I
Figure 2 . 1 Retention of standard cytokinins on HPLC using the gradient established forI
separation of cytokinins ( a) and immunohistogram of cytokinins in a sample of kiwifruitl
outer pericarp tissue separated on the HPLC (b). Fractions 3 to 1 8 wereI
radioimmunoassayed using antiserum generated to ZR. Fractions 1 9 to 2 1 and 25 to 3 12: General Materials and Methods . 2-5
2.2 Immunological Analysis of Cytokinins
Immunology is based on the ability of a pathogen to elicit an immune response in an animal. Upon infection with a pathogen, the immune system produces antibody proteins which bind to the pathogen and render it ineffective at causing damage. Collection of a blood sample from the infected animal yields semm containing antibodies to the pathogen, which are able to be used for a range of imunological based laboratory techniques.
Although immunology was developed primarily for use in medical science, its application in other fields has since been extensively developed, as it has become clear that animals can be immunised against a wide variety of substances (antigens) which may not normally be pathogenic (Galfre and Butcher, 1 986). Antibodies thus formed can be used in quantitative or qualitative immunoassays because of their ability to bind to the antigen present in biological samples of interest, such as plant extracts.
A wide variety of immunoassays have been developed, mostly based on competitive binding of the sample antigen or a labelled antigen tracer. Radioimmunoasay (RlA) techniques were developed from the work of Yalow and Berson ( 1 960) and are now widely used for analysis of plant growth regulators, including cytokinins (Weiler, 1 980; MacDonald et al., 1 98 1 ; Ernst, 1 986). RIA is based on an equilibrium between unlabelled antigen and radioactively labelled antigen (tracer) for a saturable amount of antibody binding sites (Ernst, 1 986). After equilibrium, bound and free tracer are separated and the radioactivity present (usually) in the bound form is measured. As the concentration of unlabelled antigen increases, competition with the tracer for limited binding sites will occur, leading to a decreased amount of radioactivity in the bound form. This allows a standard curve to be generated, enabling quantification of antigen concentration in samples.
The main requirements for RlA (Chard, 1 990) are:
1 . Purified antigen for use as standards.
2-6 2: General Materials and Methods. Cytokinin Analysis 3 . Radio-labelled tracer which behaves identically with the unlabelled antigen.
4. Antibodies which have high specificity (bind specifically to antigen), affinity (ability to bind antigen) and titre (concentration of binding sites).
S . An efficient and practical separation system.
2.2.1 Synthesis of 3H-cytokinin riboside dialcohol tracers
The method of MacDonald and Morris ( 1 98S) was used to prepare radio-labelled dialcohols for use in RIA. Although the riboside part of the cytokinin molecule is altered in the procedure, antibody specificity is usually directed to the opposite side of coupling to protein (Weiler, 1 980). As coupling of cytokinins to protein for generation of antiserum is through the same position as alteration of the dialcohol molecule (carbon three and four of the ribose group), cross reactivity to the cytokinin dialcohol should be similar to that of the unaltered cytokinin riboside.
t-Zeatin-riboside (ZR) or Isopentenyl-adenosine (IPA) ( I S �Mol) were individually dissolved in SOO �l HPLC grade methanol and millipure H20 (SOOul). NaI04 (Sigma S 1 878) (30�Mol) was added, vortex mixed briefly and placed at 4°C for 4S minutes to convert the riboside group to a dialdehyde. The cytokinin dialdehyde was immediately loaded onto a CI8 Sep-pak cartidge (Waters) which had been pre-conditioned with methanol (Srnl) and H20 (Srnl). Columns were washed with H20 (SmJ) to remove excess periodate. Cytokinin dialdehydes were eluted from the columns with methanol (6rnl) , dried under a stream of oxygen free nitrogen gas and re-dissolved in 1 00 �l methanol. A 20 �l sample was placed in a polypropylene micro-centrifuge tube and 1 M NaHC03 ( 1 00 �l, pH=9.3) added.
Tritiated sodium borohydride ( 1 Ci, specific activity SOCilmMol obtained from Amersham) was dissolved in NaOH ( 1 00 �l O.SM ) in a vented fume cupboard. 2S�1 was immediately added to each dialdehyde preparation and left to react for 30 minutes to form a tritiated dialcohol. Glacial acetic acid was added very slowly to degrade excess borohydride until bubbling had ceased. Each putative dialcohol was purified on a pre conditioned sep-pak column as for the dialdehyde.
2: General Materials and Methods. Cytokinin Analysis 2-7
Samples of the putative dialcohols diluted 2000: I with 20% acetonitrile in TEA were injected onto a CIS HPLC column running under isocratic conditions (20% acetonitrile, 80% TEA) connected to a �ram radioactivity monitor (see section 2. 1 .2). A single radioactive peak was found to elute for each cytokinin preparation and these were considered likely to be cytokinin dialcohols. The retention time for each putative 3H_ dialcohol was lower than for standard cytokinin ribosides and similar to that expected for the dialcohol, but in each case the radioactive peak corresponded to a single UV peak.
Specific activity was estimated for each of the dialcohols using a standard curve of UV absorbance for each cytokinin to estimate the amount of cytokinin dia1cohol and by counting radioactivity on the scintillation counter. IPA was estimated at 47 Ci/mMol and ZR was estimated at 37 CilmMol.
2.2.2 Preparation of cytokinin-protein conjugates
Low molecular weight molecules such as cytokinins will not normally induce an immunogenic response and thus need to be conj ugated to a larger molecule such as a protein prior to immunisation. When this is done, antibodies specific to both the protein and the small molecule (hapten) are generated.
ZR and IPA were linked to bovine serum albumen (BSA) using the periodate oxidation method of Erlanger and Beiser ( 1 964) modified by MacDonald et aI. ( 1 98 1 ). ZR (Sigma Z0375) or IPA (Sigma 07257) (22mg) were dissolved in 5ml millipure water. N aI04 (27 mg) was added and the mixture incubated for four hours at 4°C. BSA (60 mg of fraction V, Sigma A3350) was added and allowed to dissolve without shaking. The pH was adjusted to 9.3 with K2C03 solution (5% w/v) and the solution was incubated at 4°C for three hours. Ethylene glycol (25�1, Sigma E9 1 29) was added to destroy excess periodate and the pH was checked and readj usted to 9.3 if required before a further 2 hour incubation at 4°C. NaBH4 (35mg) was added, the vial covered and placed to incubate at 4°C for 26 hours. Excess borohydride was eliminated by adjusting to the pH to 5 . 5 with
2-8 Chapter 2 : General Materials and Methods. Cytokinin A nalysis
1 M fonnic acid, slowly to avoid excess foaming and then readjusting the pH to 8.5 with 5% K2CO:,.
The cytokinin-BSA conjugate formed was placed into wide dialysis tubing, double knotted and dialysed repeatedly against 0.05M phosphate buffered saline (PBS) pH 8.5 for four days with three changes per day, 20ml vs. 20 Htres with constant stirring at 4°C. The conjugates were stored frozen in PBS.
Binding ratios were estimated spectrophotometric ally. Protein (BSA) concentration of conjugates was estimated using the biuret assay . Absorbance of conjugate at 269nm was measured against a reference cell containing a B SA solution of the same concentration. This gave the absorbance at 269nm due to the presence of cytokinin (bound to the protein). The concentration of cytokinin was then estimated using a standard curve of absorbance of IPA or ZR at 269 nm (Figure 2.2). The binding ratio was calculated as mol-cytokinin/mol-BSA (BSA, molecular weight==68 000 g.mor1 ) and was 1 5 .6 for ZR and 1 6.5 for IP A (Figure 2.2), which is comparable to that cited in the literature (MacDonald et aI. , 1 98 1 ; Ernst, 1 986). BSA has 30-35 lysine residues available as binding sites (Harlow and Lane, 1 988). Approximately one mol of hapten per 50 amino acid residues is a minimum binding ratio for the generation of antibodies to the hapten (Harlow and Lane, 1 988). BSA contains 2 1 1 residues, thus a minimum binding ratio of 4.2 is required for suitable for generation of antiserum, which was exceeded by the conj ugates prepared.
2.2.3 Generation of polyclonal antiserum.
The use of adjuvants to enhance immune responses is widespread. Adjuvants perfonn two functions. Firstly they form a deposit to protect antigens against rapid catabolism and secondly they act to stimulate the immune system non-specifically (Harlow and Lane, 1 988). Freunds incomplete adjuvant (iFA) contains 85% mineral oil and 1 5% mannide mono-oleate emulsifier (Harlow and Lane, 1 988). Freunds complete adjuvant (cFA) is formed when iFA is mixed with heat killed mycobacteria. Although cFA has been the adjuvant of choice for immunisation, use in recent years has declined due to
2: General Materials and Methods. Cytokinin Analysis 2-9
adverse side effects such as granulomas and hypersensitivity reactions (Broderson, 1 989) and thus concern for animal welfare.