Conditions for UV-induced cross-linking of abscisic acid (ABA) through
its enone chromophore to binding proteins were evaluated. The effects
of a UV-light band between 260 and 530 nm on both unconjugated and pr
otein-conjugated ABA, as well as on anti-ABA antibodies as models of A
BA-binding proteins were determined. UV irradiation caused both isomer
ization and photolysis of ABA, but increasing the lower irradiation bo
undary to 345 nm strongly reduced photolysis and largely prevented iso
merization. When conjugated to alkaline phosphatase (AP), ABA remained
stable when using either a 320 or a 345 nm filter. At these wavelengt
hs both binding of ABA to antibodies as well as AP enzymatic activity
were maintained. UV-induced cross-linking of monoclonal anti-ABA antib
odies to immobilized ABA was analysed by immunoassays. Optimal cross-l
inking was achieved after a 5 min irradiation period at 0 degrees, usi
ng a long pass, cut-on filter to quench wavelengths below 290 nm. This
cross-linking faithfully reflected cognate binding activity.