Production and characterization of domain-specific monoclonal antibodies against tissue-type plasminogen activator

Animal Reproduction Science, 34 ( 1993 ) 69-81

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0378-4320/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved

Production and characterization of domainspecific monoclonal antibodies against tissue-type plasminogen activator M.N. Leonard*, W.L. Campbell, N. Jenkins Biological Laboratory, University of Kent, Canterbuo,, CT2 7NJ, UK (Accepted 14 December 1992)

Abstract

Two mouse hybridoma lines ( 3E6 and WA3 ) have been isolated in order to generate specific antibody probes for fibrinolysis in bovine gonadal cells. Mice were immunized with human recombinant tissue plasminogen activator (tPA) and the resultant spleen cells fused with SP2/0 myelomas. Both the resultant monoclonal antibodies recognized human recombinant tPA and bovine Sertoli cell tEA in a dose-dependent manner, and were identified as class IgG2b. The precise epitope recognized b,.' each antibody was characterized using modified enzyme-linked immunosorbent assays, immunoblotting analysis and protease assays. A synthetic inhibitor that irreversibly modifies the active site histidine of tPA (phe-pro-arg-chloromethylketone) was used to identify antibodies which bind to the t PA protease domain. Antibody 3E6 recognized this domain on the light chain of tPA, and will be useful for monitoring secreted tPA that is not complexed with its natural inhibitors. Antibody WA3 recognized an epitope which is remote from the active centre, on the heavy chain oftPA. Its binding to tPA obstructed the subsequent binding of fibrin, which indicates that WA3 binds to either the 'finger' or 'kringle 2' domain of tPA. This antibody has potential for monitoring tPA interactions with fibrin or extracellular matrix proteins. Both antibodies have been used in the accompanying paper to investigate the hormonal regulation of bovine Sertoli cell and granulosa cell fibrinolysis.

Introduction Plasminogen activators are a group of serine proteases that convert the zymogen plasminogen into the active protease plasmin, which subsequently activates fibrinolysis (Dano et al., 1985 ). Extracellular matrix proteins such as laminin (Salonen et al., 1984) and fibronectin (Salonen et al., 1985) are also cleaved by this proteolytic cascade. Plasminogen activators mediate in the control of cell migration and tissue re-modelling, particularly in the testis and ovary (reviewed by Saksela, 1985; Jenkins, 1987). The production of plasminogen activators and their specific inhibitors is controlled by hor*Corresponding author.

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mones in these organs (Ny et al., 1985; Vikho et al., 1986; Harlow et al., 1987). Tissue-type plasminogen activator (tPA) can been distinguished from the other mammalian plasminogen activator, urokinase (uPA), by its affinity for fibrin (Rijken et al., 1982). In fact, tPA and uPA are products of separate genes (Pennica et al., 1983; Riccio et al., 1985 ). Only tPA is produced in the testis of the calf (Coombs et al., 1988) and mouse (Rickles and Strickland, 1988 ), although both tPA and uPA are present in the rat testis (Vikho et al., 1986). tPA is synthesized as a single peptide (72 kDa) and cleaved by plasmin into two chains that are still connected by a disulphide bond (Van Zonneveld et al., 1986), as shown in Fig. 1. The heavy chain of tPA (38 kDa) has four distinct domains: a 'finger' domain homologous with regions of fibronectin, an epidermal growth factorlike domain (Ayub et al., 1990) and two 'kringle' domains similar to regions of prothrombin and plasminogen (Van Zonneveld et al., 1986). In plasma, fibrin binding to the 'finger' and 'kringle' domains stimulates tPA activity. Bicsak and Hseuh (1989), however, could find no fibrin stimulation of tPA isolated from rat ovaries and postulated that the enzyme was already stimulated by an endogenous factor. The light chain of tPA (34 kDa) contains its active protease site (MacGregor et al., 1985 ). All three natural PA inhibitors (PAls) bind to this domain (Fig. 1 ). These inhibitors have been classified as PAI-1 (Erickson et al., 1985), PAI-2 (Kruithof et al., 1986), and protease nexin (Howard and Knauer, 1986). A molecule resembling PAI-2 is produced by bovine Sertoli cells in culture (Coombs and Jenkins, 1988), although a PAl-l-like protein is secreted by rat peritubular cells (Hettle et al., 1988).

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Fig. 1. Diagram of the proposed tPA domain interactions with monoclonal Antibodies (Ab) WA3 and 3E6 (black arrows). The known interactions between tPA domains and fibrin, plasminogen activator inhibitors (PAI), and the peptide inhibitor phe-pro-arg-chloromethyl ketone (CMK) are depicted by white arrows.

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Several monoclonal antibodies have been raised against human tPA for its immunoassay in plasma (MacGregor et al., 1985; Holvoet et al., 1986). These immunoassays are an improvement over enzyme assays for tPA activity, which cannot discern the interactions between tPA, fibrin and PAl (Coleman and Green 1981; Jenkins and Ellison, 1986). A few of these antibodies have been mapped to specific epitopes on the human tPA molecule (Reilly et al., 1988; Wojta et al., 1989) but none has been used to assay tPA in cattle. This paper describes the production and characterization of two novel monoclonal antibodies directed against epitopes in the heavy and light chains of the tPA molecule. The accompanying paper (Leonard and Jenkins, 1993 ) reports the use of these antibodies to investigate how hormones regulate proteolysis in the bovine testis and ovary. Materials and methods

Chemicals Rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase were obtained from Dakopatts (Copenhagen, Denmark), and polyethylene glycol Grade 1500 from Boehringer-Mannheim (Lewes, UK). The tPA chromogenic Substrate $2251 and CNBr-digested fibrin fragments were gifts from Duphar B.P. (Weesp, Holland). Phe-pro-arg-chloro-methyl ketone was obtained from Boehringer Mannheim. Recombinant human tPA expressed in Chinese Hamster Ovary cells, and the hybridoma producing Antibody WA3 were gifts from Wellcome Biotechnology (Beckenham, UK). All other chemicals were of the highest purity available and were obtained from Sigma Chemical Company (Poole, UK).

Production of monoclonal antibodies Monoclonal Antibody 3E6 was produced by the following method, adapted from Gefter et al. (1977). BALB/c strain mice (6-10 weeks old) were immunized with two intraperitoneal injections of 25 #g human recombinant tPA per mouse in Freund's adjuvant at 30 and 15 days before the fusion, followed by a final injection of 10/~g tPA, 3 days before fusion. The antibody titre was measured in blood samples taken from the tail vein at 10 days before fusion, to select the best responding mice. Spleen cells from immune mice were fused to SP2/0 myelomas using polyethylene glycol, and hybridomas selected in a medium containing hypoxanthine-aminopterin-thymidine (Campbell, 1986). A similar method was used to produce Antibody WA3, using the same antigen. Antibody secreted into the medium was characterized using the three ELISA methods described below. Positive hybridomas were cloned three times by limiting dilution, and

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M.N. Leonard et al. ~Animal Reproduction Science 34 (1993) 69-81

ascites fluid was generated in BALB/c mice (Campbell, 1986). The immunoglobulin class was determined using the double immunodiffusion technique of Ouchterlony ( 1958 ).

Enzyme Linked Immunosorbent Assays (ELISA ) Three ELISA systems were employed for screening hybridomas that secreted tPA antibodies: one to identify positive hybridomas (Type I), and two others to determine which antibodies recognize the serine protease domain (Type II), or the fibrin binding domains (Type III) of human tPA. In the latter two ELISAs, a peptide or protein molecule bound to a specific tPA domain was used to modulate its subsequent interaction with the monoclonal antibody.

Type I tPA ( 10/tg m l - ~; 50 gl per well ) was plated in 50 mM Tris-HC1 buffer (pH 7.5 ) onto 96-well microtitre plates and left overnight at 4°C. The plates were then washed three times in 50 mM phosphate-buffered saline containing Tween 20 (0.1% v/v, Buffer A). To minimize non-specific binding the plates were coated with 150 #1 per well of BSA in Buffer A (2% w/v ) for 1 h at room temperature. After subsequent washing (three times in Buffer A), supernatants from hybridoma cultures were added (50/zl per well) and incubated for 1 h at room temperature. After a further three washes in Buffer A, alkalinephosphatase linked goat anti-mouse immunoglobulins were added ( 1 : 350 v/ v in Buffer A; 50/tl per well), and the plate incubated for 1 h at room temperature. After a final three washes in Buffer A each well was treated with 100/~1 of p-nitrophenol phosphate (Sigma 104 substrate; 1 mg m1-1 ) in Buffer B (100 mM glycine, 1 mM ZnC12, 1 mM Mg C12; pH 10.4) and left at room temperature for 30 min in the dark. The colour in each well was quantified using a Dynatek plate reader at 410 nm.

Type H This ELISA, based on one described by MacGregor et al. ( 1985 ) was used to identify antibodies directed against the serine protease domain oftPA. The tPA ( 10/~1 m l - 1 in 50 mM Tris-HC1 buffer, pH 7.5 ) was first incubated with a specific inhibitor of this serine protease site (see Fig. 1 ): phe-pro-arg-chloromethyl ketone (10 #g ml-~ for 1 h at 37°C), followed by plating at 50 ~tl per well overnight at 4 ° C. The remainder of the ELISA using inactivated tPA was identical to ELISA Type I.

Type III An ELISA was developed to identify antibodies recognizing the fibrin binding domains of tPA. Human tPA antigen (0.15-500 #g m l - 1 in 50 mM Tris-

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HC1 buffer, pH 7.5) was first incubated with CNBr-generated fibrin fragments (20 zig ml -~) for 1 h at 37°C. Control tubes substituting Tris-HC1 buffer for fibrin fragments were set up at each dose of tPA. Samples were loaded into microtitre plates at 50/tl per well, and incubated overnight at 4°C. The remainder of the ELISA was identical to ELISA Type I.

Enzyme activity assays The tPA protease activity in cell culture supernatants was assayed using a well-defined chromogenic substrate assay (Reilly et al., 1988). Human recombinant tPA standard was diluted in assay buffer (50 mM Tris-HC1, pH 7.4, containing 0.1% v/v Tween 80) and used in the range 1.35-5400 IU m l - 1. Standards were applied to a microtitre plate (40/~1 per well) followed by the addition of assay buffer (80/A) to each well. In most assays, CNBrdigested fibrin fragments ( 5 mg in 10 ml ) were added to each well to enhance tPA activity. These were followed by 100/A of $2251 substrate (2.5 mM ) and 20/d of purified bovine plasminogen (0.04 U m l - l ) . The plate was then incubated at 37°C for 15 min and the reaction was ended by adding 10 ¢zl of 7.5% ( v / v ) acetic acid to each well. In assays to determine the effects of monoclonal antibodies on proteolytic activity (see Fig. 4), 50/~1 of ascites fluid was included in the incubation tubes. The absorbance was then read at 41(I nm using a Dynatek plate reader.

Bh~od clot lysis assay A fibrinolysis assay was used to confirm the enzyme activity data, as described by Chohan et al. ( 1975 ). Briefly, human blood was collected and immediately diluted (1:5) with 120 mM sodium acetate buffer (pH 7.4). A 15% blood clot was prepared by addition of 50/A 125I-fibrinogen, 350/~1 of acetate buffer and 100/zl thrombin (25 NIH U ml -l ) to 1.5 ml of diluted human blood. In test samples, 5-100/~1 of ascites f u i d containing Antibody 3E6 was added before clotting. After coagulation, the tubes were placed at 37 ° C, and the supernatant was sampled for soluble 125I-labelled fibrin degradation products in the medium in a gamma scintillation counter (LKB, Sweden). The effect of Antibody 3E6 on clot lysis was compared with lysis without antibody after 5 h.

Gel electrophoresis and immunoblot analysis The gels were performed as described by Laemmli (1970) and Burnette (1980) using 14% ( w / v ) total acrylamide in the separating gels, and 4.5% ( w / v ) total acrylamide in the stacking gels. Electrophoresed proteins were transferred onto nitrocellulose paper and probed according to the method of

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M,N. Leonard et aL / Animal Reproduction Science 34 (1993) 69-81

Coombs et al. (1988 ). In order to validate the anti-human tPA Antibodies 3E6 and WA3 for use with bovine proteins, calf Sertoli cells were stimulated with 1 m M 8-Bromo-cAMP to induce tPA production, as described in Leonard and Jenkins ( 1993 ). Culture supernatants were harvested after 24 h, concentrated five-fold by ultrafiltration, and probed on an immunoblot as described above. Results Two stable mouse hybridoma lines (3E6 and WA3 ) were isolated, and the antibodies produced by both cell lines recognized h u m a n tPA, as shown by a conventional ELISA (Type I ) (Fig. 2 ). The range of both ELISAs for h u m a n tPA was 25-2000 ng m l - ~, with EDso values of 510 ng ml-1 for Antibod) 3E6 and 220 ng ml-1 for WA3. Both antibodies were found to belong to immunoglobulin Class IgG2b by the Ouchterlony double immunodiffusion procedure. The modified ELISA (Type II) was used to show which antibody recognizes the active site (serine protease d o m a i n ) of tPA (this site was blocked using the peptide CMK). The presence of 10/zg m l - ~ C M K did not significantly reduce the recognition of tPA by Antibody WA3. However, tPA binding by Antibody 3E6 was significantly reduced by 6 I% ( P < 0.001 ) under the same conditions, suggesting that only Antibody 3E6 is directed against the active site oftPA. This conclusion was supported by an immunoblot analysis

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M. N Leonard et al. / Animal Reproduction Science 34 (I 993) 69-81

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M.N. Leonard et al. / Animal Reproduction Science 34 (1993) 69-81

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o f tPA reduced into its constituent heavy and light chains by boiling in the presence o f 0.1 M dithiothreitol (Fig. 3a). Antibody 3E6 only stained the light chain of the resolved peptides (Mr 34 kDa), which is known to contain the active protease site (Holvoet et al., 1986). Antibody WA3 stained the heavy chain (Mr 38 kDa) oftPA. The enzyme activity o f tPA when combined with either Antibody WA3 or Antibody 3E6 was compared in the chromogenic substrate assay. Antibody

M.N. Leonard et al. / Animal Reproduction Science 34 (1993) 69-81

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Fig. 6. Displacement by human fibrin fragments (20 #g ml-t ) of Antibody WA3 binding to human recombinanttPA as determined by enzyme-linkedimmunosorbentassay Type III. Mean values ( +_SEM) are shown. 3E6 was seen to significantly reduce the protease activity oftPA over the range 0.02-5 HI ml-~ ( P < 0 . 0 0 1 ) ; Antibody WA3, however, was ineffective (Fig. 4). This inhibition of tPA enzyme activity by Antibody 3E6 was confirmed using a fibrinolytic assay, and was shown to be dose dependent (Fig. 5 ). Including fibrin fragments (which bind to domains within the tPA heavy chain) in the Type III ELISA abolished the binding of Antibody WA3 to tPA ( P < 0.005 ) (Fig. 6); however the binding of Antibody 3E6 was unaffected (data not shown). The cross-reactivity of both antibodies with the bovine tPA secreted by Sertoli cells is shown in Fig. 3b. 8-Bromo-cAMP is a strong inducer of tPA secretion (Ellison and Jenkins, 1989), and was used to induce sufficient bovine tPA to detect on an immunoblot. Supernatants from challenged cells were concentrated five-fold and subjected to immunoblot analysis with both monoclonal antibodies. Antibody WA3 recognized a polypeptide of Mr 38 kDa in h u m a n and bovine samples, equivalent to the heavy chain of tPA (Coombs et al., 1988 ), whereas Antibody 3E6 recognized a polypeptide of Mr 34 kDa equivalent to the light chain oftPA. Recognition of bovine tPA (Fig. 3b ) was less intense than that seen with h u m a n tPA (Fig. 3a), which probably reflects the lower concentration of bovine tPA rather than qualitative differences in the antibody recognition. No antibody binding was seen using culture super° natants from unstimulated sertoli cells. Type II and Type III ELISAs using

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M.N. Leonard et al. /Animal Reproduction Science 34 (1993) 69-81

both antibodies gave similar results when bovine tPA was used as the antigen, compared with human tPA (data not shown ). Discussion

Two monoclonal antibodies (3E6 and WA3 ) have been raised against human recombinant tPA and their binding has been characterized using ELISAs, immunoblotting analysis and proteolytic assays. Both antibodies were shown by ELISA to recognize tPA in a dose-dependent manner (Fig. 2). However, the antibodies were clearly shown to have different specificities by immunoblot analysis under reducing conditions: 3E6 recognizing the light chain and WA3 recognizing the heavy chain of human tPA (Fig. 3). Each antibody recognized bovine tPA on an immunoblot in the molecular weight range corresponding to previous studies (Coombs et al., 1988 ), and in only conditions which are known to stimulate tPA activity (Leonard and Jenkins, 1993 ). The fact that human recombinant tPA was used as an initial antigen and the results seen with supernatants taken from bovine cells in culture were essentially identical, demonstrates the useful interspecific cross-reactive properties of these antibodies. A cross-reaction with porcine tPA has been demonstrated using another anti-human tPA monoclonal antibody (Stigbrand et al., 1989). In order to identify antibodies recognizing the active protease domain of tPA, the ELISA protocol was modified by pre-incubating tPA with a peptideCMK conjugate. This inhibitor modifies the active site histidine of tPA, and its reaction is irreversible (Rijken et al., 1982; MacGregor et al., 1985 ). Binding of Antibody 3E6 was blocked by pre-incubation oftPA with CMK, whereas no significant inhibition of binding was seen with Antibody WA3. These findings suggest that Antibody 3E6 recognizes an epitope at, or near to, the active site histidine on the light chain of tPA. The fact that Antibody WA3 was not affected by this treatment suggests that it recognizes an epitope on the tPA heavy chain that is remote from the active centre (see Fig. 1 ). These results were supported in tPA amidolytic studies using a chromogenic substrate (Sugawara et al., 1988 ). Antibody 3E6 was seen to strongly inhibit tPA activity, whereas Antibody WA3 had no significant effect (Fig. 4). Furthermore, the inhibition oftPA activity by 3E6 was shown to be dose dependent when measured using the blood clot lysis assay (Fig. 5 ), again indicating that 3E6 is recognizing an epitope at, or near to, the active site of tPA. The presence of fibrin led to an almost complete abolition of tPA recognition by Antibody WA3 up to tPA concentrations of 50 ~g m l - 1 (Fig. 6 ). This suggests that Antibody WA3 is directed towards an epitope on either the 'finger' or the 'kringle 2' domain on the heavy chain of tPA (Fig. 1 ), as shown by other workers using deletion mutants of tPA (Verheijen et al., 1986 ). Since others have shown that antibodies directed either to the 'finger' domain or

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the 'kringle 2' d o m a i n both inhibit the fibrin stimulation o f tPA activity (Wojta et al., 1989), it is not possible to discriminate between these alternatives at present.

Conclusion The results presented here show that two monoclonal antibodies have been raised against tPA. A n t i b o d y 3E6 is probably directed against the active site o f t P A on the light chain, and Antibody WA3 to fibrin binding d o m a i n ( s ) on the heavy chain (Fig. 1 ). I m m u n o b l o t and ELISA data using tPA secreted by calf Sertoli cells in culture show that both antibodies are capable of specifically recognizing bovine tPA. Whilst it is possible that the degree of site specificity seen with these two monoclonal antibodies may not directly mimic that seen if they were originally raised against bovine tPA, it still seems that 3E6 appears to have a high degree of specificity for the d o m a i n on the light chain of bovine tPA as seen in ELISA Type II blocking studies (data not shown). These two antibodies were therefore used as tools for investigating the hormonal regulation o f bovine Sertoli cell and granulosa cell tPA production, as described in the accompanying paper (Leonard and Jenkins, 1993 ).

Acknowledgements We are indebted to Wellcome Biotechnology for the gift of recombinant h u m a n tPA, and to Peter Dean for supplying ascites fluid containing Antib o d y WA3. This work was supported by a project grant from the Agriculture and F o o d Research Council. Dr. W. Campbell is supported by the Wellcome Trust.

References Ayub, M., Jenkins, N. and White, J.O., 1990. Absence of specific cell surface binding of tissue plasminogen activator in uterine cells. J. Mol. Endocrinol., 5: 7-14. Bicsak, T.A. and Hseuh, A.J.W., 1989. Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. J. Biol. Chem., 264: 630-634. Burnette, W.N., 1980. Western blotting; electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem., 112: 195-203. Campbell, A.M., 1986. In: R.H. Burdon and P.H. van Knippenberg (Editors), Laboratory Techniques in Biochemistry and Molecular Biology -- Monoclonal Antibody Production. Elsevier Amsterdam, pp. 71-73. Chohan, I.S., Vermylen, J., Singh, I., Balakrishnan, K. and Verstraete, M., 1975. Sodium acetate buffer. Diluent of choice in the clot lysis time technique. Thromb. Diath. Haemorrh.. 33: 226-229.

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Coleman, P.L. and Green, G.D.J., 1981. A coupled photometric assay for plasminogen activator. In: L. Lorand (Editor), Methods in Enzymology. Vol. 80. Academic Press, New York. Coombs, R.J. and Jenkins, N., 1988. Characterisation of a plasminogen activator inhibitor induced by glucocorticoids in immature bovine sertoli cell-enriched cultures. J. Endocrinol., 117: 69-74. Coombs, R.J., Ellison, J., Woods, A. and Jenkins, N., 1988. Only tissue type plasminogen activator is secreted by immature bovine Sertoli cell-enriched cultures. J. Endocrinol., 117: 6367. Dano, K., Andreasen, P.A., Grondahl-Hansen, J., Kristensen, P., Nielsen, L.S. and Skriver, L., 1985. Plasminogen activators, tissue degradation and cancer. Adv. Cancer Res., 44:139166. Ellison, J.D. and Jenkins, N., 1989. Regulation of plasminogen activator secretion in Sertoli cells of the calf testis. Anim. Reprod. Sci., 20: 1-10. Erickson, P.A., Mekman, C. and Loskutoff, D., 1985. The primary plasminogen activator inhibitors in endothelial cells, platelets, serum and plasma are immunologically related. Proc. Natl. Acad. Sci. USA, 82: 8710-8714. Gefter, M.L., Margulies, D.H. and Scharff, M.D., 1977. A simple method for polyethylene glycol-promoted hybridisation of mouse myeloma cells. Somatic and Cell. Genet., 3:231-236. Harlow, C.R., Coombs, R.J., Hodges, J.K. and Jenkins, N., 1987. Modulation of plasminogen activation by glucocorticoid hormones in the rat granulosa cell. J. Endocrinol., 114: 207212. Hettle, J.A., Balekian, E., Tung, P.S. and Fritz, I.B., 1988. Rat peritubular cells in culture secrete an inhibitor ofplasminogen activator activity. Biol. Reprod., 34: 359-372. Holvoet, P., Lijnen, H.R. and Collen, D., 1986. Characterisation of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies. Eur. J. Biochem., 158: 173-177. Howard, E.W. and Knauer, D.J., 1986. Human protease nexin- 1: further characterization using a highly specific polyclonal antibody. J. Biol. Chem., 261: 648-689. Jenkins, N., 1987. Plasminogen activators and reproduction. Bibliogr. Reprod., 49: A1-A8. Jenkins, N. and Ellison, J.D., 1986. Corticosteroids suppress plasminogen activation in the bovine Sertoli cell. J. Endocrinol., 108: R1-R3. Kruithof, E.K.O., Vassalli, J.D., Schleuning, W.D., Mattaliano, R.J. and Bachmann, F., 1986. Purification and characterization of a plasminogen activator from the histiocytic lymphoma line U-937. J. Biol. Chem., 261:11207-11213. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Leonard, M. and Jenkins, N., 1993. Hormonal control of plasminogen activation in bovine gonadal cells: investigations using domain-specific monoclonal antibodies. Anim. Reprod. Sci., 34: 31-41. MacGregor, I.R., Micklem, L.R., James, K. and Pepper, D.S., 1985. Characterisation of epitopes on human tissue plasminogen activator recognised by a group of monoclonal antibodies. Thromb. Haemostasis, 53: 45-50. Mahmoud, M. and Gaffney, P.J., 1985. Bioimmunoassay (BIA) of tissue plasminogen activator (tPA) and its specific inhibitor. Thromb. Haemostasis, 53: 356-359. Ny, T., Bjersing, L., Hsueh, A.J.W. and Loskutoff, D.J., 1985. Cultured granulosa cells produce two plasminogen activators and an anti-activator, each regulated differently by gonadotrophins. Endocrinology, 116: 1666-1668. Ouchterlony, O., 1958. Diffusion-in-gel methods for immunological analysis. In: P. Kalbs (Editor), Progress in Allergy. Vol. 5. Karger, New York, pp. 1-78. Pennica, D., Holmes, W.E., Kohr, W.J., Harkins, R.N., Vehar, G.A., Ward, C.A., Bennett, W.F., Yelveron, E., Seeburg, P.H., Heyneker, H.L., Goeddel, D.V. and Collen, D., 1983. Cloning

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