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Preliminary immunological studies on horseradish and spinach peroxidase isoenzymes
BERNARDINI, Nicola, PENEL, Claude, GREPPIN, Hubert
BERNARDINI, Nicola, PENEL, Claude, GREPPIN, Hubert. Preliminary immunological studies on horseradish and spinach peroxidase isoenzymes. In: Hubert Greppin, Claude Penel & Thomas Gaspar. Molecular and physiological aspects of plant peroxidases . Genève : Université de Genève, Centre de botanique, 1986. p. 97-103
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N.BERNARDINI, C.PENEL and H.GREPPIN
Laboratoire de Physiologie vegetale, Universite de Geneve, 3 place de l'Universite, 1211 GENEVE 4, Switzerland
Introduction
The obtention of polyclonal or monoclonal antibodies against plant isoperoxidase is of great interest for several reasons:
1) The activity of peroxidase may be considerably modified by other substances also found in a crude plant extract and, in that case, only an immunoassay would allow to measure the amount of enzyme actually present.
2) There is a great incertitude concerning the structural identity of each isoperoxidase and the use of immunological techniques could permit to clarify the structural similarities between them. The technology of molecular biology (gene cloning, in vitro translation), which also requires the use of antibodies, could be used for the same purpose.
3) Antibodies could eventually be used for the determination of the cellular localization of isoperoxidases and their precursors by histological preparations, for either optical or electron microscopy.
These reasons led us to undertake the production and testing of monoclonal and polyclonal antibodies against horseradish and spinach peroxidases. We present here some of the preliminary data obtained.
Material and methods Isoperoxidases
Three horseradish peroxidase isoenzymes, and VIII, designated HA7 and HAS) and designated HB9) were purchased from Sigma.
two acidic (types VII one basic (type IX,
98 N BERNARDINI et al.
A basic spinach peroxidase (designated SBl) was purified from leaves by the following method: leaves (200 g) were ground in 300 ml of 25 mM Mes-Tris buffer pH 7.2, containing 50 mM NaCl and 2 mM ascorbic acid. After filtration, the extract was centrifuged during 10 minutes at 20000 g. Proteins were then precipitated by the addition of an equal volume of 50% (w/v) polyethyleneglycol 6000. After centrifugation for 10 minutes at 10000 g, the pellets were resuspended in 350 ml of the same Mes-Tris buffer. The undissolved proteins were eliminated by centrifugating 5 minutes at 8000 g and the clear supernatant was applied to a DEAE Sephacel column (10x5 cm) prealably equilibrated and then washed with the same buffer. The basic proteins in the flow-through eluate were precipitated at 90% ammonium sulfate saturation, resuspended in 25 mM acetate buffer pH 4.5 and dialyzed against 5 mM acetate buffer pH 4.5. The cleared protein solution was applied to a column of CM cellulose (CM Cellex, lOxl cm) equilibrated and washed with the dialysis buffer. Bound basic proteins were eluted with a linear gradient of NaCl (0-200 mM) in the same buffer. The effluent fractions with a high peroxidase activity were pooled, concentrated by ultrafiltration (Centricon 10; Amicon) and further purified on a Sephacryl S-200 column (100x0,8cm) equilibrated with 25 mM Mes-Tris pH 7.2, 200 mM NaCl.
Collected fractions of high peroxidase activity were further concentrated, when necessary, by ultrafiltration.
Purity control of isoperoxidases
HA7, HA8, HB9 and SBl were assessed for purity by SDS
polyacrylamide (12,5 and 15%) gel electrophoresis and isoelectric focusing in agarose gel (pH range 3.5 to 11).
Polyclonal antibodies
Antisera were raised in Balb/C and Swiss Albino mice by intraperitoneal injections at 30 days intervals of HA7, HA8, HB9 and SBl dissolved in PBS pH 7.2, emulsified with Freund complete adjuvant and with Freund incomplete adjuvant for the first two injections.
Monoclonal antibodies
Two hybridomas producing monoclonal antibodies against HB9 were obtained by fusions performed by the method of Kohler (4).
The selection and cloning of the hybridomas were done according to Davis et al.(2). Both lines produce antibodies belonging to the IgGl subclass. They have not as yet been tested for eventual differences in epitope specificity.
Concentration of monoclonal antibodies
Hybridoma culture supernatants were concentrated by two successive precipitations at 50% and, after redissolution and clearing, 40% ammonium sulfate saturation. Proteins were then dissolved in a minimum volume of PBS and dialyzed against PBS.
The final concentration of monoclonal IgG was assessed by single radial immunodiffusion using rabbit antisera against mouse IgG mixed with agarose to coat the plates and serial dilutions of mouse IgG (Sigma) as standards (3).
Elisa indirect method (5)
The antigen solutions were obtained by dissolving and/or diluting HA7, HA8, HB9 and SBl in PBS pH 7.2 containing 0.01%
Thimerosal to a heme concentration estimated at 403 nm, equivalent to a solution of HB9 of 10 µg/ml. They were adsorbed on flexible U bottom microtiter plates (Dynatech). Negative control was PBS without antigen. The wells were saturated with 1%
BSA in PBS. First antibodies were applied each to a series of 5 wells (4) coated with each antigen and 1 negative control). They consisted of antisera diluted 1/100 and 1/1000 with PBS 0, 01%
Thimerosal, 1% BSA, 0.05% Tween, pH 7.2 and of hybridoma supernatant and concentrated antibody undiluted and diluted 1/2, 1/5, 1/10 with culture medium.
The second antibody was alkaline phosphatase conjugated sheep anti-mouse immunoglobulin (NEN) and substrate was p-nitrophenyl phosphate. The reaction was stopped by addition of 0.5 N NaOH and the absorbance of each well read at 410 nm on a Dynatech minireader II spectrophotometer with a printing device (Table 1).
Results and discussion Purity of antigens
Isoelectric focusing indicated a good but not complete homogeneity for the three horseradish isoenzymes HA7, HA8, and HB9 (Fig.I), this being especially visible for HB9. Nevertheless no bands corresponding to proteins of common isoelectric point existed between acidic HA7 and HA8 and the basic HB9 that could justify cross-reactivity of antisera.
SDS-polyacrylamide gel electrophoresis of SBl applied to the gel at high concentration (Fig.2) showed one protein single band and the faint trace of a contaminating higher MW protein. The relative MW of SBl is situated between 43 and 30 KDa.
Isoelectric focusing of the purified SBl showed good isoenzyme homogeneity with traces of less basic bands which could be degradation products (Fig.I). SBl has an isoelectric point higher than 10 and a RZ of 3.0, which is comparable to the HZ of HB9 indicated by Sigma (3.2).
100 N BERNARDINI et al.
Table 1. Cross-reactivity of mouse antisera with 3 isoperoxidases from horseradish (HA7, HA8, HB9) and one from spinach (SBl). A-S:
different mice injected with the various antigens and diluted lOOx (1) or lOOOx (2). The numbers are the values of absorbance at 410 nm read after the Elisa indirect method.
Antisera
A B C
D E F G
H I J K L
M N
0 p
Q R s
1 2 1 2 1 2 1 2 2 1 2 1 2 1
1 2 2 1 1 2 1 2 1 2
1 2 1 2
2 1 1 2 1 2 2 1 2 1
HA7
1.10 0.35 0.07 0.01 0.97 0.15 0.18 0.00 0.34 0.03 0.99 0.22 0.29 0.01
1. 29 0.97 1. 24 0.87 1. 24 1.10 1. 35 0.59 0.99 0.20
1. 33 0.76 0.87 0.14
0.73 0.15 0.79 0.17 1. 06 0.69 0.06 0.00 0.12 0.02
A n t i g e n s
HA8 HB9
ANTISERA TO HB9 0. 55 1. 33
0.22 0.76
0.05 0.00 0.09 0.01 0.15 0.00 0.18 0.03 0.84 0.19 0.22 0.00
1. 29 0.59 1.45 1. 22 1.19 0.53 1. 39 0.99 1.56 1.10 1. 21 0.79 ANTISERA TO HA7
0.89 0.77
0.51 0.96 0.84 0.87 0.60 1. 01 0.25 0.37 0.06
0.36 0.66 0.55 0.65 0.60 0.79 0.20 0.11 0.01 ANTISERA TO HA8
1.38 1.21
0.98 1. 01 0.28
0.53 0.47 0.06 ANTISERA TO SBl 0. 24 0 .49 o. 03
0.25 0. 03 0.52 0.16 0.06 0.00 0.03 0.01
0.09 0.54 0.11 0.77 0.39 0.06.
0.00 0.10 0.02
SB1
0.97 0.35 0.09 0.00 0.25 0.14 0.15 0.00 0.18 0.01 0.44 0.08 0.03 0.00
0.77 0.42 0.73 0.60 0.89 0.72 0.17 0.03 0.14 0.01
0.68 0.08 0.50 0.08
1.16 0.89 1. 21 0.92 1.46 1. 08 0.96 0.29 0.91 0.53
a b
Cd e f -e
�
all) �
� I
� �
�
Fig. 1. Isoelectric focusing on agarose, pH range 3.5-11, of 20 µl samples of isoperoxidases at heme concentration equivalent to a HB9 concentration of a) HB9 10 µg/ml, b) SBl 5 µg/ml, c) HA7 lµg/ml, d) HAB 1 µg/ml, e) SBl 10 µg/ml, f) HB9 10 µg/ml. Peroxidase activity was stained with benzidine-hydrogen peroxide.
a b
Cd e f .... g
Fig. 2. SOS-polyacrylamide gel electrophoresis (15 %} of a) and g) Pharmacia L.M.W. standards, b) HB9 3.3 µg, c) and d) partially purified SB], heme concentrations equivalent to 23 and 30 µg of HB9, e) purified SBl heme concentration equivalent to 20 µg of HB9, f) contaminating protein eluted from Sephacryl column.
102 N BERNARDINI et al.
Cross-reactivity The 7 antisera L), the 2 for HA8
of antisera for HB9 (A to
(M and N) and G, Table 1), the 5 for HA7 (H to the 5 for SB1 (O to S) all cross- react to a more or less great extent with the other three isoperoxidases.
Since anti HA7 sera show the strongest cross-reaction with HA8 and vice versa it appears that these 2 acidic isoenzymes are more closely related immunologically to each other than to HB9. This result confirms that obtained by Clark et al. (1) using rabbit antisera. On the other hand mouse antisera for either HA7 or HA8 cross-react with spinach leaves basic peroxidase sometimes quite strongly while rabbit antisera against horseradish acidic isoenzymes did not react with basic turnip, radish or carrot root peroxidases (1).
Table 2. Test for the reactivity of a mouse monoclonal antibody to horseradish isopero
xidase HB9 with 3 isoperoxidases from horse
radish (HA7, HA8, HB9) and one from spinach (SB1). A: Hybridoma anti HB9 culture super
natant at various dilutions. B: Monoclonal antibodies undiluted (about 150 µg/ml) and at various dilutions. The numbers are the values of absorbance at 410 nm read after the Elisa indirect method.
HA7
A 1/1 0.00 1/2 0.00 1/5 0.00 1/10 0.00
ANTIGENS HA8 HB9
0.00 0.40
o.oo
0.470.00 0.44 0.00 0.37
SB1
0.00 0.00 0.00 0.00
---
B 1 0.00 0.00 0.35 0.00 1/2 0.01 0.00 0.34 0.00 1/5 0.01 0.00 0.38 0.00 1/10 0.00 0.00 0.38 0.02
---
HA7 seems to have the largest number of similar or identical determinants in common with the other peroxidase isoenzymes tested since it is almost invariably the antigen against which all antisera cross-react most strongly. It seems improbable that this is all due to the same similar or identical epitopes that would have to be present on HA7 and the other three antigens since this would imply just as strong cross-reactions between
them and the other antisera. Furthermore the fact that antisera against basic horseradish and spinach isoperoxidases often cross
react more strongly with HA7 than respectively with SB1 and HB9 seems to show that the degree of immunological similarity is not necessarily related to the isoelectric point of the isoperoxidases, at least for different species.
All antisera show as expected the strongest avidity for the antigen against which they were raised, but antisera for a same antigen produced by different animals, while presenting similar avidities for their own antigen, may vary in the amount and/or the avidity of their cross-reacting antibodies. This is well demonstrated by diluting the antisera, some of which, after 1/1000 fold dilution, do not show detectable cross-reactions by Elisa, while in other antisera, cross-reactions are still strong.
This different behaviour of antisera can be explained by variations in individual genetic responsiveness to epitope antigenicity which apparently exists even in inbred mice strains.
Monoclonal antibody
Supernatants of high cell density hybridoma cultures secreting antibody to HB9 do not cross-react with either HA7, HA8 or SB1 (Table 2). Diluting the supernatant has no effect (except possibly a slight increase of antibody fixation to HB9).
Concentrating the antibody (about 150 µg/ml IgG) does not change or only slightly diminish the antibody fixation. Dilutions performed on the concentrated antibody seem to reestablish the situation.
References
1 CLARK SK Jr, JM CONROY 1984 Homology of plant peroxidases:
relationships among acidic isoenzymes. Physiol Plant 60:
294-298
2 DAVIS GM, JE PENNINGTON, AM KUBLER, JF CONSCIENCE 1982 A simple single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies.
J Immunol Meth 50: 161-171
3 HUDSON L, FC HAY 1980 Practical Immunology: Single radial immunodiffusion, 2nd ed. Blackwell Sci. Publ., Oxford, pp 114-116
4 KOHLER G 1978 EMBO Laboratory Course on B. Lymphocyte Fusion. Basel Institute for Immunology.
5 VOLLER A, A BARTLETT, DE BIDWELL 1978 Enzyme immunoassays with special reference to Elisa techniques. J Clin Pathol 31: 507-510
