Enzyme immunoassay for a characteristic protein in the animal crude drug lumbricus.
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 microg/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.
Bai
G
G
Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Fujiwara
K
K
Kaminishi
Y
Y
Li
H Z
HZ
Kitagawa
T
T
D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D008516:Medicine, Chinese Traditional; D009835:Oligochaeta / Q000737:chemistry* / Q000276:immunology; D011506:Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits
D007106:Immune Sera; D011506:Proteins