Preparation of a Monoclonal Antibody against Gintonin and Its Use in an Enzyme Immunoassay

• Published in: Biological & pharmaceutical bulletin 2015/10/01, Vol.38(10), pp.1631-1637
• Main Authors: Lee, Byung-Hwan, Choi, Sun-Hye, Kim, Hyeon-Joong, Jung, Seok-Won, Kim, Hyunsook, Shin, Ho-Chul, Lee, Joon-Hee, Hwang, Sung-Hee, Kim, Hyoung-Chun, Nah, Seung-Yeol
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - immunology; Cell Line, Tumor; enzyme immunoassay; Ethanol - chemistry; ginseng; gintonin; gintonin quantitation; Horseradish Peroxidase; Immunoenzyme Techniques; Mice, Inbred BALB C; monoclonal antibody; Panax - chemistry; Plant Extracts - analysis; Plant Extracts - immunology; Water - chemistry
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b15-00171

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an [Ca2+]i transient in animal cells via activation of LPA receptors. In vitro studies have shown that gintonin regulates various calcium-dependent ion channels and receptors. In in vivo studies, gintonin elicits anti-Alzheimer’s disease activity through the activation of the non-amyloidogenic pathway and anti-metastatic effects through the inhibition of autotaxin. However, a method for gintonin quantitation in ginseng has not been developed. In the present study, we developed an enzyme immunoassay (EIA) to measure gintonin. A monoclonal antibody was raised in a mouse using gintonin as the immunogen, and an indirect competitive EIA was used to measure gintonin. The working range was 0.01–10 µg per assay. The anti-gintonin monoclonal antibody did not cross-react with the ginsenosides Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg3 or with LPAs such as LPA C16:0, LPA C18:0, LPA C18:1, and LPA C18:2. Using a standard curve, we measured the amount of gintonin in various ginseng extract fractions. Interestingly, we only detected a little amount of gintonin in conventional hot water extracts of Korean red ginseng. However, we can measure gintonin after ethanol extraction of Korean red ginseng marc. Thus, gintonin can be extracted from ginseng with ethanol but not water, and the remaining Korean red ginseng marc can be used to obtain gintonin. These results indicate that the EIA with the anti-gintonin monoclonal antibody can be used to quantify gintonin in various ginseng preparations, including commercial ginseng products.