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

• Published in: Biological & pharmaceutical bulletin Vol. 21; no. 3; pp. 197 - 201
• Main Authors: BAI, Gang, FUJIWARA, Kunio, KAMINISHI, Yoshie, LI, Hao Zhi, KITAGAWA, Tsunehiro
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.03.1998; Maruzen; Japan Science and Technology Agency
• Subjects: 70 kDa protein; Animals; Biological and medical sciences; Blotting, Western; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; General pharmacology; Immune Sera; Immunoenzyme Techniques; Lumbricus; Medical sciences; Medicine, Chinese Traditional; Oligochaeta - chemistry; Oligochaeta - immunology; Pharmacognosy. Homeopathy. Health food; Pharmacology. Drug treatments; Proteins - analysis; Proteins - immunology; Proteins - isolation & purification; Rabbits; selected antibody enzyme immunoassay; traditional Chinese medicine; Animal origin; Enzyme immunoassay; Annelida; Oligochaeta; Protein; Diuretic; Chinese medicine; Folk medicine; Antispasmodic agent; Antipyretic; Lumbricus; Qualitative analysis; Chemical composition; Invertebrata
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.21.197

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 EC50 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.