Background | This ELISA assay utilise the principle of competitive binding to measure the concentration of hormone in plant extracts. The ABA (abscisic acid C1) hormone and ABA-glucosylester specific antibodies are precoated to the surface of the reaction wells. The plant extract sample, containing an unknown amount of hormone, is mixed in the reaction well with a known amount of a tracer to react with a limited number of antibodies in the reaction wells. During incubation the hormone in the sample competes with the tracer for the antibody binding sites. Unbound hormone, tracer and plant extract are washed out of the reaction wells. Following substrate addition which reacts with a tracer bound to the antibody and produces a yellow-coloured product. The absorbance of the sample is converted to concentration of hormone by means of a standard curve which is produced by simultaneously treating standards along with the samples.

Reaction wells | 5pcs for 480 assays, 60 strips with 8 wells

Tracer | 20 – 50 µl

Tracer diluent | 5xstock + 0.02 % NaN₃

Reaction and wash solution | 10xstock+0.02 % NaN₃

Stopping reagent | 2x stock, developed colour is stable for along period and stopping reagent needs to be used to prevent colour reaction from developing further

Substrate diluent | 10xstock +0 0.02 % NaN₃

Substrate | 100 mg

Standards | 600 µl of each: 15.6 pmol, 7.8 pmol, 3.9 pmol, 1.95 pmol, 975 fmol, 488fmol, 244 fmol, 122 fmol, 61 fmol, 30.5 fmol, 15.2 fmol

Assay development time | 4-5 hours

Sensitivity |0.01 to 10 pmol/50 µl or 4 pg/50 µl

Plant extract volume | 50 µl

Refrence |  Liu et al. (2015). Physiological and comparative proteomic analysis reveals different drought responses in roots and leaves of drought-tolerant wild wheat (Triticum boeoticum). PLoS One. 2015 Apr 10;10(4):e0121852. doi: 10.1371/journal.pone.0121852. eCollection 2015.

Antibody titre | 1: 5 300Unspecific binding | 2.8 %

Midrange(B/Bo=50%) | 275 pmolDetection limit | 7.8 fmol, 2 pg

Linear range of measurment | 15-500 fmolIntraassay variability | 4.3 %

Interassay variability | 5.2 % Amount of tracer per assay | 12.4 ng

Compound  | Cross-reactivity (%)

(±)-cis,trans-abscisic acid | 100

(-)-cis,trans-abscisic acid | 47

(+)-cis,trans-abscisic acid | 52

(±)-trans,trans-abscisic acid | 3.27

(+)-trans,trans-abscicis acid | 1.06

(±)-cis,trans-abscisic acid methyl ester | 217

(±)-cis,trans-absicis acid glucosyl ester | 115

phaseic acid | 0

dihyrdophaseic acid | 0.09

xanthoxin | 0.05

lunularic acid | 0

violaxanthin | 0

farnesol | 0

vomifoliol | 0

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. 

The  samples have to be methylated by diazomethane before ELISA analysis, for example by using a commercial reagent 2.0 M Trimethylsilyl)diazomethane solution in hexane (Sigma) or using a safer alternative TMSD (Trimethylsilyl diazomethane).

Frozen  plant  tissues  are  ground  to  a  fine  powder  under  liquid  nitrogen.  The  powder  is extracted in ice-cold 70% ethanol (10 ml . g-1 FW) containing sodium diethyldithiocarbamate asantioxidant (400 ug . g-1 FW). About 420 Bq (25.000 dpm) of  [2-³H] ABA tracer is added to the extracts to monitor for losses during purification steps  and to validate the chromatographic data. After 2 h extraction, the homogenate is centrifuged (15 000 g, 4°C) and pellets re-extracted by the  same way. The  combined  extracts  are  then  purified  over  a  reversed  phase C18  column  to eliminate chlorophyll and lipids. The extracts are subsequently concentrated to approx. 1.0 ml by rotary  evaporation under vacuum  at 35°C. The  samples  are diluted  to 20 ml with ammonium acetate buffer (40 mM, pH 6.5) containing sodium diethyldithiocarbamate. For the immunoassay dilution  analysis,  the 2 ml of  eluates  is dried in  vacuo  and  re-dissolved  in Tris-buffered  saline (TBS, 50 mM Tris, 10 mM NaCl, 1 mM MgCl₂, pH 7.5). Aliquots of these solutions are eitheranalysed  in serial dilutions or mixed with known amounts of ABA standards and  then analysed by ELISAs. The extracts are further purified using combined diethylaminoethylcellulose (1.0 x 5.0 cm) - octadecylsilica  (0.5 x 1.5 cm) columns. ABA and  its amino  acid metabolites are  loaded onto a DEAE column cartridge which is then washed with 10 ml dest. water and eluted in 5 ml 6% HCOOH (v/v). The eluates are loaded onto a C18 cartridge and after washing with 5 ml dest. water eluted with 5 ml methanol. The eluates are than evaporated to dryness, dissolved in 50 ul 70% ethanol + 250 ul H2O and filtered through a HPLC pre-filter (0.22 um). The samples have to be methylated by diazomethane before ELISA analysis, for example by using a commercial reagent 2.0 M Trimethylsilyl)diazomethane solution in hexane (Sigma).

HPLC separation of samples is recommended to purify away glucosylesters and amino acid conjugates which may bind anti-ABA antibodies.