Quantitative immunochemistry using IgG and IgY against plant hormones
AbstractThe present study focuses on obtaining and comparing IgY and IgG antibodies against indoleacetic acid (IAA), zeatin (Z) and isopentenyladenine (2iP). The use of antibodies in rapid tests to analyse hormones has been developed for the immediate application of high sensitivity immunoglobulins against IAA, 2iP and Z. Each plant hormone was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA), both of animal origin, using chemical couplings to their respective functional groups. The IAA, Z and 2iP antibodies were then produced from chicken egg yolks (IgY) or from rabbit serum (IgG) that had been hyperimmunised with the respective conjugated protein. The antibodies were obtained from rabbit sera or chicken egg yolks, purified by ammonium sulphate precipitation and subsequently used in indirect analytical immunoassays (competitive enzyme-linked immunosorbent assay; ELISA) to detect and quantify these hormone molecules. During titration, differences were observed not only between the specific antibody sources but also between the different conjugated hormones obtained. Although, the IgGs failed to produce a competitive IAA detection system, the IgY antibodies proved to be suitable for quantifying the plant hormones. The immunoenzymatic assay using IgY achieved a detection limit of 3.4 nmoles (3.4 ppb) for zeatin, 6 pmoles (6 ppt) for 2iP and 480 pmoles (480 ppt) for IAA, whereas the IgG detection limits established were in micromolar (ppm) range and manifested varying results and low specificity near the limit of detection. Comparisons and discussions are presented on the impact of the hormone-protein conjugates and the potential use of antibodies in analysing zeatin and 2iP. The antibody characteristics, the presence of antibody interference in the samples and the limitations and advantages of immunochemical assays are also discussed.
Keywords: Quantification, auxin, cytokinins, antibodies
African Journal of Biotechnology Vol. 12(24), pp. 3828-3834