Veterinary Immunology and Imrnunopathology, 25 (1990) 37-46 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
37
Production and Characterization of Monoclonal Antibodies Specific for B o v i n e Gamma-Interferon P.R. WOOD 1, J.S. ROTHEL 1, P.G.D. McWATERS i and S.L. JONES 2
ICSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, PO Parkville, Vic. 3052 (Australia) 2Cornrnonwealth Serum Laboratories, 45 Poplar Rd., Parkville, Vic. 3052 (Australia) (Accepted 24 October 1989)
ABSTRACT Wood, P.R., Rothel, J.S.,McWaters, P.G.D. and Jones, S.L.,1990. Production and characterization of monoclonal antibodies specificfor bovine gamma-interferon. Vet. Irnrnunol. Irnrnunopathol., 25: 37-46. Nine stablehybridoma celllineswere establishedwhich secretedspecificmonoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-7). Specificbinding of each of the M A b s to recombinant BoIFN-7 (rBoIFN-7) was demonstrated in an indirectELISA, whilst none of the M A b s bound to rBoIFN-~ or rBoIFN-p. In a Western blot the M A b s reacted with the 16 kDa and 32 kDa polypeptidespresent in rBoIFN-7 preparations.Competitive ELISA's showed that four M A b s bound to one epitope on rBoIFN-7, and the other five M A b s bound to a separate epitope. T w o MAbs, each recognising differentepi~pes, were shown to neutralisethe anti-viralactivity of naturalBoIFN-7.
INTRODUCTION
Gamma-interferon is a potent modulator of a wide variety of immune responses (Trinchieri and Perussia, 1985). For bovines the major interest has been in the alpha and beta-interferons and their possible role as antiviral agents (Bielefeldt et al., 1984; Babiuk et al., 1985; GiUespie et al., 1985; Fulton et al., 1986). With the availability of recombinant bovine gamma-interferon (rBoIFN-~ Cerretti et al., 1986; Czarniecki et al., 1986) it has been possible to accurately study the effects of this molecule on various bovine cells (Steinbeck et al., 1986). The release of gamma-interferon by T-cells in response to specific antigen has also been a useful method for measuring cellular responses in bovines (Babiuk and Rouse, 1976; Emery et al., 1988; Townsend et al., 1988; Wood et al., 1989). In analysing the role of gamma-interferon in bovine systems we have produced monoclonal antibodies against rBoIFN-7. We have now incorporated 0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
38
P.R. WOOD ET AL.
these monoclonals into an enzyme immunoassay for BoIFN-y, which we are using for the detection of tuberculosis in cattle (Rothel ~t al., 1990). MATERIALSAND METHODS
Interferons Recombinant bovine alpha, beta and gamma-interferon, with specific activities of 1.9 × 108, 4.3 × 106 and 2.2 × 106 U/rag of protein, respectively, and polyclonal rabbit antisera to bovine alpha-interferon were obtained from Ciba Geigy Corporation (Switzerland). Plasma containing natural bovine gammainterferon was produced from whole blood cultures using the method described by Wood et al. (1989).
Culture medium Complete medium. Dulbecco's modification of Eagle's medium (Flow Laboratories, Australia) was supplemented with 20 mM HEPES, 9 mM sodium bicarbonate, 2 mM L-glutamine, 50/~/2-mercaptoethanol, 100 IU/ml penicillin, 100/lg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Flow Laboratories). Fusion medium. Complete medium contained 20% FCS, 100/d~ hypoxanthine, 10/tM aminopterin and 30/tM thymidine. Production of monoclonal antibodies Female, 12-week-old BALB/c mice were injected sub-cutaneously with 100 /~g of rBoIFN-7 emulsified in 0.1 ml of Freund's complete adjuvant (oil:water, 1 : 1 ). Subsequent sub-cutaneous injections of 50/~g of antigen emulsified in 0.1 ml of Freund's incomplete adjuvant were given 21 and 28 days after the initial injection. Four days prior to fusion, mice were injected intra-peritonealy with 100/~g of rBoIFN-? in normal saline. Spleen cells from an immunised mouse were fused with the mouse myeloma cell line NS1/1-Ag4-1 using polyethylene glycol 4000 (Merck, Darmstadt, F.R.G. ) by the method described by Hewish et al. (1984). The fused cells were plated out in six 96-well tissue culture trays (Nunclon, Nunc, Denmark) in 100/ll/well of fusion medium. Hybridomas were gradually weaned onto complete medium within 3 weeks of the fusion. Supernates from wells showing positive hybridoma growth were screened for antibodies to rBoIFN-7 using the enzyme linked immunosorbent assay (ELISA) described. Antibody-secreting hybridomas were cloned twice by limiting dilution. Monoclonal antibodies were
MONOCLONAL ANTIBODIES SPECIFIC FOR BOVINE GAMMA-INTERFERON
39
isotyped using a commercially available mouse MAb isotyping kit (Misotest; Commonwealth Serum Laboratories, Parkville, Australia). For the production of ascites fluid, BALB/c mice were injected intraperitonealy with one ml of 2,6,10,14-tetramethylpentadecane (Pristane; Aldrich Chemical Company, U.S.A. ) and after 7-10 days with another 0.5 ml of Pristane followed by approximately 10 v monoclonal hybridoma cells in 0.5 ml of complete medium. Ascites fluid was collected from the peritoneal cavity after 7-14 days, clarified by centrifugation, and Stored frozen until use.
Production of polyclonal antisera to gamma-interferon Three rabbits were injected intra-muscularly with 50 gg of rBoIFN-~ emulsified in Freund's complete adjuvant (1 ml/rabbit). Further injections of 50 /~g emulsified in Freund's incomplete adjuvant were given intra-muscularly on days 30 and 60 after the initial injection. Animals were bled 10-14 days after the third injection and the serum stored frozen.
Screening for antibodies to gamma-interferon by ELISA Recombinant gamma-interferon was bound to 96-well microtitre plates (Nunc Immuno II ) by incubating 0.1/~g per well in 100 gl of 50 mM carbonate buffer (pH 9.6) for 20 h at 20°C. The plates were then post-coated (1 h at 20°C) with 100 gl per well of phosphate buffered saline (PBS; 0.9% (w/v), pH 7.2 ) containing 1% skim milk powder. After 4 washes with phosphate buffered saline containing 0.05% Tween 20 (PBST), 100 #1 of supernate from fusion wells showing hybridoma growth was added to the wells for 1 h at 20 ° C. The plates were then washed 4 times with PBST before the addition of 100 gl per well of a 1/1000 dilution of horseradish peroxidase conjugated sheep antimouse IgG (Silenus, Australia) in P B S G d ~ 1 h at 20°C. Plates were washed 4 times with PBST and 100 #1 of tetra-methyl benzidine (TMB) substrate (Bos et al., 1981) added to each well for 30 rain at 20°C before the reaction was stopped by the addition of 50 gl of 0.5 M H~SO4 per well and the absorbance read at 450 nm.
Horseradish peroxidase conjugation of MAbs Nine MAbs (IFN-1-9) were purified from ascites fluid on a Sepharose-Protein A (Pharmacia, Uppsala, Sweden) column, according to manufacturer's directions, before conjugation to horseradish peroxidase (HRP) by the method of Wilson and Nakane 1978.
40
P.R. WOOD ET AL.
Competitive ELISA Microtitre plates were coated with rBoIFN-7 and post-coated as described above. Culture supernate (100/~I) from the nine MAbs was incubated at a set dilution in individual wells for I h at 20 ° C. After 4 washes in PBST, 100/11 of the IFN MAb-HRP conjugates, diluted in P B S T to a predetermined working concentration, was added to the wells, incubated for I h at 20°C before a further 4 washes with PBST. T M B substrate was added to each well (100/~1), incubated for 30 min at 20 °C before the reaction was stopped and read as above.
Specificity of monoclonal and polyclonal antibodies The specificity of the monoclonal and polyclonal antisera was tested by indirect ELISA using recombinant bovine alpha, beta and gamma-interferon as antigens. The method used was basically as described for screening hybridoma supernates with the exception that antisera were titred out by serial doubling dilutions, and goat anti-rabbit IgG-HRP conjugate (Sigma, U.S.A. ) was used to detect the rabbit polyclonal antisera. Titres were determined from an absorbance (O.D. 450 n m ) of 0.30. This was > 2 standard deviations above control wells containing all reagents except antisera to gamma-interferon. Recombinant gamma-interferon (2/1g/well) was electrophoresed down a 15% vertical slab sodium dodecyl sulphate discontinuous polyacrylamide gel as described by Laemmli (1970). Proteins were transferred overnight to nitrocellulose using the electroblotting method described by Towbin et al. (1979) and the nitrocellulose probed with MAbs IFN-2 and IFN-9 and with the three polyclonal antisera using the method of Wood et al. (1988).
Effect of MAbs on anti-viral activity of gamma-interferon Monoclonal antibodies IFN-2 and 9 along with an unrelated monoclonal antibody of the IgG1 isotype were tested for their ability to block the anti-viral activity of natural BoIFN-7 in the bio-assay described by Wood et al. (1989). Briefly, culture supernate from the two monoclonals was incubated with bovine plasma containing natural BoIFN-7 for 1 h at 37°C before titration in microtitre plates (Nunclon) over a confluent layer of bovine kidney cells (MDBK). After 24 h incubation at 37°C the plates were washed and 104 p.f.u. of Semliki Forest Virus added to each well for a further 48 h before again washing and staining of the MDBK cells with the vital stain neutral red.
MONOCLONAL ANTIBODIESSPECIFICFORBOVINEGAMMA-INTERFERON
41
RESULTS
Production of monoclonal antibodies to bovine rlFN One fusion was performed and wells which showed positive growth of hybridomas were screened for antibodies to rBoIFN-~ by an indirect ELISA. Of the 576 original culture weUs, 87 displayed hybrid growth, of these, 34 hybridomas secreted antibodies to rBoIFN-7. Nine of the positive hybridomas were cloned twice by limiting dilution and the resulting MAbs were termed IFN-1-9. All nine of the MAbs were shown to be of the IgG1 isotype. Titration of ascites fluid in ELISA Ascites fluid representing each of the nine MAbs and the three polyclonal antisera were titrated against rBoIFN-~ in an indirect ELISA. All nine MAbs had titres in excess of 400 000 and the polyclonal antisera demonstrated titres of > 100 000 (Fig. 1). Competitive ELISA The nine MAbs were purified from ascites fluid and conjugated to HRP. Each of the conjugates were subsequently used at a dilution of 1/100. Competition ELISA results (Table 1 ) showed that MAbs IFN-1, 2, 5 and 6 recognise one epitope on rBoIFN-~, and MAbs IFN-3, 4, 7, 8 and 9 recognise a different epitope. 2.0E--
>,
--B
1.5-
C "0
co
1.0-
0
\o
0.5-
---~/
0'.6
/s
2o'00
Dilution ( r e c i p r o c a l x 10 -3)
Fig. 1. Titration of MAbs IFN-1, 2, 3 and 9 polyclonal antisera RAB-1 in an indirect ELISA against rBoIFN-~. IFN-1, 2, 3, 9 and RAB-1 are O, &, O, D, W, respectively.
42
P.R. WOOD ET AL.
Specificity of monoclonal antibodies Ascites fluid from IFN-1, 2, 3 and 9, recognising two different epitopes on rBoIFN-7, and the four polyclonal antisera (three directed against rBoIFN-7 and one directed against rBoIFN-~) were titrated in an ELISA against recombinant bovine alpha, beta and gamma-interferon (Table 2). All ascites fluids had titres of > 400 000 against rBoIFN-7 and < 25 against the other two antigens. The three polyclonal rabbit sera to rBoIFN-7 had similar specificity to the MAbs. Polyclonal rabbit antisera to bovines alpha-interferon was used as a positive control for the homologous antigen (titre of 580 000). Antisera to BoIFN-fl was not available for this study. Western blotting of rBoIFN-7 with the three polyclonal antisera and two TABLE 1 Competition E L I S A between the monoclonal antibodies to bovine gamma-interferon
HRPMAb IFN-1 IFN-2 IFN-3 IFN-4 IFN-5 IFN-6 IFN-7 IFN-8 IFN-9
Blocking M A b NIL
IFN-1
IFN-2
IFN-5
IFN-6
IFN-3
IFN-4
IFN-7
IFN-8
IFN-9
266 228 338 464 274 206 258 234 193
0 0 10 53 316 319 327 302 301
0 0 0 0 336 289 317 311 289
0 0 5 30 289 262 264 237 247
0 0 28 0 277 206 288 245 251
375 300 376 495 0 4 78 98 69
334 300 352 473 0 0 62 45 49
370 304 333 431 0 0 1 0 0
339 296 312 468 0 0 11 0 19
350 306 394 457 0 2 0 0 0
Values shown are optical densities X 103. TABLE 2 E L I S A titres of monoclonal a n d polyclonal a n t i s e r a against r B o I F N - a , -fl, -7 Antisera
IFN-1 IFN-2 IFN-3 IFN-9 RAB-1 RAB-2 RAB-3 Anti-IFN-a
E L I S A antigen rBoIFN-a
rBoIFN-fl
rBoIFN-7
< 25 < 25 < 25 < 25 < 25 < 25 < 25 580 000
< 25 < 25 < 25 < 25 < 25 < 25 < 25 210
>2 2 >2 >2
000 450 000 000 150 265 170
000 000 000 000 000 000 000 < 25
MONOCLONALANTIBODIESSPECIFICFORBOVINEGAMMA-INTERFERON
43
32 k D a -
16 kDa --
MAb 2
MAb9
RAb 1
RAb 2
RAb3
Fig. 2. Western blot of recombinant bovine gamma-interferon probed with two MAbs (IFN-2 and 9) and three polyclonal rabbit antisera.
MAbs (IFN-2 and 9) showed that all antisera recognised two bands of approximate molecular weights 16 kDa and 32 kDa {Fig. 2).
Inhibition of antiviral activity Ascites fluid from both MAbs were titred in the biological assay and IFN-2 and 9 blocked antiviral activity at dilutions of 1/128 and 1/131 000, respectively. The control monoclonal antibody demonstrated no blocking of antiviral activity. Untreated BoIFN-7 still had antiviral activity at a dilution of 1/512. DISCUSSION
This paper describes the production and characterisation of the first monoclonal antibodies specific for BoIFN-~. These MAbs were produced by fusion of NS-1 cells with spleen cells from mice immunised with rBoIFN-~. Two separate epitopes on the BoIFN-? molecule were recognised by these MAbs which bound to both recombinant and natural BoIFN-7. Similar MAbs have been produced to human IFN-~ (Wang et al., 1984; Le et al., 1984) but not all of those MAbs bound to both natural and recombinant IFN-7 (Ichimori et al., 1985). In Western blots of rBoIFN-~, IFN-2 and 9 bound to both a 16 kDa and a 32 kDa band. This concurs with the finding of Cerretti et al. (1986) that BoIFN7 occurs as both a monomer and a dimer. It has also been shown that human
44
P.R. WOODET AL.
IFN-y exists as a dimer in solution (Yip et al., 1982; Favre et al., 1989). The recognition by these rBoIFN-y MAbs of at least two separate epitopes on the BoIFN-y molecule, parallels the finding that monoclonals to human IFN-y also recognise multiple epitopes (Wang et al., 1984; Van der Meide et al., 1995). This recognition of multiple epitopes has allowed the development of a sensitive immunoassay for BoIFN-7 (Rothel et al., 1990) similar to those produced for human IFN-y (Chang et al., 1984; Tanaka et al., 1985; Van der Meide et al., 1985; Favre et al., 1989). The finding that IFN-2 and 9 neutralised the biological activity of natural BoIFN-y will make them particularly useful for examining the biological function of BoIFN-y in a wide variety of assay systems. BoIFN-y has been shown to play an important role in the generation of cytotoxic cells in cattle infected with bovine herpesvirus (Campos et al., 1989). In the murine system, IFN-y controls the isotype of immunoglobulin secreted during an in vivo response to Brucella abortus (Finkelman et al., 1988). We are interested in using these monoclonals to examine the role of BoIFN-y in the induction of the immune response to a variety of infectious agents in cattle. ACKNOWLEDGEMENTS
This work was supported by grants from the Australian Meat and Livestock Research and Development Corporation and the National Brucellosis and Tuberculosis Eradication Campaign. The authors also gratefully acknowledge the assistance of J. Cox from the Commonwealth Serum Laboratories.
REFERENCES Babiuk, L.A. and Rouse, B.T., 1976. Immune interferon production by lymphoid cells: role in the inhibition of herpesviruses. Infect. Immun., 13: 1567-1578. Babiuk, L.A., Bielefeldt Ohmann, H., Gifford, G., Czarniecki, C.W., Scialli, V.T. and Hamilton, E.B., 1985. Effect of bovine ~ interferon on bovine herpesvirus type 1-induced respiratory disease. J. Gen. Virol., 66: 2383-2394. Bielefeldt Ohmann, H., Gilchrist, J.E. and Babiuk, L.A., 1984. Effect of recombinant DNA-produced bovine interferon alpha (BoIFN-~I) on the interaction between bovine alveolar macrophages and bovine herpesvirus type 1. J. Gen. Virol., 65: 1487-1495. Bos, E.S., van der Doelen, A.A., van Rooy, N. and Schuurs, A.H.W.M., 1981.3,3',5,5'-Tetramethylbenzidine as an Ames-test negative chromogen for horse-radish peroxidase in enzymeimmunoassays. J. Immunoassay, 2: 187-204. Campos, M., Bieleveldt Ohman, H., Hutchings, D., Rapin, N., Babiuk, L.A. and Lawman, M.J.P., 1989. Role of interferon-y in inducing cytotoxicity of peripheral blood mononuclear leukocytes to bovine herpesvirus type 1 (BHV-1)-infected cells. Cell Immunol., 120: 259-269. Cerretti, D.P., McKereghan, K., Larsen, A., Cosman, D., Gillis, S. and Baker, P.E., 1986. Cloning, sequence, and expression of bovine interferon-7. J. Immunol., 136: 4561-4564. Chang, T.W., McKinney, S., Liu, V., Kung, P.C., Vilcek, J. and Le, J., 1984. Use of monoclonal
M O N O C L O N A L ANTIBODIES SPECIFIC FOR BOVINE GAMMA-INTERFERON
45
antibodies as sensitive and specific probes for biologically active human y-interferon. Proc. Natl. Acad. Sci. U.S.A., 81: 5219-5222. Czarniecki, C.W., Hamilton, E.B., Fennie, C.W. and Wolf, R.L., 1986. In vitro biological activities of Escherichia coil-derived bovine interferons-a, -fl and -?. J. Interferon Res., 6: 29-37. Emery, D.L., Dufty, J.H. and Wood, P.R., 1988. An analysis of cellular proliferation and synthesis of lymphokines and specific antibody in vitro by leucocytes from immunized cattle. Vet. Immunol. Immunopathol., 18: 67-80. Favre, C., Wijdenes, J., CabriUat, H., Djossou, 0., Banchereau, J. and de Vries, J.E., 1989. Epitope mapping of recombinant human gamma inteferon using monoclonal antibodies. Mol. Immunol., 26: 17-25. Finkelman, F.D., Katona, I.M., Mossmsn, T.R. and Coffman, R.L., 1988. IFN-? regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol., 140: 1022-1027. Fulton, R.W., Burge, L.J. and McCraken, J.S., 1986. Effect of recombinant DNA-derived bovine and human interferons on replication of bovine herpesvirus-1, parainfluenza-3 and respiratory syncytial viruses. Am. J. Vet. Res., 47: 751-753. Gillespie, J.H., Robson, D.S., Scott, F.W. and Schiff, E.I., 1985. In vitro protective effect of bacteria-derived bovine alpha interferon I1 against selected bovine viruses. J. Clin. Microbiol., 22: 912-914. Hewish, D.R., Robin,son, C.P. and Sparrow, L.G., 1984. Monoclonal antibody studies of a-keratin low sulfur proteins. Aust. J. biol. Sci., 37: 17-23. Ichimori, Y., Kurokawa, T., Honda, S., Suzuki, N., Wakimasu, M. and Tsukamoto, K., 1985. Monoclonal antibodies to human interferon-?. I. Antibodies to a synthetic carboxyl-terminal peptide. J. Immunol. Methods, 80: 55-66. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Le, J., Barrowclough, B.S. and Vilcek, J., 1984. Monoclonal antibodies to human immune interferon and their application for affinity chromatography. J. Immunol. Methods, 69: 61-70. Rothel, J.S., Jones, S.L., Corner, L.A., Cox, J.C. and Wood, P.R., 1990. A sandwich enzyme immunoassay for bovine interferon-? and its use for the detection of tuberculosis in cattle. Aust. Vet. J., 67 (in press). Steinbeck, M.J., Roth, J.A. and Kaeberle, M.L., 1986. Activation of bovine neutrophils by recombinant interferon-?. Cell. Immunol., 98: 137-144. Tanake, E., Imai, M., Usada, S., Tachibana, K., Okamoto, H., Ohike, Y., Nakamura, T., Miyakawa, Y. and Mayumi, M., 1985. A two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies. J. Immunol. Methods, 77: 275-282. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Townsend, J., Duffus, W.P.H. and Williams, D.L., 1988. Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and P13 viruses. Res. Vet. Sci., 45: 198-205. Trinchieri, G. and Perussia, B., 1985. Immune interferon: a pleiotropic lymphokine with multiple effects. Immunol. Today, 6: 131-136. Van der Meide, P.H., Dubbeld, M. and Schellekens, H., 1985. Monoclonal antibodies to human immune interferon and their use in a sensitive solid-phase ELISA. J. Immunol. Methods, 79: 293-305. Wang, C.Y., Bushkin, Y., Chen, P-D., Platsoucas, C.D. and Long, C., 1984. Preparation and characterization of monoclonal antibodies directed at epitopes of human IFN-y. Hybridoma, 3: 321-332. Wilson, M.B. and Nakane, P.K., 1978. In: W. Knapp, K. Holubar and G. Wick (Editors), Ira-
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P.R. WOODET AL.
munofluorescence and Related Staining Techniques. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 215-224. Wood, P.R., Ripper, J., Radford, A.J., Bundesen, P.G., Rylatt, D.B., Cottis, L.E., John, M. and Plackett, P., 1988. Production and characterization of monoclonal antibodies specific for Mycobacterium bovis. J. Gen. Microbiol., 134: 2599-2604. Wood, P.R., Kopsidas, K., Milner, A.R., Hill, J., Gill, I., Webb, R,, Mack, W.N. and Coates, K., 1989. The development of an in vitro cellular assay for Johne's disease in cattle. In: A.R. Milner and P.R. Wood (Editors), Johne's Disease: Current Trends in Research, Diagnosis and Management. CSIRO Australia East, Melbourne, pp. 164-167. Yip, Y.K., Barrowclough, B.S., Urban, C. and Vilcek, J., 1982. Molecular weight of human gamma interferon is similar to that of other human interferons. Science (Washington, DC), 215: 411413.
37
Production and Characterization of Monoclonal Antibodies Specific for B o v i n e Gamma-Interferon P.R. WOOD 1, J.S. ROTHEL 1, P.G.D. McWATERS i and S.L. JONES 2
ICSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, PO Parkville, Vic. 3052 (Australia) 2Cornrnonwealth Serum Laboratories, 45 Poplar Rd., Parkville, Vic. 3052 (Australia) (Accepted 24 October 1989)
ABSTRACT Wood, P.R., Rothel, J.S.,McWaters, P.G.D. and Jones, S.L.,1990. Production and characterization of monoclonal antibodies specificfor bovine gamma-interferon. Vet. Irnrnunol. Irnrnunopathol., 25: 37-46. Nine stablehybridoma celllineswere establishedwhich secretedspecificmonoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-7). Specificbinding of each of the M A b s to recombinant BoIFN-7 (rBoIFN-7) was demonstrated in an indirectELISA, whilst none of the M A b s bound to rBoIFN-~ or rBoIFN-p. In a Western blot the M A b s reacted with the 16 kDa and 32 kDa polypeptidespresent in rBoIFN-7 preparations.Competitive ELISA's showed that four M A b s bound to one epitope on rBoIFN-7, and the other five M A b s bound to a separate epitope. T w o MAbs, each recognising differentepi~pes, were shown to neutralisethe anti-viralactivity of naturalBoIFN-7.
INTRODUCTION
Gamma-interferon is a potent modulator of a wide variety of immune responses (Trinchieri and Perussia, 1985). For bovines the major interest has been in the alpha and beta-interferons and their possible role as antiviral agents (Bielefeldt et al., 1984; Babiuk et al., 1985; GiUespie et al., 1985; Fulton et al., 1986). With the availability of recombinant bovine gamma-interferon (rBoIFN-~ Cerretti et al., 1986; Czarniecki et al., 1986) it has been possible to accurately study the effects of this molecule on various bovine cells (Steinbeck et al., 1986). The release of gamma-interferon by T-cells in response to specific antigen has also been a useful method for measuring cellular responses in bovines (Babiuk and Rouse, 1976; Emery et al., 1988; Townsend et al., 1988; Wood et al., 1989). In analysing the role of gamma-interferon in bovine systems we have produced monoclonal antibodies against rBoIFN-7. We have now incorporated 0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
38
P.R. WOOD ET AL.
these monoclonals into an enzyme immunoassay for BoIFN-y, which we are using for the detection of tuberculosis in cattle (Rothel ~t al., 1990). MATERIALSAND METHODS
Interferons Recombinant bovine alpha, beta and gamma-interferon, with specific activities of 1.9 × 108, 4.3 × 106 and 2.2 × 106 U/rag of protein, respectively, and polyclonal rabbit antisera to bovine alpha-interferon were obtained from Ciba Geigy Corporation (Switzerland). Plasma containing natural bovine gammainterferon was produced from whole blood cultures using the method described by Wood et al. (1989).
Culture medium Complete medium. Dulbecco's modification of Eagle's medium (Flow Laboratories, Australia) was supplemented with 20 mM HEPES, 9 mM sodium bicarbonate, 2 mM L-glutamine, 50/~/2-mercaptoethanol, 100 IU/ml penicillin, 100/lg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Flow Laboratories). Fusion medium. Complete medium contained 20% FCS, 100/d~ hypoxanthine, 10/tM aminopterin and 30/tM thymidine. Production of monoclonal antibodies Female, 12-week-old BALB/c mice were injected sub-cutaneously with 100 /~g of rBoIFN-7 emulsified in 0.1 ml of Freund's complete adjuvant (oil:water, 1 : 1 ). Subsequent sub-cutaneous injections of 50/~g of antigen emulsified in 0.1 ml of Freund's incomplete adjuvant were given 21 and 28 days after the initial injection. Four days prior to fusion, mice were injected intra-peritonealy with 100/~g of rBoIFN-? in normal saline. Spleen cells from an immunised mouse were fused with the mouse myeloma cell line NS1/1-Ag4-1 using polyethylene glycol 4000 (Merck, Darmstadt, F.R.G. ) by the method described by Hewish et al. (1984). The fused cells were plated out in six 96-well tissue culture trays (Nunclon, Nunc, Denmark) in 100/ll/well of fusion medium. Hybridomas were gradually weaned onto complete medium within 3 weeks of the fusion. Supernates from wells showing positive hybridoma growth were screened for antibodies to rBoIFN-7 using the enzyme linked immunosorbent assay (ELISA) described. Antibody-secreting hybridomas were cloned twice by limiting dilution. Monoclonal antibodies were
MONOCLONAL ANTIBODIES SPECIFIC FOR BOVINE GAMMA-INTERFERON
39
isotyped using a commercially available mouse MAb isotyping kit (Misotest; Commonwealth Serum Laboratories, Parkville, Australia). For the production of ascites fluid, BALB/c mice were injected intraperitonealy with one ml of 2,6,10,14-tetramethylpentadecane (Pristane; Aldrich Chemical Company, U.S.A. ) and after 7-10 days with another 0.5 ml of Pristane followed by approximately 10 v monoclonal hybridoma cells in 0.5 ml of complete medium. Ascites fluid was collected from the peritoneal cavity after 7-14 days, clarified by centrifugation, and Stored frozen until use.
Production of polyclonal antisera to gamma-interferon Three rabbits were injected intra-muscularly with 50 gg of rBoIFN-~ emulsified in Freund's complete adjuvant (1 ml/rabbit). Further injections of 50 /~g emulsified in Freund's incomplete adjuvant were given intra-muscularly on days 30 and 60 after the initial injection. Animals were bled 10-14 days after the third injection and the serum stored frozen.
Screening for antibodies to gamma-interferon by ELISA Recombinant gamma-interferon was bound to 96-well microtitre plates (Nunc Immuno II ) by incubating 0.1/~g per well in 100 gl of 50 mM carbonate buffer (pH 9.6) for 20 h at 20°C. The plates were then post-coated (1 h at 20°C) with 100 gl per well of phosphate buffered saline (PBS; 0.9% (w/v), pH 7.2 ) containing 1% skim milk powder. After 4 washes with phosphate buffered saline containing 0.05% Tween 20 (PBST), 100 #1 of supernate from fusion wells showing hybridoma growth was added to the wells for 1 h at 20 ° C. The plates were then washed 4 times with PBST before the addition of 100 gl per well of a 1/1000 dilution of horseradish peroxidase conjugated sheep antimouse IgG (Silenus, Australia) in P B S G d ~ 1 h at 20°C. Plates were washed 4 times with PBST and 100 #1 of tetra-methyl benzidine (TMB) substrate (Bos et al., 1981) added to each well for 30 rain at 20°C before the reaction was stopped by the addition of 50 gl of 0.5 M H~SO4 per well and the absorbance read at 450 nm.
Horseradish peroxidase conjugation of MAbs Nine MAbs (IFN-1-9) were purified from ascites fluid on a Sepharose-Protein A (Pharmacia, Uppsala, Sweden) column, according to manufacturer's directions, before conjugation to horseradish peroxidase (HRP) by the method of Wilson and Nakane 1978.
40
P.R. WOOD ET AL.
Competitive ELISA Microtitre plates were coated with rBoIFN-7 and post-coated as described above. Culture supernate (100/~I) from the nine MAbs was incubated at a set dilution in individual wells for I h at 20 ° C. After 4 washes in PBST, 100/11 of the IFN MAb-HRP conjugates, diluted in P B S T to a predetermined working concentration, was added to the wells, incubated for I h at 20°C before a further 4 washes with PBST. T M B substrate was added to each well (100/~1), incubated for 30 min at 20 °C before the reaction was stopped and read as above.
Specificity of monoclonal and polyclonal antibodies The specificity of the monoclonal and polyclonal antisera was tested by indirect ELISA using recombinant bovine alpha, beta and gamma-interferon as antigens. The method used was basically as described for screening hybridoma supernates with the exception that antisera were titred out by serial doubling dilutions, and goat anti-rabbit IgG-HRP conjugate (Sigma, U.S.A. ) was used to detect the rabbit polyclonal antisera. Titres were determined from an absorbance (O.D. 450 n m ) of 0.30. This was > 2 standard deviations above control wells containing all reagents except antisera to gamma-interferon. Recombinant gamma-interferon (2/1g/well) was electrophoresed down a 15% vertical slab sodium dodecyl sulphate discontinuous polyacrylamide gel as described by Laemmli (1970). Proteins were transferred overnight to nitrocellulose using the electroblotting method described by Towbin et al. (1979) and the nitrocellulose probed with MAbs IFN-2 and IFN-9 and with the three polyclonal antisera using the method of Wood et al. (1988).
Effect of MAbs on anti-viral activity of gamma-interferon Monoclonal antibodies IFN-2 and 9 along with an unrelated monoclonal antibody of the IgG1 isotype were tested for their ability to block the anti-viral activity of natural BoIFN-7 in the bio-assay described by Wood et al. (1989). Briefly, culture supernate from the two monoclonals was incubated with bovine plasma containing natural BoIFN-7 for 1 h at 37°C before titration in microtitre plates (Nunclon) over a confluent layer of bovine kidney cells (MDBK). After 24 h incubation at 37°C the plates were washed and 104 p.f.u. of Semliki Forest Virus added to each well for a further 48 h before again washing and staining of the MDBK cells with the vital stain neutral red.
MONOCLONAL ANTIBODIESSPECIFICFORBOVINEGAMMA-INTERFERON
41
RESULTS
Production of monoclonal antibodies to bovine rlFN One fusion was performed and wells which showed positive growth of hybridomas were screened for antibodies to rBoIFN-~ by an indirect ELISA. Of the 576 original culture weUs, 87 displayed hybrid growth, of these, 34 hybridomas secreted antibodies to rBoIFN-7. Nine of the positive hybridomas were cloned twice by limiting dilution and the resulting MAbs were termed IFN-1-9. All nine of the MAbs were shown to be of the IgG1 isotype. Titration of ascites fluid in ELISA Ascites fluid representing each of the nine MAbs and the three polyclonal antisera were titrated against rBoIFN-~ in an indirect ELISA. All nine MAbs had titres in excess of 400 000 and the polyclonal antisera demonstrated titres of > 100 000 (Fig. 1). Competitive ELISA The nine MAbs were purified from ascites fluid and conjugated to HRP. Each of the conjugates were subsequently used at a dilution of 1/100. Competition ELISA results (Table 1 ) showed that MAbs IFN-1, 2, 5 and 6 recognise one epitope on rBoIFN-~, and MAbs IFN-3, 4, 7, 8 and 9 recognise a different epitope. 2.0E--
>,
--B
1.5-
C "0
co
1.0-
0
\o
0.5-
---~/
0'.6
/s
2o'00
Dilution ( r e c i p r o c a l x 10 -3)
Fig. 1. Titration of MAbs IFN-1, 2, 3 and 9 polyclonal antisera RAB-1 in an indirect ELISA against rBoIFN-~. IFN-1, 2, 3, 9 and RAB-1 are O, &, O, D, W, respectively.
42
P.R. WOOD ET AL.
Specificity of monoclonal antibodies Ascites fluid from IFN-1, 2, 3 and 9, recognising two different epitopes on rBoIFN-7, and the four polyclonal antisera (three directed against rBoIFN-7 and one directed against rBoIFN-~) were titrated in an ELISA against recombinant bovine alpha, beta and gamma-interferon (Table 2). All ascites fluids had titres of > 400 000 against rBoIFN-7 and < 25 against the other two antigens. The three polyclonal rabbit sera to rBoIFN-7 had similar specificity to the MAbs. Polyclonal rabbit antisera to bovines alpha-interferon was used as a positive control for the homologous antigen (titre of 580 000). Antisera to BoIFN-fl was not available for this study. Western blotting of rBoIFN-7 with the three polyclonal antisera and two TABLE 1 Competition E L I S A between the monoclonal antibodies to bovine gamma-interferon
HRPMAb IFN-1 IFN-2 IFN-3 IFN-4 IFN-5 IFN-6 IFN-7 IFN-8 IFN-9
Blocking M A b NIL
IFN-1
IFN-2
IFN-5
IFN-6
IFN-3
IFN-4
IFN-7
IFN-8
IFN-9
266 228 338 464 274 206 258 234 193
0 0 10 53 316 319 327 302 301
0 0 0 0 336 289 317 311 289
0 0 5 30 289 262 264 237 247
0 0 28 0 277 206 288 245 251
375 300 376 495 0 4 78 98 69
334 300 352 473 0 0 62 45 49
370 304 333 431 0 0 1 0 0
339 296 312 468 0 0 11 0 19
350 306 394 457 0 2 0 0 0
Values shown are optical densities X 103. TABLE 2 E L I S A titres of monoclonal a n d polyclonal a n t i s e r a against r B o I F N - a , -fl, -7 Antisera
IFN-1 IFN-2 IFN-3 IFN-9 RAB-1 RAB-2 RAB-3 Anti-IFN-a
E L I S A antigen rBoIFN-a
rBoIFN-fl
rBoIFN-7
< 25 < 25 < 25 < 25 < 25 < 25 < 25 580 000
< 25 < 25 < 25 < 25 < 25 < 25 < 25 210
>2 2 >2 >2
000 450 000 000 150 265 170
000 000 000 000 000 000 000 < 25
MONOCLONALANTIBODIESSPECIFICFORBOVINEGAMMA-INTERFERON
43
32 k D a -
16 kDa --
MAb 2
MAb9
RAb 1
RAb 2
RAb3
Fig. 2. Western blot of recombinant bovine gamma-interferon probed with two MAbs (IFN-2 and 9) and three polyclonal rabbit antisera.
MAbs (IFN-2 and 9) showed that all antisera recognised two bands of approximate molecular weights 16 kDa and 32 kDa {Fig. 2).
Inhibition of antiviral activity Ascites fluid from both MAbs were titred in the biological assay and IFN-2 and 9 blocked antiviral activity at dilutions of 1/128 and 1/131 000, respectively. The control monoclonal antibody demonstrated no blocking of antiviral activity. Untreated BoIFN-7 still had antiviral activity at a dilution of 1/512. DISCUSSION
This paper describes the production and characterisation of the first monoclonal antibodies specific for BoIFN-~. These MAbs were produced by fusion of NS-1 cells with spleen cells from mice immunised with rBoIFN-~. Two separate epitopes on the BoIFN-? molecule were recognised by these MAbs which bound to both recombinant and natural BoIFN-7. Similar MAbs have been produced to human IFN-~ (Wang et al., 1984; Le et al., 1984) but not all of those MAbs bound to both natural and recombinant IFN-7 (Ichimori et al., 1985). In Western blots of rBoIFN-~, IFN-2 and 9 bound to both a 16 kDa and a 32 kDa band. This concurs with the finding of Cerretti et al. (1986) that BoIFN7 occurs as both a monomer and a dimer. It has also been shown that human
44
P.R. WOODET AL.
IFN-y exists as a dimer in solution (Yip et al., 1982; Favre et al., 1989). The recognition by these rBoIFN-y MAbs of at least two separate epitopes on the BoIFN-y molecule, parallels the finding that monoclonals to human IFN-y also recognise multiple epitopes (Wang et al., 1984; Van der Meide et al., 1995). This recognition of multiple epitopes has allowed the development of a sensitive immunoassay for BoIFN-7 (Rothel et al., 1990) similar to those produced for human IFN-y (Chang et al., 1984; Tanaka et al., 1985; Van der Meide et al., 1985; Favre et al., 1989). The finding that IFN-2 and 9 neutralised the biological activity of natural BoIFN-y will make them particularly useful for examining the biological function of BoIFN-y in a wide variety of assay systems. BoIFN-y has been shown to play an important role in the generation of cytotoxic cells in cattle infected with bovine herpesvirus (Campos et al., 1989). In the murine system, IFN-y controls the isotype of immunoglobulin secreted during an in vivo response to Brucella abortus (Finkelman et al., 1988). We are interested in using these monoclonals to examine the role of BoIFN-y in the induction of the immune response to a variety of infectious agents in cattle. ACKNOWLEDGEMENTS
This work was supported by grants from the Australian Meat and Livestock Research and Development Corporation and the National Brucellosis and Tuberculosis Eradication Campaign. The authors also gratefully acknowledge the assistance of J. Cox from the Commonwealth Serum Laboratories.
REFERENCES Babiuk, L.A. and Rouse, B.T., 1976. Immune interferon production by lymphoid cells: role in the inhibition of herpesviruses. Infect. Immun., 13: 1567-1578. Babiuk, L.A., Bielefeldt Ohmann, H., Gifford, G., Czarniecki, C.W., Scialli, V.T. and Hamilton, E.B., 1985. Effect of bovine ~ interferon on bovine herpesvirus type 1-induced respiratory disease. J. Gen. Virol., 66: 2383-2394. Bielefeldt Ohmann, H., Gilchrist, J.E. and Babiuk, L.A., 1984. Effect of recombinant DNA-produced bovine interferon alpha (BoIFN-~I) on the interaction between bovine alveolar macrophages and bovine herpesvirus type 1. J. Gen. Virol., 65: 1487-1495. Bos, E.S., van der Doelen, A.A., van Rooy, N. and Schuurs, A.H.W.M., 1981.3,3',5,5'-Tetramethylbenzidine as an Ames-test negative chromogen for horse-radish peroxidase in enzymeimmunoassays. J. Immunoassay, 2: 187-204. Campos, M., Bieleveldt Ohman, H., Hutchings, D., Rapin, N., Babiuk, L.A. and Lawman, M.J.P., 1989. Role of interferon-y in inducing cytotoxicity of peripheral blood mononuclear leukocytes to bovine herpesvirus type 1 (BHV-1)-infected cells. Cell Immunol., 120: 259-269. Cerretti, D.P., McKereghan, K., Larsen, A., Cosman, D., Gillis, S. and Baker, P.E., 1986. Cloning, sequence, and expression of bovine interferon-7. J. Immunol., 136: 4561-4564. Chang, T.W., McKinney, S., Liu, V., Kung, P.C., Vilcek, J. and Le, J., 1984. Use of monoclonal
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45
antibodies as sensitive and specific probes for biologically active human y-interferon. Proc. Natl. Acad. Sci. U.S.A., 81: 5219-5222. Czarniecki, C.W., Hamilton, E.B., Fennie, C.W. and Wolf, R.L., 1986. In vitro biological activities of Escherichia coil-derived bovine interferons-a, -fl and -?. J. Interferon Res., 6: 29-37. Emery, D.L., Dufty, J.H. and Wood, P.R., 1988. An analysis of cellular proliferation and synthesis of lymphokines and specific antibody in vitro by leucocytes from immunized cattle. Vet. Immunol. Immunopathol., 18: 67-80. Favre, C., Wijdenes, J., CabriUat, H., Djossou, 0., Banchereau, J. and de Vries, J.E., 1989. Epitope mapping of recombinant human gamma inteferon using monoclonal antibodies. Mol. Immunol., 26: 17-25. Finkelman, F.D., Katona, I.M., Mossmsn, T.R. and Coffman, R.L., 1988. IFN-? regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol., 140: 1022-1027. Fulton, R.W., Burge, L.J. and McCraken, J.S., 1986. Effect of recombinant DNA-derived bovine and human interferons on replication of bovine herpesvirus-1, parainfluenza-3 and respiratory syncytial viruses. Am. J. Vet. Res., 47: 751-753. Gillespie, J.H., Robson, D.S., Scott, F.W. and Schiff, E.I., 1985. In vitro protective effect of bacteria-derived bovine alpha interferon I1 against selected bovine viruses. J. Clin. Microbiol., 22: 912-914. Hewish, D.R., Robin,son, C.P. and Sparrow, L.G., 1984. Monoclonal antibody studies of a-keratin low sulfur proteins. Aust. J. biol. Sci., 37: 17-23. Ichimori, Y., Kurokawa, T., Honda, S., Suzuki, N., Wakimasu, M. and Tsukamoto, K., 1985. Monoclonal antibodies to human interferon-?. I. Antibodies to a synthetic carboxyl-terminal peptide. J. Immunol. Methods, 80: 55-66. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Le, J., Barrowclough, B.S. and Vilcek, J., 1984. Monoclonal antibodies to human immune interferon and their application for affinity chromatography. J. Immunol. Methods, 69: 61-70. Rothel, J.S., Jones, S.L., Corner, L.A., Cox, J.C. and Wood, P.R., 1990. A sandwich enzyme immunoassay for bovine interferon-? and its use for the detection of tuberculosis in cattle. Aust. Vet. J., 67 (in press). Steinbeck, M.J., Roth, J.A. and Kaeberle, M.L., 1986. Activation of bovine neutrophils by recombinant interferon-?. Cell. Immunol., 98: 137-144. Tanake, E., Imai, M., Usada, S., Tachibana, K., Okamoto, H., Ohike, Y., Nakamura, T., Miyakawa, Y. and Mayumi, M., 1985. A two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies. J. Immunol. Methods, 77: 275-282. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Townsend, J., Duffus, W.P.H. and Williams, D.L., 1988. Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and P13 viruses. Res. Vet. Sci., 45: 198-205. Trinchieri, G. and Perussia, B., 1985. Immune interferon: a pleiotropic lymphokine with multiple effects. Immunol. Today, 6: 131-136. Van der Meide, P.H., Dubbeld, M. and Schellekens, H., 1985. Monoclonal antibodies to human immune interferon and their use in a sensitive solid-phase ELISA. J. Immunol. Methods, 79: 293-305. Wang, C.Y., Bushkin, Y., Chen, P-D., Platsoucas, C.D. and Long, C., 1984. Preparation and characterization of monoclonal antibodies directed at epitopes of human IFN-y. Hybridoma, 3: 321-332. Wilson, M.B. and Nakane, P.K., 1978. In: W. Knapp, K. Holubar and G. Wick (Editors), Ira-
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munofluorescence and Related Staining Techniques. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 215-224. Wood, P.R., Ripper, J., Radford, A.J., Bundesen, P.G., Rylatt, D.B., Cottis, L.E., John, M. and Plackett, P., 1988. Production and characterization of monoclonal antibodies specific for Mycobacterium bovis. J. Gen. Microbiol., 134: 2599-2604. Wood, P.R., Kopsidas, K., Milner, A.R., Hill, J., Gill, I., Webb, R,, Mack, W.N. and Coates, K., 1989. The development of an in vitro cellular assay for Johne's disease in cattle. In: A.R. Milner and P.R. Wood (Editors), Johne's Disease: Current Trends in Research, Diagnosis and Management. CSIRO Australia East, Melbourne, pp. 164-167. Yip, Y.K., Barrowclough, B.S., Urban, C. and Vilcek, J., 1982. Molecular weight of human gamma interferon is similar to that of other human interferons. Science (Washington, DC), 215: 411413.
