Planta

Planta (1985)166:524-529

9 Springer-Verlag 1985

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzymeqinked immunosorbent assay using the avidin-biotin system B. Leroux, R. Maldiney, E. Miginiac*, L. Sossountzov and B. Sotta Phytotron, C.N.R.S., F-91190 Gif sur Yvette, France

Abstract. In this report we describe an enzymelinked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkatine phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of L ycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by highperformance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available. Key words: Abscisic acid (quantitation) - Avidinbiotin system - Enzyme-linked immunosorbent assay. * To whom correspondence should be addressed Abbreviations: ABA = abscisic acid; BSA = bovine serum albumin; ELISA=euzyme-linked immunosorbent assay; G L C = gas liquid chromatography; HPLC = high-performance liquid chromatography; IgG = Immunoglobulin G; PBS = phosphatebuffered saline

Introduction Abscisic acid (ABA), one of the five main classes of known phytohormones, may be involved in the regulation of numerous physiological processes: growth, dormancy, abscission, senescence and stress physiology (Walton 1980). In all these cases, understanding the role of ABA needs precise measurements of its inter- and intracellular levels. Numerous data have also to be obtained rapidly for the evaluation of the precise kinetics of the physiological phenomenon under study. As pointed out by Weiler (1983, 1984), immunological methods are one of the most efficient tools to fulfill these requirements. First, specific antibodies with high affinity against an antigen have to be obtained. After that the quantitation of the antigen, for instance ABA, involves a competition for these antibodies between a labelled form of the hormone and the form to be measured (standard or sample). Radioimmuno assay (RIA) can allow very sensitive and precise quantitation of an hormone, but it needs an antigen with high specific radioactivity and a quite expensive apparatus. Immuno-enzymatic techniques, especially enzyme-linked immuno sorbent assay (ELISA), are even m o r e sensitive and require relatively cheaper laboratory apparatus and compounds. The ELISA quantitation of ABA has already been presented by Daie and Wyse (1982) and by Weiler (1982) using the coupling of the hormone with alkaline phosphatase of high specific activity. This paper describes an indirect ELISA method which provides substantial amplification of the response: the avidin-biotin system. To our knowledge, this system has not yet been applied to determine low amounts of an hormone in plant extracts. The application of the avidin-biotin interaction to immunoenzymatic techniques has been demon-

B. Leroux et al. : Quantitation of abscisic acid

strated by Guesdon et al. (1979). A substantial increase in sensitivity has been obtained by this means in studies of the surface antigens of B hepatitis (Kendall et al. 1983) and for antigens of Streptococcus pneumoniae and Haemophilus influenzae (Yolken et al. 1983). We report here the capacities of this system for ABA quantitation, and confirm the ELISA results by gas-liquid chromatography (GLC). Material and methods

525 eluate was evaporated under vacuum at 30 ~ C to the aqueous phase. After acidification with acetic acid to pH 3.0, this phase was loaded again on a Sep-Pak C18 cartridge equilibrated to pH 3 with dilute acetic acid. Then, the adsorbed material was eluted with 3 ml ethyl acetate and evaporated to dryness under nitrogen. The residue was taken up with 50 ~tl methanol and 20 ~tl were injected onto the HPLC column which was eluted with an H20-methauol gradient (described in Fig. 3) adjusted to pH 2.8 with acetic acid. Fractions (0.2 ml) were collected every 0.5 min for 35 min, the ABA fraction being eluted between 26 and 29 rain. Fractions were evaporated to dryness under vacuum and methylated with etheral diazomethane. After evaporation under nitrogen they were taken up with methanol for GLC analysis or with phosphate-buffered saline (PBS) for ELISA quantitation.

Plant materials Tomatoes (Lycopersicon esculentum L.) and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) samples were collected from plants grown in the greenhouses of the Phytotron (Gif sur Yvette, CNRS, France). Tobacco (Nicotiana tabacum L.) plants were cultured in greenhouses of I N R A (Versailles, France).

Chemicals cis(+_)-Abscisic acid and cis(+)-ABA were obtained from Fluka (Buchs, Switzerland); cis(+_)-[3H]ABA(TRK644, specific activity 63.6" 10 l~ Bq. mmol-1) was purchased from the Radiochemical Centre (Amersham, Bucks. UK); bovine serum albumin (BSA), radioimmuno assay grade, caseine and biotin were obtained from Sigma (St. Louis, Mo., USA); pure avidine, affinity chromatography purified, was supplied by Reactifs IBF (Villeneuve la Garenne, France); and alkaline phosphatase from calf intestine (Grade I, specific activity >2500 U . m g 1) was from Boehringer (Mannheim, FRG). All solvents were of analytical grade.

Equipment Flat-bottom microtitration plates, Microwell 96F, were from N U N C (Denmark). Some reagents were added with an Eppendorf (Hamburg, FRG) Multipette 4780 system. Washes and application of other reagents were done with an Autodrop Flow from Titertek (Helsinki, Finland). Absorbance readings were carried out at 405 um with an EIA semi-automatic spectrophotometer from Vernon (Paris, France). Plant extracts were purified with a Beckmann (Berkeley, Calif., USA) l I 4 M high-performance liquid chromatography (HPLC) System equipped with a CPtm Spher C18 column (2 HPLC cartridges 100 mm long, 3 mm inner diameter Chrompack, Middelburg, The Netherlands). Abscisic-acid methyl ester was estimated with a GLC apparatus equipped with a 63Ni electron-capture detector (Girdel, Suresnes, France) and a 25-m CP Sil 5 capillary column (Chrompack). Calculations were done with a Sp 4200 integrator (Spectra physics, San Jose, Calif., USA).

Extraction and purification of plant material Tissues were frozen in liquid nitrogen and then homogenized in methanol: distilled water::80:20(v/v; 10ml.g -1 fresh weight) containing 40 rag. 1-1 butylated hydroxy-toluene (Milborrow and Malaby 1975). Extraction was carried out at 1~ C in the dark overnight. After filtration on a 0.45-1am Millipore filter, the methanol concentration was adjusted to 50% with water and the pH was increased to 8 with 1 M K2HPO#. The sample was then loaded on a Sep-Pak C18 cartridge (Waters, Milford, Ma., USA) to remove part of the pigments, and the

Immunoassay reagents Coupling of ( + )-ABA to BSA. The immunogenic complex was obtained by coupling 20 nag BSA with 4 mg cis( + )-ABA using 21 mg N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) according to Weiler (1980). Preparation ofanti-ABA antibodies. Antisera elicited in rabbits were obtained as recently described (Sotta et al. 1985). The immunoglobulin fraction was isolated by the standard ammonium-sulfate-precipitation method (Hurn and Chantler 1980). The immunoglobulin-G (IgG) concentration was adjusted to 1 mg-ml-1 in PBS and stored in aliquots at - 1 8 ~ C.

Coupling of ( • to goat immunoglobulins (IgG). Using N-cyclohexyl-N'-(2-morpholinoethyl)-carbodimide-metho-ptoluosulfonate (CMC), cis(+)-ABA was coupled through its carboxyl group to the protein aminoresidues of non-immunized-goat IgG, a protein which had given the lowest background among several tested in the ELISA. Eighty mg of cis( +_) -ABA were dissolved in 3 ml dimethyl formamide (DMF) and diluted with 2 ml H 2 0 ; 80 mg CMC were added and the pH was adjusted to 7.2 with 0.1 M NaOH. After stirring for I h at room temperature in the dark, the solution was added to 6 ml of 0.1 M Na-phosphate-buffered solution (pH 7) containing 30 mg goat IgG. The mixture was stirred for 24 h at room temperature in the dark and, finally, the solution was dialysed against several changes of PBS and stored at - 1 8 ~ until used. Biotinylation of goat anti-rabbit IgG. The biotinylation of goat anti-rabbit IgG (Institut Pasteur, Lyon, France) was carried out as described by Guesdon et al. (1979). Biotin was activated by N-hydroxysuccinimide according to Bayer and Wilchek (1974). The resulting compound was made 0.1 M in D M F from which 57 ~tl were added slowly to 1 ml of goat anti-rabbit IgG (10 rag) in 0.1 M sodium bicarbonate buffer, pH 8.8. After stirring for 1 h at room temperature, the reaction was stopped by addition of 1 M NH4C1, dialysed against PBS for 48 h and stored at - 18 ~ C. Preparation of avidine-phosphatase complex. The technique used was described by Avrameas and Ternynck (1971). 300 ~tl of a 10 mg. m l - 1 solution of avidine, dissolved in 0.1 M phosphate buffer supplemented with 0.01 M MgCI2 (pH 6.8), were added to 3 mg of alkaline phosphatase dissolved in the same buffer. Then 30 ~tl of 1% aqueous glutaraldehyde solution were added dropwise under gentle agitation. After 3 h, the mixture was dialysed against the same buffer and then against 0.05 M 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris)-HC1 buffer con-

526

B. Leroux et al. : Quantitation of abscisic acid

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Fig. 1. Stages in the ELISA technique for the quantitation of A B A using the avidin-biotin system. Step 1 : coating of the microtitration plate with the ABA(antigen)-goat IgG complex; step 2 and 3: A B A to be quantified and standard ABA are added to the wells with ABA (rabbit) antibodies; step 4: addition of biotin-labelled goat-antirabbit antibodies; step 5: addition of alkalinephosphatase-labelled avidin; step 6: determination of the enzymatic activity

taining 0.5 M NaC1 and 1 m M MgC12 (pH 8.8) for 24 h. The complex was stored in 50% glycerol at - 1 8 ~ C. U n d e r these conditions, 75% of the enzyme activity was retained during the coupling reaction.

Adsorption of the (+_)-ABA-goat IgG complex to polystyrene. The complex was dissolved in 50 m M NaHCOa (pH 9.6), to a concentration of 1.25 g g - m l - 1 which was the best concentration value between 0.125 gg. ml-1 to 5 gg. m l - 1 as determined by preliminary tests (data not shown). Each well of the polystyrene microtitration plates received 200 gl of the protein solution and the plates were incubated overnight at 4 ~ C. After the incubation step, they were emptied and washed four times with PBS. The plates were then ready to use (Fig. 1, step 1).

Buffers used. A: PBS + 40 txg"m l - 1 caseine + 0.04% Triton X100; B: P B S + 50 gg-m1-1 BSA; C: P B S + 0 A % Triton X-100; D: 0.05 M T r i s + 0 . 5 M NaCI+0.01 M M g C I 2 + 0 . 1 % Triton X-100 equilibrated at pH 8; E: I M diethanolamine+0.001 M MgC12+0.1% Triton X-J00 adjusted to pH 9.8; F: 1 M diethanolamine. All buffers were stored at 4 ~ C. Caseine and BSA were added at the lower concentration giving the lower background level with the higher sensitivity of the assay (data not shown).

The plates were stirred for 3 min, capped and stored at 4 ~ C for 2 h for the competition step (Fig. 1, step 3). The emptied plates were then washed four times with 250 gl of C and subsequently 200 gl of buffer C equilibrated at 37 ~ C containing the biotinylated goat anti-rabbit antibodies were added to each well. Incubation of capped plates was carried out at 37 ~ C for 1 h (Fig. 1, step 4). After this time, they were washed again four times with 250 gl of D and 20 gl of avidin phosphatase dissolved in D and previously equilibrated at 37 ~ C were added. The plates were incubated for 1 h at 37~ (Fig. 1, step 5). The plates were then washed twice with 250 ~1 of E and two times with 250 ~1 of F. The enzyme substrate p-nitrophenyl phosphate dissolved (1 mg.m1-1) in F equilibrated at 37~ was rapidly added to each well. The enzyme reaction proceeded at 37 ~ C for 1 h and was stopped with 50 gl of 5 M K O H per well (Fig. 1, step 6). After addition of 150 gl water in each well the readings were taken at 405 nm. The results were analysed with a microcomputer (Apple Cupertino, Calif., USA). A curvilinear regression of magnitude 4 was made using the average of the 3 or 4 standard curves established on each microtitration plate. The regression was calculated by Fischer's method (Snedecor and Cochran 1971).

Solid-phase immunoassay of ABA. First, 50 gl of A were added

Results

to each well and then 100 gtl of ABA standard or sample solutions dissolved in PBS. The solutions were mixed for 1 miu and stored at + 4 ~ C for 15 min. Then 50 gl of antibodies diluted in B were added to each well (Fig. 1, step 2). The dilution of anti-ABA IgGs (1 gg. m l - 1 , i.e. 1/1000 stock solution) corresponds to 50% of maximum binding of antibodies to the wells.

A B A methyl-ester could be detected and quantita-

S t a n d a r d curve.

Under the above conditions, 5 pg

tion was possible between 10 pg and 10 ng of ABA per well (Fig. 2). Three-hundred pg of methylated

B, Leroux et al. : Quantitation of abscisic acid

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