Enzyme immunoassay for the quantitative determination of ganoderic acid A from Ganoderma lucidum Enzyme immunoassay for the quantitative determination of ganoderic acid A from Ganoderma lucidum

霊芝の主成分であるganoderic acid A(GAA, 1)の定量の目的で高感度かつ特異的な酵素免疫測定法を開発した。すなわち,GAAの側鎖にglycineを付加して鎖長を伸ばし,これをN-hydroxysuccinimide esterに導いた後,β-D-galatosidase (β-Gal)やbovine serum albumin(BSA)と結合しGAA-β-Ga1(enzyme-labeled antigen, 5)及びGAA-BSA(immunogen, 6)抱合体を得た。後者をウサギに繰り返し投与しanti-GAA抗血清を得た。この抗血清は二重抗体による酵素免疫測定法でGAAに対する高感度,高特異性を示した。本酵素免疫法は0.1-1000ng/tubeの範囲内で良好な定量曲線を示した。この抗血清はganoderic acid γ(11)及びganolucidic acid A(14)(交叉率はそれぞれ52.4%及び12.9%)を除いては,霊芝から単離した種々のGAA関連化合物とは顕著な交叉反応を示さなかった。交叉率の高い2つの化合物はC-3位にカルボニル基を有する構造の類似したganoderic acidであった。ラットに静注あるいは経口投与後の血漿中のGAA濃度をこの新たに開発した酵素免疫測定法で定量した結果,5及び25mg/kg静注後のAUC_<0-480>は32.8±9.8及び201.5±38.7 μg min/mlであった。一方5及び50mg/kg経口投与後、血漿中のGAA濃度は18.1±2.5及び18.0±0.6minで最大濃度C_<max>(37及び595ng/ml)に達し,480minでは4.2及び13.3ng/mlに減少した。このことは経口投与したGAAは消化管で急速に吸収されるが,血中濃度はすぐさま減少することを示している。

For quantitative determination of ganoderic acid A (GAA, 1), a major constituent of Ganoderma lucidum, a sensitive and specific enzyme immunoassay (EIA) system was developed; the side chain of GAA was extended by introducing a glycine moiety, and this compound was coupled with β-D-galatosidase (β-Gal) and bovine serum albumin (BSA) via an N-hydroxysuccinimide ester to give GAA- β-Gal (enzyme-labeled antigen, 5) and GAA-BSA (immunogen, 6), respectively. The anti-GAA antiserum, which had been elicited in rabbits by immunization with the GAA-BSA conjugate, possessed high affinity and specificity toward GAA, when the assay was carried out with a double antibody technique. A satisfactory standard curve for EIA of GAA was explored in a range of 0.1 - 1000 ng/tube. The antiserum of GAA had no cross-reactivity with GAA-related compounds isolated from G. lucidum, except for ganoderic acid γ(11) and ganolucidic acid A (14) with the cross-reactivity of 52.4 and 12.9%, respectively, due to its close similarity in structure to GAA (ganoderic acid derivatives having a carbonyl group at C-3). The plasma concentration of GAA after its intravenous or oral administration to rats was determined by the established EIA. The AUCs after intravenous administration of GAA were 32.8±9.8 and 201.5±38.7 μg min/ml at doses of 5 and 25 mg/kg, respectively. Following oral administration of GAA at doses of 5 and 50 mg/kg to rats, the plasma concentration of GAA rapidly reached a C_<max> (37 and 595 ng/ml) at 18.1±2.5 and 18.0±0.6 min, respectively, then decreased to 4.2 and 13.3 ng/ml at 480 min, respectively, indicating that GAA was rapidly absorbed into the body fluid from the gastrointestinal tract after the oral administration, and then decreased soon.