Induction of Anti-Brassinosteroid Antibodies

Abstract

Summary Antibodies directed against brassinosteroid were induced by using a synthetic BR isomer to prepare a BR-protein conjugate and injecting it into BALB/cj mice. The conjugate was prepared by synthesizing a carboxylated BR derivative that was coupled to goat or mouse albumin. BR was carboxylated by refluxing the compound with succinic anhydride in pyridine for 12 h. The BR-monosuccinate derivative was purified from the reaction mixture by ion-exchange chromatography on QAE-Sephadex and by hydrophobic interaction chromatography on Sep Pak C-18. The succinylated compound was linked to albumin via the mixed anhydride procedure. A molar ratio of BR : protein was in the range of 15–20 : 1. After a series of injections the serum was collected from the mice, which had developed good antibody titers for the BR-protein conjugate, and was used to develop a competitive ELISA assay to quantify free BR. A standard curve for the BR isomer (30 fmol to 10 pmol) could be developed when the unconjugated compound competed with conjugated BR (2.55 pmol) for binding with the anti-BR antibodies (final serum dilution was 16,000 : 1). The antibodies showed no cross reactivity with any compound tested except those in the brassinosteroid family. A standard curve for the naturally occurring brassinosteroid, brassinolide, could also be developed when brassinolide was competed with conjugated BR (2.55 pmol) for binding with anti-BR antibodies (final dilution was 16,000 : 1) in the ELISA assay.