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Preparation of monoclonal antibodies against specific gliadin proteins and preliminary investigation of their ability to discriminate cereal cultivars
Journal of Cereal Science 10 (1989) 105-112
Preparation of Monoclonal Antibodies Against Specific Gliadin Proteins and Preliminary Investigation of their Ability to Discriminate Cereal Cultivars* M. R. DAWQODtt, N. K. HOWES§ and W. BUSHUK t
t University
of Manitoba, Food Science Department, Winnipeg, ME, Canada R3T 2N2 and §Agriculture Canada Research Station, Winnipeg, MB, Canada R3T 2M9 Received 19 December 1988
Monoclonal antibodies (MAbs) were prepared against y-gliadin 45 and Ct-gliadin 74 from the common wheat cuHivars Marquis and Marshall, respectively. Murine monoclonal antibodies were prepared and positive clones were detected using ELISA. MAbs prepared against gliadin 45 gave a low reaction in ELISA to extracts of einkorn, rye, barley and some common and durum wheats, and higher reaction to other COmmon and durum wheats. Protein blotting of total gliadins separated by SDS-PAGE showed that MAbs against gliadin 45 bound to one discrete region corresponding to the location of gliadin 45. MAbs prepared against gliadin 74 gave a low reaction to rye, a medium reaction to barley, and a high reaction to all common and durum wheats and einkorn. Protein blotting showed that these MAbs bound to the region corresponding to Ct- and ~-gliadins.
Introduction Electrophoretic patterns of gliadin components separated by polyacrylamide gel electrophoresis (PAGE) have been used for wheat cultivar identification 1 • Some disadvantages of PAGE are that it is time consuming, only a limited number of samples can be tested at one time, acrylamide poses a minor health hazard, and the patterns of protein bands are complex and sometimes difficult to discriminate. Antibodies are widely used for identification and/or detection of very low concentrations of specific antigens. Gliadin proteins have a high degree of homologous amino acid sequences 2 • 3 • Because of this, it is difficult to identify a particular gliadin using polyclonal antibodies 4 • Homogeneous monoclonal antibodies have several advantages over heterogenous polyclonal antibodies; they are of defined isotype and have a greater specificity for closely related antigens. Skerritt et al. 5 reported the preparation of MAbs against a mixture of gliadins. The resulting MAbs were active against many different gliadins as well as prolamins of barley, rice, rye, and rye grass. Freedman et al. 6 established a sandwich ELISA using murine monoclonal antibodies
* Contribution No. 139 of the University of Manitoba, Food Science Department, and No. 1346 of the Agriculture Canada Winnipeg Research Station. t Present address: Cadham Provincial Laboratory, Winnipeg, MB, Canada R3C 3YI; to whom all correspondence should be addressed. 0733-5210/89/050105 + 08 $03.00/0
© 1989 Academic Press Limited
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M. R. DAWOOD ET AL.
prepared against unfractionated wheat gliadin and purified rabbit IgG. Using their test, they were able to detect traces of gliadin in some' gluten-free' food products. Skerritt and Underwood 7 also prepared anti-gliadin (unfractionated) MAbs. Most of their MAbs bound to all gliadin bands separated by SDS-polyacrylamide gel electrophoresis. Some of the antibodies bound to a small group of gliadin proteins. Several investigators were able to produce polyclonal antibodies or monoclonal antibodies prepared against crude whole gliadin or A-gliadin (a fraction of a:-gliadin)8-1o. Different degrees of cross reactivity between the developed antibodies and a:-, p- and y-gliadins were observed. On the other hand, no cross reactivity was reported between anti-gliadin antibodies and extracts of rye, barley or oats. To prepare MAbs against epitopes that are unique to specific gliadin proteins, we investigated the use of purified gliadins for animal immunization. Experimental
Antigen preparation Gliadin bands 45 and 74 (according to the nomenclature of Zillman and Bushuk 1 ) were purified from the common wheat cultivars Marquis and Marshall, respectively, using a two step purification method as described by Howes and Kosmolak l l, except that gliadin bands after PAGE were located, cut out, and eluted with 70% ethanol. The purity of the eluted proteins was confirmed by acid PAGEl. The eluted proteins were freeze-dried then redissolved in double distilled H 2 0. The two bands, each of which may contain more than one component, will be referred to as gliadins 45 and 74. Gliadin 45 is a band in the y-gliadin region while gliadin 74 is in the a-region of the electrophoregram.
Tissue culture and media The P3X63-Ag8.653 (abbreviated as 653) cell line, a mouse myeloma cell line that does not secrete antibodies l2 , was purchased from the American Type Culture Collection (Rockville, MD 20852). RPMI 1640 cell culture medium was purchased from GIBCO/BRL Life Technology Inc. (Burlington, Canada L7P lAI). Complete RPMI 1640 medium is RPMI 1640 supplemented with 10% FCS (fetal calf serum) and penicillin-streptomycin. HT medium is complete RPMI 1640 medium supplemented with 0'1 mM hypoxanthine and 31-4 11M thymidine. HAT medium is HT medium containing O'821lM aminopterine.
Monoclonal antibody production MAbs were prepared as described by Goding l3 with modification. Female BALB/c mice were injected intramuscularly with 50 Ilg purified gliadin 45 or 74 in Freund's complete adjuvant followed by intraperitoneal injection at 4 week intervals. Three days after the last (booster) injection, spleen cells were removed. Mouse hybridomas were generated from the fusion of mouse spleen cells secreting high levels of antibodies against the desired gliadin with the 653 cell line. Cell fusion was carried out using polyethylene glycol 4000. Spleen and myeloma cells were mixed, pelleted by centrifugation and resuspended in HAT medium at a concentration of 5 x 10 5 cells/ml, plated into 96-well tissue culture plates containing feeder layer of normal mouse spleen cells (1 x 10 4 cells/well) that had been plated one day earlier. Hybridoma cells were selected by culturing in HAT medium. Two weeks later, the ceLIs were cultured in HT medium for two weeks followed by complete RPMI 1640 medium. Antibody secreting clones, identified by ELISA, were
MONOCLONAL ANTIBODIES AGAINST GLIADINS
107
subcloned at least twice at 0,5-1'0 celljwell and retested for Ab secretion. Hybridoma cells secreting MAbs that gave a differential reaction in ELISA to extracts of Marquis, Marshall, Benito and Sinton common wheats were propagated in vitro. The four wheat cultivars represent wheats with or without gliadin bands 45 andjor 74. Tissue culture media containing MAbs were aliquoted and stored at -20°C for further studies.
Enzyme-linked immunosorbent assay (ELISA) The specificities of antibodies or monoclonal antibodies produced in the supernatants of the hybridoma cells were assessed by the ELISA procedure as described by Gnann 14 • Briefly, the 70 % ethanolic extracts of whole grain meal, the protein concentrations of which were adjusted to 100 Ilgjml, were incubated in flexible 96-well microtiter plates at 100 III per well. The plates were incubated at room temperature overnight, washed, and nonspecific binding sites blocked with 1% (wjv) bovine serum albumin or 0·05 % (wjv) skim milk in phosphate-buffered saline (PBS). Appropriate dilutions of test supernatants and control serum were then incubated in the wells for 1 h at 37°C, followed by washing with PBS containing 0'05 % (vjv) Tween 20. Plates were incubated with rabbit anti-mouse F(ab')2 conjugated with peroxidase (BiojCan Scientific Inc., Mississauga, Canada L5L IC7) for 1 hat 37°C. The bound anti-antibodies were then detected by incubation with substrate, [400 mg O-phenylenediamine and I ml 30% (vjv) HP2 in II phosphate-citrate (0,1 MNaH 2PO", 0·05 Mcitric acid) buffer, pH 5] for 30 min at room temperature in the dark. The reaction was stopped by the addition of 4 M HCI (30 Ill) to each well. The color developed was measured as absorbance value at 495 nm using a multiscan microplate reader from Flow Laboratory Inc. (Mississauga, Canada LSS IA2). The test was done in four replicates and standard error (S.B.) calculated in the usual manner.
Monoclonal antibody specificity test The specificity of the developed MAbs for binding to gliadin from 16 different cereal species and cultivars was tested using ELISA. Microtiter plates were coated with 1 IIgjwell of 70 % ethanol extracts of each of the cultivars of common and durum wheats, einkorn, rye and barley. ELISA was carried as described above.
Protein blot analysis Sodium dodecyl sulfate (SDS)j2-mercaptoethanol and 70 % ethanol extracts of whole grain meal were separated by SDS-PAGE in 17·3% acrylamide gels as described by Ng and Bushuk 15 , Protein bands were electrophoretically transferred to a nitrocellulose membrane in a trans blot cell [Bio-Rad Trans-blot cell, Bio-Rad Laboratories (Canada) Ltd., Mississauga, Canada, L4X 2C8], using 0'] M Tris, 0·3 M glycine, 0·1 % (w jv) SDS buffer (pH 8'8) containing 10 % (vjv) propan-201. Transblotting was at 60 volts and 250 rnA for 4 h at 5°C. Nitrocellulose sheets (Fisher Scientific, Winnipeg, Canada, R2X 2V7), containing transferred proteins, were washed in water then blocked with blotto 16 [0'05 % (w jv) skim milk in 0·0 I M Tris, 0·15 MNaCl (pH 7·4, TBS)] and 0'05 % (vjv) Twecn 80 for I hat 25°C, rinsed in TBSjTween, and incubated for 90 min at 25°C in I : 160 dilution ofMAbs in blotto. Excess MAbs were rinsed offwith four changes ofTBSjTween and the nitrocellulose incubated 2 h at 25°C in I: 5000 dilution in blotto of goat anti-mouse (H + L) conjugated to alkaline phosphatase. Following five rinses in TBS, the bound alkaline phosphatase was detected by incubating for I h in 0·1 MTris, 0·1 M NaCl, 5 mM MnCI 2 (pH 9'5) containing 0·4 % (wjv) nitroblue tetrazolium chloride (NBT) and 0·2 % (w jv) 5-bromo-4-chloro3-indolylphosphate p-toluidine (BPIC), (GIBCO jBRL Life Technologies Inc., Burlington, Canada L7P lAt).
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TABLE 1. Binding of MAbs prepared against purified gliadin 45 to ethanol extracted proteins of cereal cultivars assessed using ELISA Absorbance at 495 nm ± S.E. Cereals/cultivars Bread wheats Marquis Marshall Benito Sinton Canthatch Gieniea Timgalen Chinese Spring Durum wheats Wakooma Medora Tetracanthatch Wascana Ward Mindum Other cereals Einkorn Rye, CY_ Prolific Barley, cy_ Bonanza
P26A Clone
P25C Clone
P24B Clone
P2IO Clone
1-3 0'5 12D Clone
P25B Clone
H5±0'13 0·86 ± 0·03 0-74±0-09 0'69±0-13 0-90±0-I2 0-87±0'05 0-40±0'05 1-11 ±0-O9
0-86±0-01 0'80±0-02 0-75±0-04 0-77±0'07 0-69±0-03 0'71 ±0'05 0-49 ± 0-02 0-84±0-05
0·71 ±0-03 0-70±0-03 0-53±0'03 0'58±0-07 0-68±0-05 0-55±0'06 0-30 ± 0-03 0'79±0-05
0-83±0-08 0-75±0-04 0'64±0'03 0'68±0-08 0-80±0-05 0'64±O'I2 0-38±0-O4 0'79±O-05
1-03±0-08 0-70±0-02 0'68±0'04 0-64±0-06 0-73±0'07 0-69±0-07 0-38 ± 0'02 0-84±O'06
0-82±0'03 0-62±0-02 0-53±0-01 0-51 ±0-05 0'75±0-05 0'52±0-01 0'29 ± 0·02 0·83±0·04
0-80±0-IO 0-79±0'03 I'26±0-27 0·29 ± 0-04 0'1l±0-03 0'30±0-03
0'63±0-03 0-65 ± 0-02 0-95 ± 0-20 0-30±0-02 0-22±0-02 0-41 ±0-03
0-48 ± 0-04 0-47±0-05 0-92±0-20 0-12±0-03 0-IO±0-03 0-20±0-05
0'59±O'05 0-53 ± 0-06 0-90±O-1l 0-31±0-05 0-10 ± 0-07 0-22±0-09
0-62±0'O8 0-56 ± 0-02 0·98±0·20 0-18±0-01 0'I8±0-02 0-25±0-03
0-51 ±0-04 0-S7 ± 0'04 0-7S±0-I7 0-22±0-OS 0-I6±0-02 0-26±0-04
-0-07±0'03 0'020±0'03 -0'076±O-03
0·03±0·02 0·05 ± 0·04 0-08 ± 0-03
0-024±0-03 -0'02±0-03 0-020±0'03
-0-07±0-04 -0-03±0'04 -0'03 ± 0-03
-0-01 ±0-02 -0-04±0'02 0'06±0'05
-0-02±0-02 -0-04±0-02 0-04±0'03
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0
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0 0 0 l'>J
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MONOCLONAL ANTIBODIES AGAINST GLlADlNS
109
Results Microscopic examination of the microtiter plates indicated the presence of 770 and 630 hybridoma clones (in 4800 wells each) prepared by the fusion of the 653 mouse myeloma 'cells with spleen cells from mice immunized with gliadins 45 and 74, respectively. The clones were tested for production of anti-gliadin antibodies using ELISA. Forty-four and 27 clones producing antibodies from animals immunized with gliadins 45 and 74, respectively, were found. Six subclones producing MAbs following immunization with gliadin 45 and 4 subclones producing MAbs following immunization with gliadin 74 were propagated in vitro in tissue culture flasks to produce MAbs for further studies. Antibody titers in tissue culture media for clone P24B and clone 7C4-203 were I: 100000 and I: 400000, respectively, using ELISA with goat anti-mouse conjugated to alkaline phosphatase as the detecting antibody. All the developed MAbs were found to be IgG class. Common wheats gave absorbance values in the range 1·148 to 0,291, durum wheats, 1·263 to O' 101, while ethanol-extracted proteins from einkorn, rye and barley showed no or very little binding (Table I). The relative binding of MAbs from different clones was the same for most extracts of different cereals. Further work is needed to produce and test more hybridoma cell lines producing MAbs with higher specific binding to gliadins from different wheat cultivars. On the other hand, binding ofMAbs to extracts of durum cultivars, Wascana and Ward, varied among clones. Wascana showed high binding to MAbs from clones P26A, P25C, P21D and P25B and low binding to MAbs from clone P24B, while Ward had a high binding to MAbs from clone P25C and a lower binding to MAbs from clones P26A, P24B and P2l D (Table I). MAbs from clones to gliadin 74 were tested for binding to ethanol extracted proteins from the same cereal cultivars. All common and durum wheats as well as einkorn gave absorbance values in the range of 0·17 to 0'27. Binding to barley prolamins was 51 to 57 % of the binding to Marshall prolamins, while binding to rye prolamins was 8 to 12 % of the binding to Marshall prolamins. Protein blots of SDSj2-mercaptoethanol extracts of wheat cultivars Ma'rquis, Marshall, Wakooma, Mindum, and Stewart and einkorn separated by SDS-PAGE (Fig. 1) showed that MAbs from clone P24B (anti-gliadin 4?) bound to one region of the Western blot, the major band having apparent molecular weight of 46400, corresponding to the position of gliadin 45, with a minor band having a molecular weight of 45600. Durum wheat cultivars Mindum and Stewart bound much less MAbs and no binding to einkorn was detected. Similar analysis of MAbs from clone 7C4-203 (anti-gliadin 74) showed that these bound to many bands corresponding in mobility to CI.- and p-gliadins and Jow molecular weight subunits of glutenin of all wheat cultivars and einkorn (Fig. 2). Neither of the two MAbs bound to high molecular weight subunits of glutenin. There was no difference in the binding of MAbs from clone P24B to protein blots of 70 % ethanol extracts or SDSj2-mercaptoethanol extracts (results not shown). By the protein blot assay, neither of the MAbs bound to alcohol extractable proteins of rye, oats, sorghum or maize (results not shown).
uo
M. R. DA WOOD ET AL.
FIGURE 1. Protein blot of SDS-ME-TRIS extracted proteins separated by SDS-PAGE and incubated with MAbs produced by clone P24B. Slots 1-6 are: Marquis, Marshall, Wakooma, Mindum, Stewart, and einkorn; slots 7-12 is a replicate gel stained with Coomassie Blue.
Discussion The use of pure gliadins for animal immunization is a new approach to the production of MAbs to cereal proteins. This procedure enabled us to produce clones that secreted MAbs that reacted with specific proteins or with a limited number of proteins. Clone P24B, produced by immunizing with gliadin 45, secreted antibodies that bound to only one region (one major and one minor band) of protein blots of cv. Marquis gliadins separated by SOS-PAGE; the major band corresponds to the mobility of gliadin 45. This indicates that gliadin 45 has an unique epitope. The higher specificity of MAbs produced in our study compared with the results of Skerritt et af.5 is presumed to be due to the use of a pure antigen in the present study. This may give the immunized animal a chance to develop antibodies against a unique epitope that may be present at only low concentration in a total gliadin extract.
MONOCLONAL ANTIBODIES AGAINST GLIADINS
111
FIGURE 2. Protein blot of SDS-ME-TRIS extractable proteins separated by SD8-PAGE and incubated with MAbs produced by clone 7C4--203. Slots 1-4 are: Marquis, Marshall, Prelude and einkom. Slots 5-8 is a replicate gel stained with Coomassie Blue. In the case of clone 7C4-203, produced by immunizing with gliadin 74, the antibodies secreted reacted with many bands corresponding in mobility to rJ.- and p-gliadins. These proteins are known to contain homologous repeating amino acid sequences 3 and are present in many other gliadins, as well as in the prolamins of other cereals such as barley but not in rye prolamins. The MAbs prepared by Skerritt et al. 5 using a mixture of gliadin proteins for immunization, reacted with prolamins from barley, rye, rye grass and rice. MAbs to gliadin 45 gave a range of reactions to common and durum wheats. Thus, these clones have potential for establishing an immune based test to differentiate wheat cultivars using a battery of MAbs prepared against several purified gliadins, as well as determining the proportions of wheat, rye or other cereal grains in various food and feed products.
112
M. R. DAWOOD ET AL.
The authors wish to thank Frank Stockl, Wayne Johnson, Susan Simpson and Elizabeth Slominski for their technical help. This work was supported by the Manitoba Strategic Research Program, Manitoba Research Council and the Natural Sciences and Engineering Research Council of Canada.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
II. 12. 13. 14. 15. 16.
Zillman, R. R. and Bushuk, W. Can. J. Plant Sci. 59 (1979) 287-298. Bietz, J. A., Huebner, F. R., Sanderson, J. E. and Wall, J. S. Cereal Chem. 54 (1987) 1070-1083. Kasarda, D. D. Ann. Technol. Agric. 29 (1980) 151-173. Dierks-Ventling, C. and Cozens, D. FEBS LeU. 142 (1982) 750-782. Skerritt, J. H., Smith, R. A., Wrigley, C. W. and Underwood, P. A. J. Cereal Sci. 2 (1984) 215-224. Freedman, A. R., Galfn!, G., Gal, E., Ellis, H. J. and Cic1itira, P. J. J. Immlillol. Methods 98 (1987) 123-127. Skerritt, J. H. and Underwood, P. A. Biochim. Biophys. Acta 874 (1986) 245-254. Windemann, H., Fritschy, F. and Baumgartner, E. Biochim. Biophys. Acta 709 (1982) 110-121. Cic1itira, P. J. and Lennox, E. S. Clill. Sci. 64 (1983) 655-659. Kagnoff, M. F., Austin, R. K., Hubert, J. J., Bernardin J. E. and Kasarda, D. D. J. Exp. Med. 160 (1984) 1544-1557. Howes, N. K. and Kosmolak, F. G. Cereal Chel11. 59 (1982) 485-488. Kearney, 1. F., Radbruch, A, Liesegang, B. and Rajewsky, K. J. Iml1lll11ol. 123 (1979) 1548-1550. Goding, J. W. Antibodies as a tool: The applications of immunochemistry. A Wiley Interscience Publication (1982) pp 273. Gnann, J. W., Nelson, J. A. and 0ldsteon, M. B. A. J. Virol. 61 (1987) 2639-2641. Ng, P. K. W. and Bushuk, W. Cereal Chem. 64 (1987) 324-327. Johnson, D. A., Gautch, J. W., Sportsman, J. R. and Helder, J. H. Gene Anal. Tech. 1 (1984) 3-8.
Preparation of Monoclonal Antibodies Against Specific Gliadin Proteins and Preliminary Investigation of their Ability to Discriminate Cereal Cultivars* M. R. DAWQODtt, N. K. HOWES§ and W. BUSHUK t
t University
of Manitoba, Food Science Department, Winnipeg, ME, Canada R3T 2N2 and §Agriculture Canada Research Station, Winnipeg, MB, Canada R3T 2M9 Received 19 December 1988
Monoclonal antibodies (MAbs) were prepared against y-gliadin 45 and Ct-gliadin 74 from the common wheat cuHivars Marquis and Marshall, respectively. Murine monoclonal antibodies were prepared and positive clones were detected using ELISA. MAbs prepared against gliadin 45 gave a low reaction in ELISA to extracts of einkorn, rye, barley and some common and durum wheats, and higher reaction to other COmmon and durum wheats. Protein blotting of total gliadins separated by SDS-PAGE showed that MAbs against gliadin 45 bound to one discrete region corresponding to the location of gliadin 45. MAbs prepared against gliadin 74 gave a low reaction to rye, a medium reaction to barley, and a high reaction to all common and durum wheats and einkorn. Protein blotting showed that these MAbs bound to the region corresponding to Ct- and ~-gliadins.
Introduction Electrophoretic patterns of gliadin components separated by polyacrylamide gel electrophoresis (PAGE) have been used for wheat cultivar identification 1 • Some disadvantages of PAGE are that it is time consuming, only a limited number of samples can be tested at one time, acrylamide poses a minor health hazard, and the patterns of protein bands are complex and sometimes difficult to discriminate. Antibodies are widely used for identification and/or detection of very low concentrations of specific antigens. Gliadin proteins have a high degree of homologous amino acid sequences 2 • 3 • Because of this, it is difficult to identify a particular gliadin using polyclonal antibodies 4 • Homogeneous monoclonal antibodies have several advantages over heterogenous polyclonal antibodies; they are of defined isotype and have a greater specificity for closely related antigens. Skerritt et al. 5 reported the preparation of MAbs against a mixture of gliadins. The resulting MAbs were active against many different gliadins as well as prolamins of barley, rice, rye, and rye grass. Freedman et al. 6 established a sandwich ELISA using murine monoclonal antibodies
* Contribution No. 139 of the University of Manitoba, Food Science Department, and No. 1346 of the Agriculture Canada Winnipeg Research Station. t Present address: Cadham Provincial Laboratory, Winnipeg, MB, Canada R3C 3YI; to whom all correspondence should be addressed. 0733-5210/89/050105 + 08 $03.00/0
© 1989 Academic Press Limited
106
M. R. DAWOOD ET AL.
prepared against unfractionated wheat gliadin and purified rabbit IgG. Using their test, they were able to detect traces of gliadin in some' gluten-free' food products. Skerritt and Underwood 7 also prepared anti-gliadin (unfractionated) MAbs. Most of their MAbs bound to all gliadin bands separated by SDS-polyacrylamide gel electrophoresis. Some of the antibodies bound to a small group of gliadin proteins. Several investigators were able to produce polyclonal antibodies or monoclonal antibodies prepared against crude whole gliadin or A-gliadin (a fraction of a:-gliadin)8-1o. Different degrees of cross reactivity between the developed antibodies and a:-, p- and y-gliadins were observed. On the other hand, no cross reactivity was reported between anti-gliadin antibodies and extracts of rye, barley or oats. To prepare MAbs against epitopes that are unique to specific gliadin proteins, we investigated the use of purified gliadins for animal immunization. Experimental
Antigen preparation Gliadin bands 45 and 74 (according to the nomenclature of Zillman and Bushuk 1 ) were purified from the common wheat cultivars Marquis and Marshall, respectively, using a two step purification method as described by Howes and Kosmolak l l, except that gliadin bands after PAGE were located, cut out, and eluted with 70% ethanol. The purity of the eluted proteins was confirmed by acid PAGEl. The eluted proteins were freeze-dried then redissolved in double distilled H 2 0. The two bands, each of which may contain more than one component, will be referred to as gliadins 45 and 74. Gliadin 45 is a band in the y-gliadin region while gliadin 74 is in the a-region of the electrophoregram.
Tissue culture and media The P3X63-Ag8.653 (abbreviated as 653) cell line, a mouse myeloma cell line that does not secrete antibodies l2 , was purchased from the American Type Culture Collection (Rockville, MD 20852). RPMI 1640 cell culture medium was purchased from GIBCO/BRL Life Technology Inc. (Burlington, Canada L7P lAI). Complete RPMI 1640 medium is RPMI 1640 supplemented with 10% FCS (fetal calf serum) and penicillin-streptomycin. HT medium is complete RPMI 1640 medium supplemented with 0'1 mM hypoxanthine and 31-4 11M thymidine. HAT medium is HT medium containing O'821lM aminopterine.
Monoclonal antibody production MAbs were prepared as described by Goding l3 with modification. Female BALB/c mice were injected intramuscularly with 50 Ilg purified gliadin 45 or 74 in Freund's complete adjuvant followed by intraperitoneal injection at 4 week intervals. Three days after the last (booster) injection, spleen cells were removed. Mouse hybridomas were generated from the fusion of mouse spleen cells secreting high levels of antibodies against the desired gliadin with the 653 cell line. Cell fusion was carried out using polyethylene glycol 4000. Spleen and myeloma cells were mixed, pelleted by centrifugation and resuspended in HAT medium at a concentration of 5 x 10 5 cells/ml, plated into 96-well tissue culture plates containing feeder layer of normal mouse spleen cells (1 x 10 4 cells/well) that had been plated one day earlier. Hybridoma cells were selected by culturing in HAT medium. Two weeks later, the ceLIs were cultured in HT medium for two weeks followed by complete RPMI 1640 medium. Antibody secreting clones, identified by ELISA, were
MONOCLONAL ANTIBODIES AGAINST GLIADINS
107
subcloned at least twice at 0,5-1'0 celljwell and retested for Ab secretion. Hybridoma cells secreting MAbs that gave a differential reaction in ELISA to extracts of Marquis, Marshall, Benito and Sinton common wheats were propagated in vitro. The four wheat cultivars represent wheats with or without gliadin bands 45 andjor 74. Tissue culture media containing MAbs were aliquoted and stored at -20°C for further studies.
Enzyme-linked immunosorbent assay (ELISA) The specificities of antibodies or monoclonal antibodies produced in the supernatants of the hybridoma cells were assessed by the ELISA procedure as described by Gnann 14 • Briefly, the 70 % ethanolic extracts of whole grain meal, the protein concentrations of which were adjusted to 100 Ilgjml, were incubated in flexible 96-well microtiter plates at 100 III per well. The plates were incubated at room temperature overnight, washed, and nonspecific binding sites blocked with 1% (wjv) bovine serum albumin or 0·05 % (wjv) skim milk in phosphate-buffered saline (PBS). Appropriate dilutions of test supernatants and control serum were then incubated in the wells for 1 h at 37°C, followed by washing with PBS containing 0'05 % (vjv) Tween 20. Plates were incubated with rabbit anti-mouse F(ab')2 conjugated with peroxidase (BiojCan Scientific Inc., Mississauga, Canada L5L IC7) for 1 hat 37°C. The bound anti-antibodies were then detected by incubation with substrate, [400 mg O-phenylenediamine and I ml 30% (vjv) HP2 in II phosphate-citrate (0,1 MNaH 2PO", 0·05 Mcitric acid) buffer, pH 5] for 30 min at room temperature in the dark. The reaction was stopped by the addition of 4 M HCI (30 Ill) to each well. The color developed was measured as absorbance value at 495 nm using a multiscan microplate reader from Flow Laboratory Inc. (Mississauga, Canada LSS IA2). The test was done in four replicates and standard error (S.B.) calculated in the usual manner.
Monoclonal antibody specificity test The specificity of the developed MAbs for binding to gliadin from 16 different cereal species and cultivars was tested using ELISA. Microtiter plates were coated with 1 IIgjwell of 70 % ethanol extracts of each of the cultivars of common and durum wheats, einkorn, rye and barley. ELISA was carried as described above.
Protein blot analysis Sodium dodecyl sulfate (SDS)j2-mercaptoethanol and 70 % ethanol extracts of whole grain meal were separated by SDS-PAGE in 17·3% acrylamide gels as described by Ng and Bushuk 15 , Protein bands were electrophoretically transferred to a nitrocellulose membrane in a trans blot cell [Bio-Rad Trans-blot cell, Bio-Rad Laboratories (Canada) Ltd., Mississauga, Canada, L4X 2C8], using 0'] M Tris, 0·3 M glycine, 0·1 % (w jv) SDS buffer (pH 8'8) containing 10 % (vjv) propan-201. Transblotting was at 60 volts and 250 rnA for 4 h at 5°C. Nitrocellulose sheets (Fisher Scientific, Winnipeg, Canada, R2X 2V7), containing transferred proteins, were washed in water then blocked with blotto 16 [0'05 % (w jv) skim milk in 0·0 I M Tris, 0·15 MNaCl (pH 7·4, TBS)] and 0'05 % (vjv) Twecn 80 for I hat 25°C, rinsed in TBSjTween, and incubated for 90 min at 25°C in I : 160 dilution ofMAbs in blotto. Excess MAbs were rinsed offwith four changes ofTBSjTween and the nitrocellulose incubated 2 h at 25°C in I: 5000 dilution in blotto of goat anti-mouse (H + L) conjugated to alkaline phosphatase. Following five rinses in TBS, the bound alkaline phosphatase was detected by incubating for I h in 0·1 MTris, 0·1 M NaCl, 5 mM MnCI 2 (pH 9'5) containing 0·4 % (wjv) nitroblue tetrazolium chloride (NBT) and 0·2 % (w jv) 5-bromo-4-chloro3-indolylphosphate p-toluidine (BPIC), (GIBCO jBRL Life Technologies Inc., Burlington, Canada L7P lAt).
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TABLE 1. Binding of MAbs prepared against purified gliadin 45 to ethanol extracted proteins of cereal cultivars assessed using ELISA Absorbance at 495 nm ± S.E. Cereals/cultivars Bread wheats Marquis Marshall Benito Sinton Canthatch Gieniea Timgalen Chinese Spring Durum wheats Wakooma Medora Tetracanthatch Wascana Ward Mindum Other cereals Einkorn Rye, CY_ Prolific Barley, cy_ Bonanza
P26A Clone
P25C Clone
P24B Clone
P2IO Clone
1-3 0'5 12D Clone
P25B Clone
H5±0'13 0·86 ± 0·03 0-74±0-09 0'69±0-13 0-90±0-I2 0-87±0'05 0-40±0'05 1-11 ±0-O9
0-86±0-01 0'80±0-02 0-75±0-04 0-77±0'07 0-69±0-03 0'71 ±0'05 0-49 ± 0-02 0-84±0-05
0·71 ±0-03 0-70±0-03 0-53±0'03 0'58±0-07 0-68±0-05 0-55±0'06 0-30 ± 0-03 0'79±0-05
0-83±0-08 0-75±0-04 0'64±0'03 0'68±0-08 0-80±0-05 0'64±O'I2 0-38±0-O4 0'79±O-05
1-03±0-08 0-70±0-02 0'68±0'04 0-64±0-06 0-73±0'07 0-69±0-07 0-38 ± 0'02 0-84±O'06
0-82±0'03 0-62±0-02 0-53±0-01 0-51 ±0-05 0'75±0-05 0'52±0-01 0'29 ± 0·02 0·83±0·04
0-80±0-IO 0-79±0'03 I'26±0-27 0·29 ± 0-04 0'1l±0-03 0'30±0-03
0'63±0-03 0-65 ± 0-02 0-95 ± 0-20 0-30±0-02 0-22±0-02 0-41 ±0-03
0-48 ± 0-04 0-47±0-05 0-92±0-20 0-12±0-03 0-IO±0-03 0-20±0-05
0'59±O'05 0-53 ± 0-06 0-90±O-1l 0-31±0-05 0-10 ± 0-07 0-22±0-09
0-62±0'O8 0-56 ± 0-02 0·98±0·20 0-18±0-01 0'I8±0-02 0-25±0-03
0-51 ±0-04 0-S7 ± 0'04 0-7S±0-I7 0-22±0-OS 0-I6±0-02 0-26±0-04
-0-07±0'03 0'020±0'03 -0'076±O-03
0·03±0·02 0·05 ± 0·04 0-08 ± 0-03
0-024±0-03 -0'02±0-03 0-020±0'03
-0-07±0-04 -0-03±0'04 -0'03 ± 0-03
-0-01 ±0-02 -0-04±0'02 0'06±0'05
-0-02±0-02 -0-04±0-02 0-04±0'03
3: :;0
0
;J;>
::E
0 0 0 l'>J
"-l
::...
r-.
MONOCLONAL ANTIBODIES AGAINST GLlADlNS
109
Results Microscopic examination of the microtiter plates indicated the presence of 770 and 630 hybridoma clones (in 4800 wells each) prepared by the fusion of the 653 mouse myeloma 'cells with spleen cells from mice immunized with gliadins 45 and 74, respectively. The clones were tested for production of anti-gliadin antibodies using ELISA. Forty-four and 27 clones producing antibodies from animals immunized with gliadins 45 and 74, respectively, were found. Six subclones producing MAbs following immunization with gliadin 45 and 4 subclones producing MAbs following immunization with gliadin 74 were propagated in vitro in tissue culture flasks to produce MAbs for further studies. Antibody titers in tissue culture media for clone P24B and clone 7C4-203 were I: 100000 and I: 400000, respectively, using ELISA with goat anti-mouse conjugated to alkaline phosphatase as the detecting antibody. All the developed MAbs were found to be IgG class. Common wheats gave absorbance values in the range 1·148 to 0,291, durum wheats, 1·263 to O' 101, while ethanol-extracted proteins from einkorn, rye and barley showed no or very little binding (Table I). The relative binding of MAbs from different clones was the same for most extracts of different cereals. Further work is needed to produce and test more hybridoma cell lines producing MAbs with higher specific binding to gliadins from different wheat cultivars. On the other hand, binding ofMAbs to extracts of durum cultivars, Wascana and Ward, varied among clones. Wascana showed high binding to MAbs from clones P26A, P25C, P21D and P25B and low binding to MAbs from clone P24B, while Ward had a high binding to MAbs from clone P25C and a lower binding to MAbs from clones P26A, P24B and P2l D (Table I). MAbs from clones to gliadin 74 were tested for binding to ethanol extracted proteins from the same cereal cultivars. All common and durum wheats as well as einkorn gave absorbance values in the range of 0·17 to 0'27. Binding to barley prolamins was 51 to 57 % of the binding to Marshall prolamins, while binding to rye prolamins was 8 to 12 % of the binding to Marshall prolamins. Protein blots of SDSj2-mercaptoethanol extracts of wheat cultivars Ma'rquis, Marshall, Wakooma, Mindum, and Stewart and einkorn separated by SDS-PAGE (Fig. 1) showed that MAbs from clone P24B (anti-gliadin 4?) bound to one region of the Western blot, the major band having apparent molecular weight of 46400, corresponding to the position of gliadin 45, with a minor band having a molecular weight of 45600. Durum wheat cultivars Mindum and Stewart bound much less MAbs and no binding to einkorn was detected. Similar analysis of MAbs from clone 7C4-203 (anti-gliadin 74) showed that these bound to many bands corresponding in mobility to CI.- and p-gliadins and Jow molecular weight subunits of glutenin of all wheat cultivars and einkorn (Fig. 2). Neither of the two MAbs bound to high molecular weight subunits of glutenin. There was no difference in the binding of MAbs from clone P24B to protein blots of 70 % ethanol extracts or SDSj2-mercaptoethanol extracts (results not shown). By the protein blot assay, neither of the MAbs bound to alcohol extractable proteins of rye, oats, sorghum or maize (results not shown).
uo
M. R. DA WOOD ET AL.
FIGURE 1. Protein blot of SDS-ME-TRIS extracted proteins separated by SDS-PAGE and incubated with MAbs produced by clone P24B. Slots 1-6 are: Marquis, Marshall, Wakooma, Mindum, Stewart, and einkorn; slots 7-12 is a replicate gel stained with Coomassie Blue.
Discussion The use of pure gliadins for animal immunization is a new approach to the production of MAbs to cereal proteins. This procedure enabled us to produce clones that secreted MAbs that reacted with specific proteins or with a limited number of proteins. Clone P24B, produced by immunizing with gliadin 45, secreted antibodies that bound to only one region (one major and one minor band) of protein blots of cv. Marquis gliadins separated by SOS-PAGE; the major band corresponds to the mobility of gliadin 45. This indicates that gliadin 45 has an unique epitope. The higher specificity of MAbs produced in our study compared with the results of Skerritt et af.5 is presumed to be due to the use of a pure antigen in the present study. This may give the immunized animal a chance to develop antibodies against a unique epitope that may be present at only low concentration in a total gliadin extract.
MONOCLONAL ANTIBODIES AGAINST GLIADINS
111
FIGURE 2. Protein blot of SDS-ME-TRIS extractable proteins separated by SD8-PAGE and incubated with MAbs produced by clone 7C4--203. Slots 1-4 are: Marquis, Marshall, Prelude and einkom. Slots 5-8 is a replicate gel stained with Coomassie Blue. In the case of clone 7C4-203, produced by immunizing with gliadin 74, the antibodies secreted reacted with many bands corresponding in mobility to rJ.- and p-gliadins. These proteins are known to contain homologous repeating amino acid sequences 3 and are present in many other gliadins, as well as in the prolamins of other cereals such as barley but not in rye prolamins. The MAbs prepared by Skerritt et al. 5 using a mixture of gliadin proteins for immunization, reacted with prolamins from barley, rye, rye grass and rice. MAbs to gliadin 45 gave a range of reactions to common and durum wheats. Thus, these clones have potential for establishing an immune based test to differentiate wheat cultivars using a battery of MAbs prepared against several purified gliadins, as well as determining the proportions of wheat, rye or other cereal grains in various food and feed products.
112
M. R. DAWOOD ET AL.
The authors wish to thank Frank Stockl, Wayne Johnson, Susan Simpson and Elizabeth Slominski for their technical help. This work was supported by the Manitoba Strategic Research Program, Manitoba Research Council and the Natural Sciences and Engineering Research Council of Canada.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
II. 12. 13. 14. 15. 16.
Zillman, R. R. and Bushuk, W. Can. J. Plant Sci. 59 (1979) 287-298. Bietz, J. A., Huebner, F. R., Sanderson, J. E. and Wall, J. S. Cereal Chem. 54 (1987) 1070-1083. Kasarda, D. D. Ann. Technol. Agric. 29 (1980) 151-173. Dierks-Ventling, C. and Cozens, D. FEBS LeU. 142 (1982) 750-782. Skerritt, J. H., Smith, R. A., Wrigley, C. W. and Underwood, P. A. J. Cereal Sci. 2 (1984) 215-224. Freedman, A. R., Galfn!, G., Gal, E., Ellis, H. J. and Cic1itira, P. J. J. Immlillol. Methods 98 (1987) 123-127. Skerritt, J. H. and Underwood, P. A. Biochim. Biophys. Acta 874 (1986) 245-254. Windemann, H., Fritschy, F. and Baumgartner, E. Biochim. Biophys. Acta 709 (1982) 110-121. Cic1itira, P. J. and Lennox, E. S. Clill. Sci. 64 (1983) 655-659. Kagnoff, M. F., Austin, R. K., Hubert, J. J., Bernardin J. E. and Kasarda, D. D. J. Exp. Med. 160 (1984) 1544-1557. Howes, N. K. and Kosmolak, F. G. Cereal Chel11. 59 (1982) 485-488. Kearney, 1. F., Radbruch, A, Liesegang, B. and Rajewsky, K. J. Iml1lll11ol. 123 (1979) 1548-1550. Goding, J. W. Antibodies as a tool: The applications of immunochemistry. A Wiley Interscience Publication (1982) pp 273. Gnann, J. W., Nelson, J. A. and 0ldsteon, M. B. A. J. Virol. 61 (1987) 2639-2641. Ng, P. K. W. and Bushuk, W. Cereal Chem. 64 (1987) 324-327. Johnson, D. A., Gautch, J. W., Sportsman, J. R. and Helder, J. H. Gene Anal. Tech. 1 (1984) 3-8.