Enzyme Immunoassay for a Characteristic Protein in the Animal Crude Drug Lumbricus

Abstract

A new method was developed to measure the content of a Lumbricus component in a traditional Chinese medicine (TCM). An antiserum specific to Lumbricus was elicited in a rabbit following immunization with a suspension of Lumbricus fragments. A characteristic antigen protein, 70 kDa, was found in Lumbricus and was purified almost to singleness using a column chromatography series of gel filtration and DEAE-Sepharose. A selected antibody enzyme immunoassay (SAEIA) was developed using the antiserum and the purified 70 kDa protein as a solid-phase antigen. The SAEIA was specific to Lumbricus species, and showed no cross-reaction with any crude drugs other than Lumbricus. This SAEIA detected 70 kDa protein in the amount of 10 ng /ml with excellent reproducibility (coefficient of variation=3.0%) and an EC5050 of 0.24 μg/ml. Using this assay, Lumbricus levels were easily determined in a Lumbricus-based TCM Kazecoll, but not in the control Kazecoll (Kakkonto) prepared without Lumbricus. The SAEIA for 70 kDa protein was simple, accurate, reproducible and may provide a general analytical method for the quality control of Lumbricus-based TCMs. © 1998, The Pharmaceutical Society of Japan. All rights reserved.