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Development and Growth Regulation

Comparison of a Commercial ELISA Assay for Indole-3-Acetic Acid at Several Stages of Purification and Analysis by Gas Chromatography-Selected Ion Monitoring-Mass Spectrometry Using a ¹³C₆-Labeled Internal Standard ¹

United States Department of Agriculture, Agricultural Research Service, Plant Hormone Laboratory, Beltsville Agricultural Research Center-West, Beltsville Maryland 20705, Department of Botany, University of Maryland, College Park, Maryland 20742, United States Department of Agriculture, Agricultural Research Service, United States Horticultural Research Laboratory, Orlando, Florida 32803, Plant Physiology Research Group, Department of Biology, The University of Calgary, Calgary, Alberta, T2N 1N4, Canada

Quantitative analysis of indole-3-acetic acid (IAA) using selected ion monitoring gas chromatography-mass spectrometry (GC-MS) with ¹³C₆[benzene ring]-IAA as the internal standard was used to compare the quantitative accuracy of commercial enzyme-linked immunoabsorbent assay (ELISA) kits. Plant materials differed in the amount of purification required prior to use of ELISA for reliable estimates to be made. Purification similar to that obtained by at least one high performance liquid chromatographic (HPLC) step was generally necessary prior to ELISA analysis of plant materials. Additional levels of purification appeared to be required for some plant materials prior to HPLC in order to obtain an accurate estimate by ELISA techniques. In no case was it possible to obtain reasonable estimates of IAA from crude extracts or even from acidic fractions of extracts of plant tissues. GC-MS techniques provide a rapid and simple method for checking the validity of ELISA techniques. Quantitative GC-MS, or a similar technique that provides an independent quantitative validation, should, whenever possible, be applied to each new plant material under study if use of the ELISA is planned.

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