (Abacus Concepts, Inc.). Data are expressed as means f SEM. Data that appeared statistically significant were compared by Student's t-test (two-tailed) for comparing the means of multiple groups, and considered significant if p values were less than 0.05.
..
RESULTS AND DISCUSSION After establishing that our ENA-78 antiserum at a dilution of 1:1,000,000 detected antigenic ENA-78 by direct ELISA, the polyclonal IgG fraction of the antiserum was purified using a protein A-agarose column and an aliquot was biotinylated for use as detection antibody. To prove whether our ENA-78 ELISA was reproducible and efficacious for the detection of antigenic ENA-78, we performed 12 independent ELISA experiments. A compilation of 12 ELISAs using ENA-78 in a
STRIETER ET AL.
594
TABLE I
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-A €TRL ENA-78 (0.1 ng/ml) ENA-78 (1ng/ml) ENA-78 (10 ng/ml) IL-la (10 ng/ml) IL-la (10 ng/ml) IL-1RA (10 ng/ml) IL-4(10 ng/ml) IL-6 (10 ng/ml) IL-7 (10 ng/ml) IL-8 (10 ng/ml) MCP-l(l0 ng/ml) TGFp (10 ng/ml) TNFa (10 ng/ml) GROa (10 ng/ml) NAP-2 (10 ng/ml) YIP-10(10 ng/ml)
0.279 0.311 0.512 1.050 0.267 0.279 0.276 0.232 0.268 0.283 0.278 0.276 0.272 0.285 0.305 0.306 0.299
concentration from 0.001 ng/ml to 50 ng/ml is shown in Figure 1. The absorbance measured in the presence or absence of ENA-78 ranged from the negative control/absence of ENA-78 (0.279kO.003) to 50 ng/ml of ENA-78 (1.46(H0.100). The lower threshold limit for significant detection of ENA-78 over either background or the next preceding absorbance was 0.100 ng/ml (p=O.OOOl), suggesting that the sensitivity for the detection of ENA-78 was 100 pg/ml. Furthermore, when antigenic ENA-78 was placed in either FCS or human plasma as compared to dilution-buffer, the detection of antigenic ENA-78 by ELISA was unaltered (data not shown). This ELISA offered the advantage of greater sensitivity of measuring ENA-78 protein as compared to a neutrophil chemotactic bioassay (5). Since it was apparent that our ENA-78 ELISA was reproducible for the detection of antigenic ENA-78. We next determined whether this ELISA was specific only for ENA-78. As shown in Table I, ENA-78, IL-la, IL-lp, IRAF', IL-4, IL-6, IL-7, IL-8, MCP-1, TGFP, TNFa,GROa, NAP-2, YIp-10 were analyzed for their ability to react with either capture or detection polyclonal antibodies in the ENA-78 ELISA (experimental n=3). All of the cytokines, with the exception of ENA-78, failed to be detected, as compared to background/negative control. These data suggested that the ENA-78 ELEA was specific for the detection of antigenic human ENA-78.
DETECTION OF NEUTROPHIL-ACTIVATING PEPTIDE
595
TABLE I1 ENA-78 (pglml)
stimulus
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1L-l400 Monocytes Neutrophils
