- Email: [email protected]
A monoclonal antibody to endogenous α-amylase inhibitor of barley
Journal of Cereal Science 15 (1992) 1-4
RAPID COMMUNICATION
A Monoclonal Antibody to Endogenous Inhibitor of Barley J. ZAWISTOWSKI*, M. ANSELL*,
u. ZAWISTOWSKAt
~-amylase
and N. K.
HOWES~
* Manitoba Research Council - Biotechnology Laboratory, University of Manitoba, Food Science Department, t Rh Pharmaceuticals Inc., 104 Chancellor Matheson Rd and ~ Agriculture Canada Research Station, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2N2 Received 27 August 1991
The endogenous ex-amylase inhibitors are present in the endosperm of barley, wheat, rye, triticale and possibly other cereals. According to Weselake et al. \ barley contains a higher level of the inhibitor than other cereals. However, studies of the variation of examylase inhibitor in barleys were limited to a few cultivars, excluding wild varieties 1 ,3,4. Systematic studies of the variation of ex-amylase inhibitor in this cereal could have practical applications. A barley line with a high ex-amylase inhibitor content may find use in the baking industry since it has been shown that addition of barley inhibitor to sprouted wheat flour improved its baking properties 2 • We describe here the first report of the production of a highly specific monoclonal antibody (4FIO-5, IgG 1) to barley endogenous ex-amylase inhibitor, which could be used to develop a quantitative assay to screen for the presence of the inhibitor in barley varieties. Four female BALB/c mice (6-8 weeks old) were immunized by four sequential subcutaneous injections of barley a-amylase inhibitor (50 Ilg/mouse) purified as described previously5. Antibody titre of tail bleedings was measured before the first injection and 7 days after each of the subsequent injections, using an indirect enzyme immunoassay (ELISA) according to the procedure described earlier 6 • All mice, except one, developed antibody titre of approximately 10-6 • Three days after final booster injection of antigen, the splenocytes of a mouse with a serum antibody titre of 1·6 x 10-6 were harvested and fused with P3 x 63-Ag 8.653. The fusion and hybridoma production was as previously described 6 • Hybridomas were screened for antibody production by an indirect ELISA using purified barley ex-amylase inhibitor as antigen. Briefly, the wells of ELISA plates (Falcon 3912, Becton Dickinson, Colorado) were coated overnight at 4°C with purified inhibitor (l Ilg/ml) in carbonate buffer, pH 9'6, blocked for 1 h at 37°C with phosphate-buffered saline (PBS) containing 2 % skim milk and incubated for 1·5 h at 37°C with spent medium (l00 ~L1/well). Then, goat anti-mouse immunoglobulin alkaline phosphatase conjugate (BioRad; diluted 1: 3000 in PBS containing 0·05 % 0733-5210/92/010001 +04 $03.00/0
© 1992 Academic Press Limited CER 15
J. ZAWISTOWSKI ET AL.
2
200,---------------,
2·0
( b)
(a) E c
lO
1·5
1·5
1·0
1·0
0·5
0·5
0
:!.
'"<.>c a
.0
L-
0
'"
.0 <{
0
3
2
4
5
0
2
3
4
5
Lag Ab dilution factor
2·0 (c) E
c
10
1·5
0
~
'"<.>c
1-0
Cl
~ a
.0
0·5
<{
o
2
3
4
5
Log Ab dilution factor
FIGURE 1. Reactivity of monoclonal antibodies: (a) 4FlO-5; (b) 4F6-1; and (c) IHI7 to endogenous ex-amylase inhibitor using ELISA. 0, purified inhibitor from barley cv. Bonanza; e, crude inhibitor from barley cv. Harrington; D, crude inhibitor from wheat cv. Columbus; _, crude inhibitor from triticale cv. T3; J::,., crude inhibitor from rye cv. Dankowskie Zlote.
Tween 20) was added (100 J.lI/well) and the plates were incubated for 1 hat 37°C. The plates were developed by adding a solution (l00 J.lI/well) of freshly prepared pnitrophenyl phosphate in diethanolamine buffer, pH 9·8 (l mg/ml), I h incubation in dark at room temperature, and reading at A 405 nm on a Titertek Multiskan (Flow Laboratories, McLean, Virginia). Negative control was made by omitting the antigen, but including both antibodies, while immune serum diluted I: 2000 in PBS was used as positive control. A fusion frequency of 6 x 10-4 was calculated on the basis of all splenocytes (4 x l0 7) used in the fusion. Initially, 90 % of hybridoma cultures were positive by ELISA for production of antibodies to inhibitor. Of these, 10 hybrids were further cloned and successfully stabilized. We report on three clones that may have different epitope specificity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed under reducing conditions by the Laemmli method 7 in a minigel system (BioRad Laboratories, Richmond, California). Electrophoresis was
MONOCLONAL ANTIBODY TO ENDOGENOUS Ci-AMYLASE INHIBITOR
3
conducted in 15 % slab gels at 200 V for I h, using appropriate pre-stained molecular weight markers (BioRad). Electrophoretic transfers onto nitrocellulose (BioRad) were performed at 100 V for I h according to the method of Towbin et at. s. Subsequently, the blots were either stained with Amidoblack or immunostained with monoclonal antibodies 6 • Protein concentration was measured with a commercial protein assay reagent (BioRad). The subisotype of MAbs was determined with a mouse subisotyping kit (BioRad) used as recommended by the manufacturer. All three clones secreted antibodies of the IgG 1 subclass with kappa light chain. Serially diluted monoclonal antibodies, obtained from all three lines, were tested against purified barley inhibitor, barley, wheat, triticale and rye aqueous extracts by ELISA assay and the results are presented in Fig. 1. MAb raised from 4FIO-5 monoclonal cell line reacted only with purified inhibitor and barley extract, while MAbs obtained from cell lines 4F6-1 and IHI-7 reacted with all cereal extracts, except rye. Although MAb 4FlO-5 gave highest reaction with purified inhibitor, the magnitude of reactions with barley extract was similar for all three MAbs. In earlier reports, comparison of inhibitors from different cereals on the basis of reaction with polyclonal antibodies suggested that inhibitors from barley, wheat, rye and triticale are immunologically similar 1 • 9 • This is presumably because of the similar structures of the individual inhibitors. In fact it has been reported that inhibitors isolated from barley and wheat have almost identical amino acid sequences 10 • However, in our investigation, the specificity of the 4FIO-5 antibody suggested the presence of a specific antigenic determinant only in barley inhibitor indicating that barley inhibitor is immunochemically different from inhibitors from the other cereals tested. To test for cross-reactivity, all MAbs were further investigated by immunoblotting after SDS-PAGE (Fig. 2). Only 4FIO-5 bound to the barley inhibitor, showing no reaction with other proteins [Fig. 2(b)]. Partial renaturation of proteins blotted after SDS-PAGE might occur during the transfer; however, it is size-dependent and less efficient for proteins of M r 20k to 15k than those of higher M r ll . Since the barley inhibitor has a M r of approximately 20k it is expected that MAb 4FIO-5 recognizes a sequential epitope rather than a conformational epitope, while the other two MAbs are specific to the native form of inhibitor. To support this hypothesis, all antibodies were titrated on ELISA plates coated with denatured preparations (boiled for 5 min) of pure inhibitor, barley, wheat, triticale or rye extracts. Of the three monoclonal antibodies, only 4FlO-5 reacted with the denaturated form of barley inhibitor, although the absorbance value with denatured inhibitor was slightly lower than with its native form (data not shown). The difference in the reactivities of the 4FIO-5 and the other two mabs in the ELISA and the fact that only 4FlO-5 reacts on immunoblots may indicate that the 4FIO-5 recognizes a unique inhibitor epitope. Moreover, because of its high specificity to barley inhibitor, this MAb may provide a suitable reagent for the detection and quantification of this protein in a variety of barley cultivars and its wild counterparts. We thank Ms Lidia Gosek for her skilful technical assistance. Contribution no. 209 of the Food Science Department, University of Manitoba. \-2
4
J. ZAWISTOWSKI ET AL. 2 110-84--
3
4
5
2
6
3
4
5
:
.'. .... , .... ':.
'
·'/.~:·~;i~~···'
4733--
24-
/6 -
~W'
(a)
(b)
FIGURE 2. Protein blot of aqueous extracts of cereal kernels separated by 80S-PAGE. (a) Blot stained with Amidoblaek. Lanes (1) M r standards; (2) purified inhibitor standard; (3) triticale ev. T3; (4) barley ev. Harrington; (5) wheat ev. Columbus; (6) rye ev. Oanksowskie Zlote. (b) Blot immunostained with MAb 4FlO5. Lanes (I) purified inhibitor standard; (2) barleyev. Harrington; (3) wheat ev. Columbus; (4) triticale ev. T3; (5) rye ev. Oankowskie Zlote.
References I. Weselake, R. J., MacGregor, A. W. and Hill, R. D. Cereal Chem. 62 (1985) 120-123. Zawistowska, D., Langstaff, J. and Bushuk, W. J. Cereal Sci. 8 (1988) 207-209. Munck, L., Mundy, J. and Vaag, P. Am. Soc. Brew. Chem. J. 43 (1985) 35-38. Henson, C. A. and Stone, J. M. J. Cereal Sci. 8 (1988) 39-46. Zawistowska, D. US Patent 4,910,297 (1990). Zawistowski, J. and Howes, N. K. J. Cereal Sci. 12 (1990) 235-244. Laemmli, U. K. Nature 227 (1970) 880--685. Towbin, R., Staehelin, T. and Gordon, J. Proc. Nat!. Acad. Sci. USA 76 (1979) 4350-4354. Sadowski, J., MacGregor, A. and Daussant, J. Electrophoresis 7 (1986) 176-179. Mundy, J., Hejgaard, J. and Svendsen, 1. FEBS Lett. 167 (1984) 210-214. II. Dunn, S. D. Anal. Biochem. 157 (1986) 144-153.
2. 3. 4. 5. 6. 7. 8. 9. 10.
RAPID COMMUNICATION
A Monoclonal Antibody to Endogenous Inhibitor of Barley J. ZAWISTOWSKI*, M. ANSELL*,
u. ZAWISTOWSKAt
~-amylase
and N. K.
HOWES~
* Manitoba Research Council - Biotechnology Laboratory, University of Manitoba, Food Science Department, t Rh Pharmaceuticals Inc., 104 Chancellor Matheson Rd and ~ Agriculture Canada Research Station, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2N2 Received 27 August 1991
The endogenous ex-amylase inhibitors are present in the endosperm of barley, wheat, rye, triticale and possibly other cereals. According to Weselake et al. \ barley contains a higher level of the inhibitor than other cereals. However, studies of the variation of examylase inhibitor in barleys were limited to a few cultivars, excluding wild varieties 1 ,3,4. Systematic studies of the variation of ex-amylase inhibitor in this cereal could have practical applications. A barley line with a high ex-amylase inhibitor content may find use in the baking industry since it has been shown that addition of barley inhibitor to sprouted wheat flour improved its baking properties 2 • We describe here the first report of the production of a highly specific monoclonal antibody (4FIO-5, IgG 1) to barley endogenous ex-amylase inhibitor, which could be used to develop a quantitative assay to screen for the presence of the inhibitor in barley varieties. Four female BALB/c mice (6-8 weeks old) were immunized by four sequential subcutaneous injections of barley a-amylase inhibitor (50 Ilg/mouse) purified as described previously5. Antibody titre of tail bleedings was measured before the first injection and 7 days after each of the subsequent injections, using an indirect enzyme immunoassay (ELISA) according to the procedure described earlier 6 • All mice, except one, developed antibody titre of approximately 10-6 • Three days after final booster injection of antigen, the splenocytes of a mouse with a serum antibody titre of 1·6 x 10-6 were harvested and fused with P3 x 63-Ag 8.653. The fusion and hybridoma production was as previously described 6 • Hybridomas were screened for antibody production by an indirect ELISA using purified barley ex-amylase inhibitor as antigen. Briefly, the wells of ELISA plates (Falcon 3912, Becton Dickinson, Colorado) were coated overnight at 4°C with purified inhibitor (l Ilg/ml) in carbonate buffer, pH 9'6, blocked for 1 h at 37°C with phosphate-buffered saline (PBS) containing 2 % skim milk and incubated for 1·5 h at 37°C with spent medium (l00 ~L1/well). Then, goat anti-mouse immunoglobulin alkaline phosphatase conjugate (BioRad; diluted 1: 3000 in PBS containing 0·05 % 0733-5210/92/010001 +04 $03.00/0
© 1992 Academic Press Limited CER 15
J. ZAWISTOWSKI ET AL.
2
200,---------------,
2·0
( b)
(a) E c
lO
1·5
1·5
1·0
1·0
0·5
0·5
0
:!.
'"<.>c a
.0
L-
0
'"
.0 <{
0
3
2
4
5
0
2
3
4
5
Lag Ab dilution factor
2·0 (c) E
c
10
1·5
0
~
'"<.>c
1-0
Cl
~ a
.0
0·5
<{
o
2
3
4
5
Log Ab dilution factor
FIGURE 1. Reactivity of monoclonal antibodies: (a) 4FlO-5; (b) 4F6-1; and (c) IHI7 to endogenous ex-amylase inhibitor using ELISA. 0, purified inhibitor from barley cv. Bonanza; e, crude inhibitor from barley cv. Harrington; D, crude inhibitor from wheat cv. Columbus; _, crude inhibitor from triticale cv. T3; J::,., crude inhibitor from rye cv. Dankowskie Zlote.
Tween 20) was added (100 J.lI/well) and the plates were incubated for 1 hat 37°C. The plates were developed by adding a solution (l00 J.lI/well) of freshly prepared pnitrophenyl phosphate in diethanolamine buffer, pH 9·8 (l mg/ml), I h incubation in dark at room temperature, and reading at A 405 nm on a Titertek Multiskan (Flow Laboratories, McLean, Virginia). Negative control was made by omitting the antigen, but including both antibodies, while immune serum diluted I: 2000 in PBS was used as positive control. A fusion frequency of 6 x 10-4 was calculated on the basis of all splenocytes (4 x l0 7) used in the fusion. Initially, 90 % of hybridoma cultures were positive by ELISA for production of antibodies to inhibitor. Of these, 10 hybrids were further cloned and successfully stabilized. We report on three clones that may have different epitope specificity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed under reducing conditions by the Laemmli method 7 in a minigel system (BioRad Laboratories, Richmond, California). Electrophoresis was
MONOCLONAL ANTIBODY TO ENDOGENOUS Ci-AMYLASE INHIBITOR
3
conducted in 15 % slab gels at 200 V for I h, using appropriate pre-stained molecular weight markers (BioRad). Electrophoretic transfers onto nitrocellulose (BioRad) were performed at 100 V for I h according to the method of Towbin et at. s. Subsequently, the blots were either stained with Amidoblack or immunostained with monoclonal antibodies 6 • Protein concentration was measured with a commercial protein assay reagent (BioRad). The subisotype of MAbs was determined with a mouse subisotyping kit (BioRad) used as recommended by the manufacturer. All three clones secreted antibodies of the IgG 1 subclass with kappa light chain. Serially diluted monoclonal antibodies, obtained from all three lines, were tested against purified barley inhibitor, barley, wheat, triticale and rye aqueous extracts by ELISA assay and the results are presented in Fig. 1. MAb raised from 4FIO-5 monoclonal cell line reacted only with purified inhibitor and barley extract, while MAbs obtained from cell lines 4F6-1 and IHI-7 reacted with all cereal extracts, except rye. Although MAb 4FlO-5 gave highest reaction with purified inhibitor, the magnitude of reactions with barley extract was similar for all three MAbs. In earlier reports, comparison of inhibitors from different cereals on the basis of reaction with polyclonal antibodies suggested that inhibitors from barley, wheat, rye and triticale are immunologically similar 1 • 9 • This is presumably because of the similar structures of the individual inhibitors. In fact it has been reported that inhibitors isolated from barley and wheat have almost identical amino acid sequences 10 • However, in our investigation, the specificity of the 4FIO-5 antibody suggested the presence of a specific antigenic determinant only in barley inhibitor indicating that barley inhibitor is immunochemically different from inhibitors from the other cereals tested. To test for cross-reactivity, all MAbs were further investigated by immunoblotting after SDS-PAGE (Fig. 2). Only 4FIO-5 bound to the barley inhibitor, showing no reaction with other proteins [Fig. 2(b)]. Partial renaturation of proteins blotted after SDS-PAGE might occur during the transfer; however, it is size-dependent and less efficient for proteins of M r 20k to 15k than those of higher M r ll . Since the barley inhibitor has a M r of approximately 20k it is expected that MAb 4FIO-5 recognizes a sequential epitope rather than a conformational epitope, while the other two MAbs are specific to the native form of inhibitor. To support this hypothesis, all antibodies were titrated on ELISA plates coated with denatured preparations (boiled for 5 min) of pure inhibitor, barley, wheat, triticale or rye extracts. Of the three monoclonal antibodies, only 4FlO-5 reacted with the denaturated form of barley inhibitor, although the absorbance value with denatured inhibitor was slightly lower than with its native form (data not shown). The difference in the reactivities of the 4FIO-5 and the other two mabs in the ELISA and the fact that only 4FlO-5 reacts on immunoblots may indicate that the 4FIO-5 recognizes a unique inhibitor epitope. Moreover, because of its high specificity to barley inhibitor, this MAb may provide a suitable reagent for the detection and quantification of this protein in a variety of barley cultivars and its wild counterparts. We thank Ms Lidia Gosek for her skilful technical assistance. Contribution no. 209 of the Food Science Department, University of Manitoba. \-2
4
J. ZAWISTOWSKI ET AL. 2 110-84--
3
4
5
2
6
3
4
5
:
.'. .... , .... ':.
'
·'/.~:·~;i~~···'
4733--
24-
/6 -
~W'
(a)
(b)
FIGURE 2. Protein blot of aqueous extracts of cereal kernels separated by 80S-PAGE. (a) Blot stained with Amidoblaek. Lanes (1) M r standards; (2) purified inhibitor standard; (3) triticale ev. T3; (4) barley ev. Harrington; (5) wheat ev. Columbus; (6) rye ev. Oanksowskie Zlote. (b) Blot immunostained with MAb 4FlO5. Lanes (I) purified inhibitor standard; (2) barleyev. Harrington; (3) wheat ev. Columbus; (4) triticale ev. T3; (5) rye ev. Oankowskie Zlote.
References I. Weselake, R. J., MacGregor, A. W. and Hill, R. D. Cereal Chem. 62 (1985) 120-123. Zawistowska, D., Langstaff, J. and Bushuk, W. J. Cereal Sci. 8 (1988) 207-209. Munck, L., Mundy, J. and Vaag, P. Am. Soc. Brew. Chem. J. 43 (1985) 35-38. Henson, C. A. and Stone, J. M. J. Cereal Sci. 8 (1988) 39-46. Zawistowska, D. US Patent 4,910,297 (1990). Zawistowski, J. and Howes, N. K. J. Cereal Sci. 12 (1990) 235-244. Laemmli, U. K. Nature 227 (1970) 880--685. Towbin, R., Staehelin, T. and Gordon, J. Proc. Nat!. Acad. Sci. USA 76 (1979) 4350-4354. Sadowski, J., MacGregor, A. and Daussant, J. Electrophoresis 7 (1986) 176-179. Mundy, J., Hejgaard, J. and Svendsen, 1. FEBS Lett. 167 (1984) 210-214. II. Dunn, S. D. Anal. Biochem. 157 (1986) 144-153.
2. 3. 4. 5. 6. 7. 8. 9. 10.