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

Abstract

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an [Ca 2 ] 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 antimetastatic 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, Rb 1 , Rb 2 , Rc, Rd, Re, Rf, Rg 1 , and Rg 3 or with LPAs such as LPA C 16:0 , LPA C 18:0 , LPA C 18:1 , and LPA C 18:2 . 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.Key words ginseng; gintonin; monoclonal antibody; enzyme immunoassay; gintonin quantitation Ginseng is an herbal medicine with a variety of physiological and pharmacological effects. 1) Ginseng is used worldwide as a tonic. Recent studies have shown that ginseng contains a novel G protein-coupled lysophosphatidic acid (LPA) receptor ligand, designated gintonin. Gintonin consists of LPAs and a ginseng protein complex.2-4) Gintonin exhibits several characteristics distinct from those other ginseng components such as ginsenosides, which are glycosides. Gintonin, but not ginsenosides, induces an intracellular free calcium concentration ([Ca 2,7) Oral administration of gintonin elicits anti-Alzheimer's disease effects in vivo by activating the non-amyloidogenic pathway through LPA receptors and induces anti-metastatic effects by inhibiting autotaxin activity. 7,8) Biochemical analysis has shown that gintonin consists of carbohydrates, lipids, and proteins.2) The protein component of gintonin consists of ginseng major latex-like protein 151 (GLP) and ginseng ribonuclease-like storage protein (GSP). 2)GLP, which belongs to the Bet v 1 protein superfamily, contains a hydrophobic ligand-binding site and a glycine-rich loop called the P-loop, which might bind nucleotides. 9) GSP is homologous to plant ribonuclease, but lacks ribonuclease activity.10) GSP expression in ginseng root vari...

Gintonin absorption in intestinal model systems

“…The amount of gintonin in the samples from the PAMPA and everted sac assay was determined by ELISA as previously described, with some modifications [17] . Briefly, each sample solution was diluted with PBS, added to the wells of a 96-well microplate, and incubated overnight at 4°C.…”

“…In contrast, the everted gut sac system is used to examine the transport of various substances via ex vivo intestinal absorption [14] , [15] , [16] . Recently, we produced a gintonin-specific monoclonal and polyclonal antibodies and developed an enzyme-linked immunosorbent assay (ELISA) for gintonin detection using the monoclonal antibody [17] . In this study, the PAMPA and the mouse everted intestinal sac model were used to investigate gintonin absorption in an artificial biological membrane and the small intestine, respectively.…”

“…In this study, the PAMPA and the mouse everted intestinal sac model were used to investigate gintonin absorption in an artificial biological membrane and the small intestine, respectively. The amount of gintonin transported through the artificial membrane or everted gut sacs was quantified by ELISA using a polyclonal antibody against gintonin [17] .…”