Matrix Vol. 12/1992, pp. 475 -480 © 1992 by Gustav Fischer Verlag, Stuttgart
Monoclonal Antibodies Against Human Fibroblast Collagenase and the Design of an Enzyme-Linked ImmunosorbentAssayto Measure Total Collagenase IAN M. CLARK, LIZ K. POWELL, JOHN K. WRIGHT, TIM E. CAWSTON and BRIAN L. HAZLEMAN Rheumatology Research Unit, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK.
Monoclonal antibodies have been raised against purified human fibroblast collagenase and characterised. One of these antibodies has been used in combination with a polyclonal anticollagenase antibody in a double antibody sandwich ELISA to measure collagenase. The assay range was 2 - 50 ng/ml collagenase. The assay measures total collagenase, i. e. pro- and active enzyme as well as collagenase in complex with TIMP. The level of collagenase has been measured in sera samples from patients with rheumatoid arthritis and compared with age- and sex-matched controls. The levels measured were: rheumatoid arthritis, 69 ± 29 ng/ml; normal, 49 ± 14 ng/ml. Key words: collagenase, enzyme immunoassay, monoclonal antibody, polyclonal antibody, tissue inhibitor of metalloproteinases (TIMP).
Vertebrate collagenase (EC 22.214.171.124, matrix metalloproteinase 1, MMP-l) is a neutral metalloproteinase capable of cleaving the collagen triple helix at a single locus to yield characteristic one-quarter - three-quarter products (Gross and Nagai, 1965). This enzyme is one of a family of socalled matrix metalloproteinases which, between them, can degrade all the components of the extracellular matrix. Consequently, these enzymes are tightly controlled by a variety of mechanisms which include: synthesis and secretion in a latent proenzyme form; extracellular activation of the proenzymes; inhibition of the active enzymes by inhibitors. A further vertebrate collagenase, from neutrophils (MMP-8), has also been described; this is a distinct gene product which has 57% amino acid homology with the fibroblast enzyme (Hasty et al., 1990). A variety of cytokines and growth factors can act upon connective tissue cells in vitro either to increase or decrease levels of secreted collagenase and/or inhibitors (e. g. Edwards et al., 1987; Golds et al., 1983). The major secreted form of collagenase is a latent proenzyme of Me
51929 which can be N-glycosylated to a minor form of Me approximately 57000 (Goldberg et al., 1986, Whitham et al., 1986). Procollagenase can be activated, at least in vitro, by proteinases (e. g. trypsin and plasmin) and mercurials (e.g. 4-aminophenylmercuric acetate, APMA); in vivo, activation may involve at least plasmin, and a second matrix metalloproteinase, stromelysin (MMP-3) (Mignatti et al., 1986; Murphy et al., 1987). Activation results in the removal of 80-amino acid residues from the N-terminal end (Goldberg et al., 1986). Once active, collagenase can be inhibited by a2-macroglobulin (Barrett and Starkey, 1973) or by the more specific tissue inhibitor of metalloproteinases (TIMP) (Cawston et al., 1981). A further inhibitor of metalloproteinases (TIMP-2) with homology to TIMP has recently been described (DeClerck et al., 1989; StetlerStevenson et al., 1989). Collagenase, and the balance between levels of this enzyme and its inhibitors, are thought to be important in diseases such as: rheumatoid and osteoarthritis (Woolley et al., 1977; Martel-Pelletier et al., 1987); tumour invasion and metastasis (Mignatti et al., 1986); lung fibrosis (Montano et al., 1989). The ability to measure accurately the
I. M. Clark et al.
level of collagenase, TIMP and enzyme inhibitor complexes is crucial to the investigation of their role in such diseases. The majority of current assays for collagenase or TIMP rely on the biological activity of either collagenase to degrade collagen, or TIMP to inhibit this degradation (henceforth termed 'bioassays', Cawston and Barrett, 1979; Cawston et aI., 1981). In order to measure procollagenase, it must first be activated e. g. with APMA or trypsin. It is often necessary to measure proenzyme in a system where inhibitor will also be present e. g. in connective tissue cell culture supernates or body fluids; when the procollagenase is activated, it can form an inactive enzyme-inhibitor complex with TIMP. Using the bioassay, the measured enzyme level will thus be lower than the true level. Moreover, the bioassay to measure collagenase cannot distinguish between the fibroblast and neutrophil enzymes; similarly, the bioassay to measure TIMP cannot distinguish between inhibitors of collagenase and hence inhibition could be due to urmacroglobulin, TIMP, or TIMP-2. Here, in the third part of a study to develop immunoassays to measure matrix metalloproteinases and their inhibitors, we report raising monoclonal antibodies against human fibroblast collagenase and the design of an ELISA to measure total collagenase.
Chemicals for electrophoresis were obtained from BDH Chemicals, Poole, Dorset, UK; prestained Mr markers were from Bio-Rad Laboratories, Watford, Herts, UK; enzymelinked antibodies were from Dako Ltd., Bucks, UK; nitrocellulose paper was from Anderman, Kingston upon Thames, Surrey, UK; 96-well EIA II plates were from ICN Flow, Rickmansworth, Herts, UK; Freunds Adjuvant was from Sigma Chemicals, Poole, Dorset, UK. All other chemicals were analytical reagent grade from FSA, Loughborough, Leics, UK. Methods Protein purification
Human fibroblast TIMP was purified from the serum free culture medium of WI38 fetal lung fibroblasts as described in Clark and Cawston (1989) (large scale cell culture was kindly performed by Dr. C. Mannix, SmithKline Beecham Pharmaceuticals, Gt. Burgh, Surrey, UK). Human fibroblast collagenase (activated by treatment with 0.67 mM APMA) was purified from the same medium using zinc-chelate chromatography followed by ionexchange chromatography on Mono-S (FPLC). Human neutrophil collagenase was kindly supplied by Prof. H. Tschesche and Dr. V. Knauper, University of Bielefeld, Germany.
Collagenase activity was measured with [3H]-acetylated type I calf skin collagen in the diffuse fibril assay (Cawston and Barrett, 1979). One unit of collagenase digests 1 f,lg collagen per min at 37°C. To assay TIMP, a standard amount of collagenase was added (approximately 0.05 units), and assayed with TIMP-containing samples. One unit of inhibitor is defined as the amount giving 50% inhibition of two units of collagenase. Electrophoresis
SDS-PAGE was performed by the method of Laemmli and Favre (1973), using a mini-gel format (Mini-Protean II, BioRad). Western blotting
Proteins were separated by SDS-PAGE and electroblotted onto nitrocellulose paper according to the method of Towbin et al. (1979). This was then incubated with anticollagenase antibody, followed by alkaline phosphataseconjugated anti-mouse immunoglobulins. Colour was then developed with using 5-bromo-4-chloro-3-indoyl phosphate and p-nitro blue tetrazolium. Raising antibodies
Polyclonal antibodies against collagenase were raised as described in Clark et al. (1991). The protocol for raising monoclonal antibodies against collagenase was essentially the same as that described for TIMP in Clark et al. (1991); the antigen was purified active human collagenase as above. Purification of IgG antibodies was by protein A-Sepharose chromatography as described in Clark et aI. (1991); purification of IgM antibodies was as follows: concentrated culture medium conditioned by the antibody secreting clone was dialysed overnight against 2 mM phosphate buffer, pH 6, to precipitate the IgM; the precipitate was centrifuged and washed in the same buffer, then redissolved in 25 mM Tris-HCI pH 7.5 containing 1 M NaCl and 0.02% NaN3. The antibody was then further purified by gel filtration on a 1.6 x 90 cm column of Ultrogel AcA22. Biotinylation ofantibodies
Antibodies were biotinylated using sulphosuccinimydyl6-(biotin amido)-hexanoate (NHS-LC-biotin, Pierce), as previously described (Clark et aI., 1991). Collection ofbody fluids
Blood for serum was allowed to clot in plastic tubes at 4°C and centrifuged at 1000 x g for 15 min to remove the clot. Aliquots were stored at - 70°C. Rheumatoid factor
Enzyme-Linked Immunosorbent Assays to Measure Metalloproteinases and Their Inhibitors was measured by rate nephelometry using a Beckman Immunochemistry System (assays kindly performed by Mrs. Greta Humm, Rheumatology Research Unit, Addenbrooke's Hospital).
Results Monoclonal antibodies Four fully cloned hybridoma cell lines secreting anticollagenase antibodies were selected, and expanded to bulk culture in the serum-free medium DCCM-2 (Biological Industries). The four secreted antibodies were designated RRU-CLl, CU, CL3 and CL4 and their isotypes were
80 c 0 .;:;
:e .J:: c
anti-collagenase IgG / IJg
A 120 100 c
assayed using a red cell agglutination assay (Serotec). RRUCLl was shown to be IgGl, whilst RRU-CL2, CL3, CL4 were IgM. The IgG was purified by protein A-Sepharose chromatography, whilst the IgM was purified by precipitation and gel filtration; both were shown to be pure by SDSPAGE (data not shown). The purified monoclonal antibodies were tested for their ability to inhibit collagenase in the bioassay. RRU-CLl and RRU-CL4 were able to inhibit collagenase showing a dosedependent inhibition from 0.15 - 30 Ilg IgG and 0.04-0.60 Ilg IgM added respectively (Fig. 1). On immunoblotting, RRU-CLl recognised both latent and active collagenase under either reducing or non-reducing conditions (Fig. 2); recognition was much stronger before reduction. The antibody also recognised the N-terminal autolysis fragment of collagenase (Clark and Cawston, 1989). A high M r band was stained in the non-reduced latent sample; this may be due to aggregation of latent collagenase, since it was not present in the same sample after reduction or after activation. The three IgM monoclonal antibodies did not recognise any forms of collagenase on the immunoblot.
Design ofELISA to measure collagenase
anti-collagenase IgM / IJg
Fig. 1: Inhibition of collagenase activity by monoclonal anti-collagenase IgG or IgM. Varying amounts of either anti-collagenase (a) RRU-CLl (lgG) or (b) RRU-CL4 (lgM) were preincubated (30 minutes, room temperature) with part-purified human skin fibroblast collagenase (0.055 units), and the sample was then assayed for remaining enzyme activity against type I calf skin collagen.
Combinations of anti-collagenase antibodies (both monoclonal and polyclonal) were tested to determine the best pair to use in a double antibody sandwich ELISA to measure collagenase. Only one pair of antibodies gave a good dose-dependent response to collagenase: RRU-CLl as a capture antibody, with polyclonal anti-collagenase as the detecting antibody. Concentrations of reagents and incubation times were optimised to give a final assay protocol as follows: (i) Microplates were coated with 100 Ill/well, 11lg/ml RRU-CLl in phosphate-buffered saline (PBS), overnight at 4°C. Free protein sites were then blocked with 10 mg/ml BSA in PBS for 1 hour at room temperature. (Plates were stored after this step at 4°C with 100 Ill/ well blocking buffer containing 0.02% NaN3 ) . (ii) Plates were washed once with PBS containing 0.05% Tween 20 (wash buffer) and incubated with 100 Ill/well samples or standards (diluted in wash buffer containing 0.5 mg/ml BSA (protein diluent) as necessary) for 2 hours at room temperature. (iii) Plates were washed three times with wash buffer and incubated with 100 Ill/well, 21lg/ml biotin-labelled anticollagenase IgG in protein diluent, for 2 hours at room temperature. (iv) Plates were washed three times with wash buffer and incubated with 100 Ill/well of 1/5000 horseradish peroxidase-linked streptavidin (Dako), for 30 minutes at room temperature. (v) Plates were washed three times in wash buffer and incubated with 100 Ill/well solution of o-pheny-
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Fig. 2: Immunoblot to demonstrate specificity of anti-collagenase RRU-CLl. Samples were electrophoresed on 12.5%. SOS-polyacrylamide gels and electroblotted onto nitrocellulose paper. Lane 1, 4, part-purified human fibroblast pro-collagenase; lane 2,5, part-purified active human fibroblast collagenase; lane 3,6, low Mr autolysis fragments of collagenase; STO, M r markers. Lane 1-3, reduced with 4 mg/ml dithiothreitol, lane 4-6, non-reduced. Blots were probed with 5 Ilg/ml monoclonal anti-collagenase RRU-CLl, then incubated with alkaline phosphataseconjugated rabbit anti-(mouse immunoglobulins) antibody. Colour was developed using 5-bromo-4-chloro-3indolyl phosphate and p-nitro blue tetrazolium. 1.6
-_....- procollagenase --.....- active collagenase
- - ~ - - procollagenase + TIMP ........... active collagenase + TIMP
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