Production of monoclonal antibody against a major purgative component, sennoside A, its characterization and ELISA

Abstract

For immunization, sennoside A was conjugated with bovine serum albumin. The hapten number in an antigen conjugate was determined to be five by matrix-assisted laser desorption/ionization TOF mass spectrometry. A hybridoma secreting monoclonal antibody against sennoside A was produced by fusing splenocytes immunized with sennoside A–bovine serum albumin and a hypoxantine–aminopterin–thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Weak cross-reactivities occurred with sennoside B which is a stereochemical isomer, and a monomer of sennoside A, rhein, but no cross-reactivities were observed with other related anthraquinones and phenolics. The full range of the assay extends from 20 to 200 ng ml⁻¹ of sennoside A. Good correlation of sennoside A concentrations in crude extract of rhubarb between ELISA and HPLC methods was obtained. The contents of sennoside A in various rhubarb roots were assayed by the newly established ELISA indicating a good agreement with the previous reports.