Development of an enzyme-linked immunosorbent assay for azadirachtins

An enzyme-linked immunosorbent assay for azadirachtin A, a bioactive tetranor-triterpenoid from the neem tree (Azadirachta indica A. Juss), was developed. The conjugates were synthesized by employing the hemisuccinate method and an ester activation method, which were previously developed. Four rabbits were immunized with either 7-hemisuccinylazadirachtin-keyhole limpet hemocyanin (KLH) or 3-hemisuccinylazadirachtin-KLH. All sera were tested against the coating antigens 7-hemisuccinylazadirachtin-bovine serum albumin (BSA) and 3-hemisuccinylazadirachtin-BSA. From this screening procedure, an assay was developed using antiserum 7056. The assay showed an IC50 value of about 100 ppb, with a range of detection from 10 to 1000 ppb for azadirachtin A and related compounds. Thus, the method presented is a step forward in terms of inexpensive, fast, and sensitive (improvement by a factor of 100) analysis of azadirachtin and should provide a method that can easily be used more widely.