Immunoaffinity techniques applied to the purification of gibberellins from plant extracts

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA₁, GA₃, GA₄, GA₅, GA₇, and GA₉ conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA₇ was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA₂₀ and GA₂₉ were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA₉ were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA₉ was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA₂₀ and GA₅₁ were also observed in testae.