Plant Science Letters, 35 (1984) 159--167 159 Elsevier Scientific Publishers Ireland Ltd.

PEROXIDASE-LINKED, SOLID-PHASE ENZYME IMMUNOASSAY FOR THE DETERMINATION OF PICOMOLE LEVELS OF LIMONIN*

E.W. WEILER a, P.S. JOURDAN a'* * and R.L. MANSELL b'** *

aLehrustuhl fur Pflanzenphysiologie, Ruhr, Universitaet, D-4630 Bochum (F.R.G.) and bBiology Department, University of South Florida, Tampa, FL 33620 (U.S.A.))

(Received December 5th, 1983) (Revision received February 22nd, 1984) (Accepted February 22nd, 1984)

A solid-phase enzyme immunoassay (EIA) for the determination of 0.1--10.0 ng/0.1 ml of the bitter triterpene- lactone, limonin, in plant extracts and juice samples is described. As little as 0.15 pmol of limonin can be detected. Quantitative results are available within 30 min of total assay time. The assay makes use of a limonin-horseradish- peroxidase tracer of high immunoreactivity and has been semi-automated using antibody-coated polystyrene optical cuvettes.

Key words: l imonin; enzyme-immunoassay; solid-phase ; bitter principle; Citrus

Introduction

Limonin is a tetracyclic tri terpenoid dilactone that occurs in Citrus spp. and has been the subject of numerous investigations because of its extremely bitter taste [1]. A concentrat ion of limonin above 7 ppm (parts per million) in citrus juices makes them unpalatable and unacceptable for commer- cial distribution [~] . Therefore, ways to reduce the levels of limonin either in the fruit or in the juice have been sought inten- sively but, as yet, no useful method exists. Attempts to control limonin production in the plant have led to biosynthetic and meta- bolic studies [3] and preliminary experiments

*Part 14 in the Series: 'Use of Immunoassay in Plant Science'. **Current address: Plant Breeding Department, 252 Emerson Hall, Cornell University, Ithaca, NY 14853, U.S.A. ***To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; EIA, enzyme immunoassay; HPLC, high-pressure liquid chromatography; HRP, horse-radish Peroxide; PBS, phosphate-buffered saline, RIA, radioimmunoassay.

have shown that limonin (or an immediate precursor) is synthesized in the leaves and translocated to the fruit where it accumulates in the seeds, carpellary membranes and core [4] . The biosynthetic capacity of fruits has not been unequivocally ruled-out, however [3] .

As in the case with many products of plant secondary metabolism, the quantification of limonin has been cumbersome and tedious until recently, when radioimmunoassay (RIA) methods for the determination of limonin were introduced by our laboratories [5,6]. This method has greatly facilitated the quantitative measurement of limonin because it is characterized by high sensitivity, specifi- city, high throughput and simplicity. Further- more, RIA can measure limonin in crude extracts of citrus tissues. The performance of the RIA and the necessary radiosynthesis, however, require specialized facilities and costly counting equipment. In addition, the involvement of isotopes precludes the use of RIAs in facilities such as food processing plants. The objective of the present work was to develop and characterize an immuno-

0304-4211/84/$03.00 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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logical method for limonin quantification without the use of radioactive tracers. Enzyme immunoassays have been developed to broaden and facilitate the application of immunological ~ assays [7] . We report here a solid-phase enzyme immunoassay for limonin which requires only ordinary laboratory equipment for performance, yet retains the sensitivity, specificity and accuracy of RIA.

Materials and methods

Apparatus An EIA colorimeter/pipetor (Gilford Instru-

ments, Model PR 50) was used for pipeting enzyme reaction mixtures and for determina- tion of optical densities (490-nm filter). Alternatively, a separate colorimeter (Gilford Instruments, EIA Manual Reader) was used for some optical density measurements. Dispensing of incubation mixtures was done with a Pipetor/Diluter (Gilford) and samples were pipeted using Eppendorf pipets.

Materials Immunoassays were routinely performed

in Gilford polystyrene Cuvette-Packs T M ; however, we found that other polystyrene surfaces could also be used: microtiter plates (Dynatech M-29-A, Cat. No. 655101 supplied by Grenier Co., F.R.G.), Removawell cups; flat bo t tom type, supplied also by Grenier, and 12 × 75 mm tubes (Sherwood Medical Industries, St. Louis, MO; lot No. 14763).

Chemicals Horse-radish Peroxidase (HRP, RZ 3.2,

Type VI) and 4-amino-antipyrine were from Sigma, St. Louis. Gelatine powder was obtained from Merck, Darmstadt and bovine serum albumin (BSA) was from Serva, Heidelberg. All other chemicals were of the highest puri ty commercially available.

The limonin-HRP conjugate was prepared as follows: 4.6 mg (9 ~mol) of limonin-O- carboxymethyl oxime, synthesized as de- scribed elsewhere [5] , were dissolved in 100 ul dimethyl formamide; 4 ~l (22 ~mol)

tri-n-butyl amine were added and the mixture cooled to - 8 ° C in a salt-ice bath. After a few minutes, 4 ul (30 ~mol) iso-butylchloro- formate were added and the reaction was allowed to proceed for 20 min. This solution was then added in 20-~1 aliquots over a period of 1 h, to the enzyme solution consis- ting of 5.3 mg (0.4 ~mol) HRP in 0.18 ml of 0.5% (w/v) NaHCO3 and 0.18 ml dioxane. The enzyme solution was stirred continuously for another hour and then diluted with 1.8 ml of 0.1 M NaH2PO4 (pH 7.0). The conjugate was dialyzed against 6 × 1 I of the same buffer for 3 days. The dialyzed solution was stored at 4°C in the presence of 0.02% (w/v) NAN3.

For the immunoassay procedure, phos- phate-buffered saline (PBS, 0.01 M K ÷ phos- phate, 0.15 M NaC1, pH 7.4) was used. For the enzyme reaction, 0.1 M NaH:PO4 (pH 7.3) was used. Standard limonin solutions and compounds fdr cross-reactivity studies were prepared as previously described [5] .

Antiserum and fractionation of IgG Limonin antibodies were raised in rabbits

as previously described [5] . The IgG fraction from the antiserum was obtained by ammon- ium sulfate precipitation as follows: to 10 ml of serum were added 5 ml of saturated (NH4):SO4 solution (pH 7.0). The mixture was stirred on ice for 30 min and centrifuged for 10 min at 35 000 X g in a refrigerated centrifuge. The precipitate was resuspended in 5 ml of H20 and 2.5 ml of saturated (NH4)2SO4 was again added. After 30 min stirring and centrifugation, the precipitate was resuspended in 5 ml of water and the precipitation procedure repeated four times. The final precipitate was dissolved in 5 ml of water, dialyzed against 4 × 10 1 water for 2 days and lyophilized. Lyophylized material (65 mg) was obtained and was stored at -18°C.

Coating of polystyrene surfaces The polystyrene surfaces were routinely

coated with a solution of 0.1 mg/ml of lyophylized IgG in 50 mM NaHCO3 (pH 9.3}.

Coating was done overnight at 4°C. Each cuvette of a Gilford Cuvette-Pack T M was coated with 0.5 ml of solution while 0.2 ml/ well were used for the microtitre plates and 1.0 ml/ tube for individual plastic tubes. After coating, the solution was decanted and saved. The surfaces were then incubated with 0.01% (w/v) BSA in PBS for 15 min at room temperature (0.7 ml/cuvette for Cuvette-Pack T M , 0.3 ml/well for microtiter plates and 1.4 ml/tube). After decanting, the cuvettes were ready for use and were stored desiccated at 4°C.

Assay for HRP activity The enzyme activity was assayed using

a modification of the method described by Gallati [8]. The reaction mixture consisted of a stock solution of 1 mM 4-aminoanti- pyrine, 25 mM phenol and 0.1 M NaH2PO4 (pH 7.3). The stock solution was stored at room temperature in the dark and was ~table for at least 2 weeks. Just prior to use, the solution was mixed with H202 to a final concentration of 0.8 mM H202. The reaction mixture was stable for at least 5 h at room temperature and for 4 h at 37°C.

Immunoassay procedure All samples and standards were assayed in

triplicate. When the immunoassay was carried out in the Cuvette-Packs, the incubation mixture consisted of 0.3 ml PBS ar~.d 0.1 ml of the limonin-HRP tracer prepared by diluting the stock limonin-HRP conjugate solution 1 : 1000 with PBS-gelatin (1 g gelatin/1 PBS). The mixture was kept cold at all times. Two modifications of the assay were used; these were based on the duration of the initial incubation step (see Results).

Long-term assay. To each cuvette were added, simultaneously, 0.4 ml of the incuba- t ion mixture and 0.05 ml of standard or sample. The cuvettes were covered with parafilm and kept at 4°C for 120 min. The solutions were then decanted and each cuvette was washed twice with 0.7 ml PBS. The cuvettes were allowed to equilibrate at

161

room temperature for a few minutes and the enzyme bound to the solid phase was assayed by adding 0.5 ml of the reaction mixture which had also been equilibrated at room temperature (22°C). The subsequent increase in absorbance was monitored at 490 nm for 2 h .

Rapid assay. For the rapid assay, the incubation mixture consisted of 0.3 ml of PBS, 0.1 ml of tracer (as above) and 0.1 ml of sample or standard. The buffer, tracer and sample/standard were pre-mixed, chilled on ice and 0.5 ml of this solution was then added to the corresponding cuvettes. The cuvettes were incubated at 4°C for 15 min and then decanted, rinsed twice with 0.7 ml of PBS and assayed for peroxidase activity after addition of 0.5 ml of reaction mixture to each cuvette. The reaction mixture was pre-warmed to 37°C, and the increase in absorbance at 490 nm was monitored at 37°C.

Radioimmunoassay for limonin The RIA for limonin was performed as

described previously [6], but modified as follows: the incubation time was 2 h at 4°C and the tracer used was of higher specific radioactivity (10 000 cpm, . 0.24 pmol of [3H]limonol; total activity per assay tube).

Preparation of fruit extracts and juice samples Fresh plant tissue (400--900 mg) was

extracted with 10 ml of MeOH by refluxing for 2 h. The solutions were filtered and the volumes of the filtrates were brought to 10 ml. Each solution was then diluted with water and 0.1 ml or 0.05 ml samples were subjected to immunoassay. Juice samples were diluted with water (500-fold) and similar aliquots were assayed directly.

Results and discussion

Characterization of the enzyme tracer The coupling of limonin-7-O-carboxy-

methyl oxime to peroxidase and the subse- quent purification by extensive dialysis

162

resulted in a 33% recovery of the original enzyme activity. When the enzyme tracer was incubated with excess antiserum, and the limonin antibodies were precipitated with a second ant ibody [9] , 65% of the enzyme activity was found associated with the anti- bodies (i.e., approx. 35% of the peroxidase molecules carried no limonin derivative). A tracer stock solution in 0.1 M Na ÷ phosphate buffer, 0.02% (w/v) NaN3 (pH 7.0) was stable for several months at 4°C without any apparent loss in activity.

Optimization of assay conditions Coating of polystyrene surfaces. Poly-

styrene cuvettes (Gilford system) were coated with 0.5 ml of a buffered anti-limonin IgG solution. Various parameters of the coating procedure have been optimized. It was found to be best to coat with a solution containing 0.1 mg of IgG/ml and 50 mM NaHCO3 (pH 9.3). Higher concentrations of IgG, up to 1 mg/ml, showed little additional effect. In contrast, lowering the IgG concen- tration in the coating solution led to a concomitant decrease in coating efficiency. Variations in pH between 8 and 10 had no effect on coating nor did variation in the NaHCO3 concentration between 1 and 200 mM at pH 9.3. Coating efficiency de- creased when concentrations higher than 0.2 M NaHCO3 were used. After incubation of the coating solution for 4 h at 4°C, the adsorption of antibodies to the polystyrene was complete; however, coating was routinely done overnight at 4°C. The IgG solution could be reused for coating 10 more times without a noticeable decrease in efficiency. The cohting capacities per unit area (c.c.) of other polystyrene solid supports was tested under the same conditions used with the Gilford system. Using the latter as refer- ence (c.c. = 1.0), we found the Microtitre plates (c .c .= 1.5) and Removawell cups ( c . c= 2.1) had higher capacities, whereas 12 × 75 mm polystyrene tubes ( c . c . - -0 .6 ) had a slightly lower capacity.

A second incubation with 0.01% (w/v)

BSA was originally performed after ant ibody coating in order to minimize any potential unspecific adsorption of the enzyme-tracer to the plastic surface. It has subsequently been found that this step can be omitted wi thout increasing unspecific binding.

Antigen-antibody reaction. The principle of the EIA is based on t h e competi t ion between the enzyme tracer and free limonin in solution for a limited number of ant ibody binding sites on the solid support. A quanti- tative relationship is then established between the amount of enzyme activity on the walls and the amount of limonin in the well. In order to insure maximum reproducibility of the assay, the dilute standard or sample was pre-mixed with the buffered tracer solution in a separate tube, and this solution was delivered into the coated cuvettes. The t ime course of limonin-peroxidase tracer binding to the antibodies is shown in Fig. 1. It can be seen that at 4°C, equilibrium is reached very slowly and would require long incubation periods. However, it was sufficient to incubate assays for 2 h at 4°C (long-term assay), or alternatively, given precise timing of the various steps in the procedure, it was possible to reduce the incubation time to only 15 min at 4°C. This shortened incuba- tion results in approx. 40% less tracer binding,

si

X

c_ 3"~ E C%1 0~2"

c~

o

3'o 6'o 9'o do Incubation Time [m in i

Fig. 1. M a x i m u m specific binding (]3o) of the l imonin -horse rad i sh pe rox idase t racer to the solid phase antibodies as a f u n c t i o n of incubation time at 4°C.

163

n, 3 -

• 2"

O 1-

H202 [mM] Fig. 2. Maximum specific binding (Bo) o f the l imonin-horseradish peroxidase tracer to the solid phase an t ibodies at 22°C as a func t ion of H:O2 concen t ra t ion .

but is still sufficient for short and convenient incubation times during the enzyme reaction step (rapid assay).

Enzyme reaction. The assay for peroxidase activity [8] was found to be extremely

dependent on the H202 concentration; slight deviations on either side of the optimal concentration resulted in significant decreases in color development (Fig. 2). The peroxi- dase reaction mixture is stable for about 2 weeks when kept in the dark and without H202. Upon addition of peroxide, the mixture is stable for only 4 h at 37°C. The time- course of the enzyme reaction for a complete standard curve is presented for both the long- term (Fig. 3) and the rapid assay (Fig. 4). The long-term assay was performed at 22°C without mixing the solution in the cuvettes, whereas the rapid assay was performed at 37°C with frequent overhead shaking. This rapid assay system resulted in a 2.5-fold increase in the rate of peroxidase reaction due to the higher temperature. Thus, it is possible to obtain precise readings with this latter method after an incubation time of 15 min (a maximum absorbance of 0.45-- 0.50). As can be seen in Fig. 3, for the long-term assay, when the solutions in the cuvettes are not mixed, the system requires about 15 min to give consistent readings

0g

0.7

0.5-

O 0.3"

0.1"

3

~ 0.25

O.S

2.5

~'s 3'o is ~ 7's io ~6s lio R e a c t i o n t i m e [min]

~ 0.8' S"

E 0.6

"~ - 0.4' ~ m

0 0.2,

I - o3 $

1'5 3'0 Reaction time [min I

Fig. 3. Time course o f t he peroxidase reac t ion for a co mp l e t e l imonin s tandard curve. The en zy me react ion was performed in the coa ted cuvet tes at 22°C wi thou t mixing. Readings were made automat ica l ly wi th an enzyme immunoassay PR 50 color imeter .

Fig. 4. Time course of t he peroxidase reac t ion for a co mp l e t e l imonin s tandard curve. The enzyme react ion was pe r fo rmed as in Fig. 3 but at 37°C with overhead mixing. Readings were done with a manual enzyme immunoassay reader.

164

because the light path of the colorimeter goes through the center of the cuvettes while the enzyme reaction takes place on the walls. Several minutes are therefore required to equilibrate the system by diffusion. This effect can be minimized by inverting the cuvettes several times; however, this mixing does not change the overall rate of color formation. Diffusion of the substrates and product apparently is not rate-limiting in this system. When assays are evaluated by endpoint determination, mixing would be unnecessary, but assays evaluated by initial

reaction rates would require continuous shaking. In this study, calculations were based on endpoint determination taken 2 h after starting the reaction for the long- term assay, and after 15 min reaction time for the rapid assay.

Assay characteristics Standard curve and measuring range. The

principal assay characteristics have been summarized in Table I. Average standard curves, constructed from several assays performed on different days for both the

Table I. Characteristic assay parameters of the limonin enzyme immunoassay.

Long-term assay Rapid assay

Total activity per tubea: O.D. × 10-3/rain 19 (22°C) 44 (37°C) Relative (%) 100 100

Non-specific binding b : O.D. × 10-3/min 0.4 0.1 Relative (%) 2 0.3

Maximum specific binding (Bo)C: O.D. × 10-3/min 2.7 2.8 Relative (%) 13 6

Measuring range: ng 0.1--10 0.1--10 pmol 0.2--20 0.2--20 ppb 1--100 1--100

Detection l imit: ppb < 1

Maximum color development : O.D. after 2 h at 22°C O.D. after 15 min at 37°C

Recovery (%) of lirnonin added to sample

Assay precision through measuring range: Coefficient of variation % + S.D. of

triplicate determinations

1.1

90+ 2

0.45

ND d

2 . 5 ± 1 . 2 3 . 0 ± 1 . 8

aAmount of limonin-horseradish peroxidase tracer activity that was added to each reaction vessel (cuvette). b Activity of the limonin-horseradish peroxidase tracer bound to the cuvette in the absence of limonin antibody. CActivity of the limonin-horseradish peroxidase tracer which was bound to the antibody coated cuvettes in the absence of limonin antigen.

d Not determined.

-2"

-3- 3-

5A

2-

-2"

I "

r n

.~. 0-

o~-I. m

0:1 i 1'0 Lim0nin [ng/assay]

-3

t ~ J .._ 0 O~ O

- I "

165

2.0-

In in

O1

c 1.0" C

E

.2"" EIA [Lim0nin ng/assay]

Fig. 5. Standard curves for the limonin enzyme immunoassay, plotted in linearized form after transformation of relative binding (B/Bo) according to: logit (B/Bo)= In[(% B/Bo)/(100 - % B/Bo)]. A: Long-term assay, curve constructed from 7 consecutive standard curves. B: Rapid assay, curve constructed from 5 consecutive standard curves. The bars represent + S.D. and indicate day-to-day reproducibility.

Fig. 6. Correlation of the results using the enzyme immunoassay with those obtained using the radioimmuno- assay for plant extracts (e) and juice samples (o) covering the whole measuring range of the enzyme immuno- assay.

long-term and the rapid assay, are given in Fig. 5. Both assays yield essentially the same standard curve and the measuring ranges extend from 0.1--10 ng/assay. The day-to<lay reproducibility of the assay is demonstrated by coefficients of variation throughout the measuring range of 2.5% (long-term assay) and 3.0% (rapid assay). This EIA is capable of detecting as little as 0.1 ng/0.1 ml (1 part per billion) of limonin with a total assay t ime of only 45 min.

Assay specificity. Table II compares the cross-reactivities of various naturally-occurring limonoids in both the EIA and RIA. The cross-reactivities are essentially identical for

Table II. Cross-reactivities of various limonoids with the limonin antibody in the EIA as compared with the RIA.

Compound Cross reactivity (%)

EIA RIA a

Limonin 100 100 Deoxylimonin 30 27 Deacetylnomilin 4.7 6.6 Nomilin 1.0 0.9 Obacunone 0.2 0.4 Nomilinic acid 0 0 Isoobacunoic acid 0 0

aSource: [5].

166

both assays. Thus, as in the RIA, the EIA is applicable to the direct analysis of limonin in crude extracts or juice samples of citrus.

Assay accuracy. The validity of the assay for limonin quantification has been deter- mined in several ways. Recovery of limonin added to extracts prior to assay was 90% + 2%. Extracts prepared from leaves and fruit of Citrus paradisi, from fruits of C. sinensis and C. limon as well as various commercial grape- fruit juices and drinks have been assayed by both EIA and RIA (Fig. 6) [6]. Both methods yield identical results as exemplified by a regression equation of y = 1.04x - 0.015, r= 0.955. It has previously been demonstrated that there is also close corre- lation between the RIA and high-pressure liquid chromatography (HPLC) [10]. Limonin determination within the entire measuring range is done with high precision. Expressed as percent coefficient of variation, variability of triplicate samples equals 2.5 -+ 1.5% for the long-term assay and 3.0 + 1.8% for the rapid assay.

Processing capacity. The long-term assay is more practical for the analysis of large batches of samples than the rapid assay which was designed to rapidly quantitate limonin in a few samples at a time. With the long-term assay, 150--200 samples may be conveniently assayed in triplicate within a working day (5--6 h of assay time), whereas the rapid assay permits duplicate or triplicate analyses of 5--10 samples within 30--45 rain.

Limonin concentration in plant tissues. Samples of grapefruits, oranges and lemons, obtained from local stores were dissected into their individual tissues and analysed separately by RIA and by EIA. As Table III shows, both methods yield identical results in all of the tissues assayed. High levels of limonin were found in grapefruit and signifi- cant amounts were also found in lemon, whereas in orange virtually no limonin was detected. These findings are in good agree- ment with data from the literature [1]. In the grapefruit variety analysed here, the same pattern of limonin distribution within

Table III. Limonin concentrations in various plant tissues as determined by EIA and RIA.

Limonin (ng/mg fresh wt. )

EIA RIA

Grapefruit (Dole-Honduras) peel 41 46 albedo 240 234 membranes 529 544 juice vesicles 2 3 seed 2670 2790

Orange (Jaffa) peel 1.2 1.8 albedo 2.1 2.4 membranes 1.6 2.4 juice vesicles 2.0 1.9

Lemon (source not specified) peel 18.0 17.7 albedo 13.8 11.7 membranes 111 111 juice vesicles 3.8 3.4

C. paradisi leaf 155 122

the fruit was found as in an earlier s tudy [5]; the compound is concentrated in the seeds and in the intercarpellary membranes, whereas the juice vesicles are nearly free of limonin.

Limonin levels in juice and drinks. Three commercial grapefruit drinks and 8 grapefruit juices were obtained from local markets. The direct analysis of these samples with both EIA variations revealed that in two of the drinks, no limonin was present, whereas one had 2 #g/ml. The grapefruit juices had between 6 and 11 ppm, levels which in organoleptic studies [2] were considered to be non-bitter (6 ppm), slightly bitter (7--9 ppm), or bitter (10--16 ppm). The correlation of the EIA data with the RIA in this experiment was r = 0.96 (long-term assay) and r = 0.91 (rapid assay).

The solid-phase EIA reported here is a simple and versatile technique and the general principles demonstrated should be applicable to many other natural products. The sensi- tivity and precision of the assay permits the

rapid quan t i f i c a t i on o f l imon in in c rude ex t r ac t s o f small ( 5 0 - - 1 0 0 mg fresh wt . ) samples o f t issue. This abi l i ty to specif ical ly measu re a c o m p o u n d in such m i n u t e samples would fac i l i ta te phys io log ica l and b iochemica l e x p e r i m e n t s on its b iosynthes i s , t r anspor t , a c c u m u l a t i o n and me tabo l i sm . F u r t h e r m o r e , the versa t i l i ty o f the l imon in EIA is also exempl i f i ed b y a m od i f i ed E I A we have deve loped which uses a d i f fe ren t e n z y m e t racer , lower a n t i b o d y coa t ing concen t r a t i ons , higher i ncuba t ion t e m p e r a t u r e s and is specifi- cally designed to quan t i f y l imonin in un- d i lu ted g rapef ru i t juices [ 11 ] .

A c k n o w l e d g e m e n t s

This research was s u p p o r t e d in pa r t b y a U S D A / S E A grant to R.L.M.

References

1 T.W. Goodwin and L.J. Goad, Carotenoids and

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terpenoids, in: A.D. Hulme (Ed.), The Biochem- istry of Fruits and Their Products, Vol. I, Aca- demic Press, New York, 1970, p. 339.

2 V.P. Maier, R.D. Bennet and S. Hasegawa, Limonin and other limonoids, in" S. Nagy, P.E. Shaw and M.K. Velhuis (Eds.), Citrus Science and Technology, Vol. 2, Avi Publishing, Westport, 1977, p. 482.

3 S. Datta, H.J. Nicholas, Phytochemistry, 7 (1968) 955.

4 S. Hasegawa and J.E. Hoagland, Phytochemistry, 16 (1977) 469.

5 R.L. Mansell and E.W. Weiler, Phytochemistry, 19 (1980) 1403.

6 E.W. Weiler and R.L. Mansell, J. Agric. Food Chem., 28 (1980) 543.

7 A.H.W.M. Schuurs and B.K. Van Weemen, Clin. Chim. Acta, 81 {1977) 1.

8 H. Gallati, J. Clin. Chem. Biochem., 15 (1977) 699.

9 T. Chard, An Introduction to Radioimmunoassay and Related Techniques, Elsevier, Amsterdam, 1978, p. 413.

10 R.L. Rouseff and R.L. Mansell, Proc. Fla. State Hortic. Soc., 95 (1982) 249.

11 P.S. Jourdan, R.L. Mansell, D.G. Oliver and E.W. Weiler, Anal. Biochem., (1984} in press.