We are thinking on working on ABA detection in plant tissues. We have HPLC with various detectors, but we are looking also on Elisa kits. Please feel free to share any thought on detecting ABA either by HPLC or Elisa kit and which Elisa kit works better and is easier to handle.
Dear Sir. Concerning your issue about the detection of Abscisic acid plant tissues either by HPLC or Elisa kit. Abscisic acid is a single compound unlike the auxins, gibberellins,
and cytokinins. It was called "abscisin II" originally because it was thought to play a major role in abscission of fruits. The microtiter plate provided in ELISA kit has been pre-coated with 4 goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated ABA and antibody preparation specific for ABA, and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of ABA in the samples is then determined by comparing the O.D. of the samples to the standard curve. The detection range was 0.4ng/ml-100ng/ml. The standard curve concentrations used for the ELISA’s were 100ng/ml, 25ng/ml, 6.25ng/ml, 1.55ng/ml, 0.4ng/ml. This assay recognizes plant ABA. No significant cross-reactivity or interference was observed. The minimum detectable dose of plant ABA is typically less than 0.25ng/ml . The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Processing of plant extracts required prior ELISA analysis may vary from plant to plant and will depend upon the actual research objective. In most cases pigments and lipophilic material needs to be removed by C18 reversed phase chromatography and subsequently by combined DEAE-cellulose-reversed phase C18 columns. No sample purification is required for algae, cyanobacterial and mosses. For detection of ABA from cereal grains use: extraction in 80% methanol drying to water phase, dilution by water, purification via C18 column, elution from the column by 100% methanol followed by drying, methylation and analyses. Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-14C]ABA, of which 98.9% was recovered. Following purification by the preparative HPLC procedure, the ABA in a soybean seed extract was quantified using either GLC with an electron capture detector (GLC-EC) or by analytical HPLC with a UV detector. For soybean seed extracts, two analytical HPLC column packing materials were found adequate: μPorasil and μBondapak-NH2 (Waters Associates). However, with complex tissue extracts, such as soybean leaf and nodule tissues, only GLC-EC had the necessary selectivity and sensitivity. I think the following below links may help you in your analysis:
https://www.biotrend-usa.com/spec/CSB-E09159Pl.pdf
http://www.pnas.org/content/104/14/5759.long
http://www.agrisera.com/cgi-bin/ibutik/SkapaFaktura.pl?SkrivPDF=J&funk=visa_artikel&artnr=AS11%201748&Friendly_Grupp=elisa-kits&Friendly=aba-elisa-quantitation-kit-2&artgrp=367&Sprak=en
Thanks
Dear Isam thank you for your prompt reply. I have checked all these but I would appreciate your experience on ABA analysis and your suggestion. Is it the kit from Agrisera?
hi dear
you can check the manual attached below
for more information you can contact +86 15968300958 whats app
thank-you
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