Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56961—Plant cells or fungi

Definitions

• 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.
• LMWOCs low molecular weight organic compounds
• LMWOCs LMWOCs
• concentration and/or activity of these LMWOCs is more pronounced in the meris tematic regions of the plant.
• WO 86/04096 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.
• 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.
• a method for sample extraction, purification, concentration and quantification of low molecular weight organic compounds from biological sources 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 ⁇ ompounds reversibly to an immuno-affinity column using aqueous
• 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.
• 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.
• the antibody is covalsnt- ly bound to the matrix via an amide bond formation preferably introduced by using a succinyl-activated carbonyl gel.
• mild conditions can be used for preparing the immuno-affinity material.
• 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.
• 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.
• the LMWOCs are specifically eluted using an aqueous solvent. It is especially preferred to use 0.1 M phosphoric acid, pH
• the present method depends therefore for its successive 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.
• a sugar is included during the preparation of the ELISA plates.
• 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.
• 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.
• 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.
• LMWOCs such as zeatin and N-9 substituted derivatives of zeatin
• ELISA enzyme- linked immunosorbent assay
• 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.
• 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.
• cytokinin e.g. zeatin
• 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.
• 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.
• 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.
• 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., R ⁇ delsheim, P. and van Onckelen, H.: Immuno-affinity purification of indole- 3 -acetamide using monoclonal antibodies. Plant Cell Physiol., vol. 28,
• 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.
• 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.
• 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.
• a sugar solution e.g. 5% (v/v) of sucrose
• sucrose a sugar solution
• 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.
• a LMWOC-enzyme conjugate e.g.
• 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.
• novel analytical kit according to the invention useful for carrying out the method according to the Invention comprises
• 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
• 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. NH 4 OH)
• 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
• PBS phosphate-buffered saline
• Tween R -20 O substrate diluent (containing e.g. diethanolamine or TRIS buffer)
• kits 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.
• 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.
• kit can be used without using toxic organic liquids for elution purposes.
• 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.
• 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.
• 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.
• 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
• 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 ⁇ l of this solution were then added to 48.9 ml of 96% ethanol followed by 250 ⁇ 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 powerbeet (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
• 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
• 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 ⁇ l/100 ⁇ l 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.
• a microtiter plate was coated by overnight incubation with 200 ⁇ l of a polyclonal anti-zeatin riboside antibody per well diluted 1 : 15,000 times at 4°C. Then
• the ELISA-plate was washed 4 times with PBS/Tween R washing buffer. At least 225 ⁇ l per well per wash was used. Excess wash solution was removed by patting the plate against dry blotting paper.
• the plate was then covered with a lid and incubated for 3 h at room temperature
• B antioxidant: butylated hydroxy buffer
• C salt adjustment buffer: 3 M NaCl, 0.75 M Hepes,. pH 7.5
• pre-immune pre-column immobilised immuno- globulin
• immuno-affinity column immobilised monoclonal antibody
• K enzyme conjugate: alkaline phosphatase conjugated zeatin riboside
• L diluent for enzyme conjugate: PBS or TBS with 4% polyethyleneglycol
• P substrate: p-nitrophenyl phosphate
• Q blocking buffer: 1 nM adenosine dissolved in component G (10 times diluted)
• 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.
• BSA bovine serum albumin
• 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 (200 ⁇ l 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 ⁇ l 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.
• 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
• Two immuno-affinity column A and B 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.
• Effluent 250 200 Radioactivity eluted with NaCl-solution ⁇ 100 ⁇ 100
• 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. H 2 O. Elution was performed with 2 ml. icecold 100% methanol. The radioactivity in the eluate was determined.

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