A monoclonal antibody to (S)-abscisic acid: its characterisation and use in a radioimmunoassay for measuring abscisic acid in crude extracts of cereal and lupin leaves |
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| Authors: | S A Quarrie P N Whitford N E J Appleford T L Wang S K Cook I E Henson B R Loveys |
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| Institution: | (1) Cambridge Laboratory, AFRC-IPSR, Maris Lane Trumpington, CB2 2LQ Cambridge, UK;(2) Department of Agricultural Sciences, University of Bristol, Long Ashton Research Station, Long Ashton, BS18 9AF Bristol, UK;(3) AFRC-IPSR, John Innes Institute, Colney Lane, NR4 7UH Norwich, UK;(4) CSIRO Division of Plant Industry, Private Bag, P.O., 6014 Wembley, W.A., Australia;(5) CSIRO Division of Horticultural Research, Box 350, G.P.O., 5001 Adelaide, S.A., Australia |
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| Abstract: | A monoclonal antibody produced to abscisic acid (ABA) has been characterised and the development of a radioimmunoassay (RIA) for ABA using the antibody is described. The antibody had a high selectivity for the free acid of (S)-cis, trans-ABA. Using the antibody, ABA could be assayed reliably in the RIA over a range from 100 to 4000 pg (0.4 to 15 pmol) ABA per assay vial. As methanol and acetone affected ABA-antibody binding, water was used to extract ABA from leaves. Water was as effective as aqueous methanol and acetone in extracting the ABA present. Crude aqueous extracts of wheat, maize and lupin leaves could be analysed without serious interference from other immunoreactive material. This was shown by measuring the distribution of immunoreactivity in crude extracts separated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), or by comparing the assay with physicochemical methods of analysis. Analysis of crude extracts by RIA and either, after TLC purification, by gas chromatography using an electron-capture detector or, after HPLC purification, by combined gas chromatography-mass spectrometry (GC-MS) gave very similar ABA concentrations in the initial leaf samples. However, RIA analysis of crude aqueous extracts of pea seeds resulted in considerable overestimation of the amount of ABA present. Determinations of ABA content by GC-MS and RIA were similar after pea seed extracts had been purified by HPLC. Although the RIA could not be used to analyse ABA in crude extracts of pea seeds, it is likely that crude extracts of leaves of several other species may be assayed successfully.Abbreviations ABA
abscisic acid
- DW
dry weight
- FW
fresh weight
- GC-ECD
gas chromatography using an electron capture detector
- GC-MS
combined gas chromatographymass spectrometry
- HPLC
high-performance liquid chromatography
- McAb
monoclonal antibody
- PVP
soluble polyvinylpyrrolidone
- RIA
radioimmunoassay
- TLC
thin-layer chromatography |
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| Keywords: | Abscisic acid (assay) Cereals Legumes Monoclonal antibody Radioimmunoassay (abscisic acid) |
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