Application of polyclonal antibodies for ABA measurement in unicellular alga Dunaliella salina

The measurement of the phytohormone ABA was determined following to study of sensitivity, validity and reproducibility of different techniques. An indirect competitive enzyme-linked immunosorbent assay (ELISA) to the quantitative analysis of ABA in Dunaliella salina was selected. Because of its small size, ABA is not itself immunogenic. Therefore it counts as a Haptenn. The bovine serum albumin (BSA) as a protein carrier can be used for preparation of suitable immunogen. The ABA was coupled to bovine serum albumin. The evaluation of conjugates was determined by UV-spectroscopic analysis. Then the immunizations of rabbits were performed. Immune rabbits were bled after booster injection. The immunoglobulin fraction was isolated from other proteins of serum. In competitive Elisa, free and coated antigens compete for binding sites on the antibody. The second antibody enzyme labeled with peroxidase was added. Bound enzyme activity was calorimetrically determined after addition of substrate (TMB). The standard curve of Elisa was obtained by plotting absorbance at 450 nm versus the log of ABA concentration in the assay. Absorbance was linear between 100 pg and 1000 ng ABA concentration. The evaluation of coefficient of variation (%CV) in inter and intra assay of standard curves were accepted in the range (below 10). When known amounts of ABA were added as internal standard to purified alga extracts, the recovery was evaluated about 109%. This high recovery confirms both the specificity and accuracy of the assay. When the parallelism test was performed in purified alga extracts and in a serial diluted samples, the parallel pattern to the standard curve was observed. The Ka (affinity coefficient) values of antibodies were found to be 0.32×109 L.mol-.1. In conclusion, our results showed the accuracy of the Elisa assay for measurement of ABA content of D.salina. Then the ABA contents of algal samples were measured in control (1.5M NaCl) and stressed (3.5M NaCl) Dunaliella cells. Results indicated that ABA was found to be as an endogenous compound and also as a stress hormone in D.salina. In D. salina cells, the ABA content was low and the rate of ABA production was increased when the cells were exposed to 3.5 M NaCl in the medium.