Journal of Experimental Botany, Vol. 51, No. 343, pp. 305-307,
February 2000
© 2000 Oxford University Press
Methods Papers |
The monoclonal antibody MAC252 does not react with the (-) enantiomer of abscisic acid
Ecophysiologie des Plantes sous Stress Environnementaux, INRA, 2 place Viala, F-34060 Montepellier cedex 1, France
Received 14 June 1999; Accepted 30 September 1999
Abstract
An RIA procedure has been developed for ABA quantification using MAC62, a monoclonal antibody raised against (+)-cis, trans-ABA. This widely used method now relies on MAC252, a recloned version of the exhausted MAC62. Recently, it has been suggested that MAC252 was not able to discriminate between the (+) and (-) enantiomers of ABA. As this can be misleading when interpreting RIA results, it has been carefully examined here whether MAC252 reacts with (-)-ABA. MAC252 exhibited negligible cross-reactivity with (-)-ABA, which was confirmed with commercial mixtures of ABA isomers. It is concluded that the RIA protocol can continue to be used with MAC252 as it was with MAC62.
Key words: RIA, MAC252, ABA isomers
Introduction
During the past few years, monoclonal antibodies have become widely used to quantify endogenous ABA concentrations in plant extracts, enabling large-scale ABA analyses (Tuberosa et al., 1994
; Pekic et al., 1995). Immunological methods (for a review, see Walker-Simmons and Abrams, 1991) include enzyme-linked immunosorbent assays, fluoroimmuno assays and radioimmuno assays (RIA). Many groups (Wang et al., 1987; Saab et al., 1990; Tuberosa et al., 1994; Jackson et al., 1995; Dodd and Davies, 1996; Cramer et al., 1998; Simonneau et al., 1998) have adopted the RIA protocol (proposed by Quarrie et al., 1988) using MAC62, a highly specific monoclonal antibody raised against (+) 2 cis-4 trans-ABA (Quarrie and Galfre, 1985). MAC62 has an affinity constant of c. 5x108 l mol-1 for (+)-ABA, and does not bind (-)-ABA or the large majority of ABA metabolites and derivatives (Quarrie et al., 1988).
In 1998, results were published (Cramer et al., 1998) based on the procedure of Quarrie et al. (Quarrie et al., 1988), but with two versions of the antibody: MAC62, that was extensively characterized by Quarrie et al. and MAC252, a recloned version later supplied by the John Innes Centre, Norwich UK. Surprisingly, it was found that, in contrast to MAC62, MAC252 could not discriminate between the natural (+)- and unnatural (-)-cis, trans ABA enantiomers, though it did not react with the trans, trans isomer (Cramer et al., 1998). It is clear that the sensitivity for ABA isomers varies with the nature of the antibody (Walker-Simmons et al., 1991), but it was not expected to change between MAC62 and its recloned version MAC252 (SA Quarrie, personal communication). Since MAC252 is now substituted for the exhausted MAC62 for use in RIA, the results of Cramer et al. need to be carefully checked.
The specificity of the antibody to (+)-ABA compared to (-)-ABA is particularly crucial where racemic mixtures of (±) 2 cis-4 trans-ABA are prepared as internal standards. It has also important implications when drawing dose–response curves with (±)-ABA-fed plants.
Therefore, a set of controls was performed with different ABA isomers, either purified or mixed, to check for the specificity of MAC252. Special attention was paid to (-)-ABA reactivity with this antibody.
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Materials and methods
ABA and antibody preparations
Purified enantiomers ((+)-ABA, ref. A4906 and (-)-ABA, ref. A8451), racemic mixture ((±)-ABA, ref. A1012) and mixed isomers (ref. A7383) of ABA were obtained from Sigma (Sigma Chemical Co, St Louis, MO, USA).
A stock of lyophilized MAC252 was purchased from Dr SA Quarrie (John Innes Centre, Colney, Norwich, UK). MAC252 was rehydrated with water (following indications provided with the stock of MAC252) and diluted 1 : 20 in phosphate buffer saline (PBS, 100 mM sodium phosphate and 200 mM sodium chloride) containing 5 mg ml-1 bovine serum albumin (BSA, Sigma) and 4 mg ml-1 soluble polyvinylpyrrolidone (PVP, MW 40 000, Sigma). This working stock solution was stored at -20 °C. For the assay, a further dilution of about 1 : 120 in buffered BSA/PVP was made, giving a total dilution of 1 : 2400.
Radioactive abscisic acid preparation
(±)-cis,trans-[G-3H]-Abscisic acid (TRK 644 Amersham Pharmacia Biotech, UK) in ethanol at an approximate concentration of 1.85–3.7 1012 Bq mmol-1 was diluted 1 : 10 in water and frozen in 50 µl aliquots (working stock). For the assay, working stock aliquots were further diluted at about 1 : 250 in PBS containing 2.5 mg ml-1 bovine 
-globulin (Sigma).
Radioimmunoassay protocol
ABA or water (for determination of maximum binding with 3H-ABA) were mixed with 100 µl [G-3H]-abscisic acid, (approximately 310 Bq), 100 µl MAC252 and 200 µl 50% PBS in 2 ml Eppendorf vials. Non-specific binding (revealed by the co-precipitation of radioactive material into the pellets) was determined by replacing water with ABA 100 µg ml-1, to have the greatest binding of the MAC252 with unlabelled ABA. The assay mixture was incubated at 4 °C in darkness for 45 min. A saturated solution (500 µl) of ammonium sulphate (Sigma) was then added and, after brief shaking, the mixture was left at room temperature for 30 min. Free antibodies and those that bound to ABA in the reaction mixture were precipitated and pelleted by centrifugation for 4 min at 8800 g, then the supernatant was discarded. The pellet was washed by resuspending in 1 ml of a 1 : 1 mix of water and saturated ammonium sulphate solution, and centrifuged again for 5 min at 8800 g. The supernatant was discarded and the pellet finally resuspended in 100 µl deionized water. After adding 1.2 ml scintillation cocktail (Emulsifier Safe, Packard Instrument Company, USA), radioactivity was quantified in a liquid-scintillation counter (Tri-carb, Packard). A logit transformation was applied to the results as follows:
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Results and discussion
Figure 1
clearly shows that (-)-ABA hardly binds to MAC252. With added (-)-ABA concentrations as high as 8000 pg/50 µl, resulting counts did not exceed an equivalent of 200 pg (+)-ABA/50 µl, indicating that, in the absence of non-labelled (+)-ABA, less than 3% of the added (-)-ABA reacted with MAC252 as (+)-ABA did. This is very different from the results of Cramer et al., who found that both enantiomers bound to the antibody equally (Cramer et al., 1998). The slight binding of (-)-ABA with MAC252 reported in Fig. 1 was estimated in the absence of competition with non-radioactive (+)-ABA. This should have overestimated the binding of (-)-ABA when the (+)-enantiomer was also added to the vial, for example, as racemic standards. This gives confidence that ignoring the cross-reactivity of MAC252 with (-)-ABA leads to negligible error.
The reactivity of the racemic ABA is consistent with a negligible binding of (-)-ABA. Compared to purified (+)-ABA, the percentage of (±)-ABA that bound MAC252, was close to 50%, though slightly but significantly higher (57.4±2.5%). Furthermore, reactivity of mixed isomers with MAC252 was even more reduced (39.7±0.8%). The proportions of each isomer in the mixture are not strictly equal (Sigma-Aldrich France, personal communication). This can partly explain why the cross-reactivity of the mixed isomers with MAC252 was greater than 25%. However, a weak binding of trans ABA enantiomers with MAC252 cannot be excluded and merits further checking, notably if mixed isomers are used as internal standards or as a source of exogenous ABA for feeding plants.
These results suggest that the recloned version of MAC62 kept the same immunological properties as its initial version. It is therefore unlikely that ABA derivatives which did not bind MAC62 could react with MAC252. However, as for MAC62, the present results do not exclude cross-reactivity with immunoreactants other than those mentioned here, particularly in crude plant extracts. As an example, it was found by thin layer chromatography that 52% of MAC252 reactivity with crude leaf extracts of perennial forage grasses were with immunoreactive compounds other than (+)-ABA (P Barrieu, unpublished data). In the same way, cross-reactivities up to 36.4% were found with some artificial ABA analogs like the C-4 C-5 acetylenic acid (Walker-Simmons et al., 1991).
Recloning of the cell line to get MAC252 was not expected to modify the antibody specificity to (+)-ABA, neither its cross-reactivity for ABA analogues. Notably, it is unlikely that MAC252 had lost the very high specificity of MAC62 for ABA analogues having the same absolute configuration at the chiral centre C-1' as (+)-ABA (Walker-Simmons et al., 1991). It is thus not known how cross-reactivity for (-)-ABA could be achieved with MAC252 as reported earlier (Cramer et al., 1998). Optically pure enantiomers of ABA were obtained by these authors from racemic methyl abscisate that was resolved to pure methyl esters of ABA (Dunstan et al., 1992). It would be helpful to know whether Cramer et al. obtained their (-)-ABA as a crystalline solid, in which case it is likely to be sufficiently pure (Cramer et al., 1998). If it was not crystallized, then it may have contained solvent residues or other substances that could have cross-reacted with MAC252. This is, however, unlikely to explain the huge differences between the present findings (Fig. 1), and those reported previously (Cramer et al., 1988).
In conclusion, ABA can be reliably assayed by RIA with MAC252 as it was with MAC62. Notably, constructing standard curves with racemic (±)-ABA and, considering that only half the (±)-ABA added binds MAC252, yields similar results to those for purified (+)-ABA standards. MAC252 can also be used to discriminate between (+)- and (-)-ABA in physiological studies based on (±)-ABA-fed plants.
Acknowledgments
The authors wish to thank Dr SA Quarrie for continuing help and advice concerning the use of MAC62 and MAC252 antibodies in RIA.
Notes
1 To whom correspondence should be addressed. Fax: +33 4 67 52 2116. E-mail:simonneau{at}ensam.inra.fr 
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