IMMUNOLOGICAL INVESTIGATIONS, 21(1),
39-45 (1992)
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A SENSITIVE ELISA FOR T H E DETECI'IONOF HUMAN MONOCYI'ECHEMOATIXACI'AN" PROTEIN-1 (MCP-1) H. L. Evanoff, M. D. Burdick, S. A. Moore, S. L. Kunkel, and R M. Strieter' Departments of Pathology and *Internal Medicine, Division of Pulmonary and Critical Care Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0360
ABSTRACT
The recruitment of monocytes into tissue is associated with both acute and chronic inflammation. Although monocyte migration is measured in vitro by monocyte chemotaxis, this technique is often difficult to determine the specific quantitative contribution of a monocyte chemotaxin. We have developed a sensitive sandwich ELISA for the detection of monocyte chemoattractant protein-1 (MCP-l), a highly specific monocyte activating/chemotactic peptide. Polyclonal antibodies were generated from rabbits. The IgG fraction of the antiserum was isolated by a protein A column, with a portion of the antibodies biotinylated. Avidin-conjugated horse radish peroxidase was used for enzymatic, colorimetric analysis. The lower threshold for detection of MCP-1 was 50 pg/ml, and the ELSA was specific for MCP-I, since it failed to recognize other cytokines in a dosedependent fashion. Furthermore, this ELISA had the capacity to measure endothelial cell and pulmonary fibroblast-derived MCP-I. The development of a sensitive ELISA for the detection MCP-1 is significant, since it will allow the measurement MCP-1 from biologically relevant fluids, and aid in establishing whether MCP-1 is present in disease.
INTRODUCTION The elicitation of peripheral blood monocytes to sites of tissue injury is an essential element to the pathogenesis of the inflammatory response. The tissue histiocyte or macrophage, an important resident mononuclear phagocyte of tissue, is derived predominantly from differentiated peripheral blood monocytes and to a limited extent from the local replication of these cells (1-3). The events that lead to monocyte recruitment and activation are complex, as they must first adhere to vascular endothelium and then undergo directed migration in response to a gradient of chemotactic factor(s). Recent studies have identified a novel monocyte-specific cytokine, monocyte chemoattractant protein-1 (MCP-I). MCP-1 is a 76-amino acid peptide that not only has chemotactic activity for monocytes at nM concentrations, but also activates monocytes, resulting in
39 Copyright 0 1992 by Marcel Dekkcr, Inc.
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40
EYANOFF ET AL.
augmented cytostatic activity of monocytes against several tumor cell lines, stimulation of the respiratory burst, and release of lysosomal enzymes (4,5). Although MCP-1 was initially isolated from human "-1 myelomonocytic and glioma cell lines (4-81, it is produced by a variety of immune and nonimmune cells, including blood mononuclear leukocytes, endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, and several tumor cell lines (5,6,9-12). In this manuscript, we demonstrate the rapid, simple method for the quantitation of MCP-1 using a sandwich enzyme-linked immunosorbent assay (ELSA) specific for the detection of MCP-1 from supernatants of cells conditioned with either lipopolysaccharide (LPS) or interleukin-1-beta (K-1p). The MCP-1 ELISA had a threshold sensitivity of 50 pg/ml. Furthermore, this MCP-1 ELISA was specific for MCP-1 and failed to detect It-1p, TNF, interleukin-4 (IL-4), interleukin-6 (ILd), interleukin-7 (IL-71, or interleukin-8 (IL-8) in a dose-dependent fashion. This immunoassay is superior to chemotactic bioassays since it provides a rapid quantitation of MCP-1 from cellular/biological sources that may contain more than one monocyte chemotactic factor. MATERIALS AND METHODS
ReaPents:
Human IL-IP was the generous gift of UpJohn Co. (Kalamazoo, MI) Human TNF-alpha was the generous gift of Genentech (San Francisco, CA). Human IL-4 and IL-6 were purchased from R&D Systems (Minneapolis, MN). Human IL-7, K-8, and MCP-1 were purchased from PeproTech, Inc. (Rocky Hill,NJ). LPS (Olll:B4) was purchased from Sigma Co. (St. Louis, MO). PopHuman umbilical endothelial cells were isolated as previously described (9), cultured to confluence in 60 mm culture plates (Coming Glass Works, Corning, NJ) in medium 199, supplemented with 100 wg/ml endothelial cell growth supplement, 20% fetal bovine serum, and 100 pg/ml bovine lung heparin, and used prior to passage 4. Human pulmonary fibroblasts (CCDlSlu) were purchased from ATCC (Rockville, MD), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, N J) in EMEM (Whitaker Biomedical Products, Walkersville, MD) containing 1 mM glutamine, 25 mM HEPES, 100 units/ml penicillin 100 ng/ml streptomycin, 1% non-essential amino acids (Gibco Laboratories, Grand Island, NY), and 10% fetal calf serum (FCS), and used prior to passage 18. On day of use both endothelial cells and pulmonary fibroblasts were cultured in the presence of 0.5% and 0% serum, respectively. Human monocytes (106 cells/ml) and neutrophils (5 X 106 cells/ml) were isolated from peripheral blood as previously described (9,13,14), cultured in 35 mm plates (Corning Glass Works, Corning, NJ) with RF'MI-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%. of MCP-1 Anttbodles: Rabbit Anti-human MCP-1 antibodies were produced by first administering multiple intradermal injections of 20 pg of recombinant human MCP-1 (total) emulsified with complete Freund's adjuvant. This procedure was repeated in 10 days using 20 pg of recombinant MCP-1 emulsified in incomplete Freunds 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 recombinant human MCP-1 as follows: ELISA (96 wells) plates (Nunc-Immuno Plate Maxisorb, Neptune, NJ) were coated
. .
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DETECTION OF HUMAN MCP-1
41
with 50 ul of MCP-1 diluted 1 Wg/ml in coating buffer [borate-buffered saline (50 mM H3B03,120 rnM NaC1, pH 8.611 overnight at 4OC. The plates were then washed three times with 200 11 of wash-buffer (phosphatebuffered saline [PBSI containing 0.05% v/v Tween-ZO), followed by blocking the plates with 200 p1 of a solution containing PBS and 2% v/v goat serum for 1 hr at 37% The plates were washed three times with wash-buffer and 50 p1 of rabbit anti-human MCP-1 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 370C. 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 37%. The plates were washed three times with washbuffer followed by the addition of 100 p1 of 1:400 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 wash-buffer, followed by the addition of 100 pl of peroxidase substrate [Oh7 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 jd of 3M H2SO4. The absorbance was measured at 490 nm on a Biotek-ELSA reader. A 1:1,000,000 titer of rabbit anti-human MCP-1 serum resulted in a significant absorbance over the negative control. The polydonal antibodies were purified from this high titer serum using a protein A-agarose column (Pierce, Rockford, IL), 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). MCP-1ELISA: The sandwich MCP-1 ELISA was preformed using a modification of the above direct ELISA. Concentrations of both purified capture and detection antibodies were varied until optimal concentrations were determined for the detection of a fixed concentration of human MCP-I. 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 MCP-1 antibody diluted to 3.2 pg/ml in coating buffer and incubated overnight at 4OC. The plates were then washed three times with 200 J/well of washbuffer, followed by blocking the plates with 200 pl/well of a solution containing PBS and 2% bovine serum albumin for 1 hr at 37OC. The plates were washed three times with 200 pl/well of wash-buffer,followed by the addition of 50 pl in duplicate of either MCP-1 standards (1 pg/ml to 1000 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 J/well of wash-buffer, followed by the addition of 50 $/well of biotinylated MCP-1 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 d/well of 1:5000 diluted avidin-HRP (Dako, Carinteria, CA) in washbuffer supplemented with 2% FCS, and incubated for 30 mins. at 37OC. The plates were washed three times with 200 pl/well of wash-buffer, followed by the addition of 100 p1 of peroxidase substrate [0.67mg/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 H2SO4. The absorbance of the color development was then measured on a Biotek-ELISA reader, and the concentration of MCP-1 was determined from the standard curve of recombinant MCP-I. Data were analyzed by MacIntosh II computer using Statview 11 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.
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EVANOFF ET AL.
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Immunol Invest Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
z
0.1 i
0.0 - 4
- 3
- 2
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3
MCP-1 (Log ng/ml)
FIGURE 1. Recombinant MCP-1 measured by sandwich ELISA in a dose-dependent fashion. MCP-1 from 0.001 ng/ml to 1000 ng/ml was measured for enzymatic, colorimetric reaction, with absorbance measured at 490 nm. The lower threshold of detection of MCP-1 was 50 pg/ml. *=negative control/absence of MCP-1.
RESULTS AND DISCUSSION
After establishing that our MCP-1 antiserum in a dilution of 1:1,000,000 detected recombinant MCP-1 by direct ELISA, we next purified the polyclonal IgC fraction of the antiserum using a protein A-agarose column and biotinylated a portion for use as our detection antibody. To prove whether our MCP-1 ELISA was reproducible and efficacious for the detection of recombinant MCP-I, we performed a variety of experiments. Figure 1, contains a compilation of 10 separate ELISA experiments using recombinant MCP-1 in a concentration from 0.001 ng/ml to 1000 ng/ml. The absorbance measured in the presence or absence of MCP-1 ranged from the negative control/absence of MCP-1 (0.0869M.003) to 1000 ng/ml of MCP-1 (0.601M.051). The lower threshold limit for significant detection of MCP-1 over either background or the next preceding absorbance was 0.050 ng/ml (p=0.005), demonstrating that the sensitivity for the detection of MCP-1 was 50 pg/ml. In contrast, the upper limit of detection was 50 ng/ml, with an absorbance of 0.45W.033. Furthermore, when recombinant MCP-1 was placed in either FCS or human plasma as compared to dilution-buffer, the detection of antigenic MCP-1 by ELISA was unaltered (data not shown). This EWSA offers the advantage of greater sensitivity of measuring MCP-1 as compared to monocyte chemotactic analysis (4,612).
43
DETECTION OF HUMAN MCP-1
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TABLE I
MCP-1 (0.1 ng/ml) MCP-1 (1ng/ml) MCP-I(l0 ng/ml) IL-lP (0.1 ng/ml) IL-lP (1 ng/ml) IL-lp (10ng/ml) TNF (0.1 ng/ml) TNF (1 ng/ml) TNF (10ng/ml) IL-4 (0.1 ng/ml) IL-4 (1 ng/ml) IL-4 (10ng/ml) IL-6 (0.1 ng/ml) I L 4 (1 ng/ml) IL-6 (10 ng/ml) IL-7(0.1 ng/ml) IL-7 (1 ng/ml) IL-7 (10ng/ml) IL-8 (0.1 ng/ml) IL-8 (1ng/ml) IL-8 (10ne/ml)
Absorbance (490 nm) 0.078 0.110 0.214 0.356 0.075 0.070 0.068 0.065 0.075 0.065 0.065 0.069 0.079 0.085 0.062 0.067 0.080 0.079 0.067 0.082 0.079 0.080
Since it was apparent that OUT MCP-1 ELISA was reproducible for the detection of recombinant MCP-I. We next determined whether this ELISA was specific for only MCP-I. As shown in Table I, MCP-I,IL-lp, TNF, IL-4, IL-6, IL-7, or IL-8 were analyzed for their ability to react with either capture or detection polyclonal antibodies in the MCP-1 ELISA (experimental n=3). All of the cytokines, with the exception of MCP-I, failed to be detected in a dose-dependent manner as compared to background/negative control. These data suggested that the MCP-1 ELISA was specific for the detection of recombinant human MCP-I. Our laboratory and others have previously demonstrated the ability of endothelial cells, smooth muscle cells, fibrobIasts, and epithelial cells to express specific mRNA for MCP-1 in response to either exogenous or endogenous stimuli (912). In contrast, neutrophils and mononuclear phagocytes, monocytes and alveolar macrophages, have failed to show inducible mRNA for MCP-1 (13,141. Based on these observations we determined whether LPS- or IL-p-stimulated conditioned media from endothelial cells, pulmonary fibroblasts, monocytes, or neutrophils would have measurable levels of MCP-I. As shown in Table 11, endothelial cells, pulmonary fibroblasts, monocytes, or neutrophils were stimulated in the presence or absence of either Lps (100 ng/ml) or IL-1P (2 ng/ml) for 24 hrs, supernatants were isolated and analyzed for the presence of MCP-1 (experimental n=3). Endothelial cells stimulated with either LPS or IL-1s
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EVANOFF ET AL.
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TABLE I1
Cell Endothelial cells
-CTRL 41W
MCP-1 bplml) Stimulus LPS (100 nv/ml) 327Oi40
Fibroblasts
648110
622330
220M35
Blood Monocytes ND
ND
ND
Neu trophils
ND
ND
ND
n-lB(2 n d m l ) 314Ok30
resulted in significant levels of MCP-1 (pc0.001) as compared to unstimulated negative control, 3270f40 pg/ml and 3140+30 pg/ml, respectively. In addition, pulmonary fibroblasts treated with IL-1p produced significant levels of MCP-I, 2200f35 pg/ml, as compared to either the LPS-stimulated (622130 pg/ml) or unstimulated controls (648f10 pg/ml), ~ ~ 0 . 0 1These . findings corroborated our previous studies of endothelial cell and fibroblast-derived MCP-1 gene expression, demonstrating the importance of signal specificity for the induction of MCP-1 mRNA (9). Endothelial cells have been shown to respond to either an exogenous or endogenous stimuli, LPS and IL-lp, respectively, whereas fibroblasts require a specific host-derived stimulus, such as IL-lp, prior to their full expression of MCP-I. In addition, it was interesting to note the finding that both endothelial cells and fibroblasts have the capacity to constitutively produce MCP-I, 41250 pg/ml and 648f30 pg/ml, respectively. In contrast to the presence of antigenic MCP-1 from stimulated endothelial cells and fibroblasts, monocytes and neutrophils were not found to have detectable levels of antigenic MCP-I. Although monocytes, alveolar macrophages, and neutrophils have been shown to produce IL-8 05-17], the absence of antigenic MCP-1 production by either monocytes or neutrophils corroborates our previous studies that have demonstrated these cells do not have inducible levels of MCP-1 mRNA in response to either LPS or IL-lp (14). These studies have demonstrated the efficacy and reproducibility of a novel MCP-1 ELISA utilizing polyclonal capture and biotinylated antibodies with the detection sensitivity of 50 pg/ml. The ability to measure specific antigenic MCP-1 from cellular cultures is important since this will allow future studies to begin to understand potential pharmacologic regulation of MCP-1 production by these cells. Furthermore, the use of a MCP-1 ELISA will allow the detection of antigenic MCP-1 from biologically relevant fluids, such as bronchoalveolar lavage fluid from patients with interstitial lung disease or from synovial fluid from patients with rheumatological diseases, further establishing the role of MCP-1 in disease states.
45
DETECTION OF HUMAN MCP-1
ACKNOWLEDGEMENTS
This research was supported in part by National Institutes of Health grants HL02401, HL31693, HL35276, and DK38149, an American Lung Association Research Grant, an American Heart Association of Florida Grant, and a Council for Tobacco Research Grant. Dr.Strieter is a RJR Nabisco Research Scholar.
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276-282 J. Shellito, C. Esparza, and C. Armstrong, Am. Rev. Respir. Dis., (1987). A. B. van Oud Albus, H. Mattie, and R A. van Furth, Cell Tissue Kinet., & 211219 (1983). P. B. Bitterman, L. E. Saltzman, S. Adelberg, V. J. Ferrans, and R. G.Crystal, J. Clin. Invest., 460-469 (1984). K. Matsushima, C. G. Larsen, G. C. DuBois, and J. J. Oppenheim, J. Exp. Med., 169.1485-1490 (1989). K. Matsushima and J. J. Oppenheim, Cytokine, L 2-13 (1989). T. Yoshimura, E. A. Robinson, S. Tanaka, E. Appella, and E. J. Leonard, J. Immunol., 142, 1956-1962 (1989). Y. Furutani, H. Nomura, M. Notake, Y. Oyamada, T. Fukui, M. Yamada, C. G. Larsen, J. J. Oppenheim, and K. Matsushima, Biochem Biophys. Res. COIIUINUI., 249-255 (1989). T. Yoshimura, E. A. Robinson, S. Tanaka, E. Appella, E. Kuratsu, and E. J. Leonard, J. Exp. Med., 169,1449-1459 (1989). R. M. Strieter, R Wiggins, S. H. Phan, 8. L. Wharram,H. J. Showell, D. G., Remick, 694-700 S.W. Chensue, and S. L. Kunkel, Biochem Biophys. Res Commun., (1989). A. Sica, J. M. Wang, F. Colotta, E. Dejana, A. Mantovani, J. J. Oppenheim, C. G. Larsen, C. 0. Zachariae, and K. Matsushima, J. Immunol. 144.3034-3038 (1990). D. T. Graves, Y. L. Jiang, M. J. Williamson, and A. J. Valente, Science, 245.14901493 (1989). S. G. Elner, R. M. Strieter, V. M. Elner, B. J. Rollis, M. A. Del Monte, and S. L. Kunkel. Lab Invest. (in press). R. M. Strieter, S. W. Chensue, T. J. Standiford, M. A. Basha, H. J. Showell, and S. L. Kunkel, Biochem. Biophys. Res. Commun., 166,886-891 (1990). R. M. Strieter, K. Kasahara, R Allen, H. J. Showell, T. J. Standiford, and S. L. Kunkel, Biochem Biophys. Res. Commun., 173.725-730(1990). T. Yoshimura, K. Matsushima, J. J. Oppenheim and E. J. Leonard, J. Immunol., 139.788-793 (1987). P. Peveri, A. Walz, B. DeWald, and M. Baggiolini, J. Exp. Med.,167. 1547-1559 (1988). R. M. Strieter, S. W. Chensue, M. A. Basha, T. J. Standiford, J. P.Lynch III, M. Baggiolini, and S. L. Kunkel, Am. J. Respir. Cell. Mol. Biol., 2 321-326 (1990).
39-45 (1992)
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A SENSITIVE ELISA FOR T H E DETECI'IONOF HUMAN MONOCYI'ECHEMOATIXACI'AN" PROTEIN-1 (MCP-1) H. L. Evanoff, M. D. Burdick, S. A. Moore, S. L. Kunkel, and R M. Strieter' Departments of Pathology and *Internal Medicine, Division of Pulmonary and Critical Care Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0360
ABSTRACT
The recruitment of monocytes into tissue is associated with both acute and chronic inflammation. Although monocyte migration is measured in vitro by monocyte chemotaxis, this technique is often difficult to determine the specific quantitative contribution of a monocyte chemotaxin. We have developed a sensitive sandwich ELISA for the detection of monocyte chemoattractant protein-1 (MCP-l), a highly specific monocyte activating/chemotactic peptide. Polyclonal antibodies were generated from rabbits. The IgG fraction of the antiserum was isolated by a protein A column, with a portion of the antibodies biotinylated. Avidin-conjugated horse radish peroxidase was used for enzymatic, colorimetric analysis. The lower threshold for detection of MCP-1 was 50 pg/ml, and the ELSA was specific for MCP-I, since it failed to recognize other cytokines in a dosedependent fashion. Furthermore, this ELISA had the capacity to measure endothelial cell and pulmonary fibroblast-derived MCP-I. The development of a sensitive ELISA for the detection MCP-1 is significant, since it will allow the measurement MCP-1 from biologically relevant fluids, and aid in establishing whether MCP-1 is present in disease.
INTRODUCTION The elicitation of peripheral blood monocytes to sites of tissue injury is an essential element to the pathogenesis of the inflammatory response. The tissue histiocyte or macrophage, an important resident mononuclear phagocyte of tissue, is derived predominantly from differentiated peripheral blood monocytes and to a limited extent from the local replication of these cells (1-3). The events that lead to monocyte recruitment and activation are complex, as they must first adhere to vascular endothelium and then undergo directed migration in response to a gradient of chemotactic factor(s). Recent studies have identified a novel monocyte-specific cytokine, monocyte chemoattractant protein-1 (MCP-I). MCP-1 is a 76-amino acid peptide that not only has chemotactic activity for monocytes at nM concentrations, but also activates monocytes, resulting in
39 Copyright 0 1992 by Marcel Dekkcr, Inc.
Immunol Invest Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
40
EYANOFF ET AL.
augmented cytostatic activity of monocytes against several tumor cell lines, stimulation of the respiratory burst, and release of lysosomal enzymes (4,5). Although MCP-1 was initially isolated from human "-1 myelomonocytic and glioma cell lines (4-81, it is produced by a variety of immune and nonimmune cells, including blood mononuclear leukocytes, endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, and several tumor cell lines (5,6,9-12). In this manuscript, we demonstrate the rapid, simple method for the quantitation of MCP-1 using a sandwich enzyme-linked immunosorbent assay (ELSA) specific for the detection of MCP-1 from supernatants of cells conditioned with either lipopolysaccharide (LPS) or interleukin-1-beta (K-1p). The MCP-1 ELISA had a threshold sensitivity of 50 pg/ml. Furthermore, this MCP-1 ELISA was specific for MCP-1 and failed to detect It-1p, TNF, interleukin-4 (IL-4), interleukin-6 (ILd), interleukin-7 (IL-71, or interleukin-8 (IL-8) in a dose-dependent fashion. This immunoassay is superior to chemotactic bioassays since it provides a rapid quantitation of MCP-1 from cellular/biological sources that may contain more than one monocyte chemotactic factor. MATERIALS AND METHODS
ReaPents:
Human IL-IP was the generous gift of UpJohn Co. (Kalamazoo, MI) Human TNF-alpha was the generous gift of Genentech (San Francisco, CA). Human IL-4 and IL-6 were purchased from R&D Systems (Minneapolis, MN). Human IL-7, K-8, and MCP-1 were purchased from PeproTech, Inc. (Rocky Hill,NJ). LPS (Olll:B4) was purchased from Sigma Co. (St. Louis, MO). PopHuman umbilical endothelial cells were isolated as previously described (9), cultured to confluence in 60 mm culture plates (Coming Glass Works, Corning, NJ) in medium 199, supplemented with 100 wg/ml endothelial cell growth supplement, 20% fetal bovine serum, and 100 pg/ml bovine lung heparin, and used prior to passage 4. Human pulmonary fibroblasts (CCDlSlu) were purchased from ATCC (Rockville, MD), cultured to confluence in 60 mm culture plates (Corning Glass Works, Corning, N J) in EMEM (Whitaker Biomedical Products, Walkersville, MD) containing 1 mM glutamine, 25 mM HEPES, 100 units/ml penicillin 100 ng/ml streptomycin, 1% non-essential amino acids (Gibco Laboratories, Grand Island, NY), and 10% fetal calf serum (FCS), and used prior to passage 18. On day of use both endothelial cells and pulmonary fibroblasts were cultured in the presence of 0.5% and 0% serum, respectively. Human monocytes (106 cells/ml) and neutrophils (5 X 106 cells/ml) were isolated from peripheral blood as previously described (9,13,14), cultured in 35 mm plates (Corning Glass Works, Corning, NJ) with RF'MI-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%. of MCP-1 Anttbodles: Rabbit Anti-human MCP-1 antibodies were produced by first administering multiple intradermal injections of 20 pg of recombinant human MCP-1 (total) emulsified with complete Freund's adjuvant. This procedure was repeated in 10 days using 20 pg of recombinant MCP-1 emulsified in incomplete Freunds 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 recombinant human MCP-1 as follows: ELISA (96 wells) plates (Nunc-Immuno Plate Maxisorb, Neptune, NJ) were coated
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DETECTION OF HUMAN MCP-1
41
with 50 ul of MCP-1 diluted 1 Wg/ml in coating buffer [borate-buffered saline (50 mM H3B03,120 rnM NaC1, pH 8.611 overnight at 4OC. The plates were then washed three times with 200 11 of wash-buffer (phosphatebuffered saline [PBSI containing 0.05% v/v Tween-ZO), followed by blocking the plates with 200 p1 of a solution containing PBS and 2% v/v goat serum for 1 hr at 37% The plates were washed three times with wash-buffer and 50 p1 of rabbit anti-human MCP-1 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 370C. 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 37%. The plates were washed three times with washbuffer followed by the addition of 100 p1 of 1:400 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 wash-buffer, followed by the addition of 100 pl of peroxidase substrate [Oh7 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 jd of 3M H2SO4. The absorbance was measured at 490 nm on a Biotek-ELSA reader. A 1:1,000,000 titer of rabbit anti-human MCP-1 serum resulted in a significant absorbance over the negative control. The polydonal antibodies were purified from this high titer serum using a protein A-agarose column (Pierce, Rockford, IL), 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). MCP-1ELISA: The sandwich MCP-1 ELISA was preformed using a modification of the above direct ELISA. Concentrations of both purified capture and detection antibodies were varied until optimal concentrations were determined for the detection of a fixed concentration of human MCP-I. 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 MCP-1 antibody diluted to 3.2 pg/ml in coating buffer and incubated overnight at 4OC. The plates were then washed three times with 200 J/well of washbuffer, followed by blocking the plates with 200 pl/well of a solution containing PBS and 2% bovine serum albumin for 1 hr at 37OC. The plates were washed three times with 200 pl/well of wash-buffer,followed by the addition of 50 pl in duplicate of either MCP-1 standards (1 pg/ml to 1000 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 J/well of wash-buffer, followed by the addition of 50 $/well of biotinylated MCP-1 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 d/well of 1:5000 diluted avidin-HRP (Dako, Carinteria, CA) in washbuffer supplemented with 2% FCS, and incubated for 30 mins. at 37OC. The plates were washed three times with 200 pl/well of wash-buffer, followed by the addition of 100 p1 of peroxidase substrate [0.67mg/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 H2SO4. The absorbance of the color development was then measured on a Biotek-ELISA reader, and the concentration of MCP-1 was determined from the standard curve of recombinant MCP-I. Data were analyzed by MacIntosh II computer using Statview 11 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.
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0.5
-
0.4
-
0.3
-
0.2
-
L5
E
C
0
UJ
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z
0.1 i
0.0 - 4
- 3
- 2
- 1
0
1
2
3
MCP-1 (Log ng/ml)
FIGURE 1. Recombinant MCP-1 measured by sandwich ELISA in a dose-dependent fashion. MCP-1 from 0.001 ng/ml to 1000 ng/ml was measured for enzymatic, colorimetric reaction, with absorbance measured at 490 nm. The lower threshold of detection of MCP-1 was 50 pg/ml. *=negative control/absence of MCP-1.
RESULTS AND DISCUSSION
After establishing that our MCP-1 antiserum in a dilution of 1:1,000,000 detected recombinant MCP-1 by direct ELISA, we next purified the polyclonal IgC fraction of the antiserum using a protein A-agarose column and biotinylated a portion for use as our detection antibody. To prove whether our MCP-1 ELISA was reproducible and efficacious for the detection of recombinant MCP-I, we performed a variety of experiments. Figure 1, contains a compilation of 10 separate ELISA experiments using recombinant MCP-1 in a concentration from 0.001 ng/ml to 1000 ng/ml. The absorbance measured in the presence or absence of MCP-1 ranged from the negative control/absence of MCP-1 (0.0869M.003) to 1000 ng/ml of MCP-1 (0.601M.051). The lower threshold limit for significant detection of MCP-1 over either background or the next preceding absorbance was 0.050 ng/ml (p=0.005), demonstrating that the sensitivity for the detection of MCP-1 was 50 pg/ml. In contrast, the upper limit of detection was 50 ng/ml, with an absorbance of 0.45W.033. Furthermore, when recombinant MCP-1 was placed in either FCS or human plasma as compared to dilution-buffer, the detection of antigenic MCP-1 by ELISA was unaltered (data not shown). This EWSA offers the advantage of greater sensitivity of measuring MCP-1 as compared to monocyte chemotactic analysis (4,612).
43
DETECTION OF HUMAN MCP-1
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TABLE I
MCP-1 (0.1 ng/ml) MCP-1 (1ng/ml) MCP-I(l0 ng/ml) IL-lP (0.1 ng/ml) IL-lP (1 ng/ml) IL-lp (10ng/ml) TNF (0.1 ng/ml) TNF (1 ng/ml) TNF (10ng/ml) IL-4 (0.1 ng/ml) IL-4 (1 ng/ml) IL-4 (10ng/ml) IL-6 (0.1 ng/ml) I L 4 (1 ng/ml) IL-6 (10 ng/ml) IL-7(0.1 ng/ml) IL-7 (1 ng/ml) IL-7 (10ng/ml) IL-8 (0.1 ng/ml) IL-8 (1ng/ml) IL-8 (10ne/ml)
Absorbance (490 nm) 0.078 0.110 0.214 0.356 0.075 0.070 0.068 0.065 0.075 0.065 0.065 0.069 0.079 0.085 0.062 0.067 0.080 0.079 0.067 0.082 0.079 0.080
Since it was apparent that OUT MCP-1 ELISA was reproducible for the detection of recombinant MCP-I. We next determined whether this ELISA was specific for only MCP-I. As shown in Table I, MCP-I,IL-lp, TNF, IL-4, IL-6, IL-7, or IL-8 were analyzed for their ability to react with either capture or detection polyclonal antibodies in the MCP-1 ELISA (experimental n=3). All of the cytokines, with the exception of MCP-I, failed to be detected in a dose-dependent manner as compared to background/negative control. These data suggested that the MCP-1 ELISA was specific for the detection of recombinant human MCP-I. Our laboratory and others have previously demonstrated the ability of endothelial cells, smooth muscle cells, fibrobIasts, and epithelial cells to express specific mRNA for MCP-1 in response to either exogenous or endogenous stimuli (912). In contrast, neutrophils and mononuclear phagocytes, monocytes and alveolar macrophages, have failed to show inducible mRNA for MCP-1 (13,141. Based on these observations we determined whether LPS- or IL-p-stimulated conditioned media from endothelial cells, pulmonary fibroblasts, monocytes, or neutrophils would have measurable levels of MCP-I. As shown in Table 11, endothelial cells, pulmonary fibroblasts, monocytes, or neutrophils were stimulated in the presence or absence of either Lps (100 ng/ml) or IL-1P (2 ng/ml) for 24 hrs, supernatants were isolated and analyzed for the presence of MCP-1 (experimental n=3). Endothelial cells stimulated with either LPS or IL-1s
44
EVANOFF ET AL.
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TABLE I1
Cell Endothelial cells
-CTRL 41W
MCP-1 bplml) Stimulus LPS (100 nv/ml) 327Oi40
Fibroblasts
648110
622330
220M35
Blood Monocytes ND
ND
ND
Neu trophils
ND
ND
ND
n-lB(2 n d m l ) 314Ok30
resulted in significant levels of MCP-1 (pc0.001) as compared to unstimulated negative control, 3270f40 pg/ml and 3140+30 pg/ml, respectively. In addition, pulmonary fibroblasts treated with IL-1p produced significant levels of MCP-I, 2200f35 pg/ml, as compared to either the LPS-stimulated (622130 pg/ml) or unstimulated controls (648f10 pg/ml), ~ ~ 0 . 0 1These . findings corroborated our previous studies of endothelial cell and fibroblast-derived MCP-1 gene expression, demonstrating the importance of signal specificity for the induction of MCP-1 mRNA (9). Endothelial cells have been shown to respond to either an exogenous or endogenous stimuli, LPS and IL-lp, respectively, whereas fibroblasts require a specific host-derived stimulus, such as IL-lp, prior to their full expression of MCP-I. In addition, it was interesting to note the finding that both endothelial cells and fibroblasts have the capacity to constitutively produce MCP-I, 41250 pg/ml and 648f30 pg/ml, respectively. In contrast to the presence of antigenic MCP-1 from stimulated endothelial cells and fibroblasts, monocytes and neutrophils were not found to have detectable levels of antigenic MCP-I. Although monocytes, alveolar macrophages, and neutrophils have been shown to produce IL-8 05-17], the absence of antigenic MCP-1 production by either monocytes or neutrophils corroborates our previous studies that have demonstrated these cells do not have inducible levels of MCP-1 mRNA in response to either LPS or IL-lp (14). These studies have demonstrated the efficacy and reproducibility of a novel MCP-1 ELISA utilizing polyclonal capture and biotinylated antibodies with the detection sensitivity of 50 pg/ml. The ability to measure specific antigenic MCP-1 from cellular cultures is important since this will allow future studies to begin to understand potential pharmacologic regulation of MCP-1 production by these cells. Furthermore, the use of a MCP-1 ELISA will allow the detection of antigenic MCP-1 from biologically relevant fluids, such as bronchoalveolar lavage fluid from patients with interstitial lung disease or from synovial fluid from patients with rheumatological diseases, further establishing the role of MCP-1 in disease states.
45
DETECTION OF HUMAN MCP-1
ACKNOWLEDGEMENTS
This research was supported in part by National Institutes of Health grants HL02401, HL31693, HL35276, and DK38149, an American Lung Association Research Grant, an American Heart Association of Florida Grant, and a Council for Tobacco Research Grant. Dr.Strieter is a RJR Nabisco Research Scholar.
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