WO1989000293A1 - A method for sample extraction, purification, concentration and quantification of low molecular weight organic compounds and an analytical kit for carrying out said method - Google Patents

Abstract

A method for sample extraction, purification, concentration and quantification of low molecualr weight organic compounds from biological sources, such as plants, specifically the hormones cytokinins and auxins comprising submitting the sample in the form of an isolated extract of biological material applied via a pre-immune pre-column or directly to an immuno-affinity purification step using a selective antibody followed by an enzyme-linked immunosorbent assay utilising said or a different antibody stabilised by sugar wherein the selective antibody displaying group specificity is used in the purification step; the antibody is covalently attached to a matrix without loss of binding capacity for the appropriate antigen; and the low molecular weight organic compounds are bound reversible to an immuno-affinity column using aqueous reagents and eluted using organic or aqueous solvents. The method is preferably carried out using a novel kit comprising (i) components required for sample extraction and purification and (ii) components necessary for use in an enzyme-linked immunosorbent assay.

Description

Title: A method for sample extraction, purification, concentration and quantification of low molecular weight organic compounds and an analytical kit for earrying out said method.

Technical Field

This invention relates to a method for sample extraction, purification, concentration and quantification of low molecular weight organic compounds (LMWOCs) from biological sources, such as plants, specifically plant hormones selected from the group consisting of cytokinins, such as zeatin and N-9 substituted derivatives of zeatin, and auxins, such as indol- 3 -acetic acid, and an analytical kit for carrying out said method.

Background Art

Interest in the quantification of LMWOCs stems from the premise that changes in their concentration influence the physiological state of the plant material. It is generally accepted, however, that the concentration and/or activity of these LMWOCs is more pronounced in the meris tematic regions of the plant.

Irrespective of the nature of the interplay between the plant growth substances and morphogenesis it is evident that the number of cells involved is small in comparison to the bulk of the tissue. Thus meaningful differences will not be obtained by analysing whole or large pieces of tissue/organ of the plant.

Existing methods for quantification of LMWOCs are not completely satisfactory in that:

1. They consume a large amount of biological material since the known methods require several different chromatographic procedures cf. Scott, I.M. and Horgan, R.: Quantification of cytokinins by selected ion monitoring using ¹⁵N-labelled internal standard. Biomed. Mass Spectrometry, vol. 7, no. 10, pp. 446- 449, 1980.

2. They thus consume a large amount of laboratory time and skilled labour.

In DE PS 29 10 707 (Abbott Laboratories) is reported the use of sugar for the maintenance of the biological activity of immunoreagents in a dried form.

WO 86/04096 (Drake et al.) discloses a dry preparation of enzymatically active reagent material, e.g. reagents suitable for enzyme-linked, specific binding assays, such as ELISA and reagent kits including such dried preparations. A preparation in which the enzymatic activity is maintained is thus provided. In the present invention a preservation of antibody-activity is achieved by means of a sugar-coating procedure.

NO PS 146.747 discloses the isolation and further purification of the placenta-specific protein PP5 by means of affinity chromato graphy , where the pro te in i s revers ib ly bound in an aqueous medium. Subsequently the protein is eluted with an aqueous solution with a pH value of 2-4. This method is, however, complicated, as it is necessary to precipitate undesired compounds with diaminoethoxy- acridine lactate during the purification and then isolate PP5 by precipitation with ammonium sulphate. Moreover, the method is especially adapted to the large molecule PP5, and cannot be used for low molecular weight organic compounds , such as plant ho rmone s s e l ec ted from the group consisting of cytokinins and auxins.

There is currently a kit on the market that concentrates entirely on the quantification of LMWOCs. It is marketed by Idetek, Inc. USA under the trade mark PHYT0DETEK^R. Users of this known kit are, however, required to isolate and purify the LMWOCs either by the method according to MacDonald, E.M.S., Akiyoshi, D.E., and Morris, R.O.: Combined high-performance liquid radioimmunoassay for cytokinins. Journal of Chromatography, vol. 214, pp. 101- 109, 1981 or by using very expensive and elaborate equipment (see e.g. Scott, I.M. and Horgan, R.: Mass spectro- metric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue. Planta, vol. 161, pp. 345 - 354, 1984). Sample purification by the traditional way necessitates the use of a number of chroma- tographic steps. This is undesirable due to an inherent loss with each step. To compensate for this loss an investigator often isolates the LMWOCs from a relatively large sample size. The use of a large sample size, is disadvantageous as the rationale in the analysis of plant growth regulators states that local differences in c on - c entrat i on ar e c r i t i c a l wi th re sp e c t to di f f e rent i at i on . The larger the sample size, the larger the sampling error.

Preparation of antibodies against LMWOCs have been reported (for reveiw see Weiler, E.W.: Immunoassay of plant growth regulators. Ann. Rev. Plant Physiol., vol. 35, pp 85-95, 1984). These antibodies have been used for the preparation of immuno-affinity columns with the stated purpose to purify the corresponding low molecular organic substance of interest, e.g. a plant hormone. Hitherto the use of such columns relied on the use of organic solvents known to be toxic and are thus unsuitable for inclusion in analytical kits that have to be shipped and shelved safely.

From EP 153 875/A2 is known to alleviate difficulties of non-specific binding, an edge effect in ELISA, by submitting a sample to an enzyme-linked immunoadsorbent assay in the presence of an effective amount of sugar. The sugar is thus present during the antigen-antibody-reaction. As (mentioned the sugar is used to omit non-specific binding reagents and to alleviate edge effects. The purpose of the present invention with respect to the inclusion of sugar is completely different, namely to preserve the biological activity of the antibody during the freeze- drying process as well as during the storage period later on.

Surprisingly, however, there has now been developed a method, which is far more useable in a kit in that the use of organic solvents is replaced by an aqueous system. By employing this method LMWOCs, e.g as described in Example 1, zeatin and N-9 substituted derivatives of zeatin may be isolated, purified and quantified in a highly sensitive, reliable, reproduceable, and fast test, which is also easily automated.

The rationale in the development of an analytical test for LMWOC relies on simple extraction procedures involving a few simple steps. This avoids the requirement for large sample size for the extraction.

Disclosure of the Invention

According to the invention there is thus provided a method for sample extraction, purification, concentration and quantification of low molecular weight organic compounds from biological sources, such as plants, specifically the hormones selected from the group consisting of cytokinins or auxins, comprising submitting the sample in form of an isolated extract of biological material applied via a pre-immune precolumn or directly to an immuno-affinity purification s tep us ing a f irs t s e le c t ive antib o dy fo l l owe d by an enzyme - linked immunosorbent assay (ELISA) utilising an antibody stabilized by sugar, said method being charac- terised by using as the selective first antibody in the purification step an antibody having group specificity; the first antibody being covalently attached to a matrix without loss of binding capacity for the appropriate anti- gen (hapten); binding the low molecular weight organic άompounds reversibly to an immuno-affinity column using aqueous reagents; eluting by means of a solvent selected among organic or aqueous solvents; and using a second antibody different from the first antibody as the antibody in the ELISA.

The important characteristics of the present invention are:

1. A first selective antibody having group specificity, preferably a monoclonal antibody is used for the immunoaffinity purification. Using this antibody a group of closely related compounds, such as zeatin and N-9 substituted derivatives of zeatin, are selectively bound.

2. A second antibody, preferably a polyclonal antibody is used for the quantification by means of ELISA. The antibody used includes antibodies with an extremely high affinity (high Ka-value) but with a low affinity to cross - reactive compounds. This is a decisive factor for achieving a low detection limit and simultaneously obtaining a very specific assay.

The combination of two different antibodies ensures a good recovery and high purity during the purification as well as a high specificity and sensitivity during all quantifications.

These advantages are especially ensured, when using a monoclonal antibody as the first antibody for the purification; and a polyclonal antibody as the second antibody in the ELISA. In a further preferred embodiment the antibody is covalsnt- ly bound to the matrix via an amide bond formation preferably introduced by using a succinyl-activated carbonyl gel. Thus mild conditions can be used for preparing the immuno-affinity material.

By the method according to the invention it is especially preferred that during the immuno-affinity purification step any non-specific adsorption to the immuno-affinity column is circumvented by coating with adenosine or similar organic molecules having no significant cross-reactivity with the selective antibody and/or other reagents used in the extraction, purification, concentration and quantifi- cation of LMWOCs, especially cytokinins or auxins. This ensures that only the components of interest are retained by the column. More precisely, the specificity obtained by restricting the binding to said monoclonal antibodies ensures that the eluted LMWOCs will be of high purity. In essence non-specific. adsorption to the column is avoided.

In relation to the present invention the following elution systems are found to be very effective in disrupting the antibody-antigen interaction leading to the elution of the affinity-purified antigen:

(i) 0.1 M glycine-HCl, pH 2.2, 20 mM magnesium acetate, 50 mM KCl (ii) 0.1 M propionic acid, 10% v/v dioxane, pH 2.5 (iϋ) 0.1 M glycin-HCl, pH 2.5, 25% v/v ethylene glycol (iv) 0.1 M phosphoric acid, pH 2.5

In a further preferred embodiment the LMWOCs are specifically eluted using an aqueous solvent. It is especially preferred to use 0.1 M phosphoric acid, pH

2.5, since this system does not interfere in the subsequent ELISA quantification. Thus a specific elution of LMWOCs by disruption of the antibody-antigen interaction is obtained.

The present method depends therefore for its succesful practice on the use of a hitherto unknown analytical kit for carrying out said method using a chromatographic matrix incorporating immobilized monoclonal antibodies plus an ELISA detection system based on the use of microtiter wells. To prepare a kit having the desired shelf life characteristics a sugar is included during the preparation of the ELISA plates.

As suitable sugars can be mentioned fructose, sucrose, sorbitol and inositol, preferably sucrose, said sugars possessing an excellent stabilising effect on native antibodies.

This novel analytical kit according to the invention has two distinct functions. Firstly, simple and effective isolation and purification steps. Secondly, quantification of the extracted components.

Thus the kit according to the invention provides a rapid and efficient isolation and quantification system for LMWOCs, such as zeatin and N-9 substituted zeatin derivatives. Immuno-affinity adsorbance columns consisting of monoclonal anti-zeatin riboside antibodies coupled to an agarose gel are used for samples preparation. The column material has a high selective adsorption for zeatin and N-9 substituted derivatives of zeatin combined with a low non-specific binding capacity.

Fractions eluted from the column are of high purity and an excellent recovery is obtained.

The stability of the column matrix and the monoclonal antibodies permit repeated elutions.

Quantification of LMWOCs, such as zeatin and N-9 substituted derivatives of zeatin is based on an enzyme- linked immunosorbent assay (ELISA) system. The ELISA component of the kit uses monoclonal or polyclonal antibodies. In both cases the sensitivity is very high, for example a polyclonal antibody having a high association constant gives femtomolar sensitivity.

An essential feature of the present analytical kit is that it allows a selective isolation and purification of LMWOCs, such as plant hormones, e.g. cytokinins, such as zeatin and N-9 substituted derivatives of zeatin, or auxins, such as indol-3-acetic acid. In the case of a cytokinin, e.g. zeatin, it is desirable to have the means of isolating the entire group of substances (i.e. zeatin plus N-9 substituted derivatives of zeatin) in a single step. At the moment the interrelationship of these substances is not fully resolved. Separation of the different cytokinins can be achieved by HPLC . The present kit is thus of special interest as it is now possible to purify and isolate zeatin and N-9 substituted derivatives as one major group of cytokinins, as the latter is an important class of biologically active substances. Herein the purification and/or concentration is effected by immuno-affinity adsorption of the tissue extract using an immuno- affinity column wherein a specific monoclonal antibody is covalently attached to an i-soluble carrier. The purpose of monoclonal antibodies during the purification step is to create an immuno-affinity adsorbant characterised by only one association constant selected to give complete and reproduceable elution of the bound sample. In other applications of the method a monoclonal antibody not necessarily the same as on the column is used on the ELISA microtiter plate. Binding of the low molecular organic compounds of interest herein is specific and reversible. Elution of the bound molecules is achieved by altering the medium so that the specific interaction involving the immobilised antibody and the components of interest is less stable and dissociates. After re-equilibration of the immuno-affinity matrix the system can be re-used many times.

Development of an effective method for sample "clean-up" based on the above principle involves:

1. Having an effective extraction procedure giving a quantitative recovery of all components of interest.

2. The proper selection of the monoclonal antibody.

3. The proper selection of the insoluble matrix.

4. The proper condition for the covalent attachment of the monoclonal antibody to the carrier.

5. The proper conditions for the adsorption and e lut i on of components of interest.

6. Quick turn around time with respect to the re-use of the immuno-affinity matrix, i.e. regeneration of the matrix requires only a single washing step.

The methods according to the invention for extraction, purification, concentration and quantification of LMWOCs comprises as described above the following main steps for illustrative purposes explained specifically in relation to the isolation of zeatin and N-9 substituted derivatives of zeatin:

Step I: Maceration of tissue to obtain a raw extract of tissue/material containing low molecular weight organic compounds as defined above.

Step II: Pre -purification treatment only necessary when using particularly "dirty samples" of the plant extract. Such samples are extracted with n-pentane prior to application to the pre-column of step III.

Step III: Purification of LMWOCs by use of a pre-immune pre-column whereby non-bound fractions are collected (see Ulvskov, P., Marcussen, J., Rajagopal, R., Prinsen, E., Rύdelsheim, P. and van Onckelen, H.: Immuno-affinity purification of indole- 3 -acetamide using monoclonal antibodies. Plant Cell Physiol., vol. 28,

No. 5, pp. 937-945, 1987).

Step IV: Purification of LMWOCs of interest by an immuno-affinity column utilising a highly specific and well characterized monoclonal antibody for said LMWOC(s) including collection of the eluate.

Step V: Effective measurement of eluted compounds with the ELISA. If a HPLC fractioning is not used before step V, a quantification of the group of substances is obtain against which the specific antibody was raised. If, however, such a HPLC fractioning is used it is possible to separate and quantify each of the isolated components, namely zeatin, zeatin riboside, zeatin glucoside, 9-alanyl- zeatin and zeatin-9-ribosid-5'-monophosphate.

Re Step I: For the maceration of the tissue material to be analysed, e.g. calli, cell suspension and leaves, a mechanical disruption of tissue in liquid nitrogen may be used, followed by extraction of disrupted tissue ac room temperature with an excess amount of an ethanol or methanol based extraction system. The extraction system should preferably be prepared just prior to use. The organic solvent is normally removed by evaporation at reduced pressure.

Re Step II: The tissue extract from step I is mixed with a suitable amount (generally 1/3 vol.) of n-pentane. After standing at room temperature separation of the phases occurs. The compounds of interest are found in the lower, aqueous phase and most of the impurities, e.g. pigments are found in the upper, organic phase. The aqueous phase is used in step III.

Re Step III: Further purification of the raw extract resulting from step I and/or II takes place on a pre-immune pre-column. The above fraction is added to the pre-column at a suit- able flow rate. The effluent contains LMWOCs and is collected. The pre-column is disposable. The purpose of the pre-column is to remove compounds binding non- specifically either to the matrix or to the ligand. In such an approach a purification step is introduced prior to the immuno-affinity chromatography. This improves the performance of the immuno-affinity column and increases the number of times the matrix can be re-used.

The pre-column contains immobilized immuno- globulin proteins covalently coupled to a matrix having a high non-specific sorption, e.g. on agarose gel, cellulose based derivatives, nylons, dextrans, and glass.

Re Step IV: The tissue extract resulting from step III is purified on an immuno-affinity column. The immuno-affinity column utilizes monoclonal antibodies raised against the antigen, e.g. plant hormone carrier or plant hormone protein conjugates. In such an approach the antibody raised against such an antigenic conjugate is covalently attached to the matrix via non-essential amino acid resi- dues of the antibodies. The matrix, an agarose gel or any matrix having similar characteristics generally displays a very low non-specific adsorption and has ideal physical and flow characteristics. (See Cua- tricasa, P., Parikh, J.: "A sorbent for affinity chromatography. Use of N-hydroxy- succinamide esters of agarose", Biochemistry, vol. 11, no. 12, pp. 2291-2298, 1972). The LMWOCs are eluted by changing the environment so that the interaction between the low molecular organic substances and the antibody becomes unstable (e.g. by addition of an organic solvent, such as methanol or by altering the pH of the medium, ideally by decreasing the pH of the medium). The eluate is collected in one or more fractions.

Re Step V: If the bound fractions were eluted by altering the pH, the eluate is neutralized before further analyses. ELISA is used for quantification. Alternatively the investigator may require fractionation of the above eluted fractions containing a mixture of zeatin and N-9 substituted derivatives. Microtiter wells are coated with polyclonal antibody raised against LMW0C- carrier, e.g. protein conjugate. The purpose of the polyclonal antibody on the microtiter plates is to have the highest possible association constants for reliable quantification of the purified components. Antiserum appropri- ately diluted in a basic buffer is added to each well of the microtiter plate and the plates are incubated for a suitable time t o allow adsorption of the antibody onto the plate. After the plates are coated with an antibody, a sugar solution, e.g. 5% (v/v) of sucrose, is added to each well of the microtiter plate, which is then freeze-dried. For determining the concentration of the LMWOCs in the test sample a standard curve is prepared. Thus a range of standard LMWOC solutions is added to some of the wells. The remaining wells are used for addition of a test sample at varying dilutions. Next a LMWOC-enzyme conjugate (e.g. zeatin alka- line phosphate) is added. Using a "competitive ELISA" as well known by people skilled in the art (see Weiler, E.V.: "Immunoassays of plant growth regulators", Ann. Rev. of Plant Physiol., vol. 35, pp. 85-95, 1980) this conjugate and the LMWOC (e.g. zeatin) in the test sample then compete for a limited number of antibody binding sites. The plates are then incubated for e.g. 3 hours at room temperature or overnight at 4ºC, and afterwards washed. An enzyme substrate, e.g. p - nitropheny 1 phosphate, is then added to each well and the microtiter plate is incubated to enable enzyme substrate reaction to occur. Finally the extinction is determined spectrophotometrically. The calibration curve constructed is used for determining the concentration in the test sample.

An analytical kit comprises normally reagents and materials permitting the determination of LMWOCs by ELISA based on the use of microtiter wells coated with polyclonal or monoclonal antibodies raised against specific compounds.

The novel analytical kit according to the invention useful for carrying out the method according to the Invention comprises

i) components A, B, and C of Table 1 (see below) required for extraction of the desired low molecular organic compounds (LMWOC),

ii) for pre-purification of tissue extract a pre- immune pre-column, component D of Table 1 containing immobilized pre- immunoglobulin proteins covalently .coupled to a matrix having a high non-specific sorption plus component G of Table 1

iii) component E of Table 1, containing monoclonal antibody directed against the desired LMWOC, elution buffer, F of Table 1, equilibration buf- fer, G of Table 1, and washing buffer, H of Table

1,

Iv) dry ELISA plates, component J of Table 1, coated with polyclonal antibody raised against LMWOC- carrier conjugate and preserved with a sugar solution, enzyme conjugate, K of Table 1, enzyme substrate, P of Table 1, plus neutralisation buffer, I of Table 1, diluent for enzyme conjugate, L of Table 1, phosphate -buffered saline, M of Table 1, Tween^R-20, N of Table 1 and enzyme- substrate diluent, 0 of Table 1.

Table 1 Reagents/materials A: homogenization buffer B: an antioxidant C: salt adjustment buffer D: a vial containing material for a pre-immune pre-column (preseved by e.g. sodium benzoate or sodium azide) E: a vial containing material for an immuno- affinity column (preserved by e.g. sodium benzoate or sodium azide)

F: elution buffer G: equilibration buffer H : washing buffer I: neutralising buffer (containing e.g. NH₄OH) J : ELISA plates, lid for an ELISA plate, plate sealer for an ELISA plate

K: a vial of LMWOC-enzyme conjugate L: diluent for enzyme conjugate

M: phosphate-buffered saline (PBS) or TRIS- buffered saline (TBS)

N: Tween^R-20 O: substrate diluent (containing e.g. diethanolamine or TRIS buffer)

P : substrate, p -nitrophenyl phosphate Q : blocking buffer

A novel and extremely valuable feature of the above kit is that it comprises a specific pre-immune pre-column to be used before the immuno-affinity purification step using the immuno-affinity column (E). The Inclusion of the said pre-column in the kit according to the invention makes it possible to purify the tissue extracts in such a way that most of the compounds which would interfere with the following Immuno-affinity purification step and/or the ELISA step are retained by the pre-column. The compounds of interest to be analysed are not retarded by the pre- column thus ensuring the obtainment of reliable test results quantitatively.

The inclusion of the pre-column in the said kit results in a better performance of the immuno-affinity column caused by the removal of interfering substances which could influence the binding of the antigen and/or the number of times the column can be reused.

A further especially valuable feature of the novel kit is the inclusion of sugar preserved and stabilised ELISA plates coated with polyclonal antibodies. This ensures a long storage time of the immobilised antibodies on the surface. The kits according to the invention thus solves the known problems of maintaining the activity of a kit even after shipment and storage for prolonged times.

As a further improvement obtained by the novel kit should be mentioned that said kit can be used without using toxic organic liquids for elution purposes.

Beyond the health- and security-related advantages obtained the described method an additional advantage should be mentioned, namely that elution by means of aqueous solutions overcomes the physical and practical problems caused by the use of known organic solvents, such as swelling and shrinkage of the column material in such a manner that the reliability and the stability of the columns is considerably prolonged.

As a further improvement achieved by the novel kit the use of an aqueous elution solvent disrupting the antigen/antibody interaction ensures that only the LMWOCs specifically attached to the immobilised monoclonal antibody are eluted. A further improvement achieved by the novel kit is the absence of interference during the quantification in the subsequent ELISA.

Best Mode for Carrying out the Invention

When performing the method according to the invention and/or using the kit according to the invention some problems may arise for those not trained or specifically skilled in the field. Assistance in such cases can be found in the following, where zeatin is used as a non- restrictive example.

Interference and precision in immunoas say.

The quantifying signal in competitive immunoassays is a tracer-displacemnt (whether radioactive, fluorescent or enzymatic) from the antibody (see Scheme 1 below). The signal does not provide information on the source of displacement. Two types of interference are encountered in competitive immunoassays: Interference from compounds which are structurally related to the analyte, and interference due to solvent effect.

Hansen et al. have demonstrated that zeatin ELISA will not yield consistent quantifications of cytokinins in crude extracts of stem segments of Zea maise (Hansen, C.E., Wenzler, H. and Meins jr., F.: Concentration gradients of trans-zeatin riboside and trans-zeatin in the maize stem. Measurement by a specific enzyme immuno- assay. Plant Physiol., vol. 75, pp. 959-963, 1984). Sufficient purification was obtained by the introduction of a butanol extraction step. Sample dilution reduces interference when solvent effects are the prevailing source of interference and also when the presence of cross- reactive compounds is the problem, as dilution of the sample will often bring down the concentration of the interfering substance below the detection limit of the assay (which is high due to the low reactivity with most potential cross - reactants), while the concentrations of zeatin and zeatin riboside are still within the measuring range. Interference is detected as an unlinearity of assay response upon sample dilution or a lack of parallellism with the standard curve. Samples with high contents of cytokinins can be diluted more prior to analysis with an assay of a given sensitivity.

Scheme 1:

-< = antibody <> = zeatin-N9 riboside <>E = enzyme conjugate

Antibodies in an ELISA well

Zeatin compound in sample competes with the enzyme conjugate for the limiting number of antibodies present

Non-bound enzyme conjugate is removed by washing

Bound enzyme conjugate reacts with the substrate (p-nitrophenyl phosphate). The product is p- nitrophenolate and is measured at 405 nm.

Quantification is made by making a zeatin-Nn riboside standard Validation bv successive approximat ion

Interference in immunoassays leads to cveras timaticns of the level growth substance in plant extracts. Such interference may be eliminated by purifying the sample successively until the last estimate of the plant hormone content is not lower than the preceding one. The term "successive approximation" has been introduced for this strategy by Reeve and Crozier in Encylopedia of Plant Physiology, New Series (J. MacMilland ed.), vol. 9, p. 203, 1980, and it has been used successfully for the analysis of IAA in cambial tissue of Pinus sylvestris (Sundberg, B., Sandberg, G. and Crozier, A. Purification of indole-3-acetic acid in plant extracts by immunoaffinity chromatorgraphy. Phytochemistry, vol. 25, no. 2, pp. 295- 298, 1986) and for the analysis of indole-3-acetamide in tobacco crown gall tissue (Ulvskov, P., Marcussen, J., Rajagopal, R., Prinsen, E., Rύdelsheim, P. and van Onckelen, H.: Immunoaffinity purification of indole-3- acetamide using monoclonal antibodies. Plant Cell Physiol., vol 28, no. 5, pp. 1-9), both cases in combination with immuno-affinity purification.

Some plant materials can be analysed in ELISA in a re- latively crude state. Callus and suspension cultured cells of carrots are materials of said type.

The Invention is further illustrated in the following non-limiting Examples:

Example 1

Extraction, isolation and purification of zeatin and N-9 substituted derivatives of zeatin bv said method

Step I: An extraction buffer was prepared just before use by dissolving 10 mg of the antioxidant, butylated hydroxy toluene (component B In the kit) in 1 ml of 96% ethanol. 100 μl of this solution were then added to 48.9 ml of 96% ethanol followed by 250 μ l of homogenization buffer (1.0 M Hepes, pH 7.5, component A in the kit). The volume was adjusted to 50 ml with water.

Step II 0.5 g of leaf tissue from sugerbeet (Beta vulgaris L.) plantlets, grown under sterile conditions were homogenised to a fine powder in a mortar under liquid nitrogen. 30 ml of the extraction buffer were then added and the resulting slurry allowed to attain room temperature. An aliquot of tritiated zeatin di-alcohol corresponding to 17,000 cpm was then added and the mixture centrifuged at 12,000 g for 1.5 min.

Step III After centrifugation the supernatant (fraction 1) was transferred to a siliconised glass container and the pellet further extracted by resuspension in 10 ml of the extraction buffer. After vigorous mixing the extract was centrifuged at 12,000 g for 15 min and the resulting supernatant combined with fraction 1. The pellet was again extracted using a further 10 ml of extraction buffer. After centrifugation at 12,000 g for 15 min the supernatant was again combined with fraction 1. After the third centrifuga- tion no radioactivity was found to be associated with the pellet, which was then discarded.

Step IV The combined supernatants were then evaporated under reduced pressure at 40ºC to remove ethanol. After removal of ethanol the remaining extract was transferred to a MInisorb^R plastic tube. The glass container was washed twice with 1 ml of de-ionised, distilled water and the washings were added to a Minisorb^R plastic tube. The extract was then transferred to a separation funnel and 3.3 ml of n-pentane (1/3 vol.) were added. After thorough mixing the phases were allowed to separate at room temperature. The upper organic phase was removed and discarded. An additional 3.3 ml of n-pentane was then added and the extraction repeated.

Step V: The aqueous phase was then transferred to a siliconised glass tube and a single freeze

(-80°C)/thaw cycle carried out. After thawing the extract was centrifuged at 12,000 g for 15 min. Traces of n-pentane contaminating the aqueous phase were now concentrated at the top of the aqueous phase and were removed. The aqueous phase containing the LMWOCs was then removed without disturbing the pellet.

Step VI: A pre-immune pre-column (0.3 ml column volume, prepared using component D of the. kit) was equilibrated with 1.5 ml of component G (10 times diluted). The immuno- affinity column (0.3 ml. column volume, prepared using component E of the kit) was equilibrated by applying 1.5 ml of component F (10 times diluted) followed by 4.5 ml of component G (10 times diluted).

Step VII: The column was then washed in the following order: (i) 10 ml of component G (10 times diluted) containing 1 nM adenosine, i.e. component Q of the kit

( ii ) 4.0 ml of component G (10 times diluted)

( i i l ) 2.0 ml of 1 M NaCl-solution

( iv) 2.0 ml of H₂O

(v) 4.0 ml of component F (10 times diluted)

(vi ) 4.5 ml of component G (10 times diluted)

S tep VI I I : The aqueous extract containing the LMWOCs of interest was then applied to the pre- immune pre-column connected in series to the immuno-affinity column. Thus the effluent from the pre-column was allowed to pass directly onto the immuno-affinity column at a flow rate not exceeding 0.5 ml/min. The pre-immune pre-column was then washed with 0.6 ml (2, times column volume) of component G (10 times diluted). This effluent was also applied slowly (less than 0.5 ml/min) to the immuno-affinity column.

Step IX: Next the Immuno-affinity column was washed with 1.5 ml of component H (2 times diluted) followed by 1.5 ml of glass distilled water. Both eluents were discarded.

Step X: The bound LMWOCs were then eluted by washing the column 4 times, each time with 1 ml of component F (10 times diluted) and the eluate was collected (fractions 1-4) and neutralised by adding component I (8 μl/100 μl of eluate). The samples were then stored on ice until further analysed. The immuno- affinity column was then equilibrated by washing with 3.0 ml of component G (10 times diluted). Radioactivity applied to macerated tissue 16,700 cpm Radioactivity remaining in pellet after 3rd extraction 3 cpir.

Radioactivity applied to immuno-affinity column 11,800 cpm

Effluent 850 cpm

Radioactivity eluted with NaCl - s o lution 0 cpm

Radioactivity eluted with H₂O 0 cpm

Radioactivity eluted with phosphoric acid fraction 1 6,400 cpm fraction 2 3,600 cpm fraction 3 750 cpm fraction 4 450 cpm

Fractions 1 and 2 were combined and the amount of zeatin and N-9 substituted derivatives of zeatin determined by ELISA.

A microtiter plate was coated by overnight incubation with 200 μl of a polyclonal anti-zeatin riboside antibody per well diluted 1 : 15,000 times at 4°C. Then

1. the ELISA-plate was washed 4 times with PBS/Tween^R washing buffer. At least 225 μl per well per wash was used. Excess wash solution was removed by patting the plate against dry blotting paper.

2. To some of the wells samples were added, whereas standards or PBS (background) were added to the others. In all cases 100 μl were used. All samples were in triplicate. At least two dilutions of each sample were used to obtain results within the best region of the standard curve (0.05-2 pmol/100 μl) Scheme 2

The range of samples including standards were applied to the ELISA plate as depicted in Scheme 2 above. In row 1, wells A-H, 250 pmol zeatin/100 μl was added. In rows 2 and 12, wells A-H, PBS was added. In rows 3-5, wells A-H, zeatin standards were added in tri- plicate, corresponding to 0.010, 0.025, 0.06, 0.20, 0.40, 1.00, 2.50 and 6.0 pmol/100 μl in wells A-H respectively. Rows 6-11 were used for the test samples.

3. 100 μl enzyme - conj ugate solution per well were then added.

4. The plate was then covered with a lid and incubated for 3 h at room temperature

5. After incubation the plate was washed with glass distilled water 4 times. At least 225 μl H₂O per well per wash were used. Excess solution was removed by blotting against paper.

6. 50 mg p -nitrophenyl phosphate was solubilised in 25 ml substrate buffer just before use, and 200 μl of this solution were added to this well. 7. The plate was covered with plate sealer and incubated for 90 min/37ºC.

8. The adsorbance at 405 nm (620nm as reference) was read in an ELISA spectrophotometer. The spectrophoto- meter substracted the "background" in row 1 (A-H).

From the ELISA measurements a value of 20 pmol zeatin and N-9 substituted derivatives of zeatin per gram fresh weight of leaf tissue was determined.

Example 2

Effect of sugar-coating on shelf-life character is t i c s of a kit according to the invention

In this example the reagents and materials contained in a kit according to the invention are described.

A: homogenised buffer: 1.0 M Hepes, pH 7.5 B : antioxidant: butylated hydroxy buffer C : salt adjustment buffer: 3 M NaCl, 0.75 M Hepes,. pH 7.5

D : pre-immune pre-column: immobilised immuno- globulin E: immuno-affinity column: immobilised monoclonal antibody

F: elution buffer: 1.0 M H₃PO₄, pH 2.5 G: equilibration buffer: 1.5 M NaCl, 500 mM Hepes

H: Washing buffer: 2.0 NaCl, 100 mM Hepes I : neutralisation buffer: 1.0 M NaOH J : ELISA plates, lid for an ELISA plate, plate sealer for an ELISA plate

K: enzyme conjugate: alkaline phosphatase conjugated zeatin riboside L: diluent for enzyme conjugate: PBS or TBS with 4% polyethyleneglycol

M : PBS or TBS buffer powder N : Tween^R-20 O Substrate diluent: diethanolamine or TRIS with Mg⁺⁺, pH 9.8

P: substrate: p-nitrophenyl phosphate Q: blocking buffer: 1 nM adenosine dissolved in component G (10 times diluted)

The above ELISA plates are prepared as follows

Step I Trans-zeatin-N- 9 - glycos ide is conjugated to bovine serum albumin (BSA). Rabbits are immunised with these antigenic substances and serum from the immunised rabbits containing immunoglobulins with high affinity against zeatin and zeatin-N-9 substituted derivatives are collected.

Step II The obtained antiserum is diluted 1:15,000 (v/v) with sodium carbonate buffer (pH 9.2). The protein components in this serum are adsorbed onto the smooth surface of the wells (200μl per well) an the microtiter ELISA plates by incubation at 4°C for 16 h.

Step III Non-bound proteins are removed by washing 4 times with PBS containing 0.1% Tween^R-20.

Step IV The adsorbed proteins are dried. Before drying 200 μl of a 5% (v/v) sugar solution in water is added to each well. Thereafter the plates are allowed to stand for 1 h at 4°C. Optionally the plates may then be partly emptied. Step V: The plates are cooled down to -20°C and allowed to stand for an additional hour at this temperature.

Step VI: Then the temperature is decreased to -80°C for an additional hour and afterwards the plates are freeze-dried for 16 h.

In order to demonstrate the favourable effect of sugar preservation, ELISA plates without sugar coating are prep ared fo r comparis on .

Uncoated plates and the sugar-preserved plates were monitored over a period of 16 weeks, using ELISA. 0 pmol zeatin (max. tracer binding, I.e. only enzyme conjugate is adsorbed to the antibodies) were added to the wells and the results recorded as described in Example 1.

The results are shown in Table 2 below:

Table 2 : Comparison of storability of antibody-coated

ELISA plates with and without sugar-treatment and stored at +4°C and -20°C.

The antibody activity is expressed as a percentage of the activity on fresh plates. Results are mean values and three independant experiments with the same antibody p rep arat ion were executed

A clear advantage of sugar-coating before freeze-drying is seen from the table at both temperatures. Almost no loss of activity is observed after 4 months of storage, whereas no sugar-coating results in a loss of about 2/3 of the activity at +4°C and about 1/2 of the activity at -20°C after 4 months of storage.

Example 3

Nature of the binding of the tritiated zeatin di-alcohol to the immuno-affinity matrix as revealed bv the effec- tivitv of two different elution systems

Two immuno-affinity column A and B (each 0.2 ml column volume) were prepared as described in Example 1, Step VI with the exception that the columns were not equilibrated with the blocking buffer (Step VII in Example 1). A stipulated amount of radioactivity (see below) was applied and the effluent determined. Each column was then sequentially washed with 5 times column volume of 1.0 M NaCl solution followed by 5 times column volume of water. Next the columns were washed with 10 times column volume of 0.1 M phosphoric acid (pH 2.5) followed by 10 times column volume of methanol.

Colunms A B Radioactivity (cpm) applied to immuno-affinity columns 16,800 15,500

Effluent 250 200 Radioactivity eluted with NaCl-solution < 100 < 100

Radioactivity eluted with H₂O < 100 < 100 Radioactivity eluted with phosphoric acid (pH 2.5) 8,300 5 ,400

Radioactivity eluted with methanol 7,500 9,800

As can be seen a considerable proportion of radioactivity was released by the phosphoric acid. Further washing with phosphoric acid, even at 0.25 M (pH 2.5) did not elute the remaining radioactivity.

It should be emphasised that the immobilisation of monoclonal antibodies on the column having the same association constant for the substrate should ensure a more uniform elution. The necessity to use methanol to elute the re- maining radioactivity is indicative for that this contribution of radioactivity is retained by the column in a different manner compared to that elutable by phosphoric acid.

Example 4

Specificity of column binding sites

The effect of blocking non-specific binding sites on the immuno-affinity column is exemplified in this example. Here three immuno-affinity columns (0.2 ml column volume) were prepared exactly as described in Example 1. Columns

A B C

Radioactivity (cpm) applied to immuno-affinity columns 10,500 9,900 9,800

Effluent 600 500 650

Eluate (0.1 M phosphoric acid, pH 2.5) 8,500 9,300 9,150

It can be seen that equilibration of the column with 1 nM adenosine (in 10 times diluted equilibration buffer G) ensures that most, if not all, of the applied radioactivity is specifically immobilised on the monoclonal antibodies and is accordingly eluted by the phosphoric acid.

Example 5

Performance of the zeatin-kit.

1 g of cell - suspension were extracted overnight at -80°C with 9 ml of methanol, 20 mg/1 butylated hydroxy toluene. An aliquot of tritiated zeatin- di - alcohol corresponding to about 13,000 cpm (counts per minute) was applied to the extract.

The extract was centrifuged at 2,000 x g for 10 min, and the supernatant filtered and transferred to a siliconised container. The pellet was washed with 10 ml cold methanol, centrifuged at 2,000 x g for 10 min and the supernatant filtered and pooled with extract from the first centrifuga- tion.

The methanol was evaporated to almost dryness at 35°C, and the residue transferred to a minis orb - tube with 2.5 ml PBS. The sample was then extracted twice with 1 ml n- pentane. The sample was then brought to -80°C, thawed and centrifuged at 2,000 x g for 10 min and the rest of the n-pentane phase was completely removed. PBS was added to the mark, i.e. 2.5 ml.

The extract was then applied to 0.25 ml immuno-affinity column equilibrated with PBS.

The amount of radioactivity in the effluent was determined.

The column was then washed with 5 ml PBS and 2.5 ml. H₂O. Elution was performed with 2 ml. icecold 100% methanol. The radioactivity in the eluate was determined.

The methanol was evaporated under a stream of nitrogen and the sample resolubilised in 1 ml PBS and the sample analysed in ELISA. The results were given in zeatin- equivalents.

The results are given with correction for aliquots taken out for determining the radioactivity.

Radioactivity applied to cellsuspension sample 13,020 cpm

Radioactivity applied to the column 10,321 cpm

Radioactivity found in the effluent 616 cpm Radioactivity found in the eluate 8,889 cpm

Zeatin equivalents in ELISA (corrected according to a total recovery of 68%) 150 pmol

Claims

Claims

1. A method for sample extraction, purification, concentration and quantification of low molecular weight organic compounds from biological sources, such as plants, specifically plant hormones selected from the group consisting of cytokinins and auxins, said method comprising submitting the sample in the form of an isolated extract of biological material applied via a pre-immune pre-column or directly to an immuno-affinity purification step using a selective first antibody followed by an enzyme-linked immunosorbent assay (ELISA) utilising an antibody stabilised by sugar c h a r a c t e r i s e d by using as the selective first antibody in the purification step an antibody having group specificity; the first antibody being covalently attached to a matrix without loss ofbinding capacity for the appropriate antigen; binding the low molecular weight organic compounds reversibly to an immuno-affinity column using aqueous reagents; eluting by means of a solvent selected among organic or aqueous solvents; and using a second antibody different from the first antibody as the antibody in the ELISA.

2. A method as claimed in claim 1 c h a r a c -a t e r i s e d by using a monoclonal antibody as the first antibody for the purification; and a polyclonal antibody as the second antibody in the ELISA.

3. A method as claimed in claims 1 or 2 c h a r a c t e r i s e d by using a monoclonal antibody covalently attached to the matrix via an amide bond formation as the first antibody for the purification.

4. A method as claimed in claim 3 c h a r a c t e r i s e d by introducing the amide bond formation by using a succinyl-acti ated carboxyl gel.

5. A method as claimed in claims 1-4 c h a r a c t e ri s e d by specifically eluting the low molecular weight organic compounds by means of a solvent selected from the group consisting of aqueous solvents and organic solvents.

6. A method as claimed in claim 5 c h a r a c t e r i s e d by using a solvent selected from the group consisting of 0.1 M phosphoric acid, pH 2.5, and methanol.

7. A method as claimed in claims 1-6 c h a r a c t e ri s e d by removing interfering substances by passing the extract through a pre-immune pre-column comprising a suitable matrix to which a pre- immunoglobulin fraction is covalently immobilised.

8. A method as claimed in claims 1-7 c h a r a c t e ri s e d by using ELISA plates, whereon the polyclonal antibodies are stabilised by a sugar.

9. A method as claimed in claim 8 c h a r a c t e r i s e d by using a sugar selected from the group consisting of fructose, sucrose, sorbitol and inositol, preferably sucrose.

10. A method as claimed in claims 1-9 c h a r a c t e ri s e d by preventing any non-specific adsorption onto the immuno-affinity column during the immuno-affinity purification step by coating any remaining non-specific binding sites with molecules selected from the group consisting of adenosine and similar organic molecules having no significant cross-reactivity with the selective antibody and/or other reagents used in the extraction, purification, concentration and quantification of LMWOCs, especially cytokinins and auxins.

11. An analytical kit for carrying out the method as claimed in any of the preceding claims 1-10 c h a r a c t e r i s e d in that it comprises i. components required for sample extraction and purification and ii. components necessary for use in an enzyme-linked immunosorbent assay.

12. An analytical kit as claimed in claim 11 c h a r - a c t e r i s e d in that it comprises I. components required for sample extraction, purification and concentration comprising components chosen from the group consisting of A, B, C, D, E, F, G, H, I, Q: component A: homogenisation buffer ( for use in tissue extraction) component B: an antioxidant (for use in tissue extraction) component C: salt adjustment buffer (for use in sample extraction) component D: a vial containing an appropriate matrix to which a pre-immune globulin fraction is covalently immobilised; said matrix being used for the preparation of a pre-immune pre-column component E: a vial containing an appropriate matrix to which a selective antibody is covalently immobilised, said matrix being used for the preparation of the immuno- affinity column component F: elution buffer (for use in sample extraction) component G: equilibration buffer (for use in sample extraction) component H: washing buffer (for use in sample extraction) component I: neutralising buffer (for use in sample extraction) component Q: blocking buffer (for use in sample extraction). II. components required for sample quantification comprising components chosen from the group consisting of J, K, L, M, N, O, P: component J: a suitable vessel coated with a suitable antibody for sample quantification, lid for an ELISA plate, plate sealer for an ELISA plate component K: a vial of LMWOC-enzyme conjugate component L: diluent for enzyme conjugate component M: phosphate-buffered saline or TRIS- buffered saline component N: Tween^R-20 component O : substrate diluent component P : substrate, p -nitrophenyl phosphate

13. A kit as cl&imed in claim 12- - c h a r a c t e r - i s e d in that component K is a conjugate of zeatin riboside and alkaline phosphatase.

14. A kit as claimed in claims 11-12 c h a r a c t e ri s e d in that component J is a microtiter plate.

15. A kit as claimed in claim 12 c h a r a c t e r - i s e d in that it comprises the components B, E, J, K, M, N, O and P

16. A kit as claimed in claim 15 c h a r a c t e r i s e d in that component B is butylated hydroxy toluene component E is a matrix to which a monoclonal antibody is covalently immobilised component J is an ELISA plate coated with a polyclonal antibody with a very high speci- ficity for zeatin and zeatin-N9-deri- vatives component K is an enzyme -conjugate of alkaline phos- phatase conjugated with zeatin-N9-ribo- side component M is phosphate - buffered saline or TRIS- buffered saline, pH 7.0 component N is Tween^R-20 component O is diethanolamine - or TRIS-buffer, pH 9.8, including M⁺⁺ component P is p-nitrophenyl-phosphate

17. A method using a kit as claimed in the preceding claims 11-16 for carrying out the following steps: i. tissue maceration in an extraction medium selected fromthe group consisting of methanol-based extraction medium and ethanol-based extraction medium, ii. application of sample to a pre-immune pre-column, iii. blocking any non-specific adsorption sites on the immuno-affinity column by using an appropriate LMWOC not reactive with the selective antibody or any of the reagents used in the purification and quantifiactaion of the LMWOCs of interest, iv. application of sample to the immuno-affinity column, v. quantification using an ELISA where an aliquot of an appropriately diluted sample containing the affinity-purified LMWOCs is added to some of the wells of the microtiter plate, vi . adding an aliquot of a zeatin riboside standard to at least one well of the microtiter plate, vii. adding an aliquot of diluted LMWOC-enzyme conjugate to each well of said microtiter plate containing aliquots of the standard zeatin riboside and aliquots of the test samples, viii. incubating said microtiter plate for a suffi- ciently long period of time as to permit a competition between antigen and LMWOC-enzyme conjugate for the limited amount of antibody immobilised on the microtiter plate, ix. rinsing each well of said microtiter plate with an appropriate rinsing solution to remove any unbound LMWOC-enzyme conjugate, x. adding an aliquot of a suitable solution to each well of said microtiter plate containing said LMWOC-enzyme conjugate, said substrate being reactive with said LMWOC-enzyme conjugate, xi. incubating for a sufficiently long period of time and under suitable conditions as to permit said enzyme to react, giving a measurable change in adsorbance, xii. measuring the adsorbance of the reacted product by a spectropho tometric method, xiii. quantification of the LMWOCs present in the test sample by using the adsorbance measurements obtained by said spectrophotometric methods.

18. A method as claimed in claim 1 using a kit as claimed in claim 16, c h a r a c t e r i s e d in the following steps: i. tissue-maceration in a methanol-based extraction medium, ii. application of sample extract solubilised in component M to component E, iil. elution from component E with 100% methanol, iv. removal of methanol and transfer of sample Into component M, v. quantification using an ELISA procedure where an aliquot of an appropriately diluted sample containing the affinity-purified zeatin derivatives is added to some of the wells of component J, vi. adding an aliquot of appropriately diluted zeatin standards to some of the other wells of component J, vii. adding an aliquot of appropriately diluted component K to each well of component J, viii. incubating said ELISA-plate for a sufficiently long period of time as to permit competition between antigen and component K for the li.niced number of antibodies immobilised in the ELISA- plate, ix. rinsing each well with a appropriate rinsing solution to remove any unbound enzyme conjugate, x. adding an aliquot of component P diluted in component 0 to each well, xi . incubating for a sufficiently long p e r i o d of time and under suitable conditions as to give a measurable change in absorbance, xii. measuring the absorbance of the reacted product by a spectrophotometric method, and xiii. quantification of the zeatin derivatives present in the test sample by using the absorbance data obtained.