Planta (Berl.) 103, 117-125 (1972) 9 by Springer-Verlag 1972

Detection and Quantitative Determination of Abscisic Acid by Immunological Assay YO~A~ F u c ~ s and SHL~ON MAYAK The Volcani Institute of Agricultural Research, Bet Dagan, Israel SA~A FUCHS :Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel Received September 20, 1971 Summary. Antibodies with specificity towards abscisie acid (ABA) were produced in rabbits. These antibodies were used for assaying ABA by the inhibition of inactivation of modified bacteriophage. For this assay conjugates of ABA with bacteriophage T4 were prepared and characterized. Such chemically modified bacteriophages were completely inactivated by the specific anti-ABA serum and this inactivation was inhibited by free ABA. The identification and quantitative determination of ABA in plant extracts by this method are demonstrated and the method is compared with a common bioassay.

Introduction The most common bioassay for ABA is the inhibition of the germination of wheat coleoptiles (Nitseh and Nitsch, 1956), other methods of determination are based on spectropolarimetry (Milborrow, 1967), gas liquid chromatography (GLC) (Davis et al., 1968; Lenten et al., 1971), and a combination of GLC and mass spectroscopy (Browning et al., 1970). I n view of the successful development of immunoassays for animal hormones (Yalow and Berson, 1960) and other substances of biological importance, the detection and quantitative determination of I A A and GA b y immunoIogieai methods have recently been investigated and reported (Fuchs and Fuchs, 1969; Fuchs et al., 1971). Plant hormones, being small organic molecules, are not antigenic per se. They can, however, be linked as haptens to protein carriers to give conjugates which m a y yield, upon immunization, antibodies with specificity towards the hapten. I n a similar manner to their conjugation to protein carriers, haptens can be conjugated to bacteriophage T4. The hormone-bacteriophage conjugates are inactivated b y antibodies against the hormone. I n turn, the ability of the antibodies to inactivate the modified phage m a y be inhibited b y the hormone (Haimovich et al., 1970). Similar methods are applied here, in which modified bacteriophage (ABA-phage)

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Y. Fuchs, S. M~vak, and S. Fuchs:

is used for i m m u n o a s s a y of A B A . T h e i d e n t i f i c a t i o n a n d q u a n t i t a t i v e d e t e r m i n a t i o n of A B A in p l a n t e x t r a c t s b y this m e t h o d are d e m o n strated, a n d t h e m e t h o d is c o m p a r e d w i t h a c o m m o n bioassay.

Materials and ~lethods Antigens. RS-Abscisie acid (ABA) was a gift from F. Hoffmann La Roche and Co. Ltd.; bovine serum albumin was purchased from Armour Pharmaceutical Company and Keyhole limpet hemocyanin from Mann Research Laboratories. Conjugation of ABA to either hemoeyanin or bovine serum albumin was done according to the procedures described previously (Fuehs and Fuchs, 1969; Ranadive and Sehon, 1967). Fifty mg of ABA was dissolved in 1 ml of dry dioxan; 27 mg of N,N-dicyelohexyl earbodiimide was added and the solution was stirred for 30 min at room temperature. The precipitate formed was removed by centrifugation. The supernatant solution containing the acid anhych-ide was added, dropwise, with stirring to 3 ml of 0.1 M borate buffer, pH 8.5, containing 30 mg of hemocyanin or bovine serum albumin. Stirring was continued overnight at 4~ The mixture was dialyzed against 0.1 M borate buffer, pH 8.0. Any precipitate formed was removed by eentrifugation. The superuatant was dialyzed against distilled water and lyophilized. The number of haptenic groups attached to the carrier proteins was determined from the amino acid analysis of the conjugated proteins before and after deamination (Anfinsen et al., 1962). There were 18 moles of ABA attached per one mole of BSA and 25 haptenic groups attached per 100 lysines of HC. Abscisyl Bacteriophage ~ Conjugate. The bacteriophage T 4 was grown and purified as described previously (Haimovich and Sela, 1966; Haimovich et al., 1970). ABA was conjugated to bacteriophage T 4 in a manner similar to the conjugation of proteins; 40 mg of ABA dissolved in 0.5 ml of dioxane was reacted, with stirring, for 30 rain, at room temperature, with 30 mg of dicyclohexyl carbodiimide. The mixture was centrifuged and 0.05 ml of the supernatant was added dropwise to 0.5 ml of bacteriophage T a suspension containing 10TM plaque-forming units per ml of 0.1 M borate buffer, pH 8.5. The reaction was allowed to continue for 10 min with stirring at 4~ and was stopped by diluting the mixture 100-fold in 0.5 M phosphate buffer, pH 6.8. The dilute solution was dialyzed for two days against the latter buffer and the surviving bacteriophage concentration was determined. Immunization Procedure. Four rabbits were immunized with abscisyl-hemoeyanin (AB-HC) by two injections, each of 1 mg per animal, at 10-day intervals. The antigen was incorporated in a water-in-oil emulsion containing one part of antigen solution and two parts of complete Freund's adjuvant (Difeo Laboratories, Detroit, Mich.). The mixture was injected intradermally at multiple sites all over the body. Assay/or Detection o] Antibodies and Antigens. Quantitative precipitin reactions and inhibition of precipitin reactions were done as described previously (Fuchs and Sela, 1963). Increasing amounts of the antigen dissolved in 0.5 ml of phosphatebuffered saline (PBS, i.e., 0.01 M phosphate buffer pH 7.4, 0.15 M NaC1) were added to 0.3 ml aliquots of the tested antiserum. The solutions were mixed and the tubes were placed in a water bath at 37~ for 30rain and then at 4~ for an additional 24 h. The resulting precipitates were centrifuged, washed twice with chilled PBS and dissolved in 1 ml of 0.1 N NaOH. The absorbance of the dissolved precipitates was read at 280nm, in a Zeiss Model P ~ Q II spectrophotometer. Inhibition of the precipitin reaction was performed as follows: increasing amounts of the inhibitor dissolved in 0.5 ml PBS were added to 0.1 ml aliquots of antiserum.

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After an incubation for 30 min at 37~ the amount of the antigen which gave optimum precipitation in the precipitin reaction was added. The mixtures were incubated for 30 min at 37~ and for 24 h in the cold room. Any precipitates formed were investigated quantitatively as described above. Inactivation of abscisyl bacteriophage T 4 by anti-ABA serum and the inhibition of the inactivation by free ABA or by other compounds were performed according to the method described by Haimovich et al. (1970). The inactivation was performed in the following way: anti-ABA serum at several dilutions (0.2 ml aliquots) and abscisyl bacteriophage solutions (0.2 ml containing 300-600 plaque-forming units) were mixed and kept at 37 ~C for 1 h. At the end of the inactivation reaction, 2.5 ml of soft agar containing about 3 X 109 bacteria (Eschcrichia coil) was added to the test tube and the whole mixture was poured onto plates of bottom layer-agar. Plaques were counted after incubating the plates for 18 h at 37~ Inhibition of the inactivation was performed in the following way: anti-ABA serum (0.2 ml), at the dilution which causes 90-95% inactivation of abscisyl-bacteriophage was mixed with various concentrations of inhibitor (0.2 ml) and the mixtures were incubated at 37~ for 1 h. Then the bacteriophage solution was added (0.2 ml containing 300-600 plaque-forming units), and the test was continued as in the inactivation procedure described above. Plant Material. Alaska peas (Pisum sativum vat. Alaska) were grown in vermiculite for 7 days in the dark. The seedlings were freeze-dried and stored at 20 oC until extraction. Rose petals var. Golden Wave (Dr. Verhage) were collected and extracted on the same day. Extraction and Chro~atography. Alaska peas were extracted as described by Goren and Goldschmidt (1970). The diisopropyl ether (DipE) fraction was chromategraphed by TLC (silica gel G) with isopropanol, ammonia, water (80:0.1:19,9, v/v). In this system, authentic ABA traveled to the 0.7-0.9 Rf zone in different runs. Plates were divided into ten Rf zones, and the silica gel of each zone was eluted separately, overnight in the cold, with i ml of PBS (0.01 M phosphate buffer, p H 7.4, 0.15 M NaC1). This eluate was used for the immunoassay, 0.2 ml per test tube. The rose petals were extracted with 80% methanol at 4~ for 24 h, during which time the alcohol was changed twice. The methanolic extracts were combined, and evaporated to the aqueous phase under vacuum. After acidification to pI-I 3.0 with tiC1, the water phase was partitioned eight times with chloroform. (In other experiments--unpublished d a t a - - i t was found t h a t most if not all of the ABA-like inhibitors are extracted into the chloroform phase.) The combined chloroform fraction was streaked on plates coated with a 250 layers of silica gel G (Merck). The plates were developed by ascending chromatography using n-propanol, n-butanol, water and ammonia (6:2:2:1, v/v). In this chromatographic system, authentic ABA traveled to the 0.6-0.7 Rf zone. To eliminate any interference from the solvents used, the plates were dried at room temperature in a forced air cabinet for 10 h. Plates were divided into ten Rf zones, and the silica gel of each zone was eluted separately with absolute methanol; this was divided into two portions, one of which--equivalent to 8 g fresh weight--was transferred to three vials used in the wheat bioassay. The other portion, equivalent to 10 g fresh weight, was transferred to the immunoassay. A TLC plate developed but unspotted was used as a control. In each experiment, after the petals were macerated, extracted and evaporated, the aqueous phase was divided into two portions, as true replicates, and processed separately. -

-

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.Bioassay. The presence of plant growth inhibitors (ABA-like substances) was studied by the wheat coleoptile assay as described by Nitsch and Nitsch (1956). Results

Antibodies with Speci/icity to A B A . Three rabbits were immunized with abscisyl-hemocyanin and all of t h e m produced specific antibodies to ABA. Blood was collected weekly, starting one week after the second injection. Studies were carried out with sera of one of the rabbits. Preeipitin reaction were carried out using abscisyl-bovine serum albumin as antigen; the results are given in Fig. 1.

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The formation of precipitates of abscisyl-BSA with anti-ABA serum was inhibited b y free ABA (Fig. 2). Inhibition of 50% was achieved with about 0.1 fzmoles of ABA. Inactivation of abscisyl-T, phage b y anti-abscisyl-hemocyanin serum is described in Fig. 3. The reaction followed first order kinetics. Inactivation of 83% was achieved b y reacting abscisyl-T~ phage for 1 h at 37~ with anti-abscisyl-hemocyanin at a final serum dilution of 1:30 000. Pre-incubating the serum with ABA at different concentrations prior to the addition of the phage, reduced the extent of inactivation, corresponding to a lower concentration of antibodies than initially present in the reaction mixture. The extent of inhibition was calculated b y the aid of the inactivation curve. Experiments concerning the inhibition of the inactivation of abscisyl T 4 phage (Fig. 4), m a y be used as an

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assay for the detection a n d q u a n t i t a t i v e d e t e r m i n a t i o n of A B A i n p l a n t extracts. I A A , k i n e t i n or GA a t a c o n c e n t r a t i o n of 10 -5 M did n o t interfere with the i m m u n o a s s a y of ABA.

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Determination o / A B A in Plant Extracts. In different extracts, each of 1 g, of seven-day-old Alaska pea stems, ABA-like substances were detected at R r zones 0.7 and 0.8 (Fig. 5) by the immunoassay method. From the inhibition curves it was calculated that the DipE fraction of 1 g fresh weight contained about 25 ~g ABA. Results of the wheat coleoptile assay of the chloroform fraction appear in the histograms in Fig. 6. A peak of inhibitory activity was found at Rf 0.6-0.7. Comparison of the immunoassay with the common bioassay (Fig. 6) using the same extracts, indicated similar patterns; in both tests Rf zone 0.6 showed the highest ABA concentration. However, in some oI the chromatograms, in which there were large amounts of yellow substances in l~f zones 0.7 to 1.0 we obtained a cross-reaction of some substances with the anti-ABA antibodies, in the immunoassay, in these Rf zones while no ABA activity was evident, in these Rf zones, in the wheat bioassay (Fig. 6). Discussion

In this work we describe the preparation of specific antibodies to ABA and their application to a quantitative immunoassay for ABA. Since ABA is too small to be immunogenic per se, it was necessary to attach it to a carrier protein for the production of anti ABA antibodies. The specific binding of free ABA to the antibodies was used for assaying it b y using the sensitive modified bacteriophage technique (Haimovich et al., 1970). The anti-ABA sera obtained are specific for ABA and they do not cross react with other hormones such as IAA, GA and kinetin ; therefore the presence of native plant hormones other than ABA do not interfere with the immunoassay. The sensitivity of the immunoassay is in the same range as that of the wheat bioassay. We tried to improve the sensitivity of the immunoassay b y using different bleedings, different schedule of immunization and b y trying to ffactionate the high affinity antibodies from the antiserum by preferential absorption with the antigen. Neither of these experiments resulted in higher sensitivity. I t seems that the limiting factor of the sensitivity is not the affinity of the antibodies but rather the size of the hapten (ABA) which is probably smaller than the full antigenic determinant (Kabat, 1962; Schechter eta[., 1966). The same antibodies can show much stronger binding if a larger part of the antigenic determinant is tested. I t was shown previously (Fuchs et al., 1970) for anti-IAA antibodies that indoleacetyt-amino caproie acid was a much better inhibitor than IAA (about three orders of magnitudes).

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Y. Fuchs, S. Mayak, and S. Fuchs:

The a m o u n t s of A B A in crude extracts were too low to be determined by the immunoassay. However, when such extracts were concentrated t h e y were too " m u d d y " to he handled and therefore the regular procedures of fractionation and c h r o m a t o g r a p h y were applied. This pre-purification is required in other methods used for determination of A B A such as the bioassay (Nitseh a n d Nitsch, 1956) or gas chromatog r a p h y (Lenten et al., 1971). I t should be pointed out t h a t in testing extcacts of rose petals activity was detected b y the i m m u n o a s s a y in the 0.7-1.0 Rf (Fig. 6b). I t m a y be t h a t this a c t i v i t y is due to the presence of or related substances derived b y the effect of light on carotenes chromatography), as suggested b y Taylor (1968).

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The advantages of the i m m u n o a s s a y are several. As mentioned above the assay is n o t influenced b y other growth hormones and thus can work in their presence. The variability in this system seems to be less t h a n t h a t of wheat seedlings. The i m m n n o a s s a y does n o t require a n y expensive i n s t r u m e n t a t i o n and the antiserum and the modified bacteriophage can be stored and give reproducible results for at least one year. One bleeding of an immunized rabbit a n d one preparation of modified phage is sufficient for thousands of assays. Antibodies to plant hormones might perhaps be used also in combination with bioassays or in order to obtain some other information in plant h o r m o n e research. This research was financed in part by grant No. FG-IS 296 from the United States Department of Agriculture, Agricultural Research Service, under Public Law 480. Contribution from The Volcani Institute of Agricultural Research, Bet Dagan, Israel. 1971 Series No. 1976-E. We are grateful to F. Hoffmann La Roche and Co. Ltd. for supplying the abseisie acid. We wish to thank Mrs. Ruth Maron, Mrs. Edna Pesis and Mrs. Lea Schlituer for their competent technical assistance.

References Anfinsen, C. B., Sela, M., Cooke, J. P. : The reversible reduction of disulfide bonds in polyalanyl ribonuclease. J. biol. Chem. 237, 1825-1831 (1962). Browning, G., Hood, G.V., Gaskin, P.: Identification of abscisic acid in flower buds of Co]lea arabica (L.). Planta (Berl.) 94, 213-219 (1970). Davis, L. A., Heinz, D. E., Addicot, F. T. : Gas liquid chromatography of trimethylsilyl derivatives of abseisic acid and other plant hormones. Plant Physiol. 43, 1389-1394 (1968). Fuchs, S., Fuchs, u Immunological assay for plant hormones using specific antibodies to indoleacetie acid and gibberellie acid. Biochim. biophys. Acta (Amst.) 192, 528 (1969). - - Haimovich, J., Fuchs, u : Immunological studies of plant hormones: detection and estimation by immunological assays. Europ. J. Bioehem. 18, 384-390 (1971). - - Sela, M. : Studies on the chemical basis of the antigenicity of proteins. Biochem. J. 87, 70-79 (1963).

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Goren, R., Goldscbmidt, E. E. : Regulative systems in the developing citrus fruit. I. The hormonal balance in orange fruit tissues. Physiol. Plantarum (Cph.) 28, 937-947 (1970). Haimovich, J., Hurowitz, E., Novik, N., Sela, M. : Preparation of protein-bacteriophage conjugates and their use in detection of anti-protein antibodies. Biochim. biophys. Aeta (Amst.) 207, 115-125 (1970). - - Sela, M. : Inactivation of poly-DL-alanyl bacteriophage Ta with antisera specific toward poly-DL-alanine. J. Immunol. 97, 338-343 (1966). Kabat, E . A . : Antigenic determinants of dextrans and blood group substances. Fed. Proe. 21, 694-701 (1962). Lenton, J. R., Perry, V. M., Saunders, P. F. : The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography. Planta (Berl.) 96, 271-290 (1971). Milborrow, B.V.: The identification of ~ abscisin I I ( • dormin) in plants and measurement of its concentrations. Planta (Berl.) 76, 93-113 (1967). Nitsch, J . P . , Nitsch, C. : Studies on the growth of coleoptile and first internode sections. A new, sensitive straight growth test for auxins. Plant Physiol. 81, 94-111 (1956). Ranadive, N. S., Sehon, A . H . : Antigenieity of 5-hydroxyindol-3-acetic acid, a derivative of serotonin. Canad. J. Bioehem. 45, 1681-1699 (1967). Schechter, I., Schechter, B., Sela, M. : Combining sites of antibodies with L-alanine and D-alanine peptide specificity and the effect of serum proteolytic activity on their estimation. Biochim. biophys. Acta (Amst.) 127, 438-456 (1966). Taylor, H . F . : Carotenoids as possible precm'sors of abscisic acid in plants. In: Plant growth regulators, Monograph No 31, p. 22-36. London: Society of the Chemical Industry 1968. Yalow, R. S., Berson, S. It. : Immunoassay for endogeneous plasma insulin in man. J. olin. Invest. 89, 1157 (1960). Dr. Yoram Fuchs The Volcani Institute of Agricultural Research Bet Dagan, Israel