title: Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel.
abstract: Paclitaxel, the major active component of the yew tree, is used as an important anti-cancer agent. To obtain the monoclonal antibody (MAb) against paclitaxel for paclitaxel determination using immunoassay, 7-xylosyltaxol was conjugated to the carrier protein bovine serum albumin (BSA) to construct the immunogen, and the ratio of hapten in XylTax-BSA conjugate was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. After immunization of mice with this conjugate, hybridomas secreting MAbs against paclitaxel were obtained by fusing the splenocytes with the mouse myeloma cell line SP2/0. After hybridoma screening, the anti-paclitaxel MAb 3A3 was obtained, which showed a relatively high specificity to paclitaxel (cross-reactivities against other naturally occurred taxanes: 7-xylosyltaxol, 31.8%; cephalomannine, 6.17%; baccatin III, 10-deacetyl-baccatin III, 1-hydroxybaccatin I, 13-acetyl-9-dihydrobaccatin III and 1-acetoxyl-5-deacetyl-baccatin I, <0.11%). Using the MAb 3A3, we established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for paclitaxel determination with a detection range of 0.098-312.5 μg ml(-1). Determination of paclitaxel contents in various yew tree samples with this icELISA resulted in recovery rates ranging from 92 to 94.8%, and intra- and inter-assay variations of 3.6 and 4.7%, respectively. This icELISA provides a valuable method of paclitaxel determination for various purposes.
authors.affiliation: School of Traditional Chinese Medicine, Southern Medical University, 1023 Shatai Road South, Guangzhou, 510515, People's Republic of China. chaozhi1971@yahoo.com.cn
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chao
authors.forename: Mingming
authors.initials: M
authors.lastname: Tan
authors.forename: Madan Kumar
authors.initials: MK
authors.lastname: Paudel
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Liling
authors.initials: L
authors.lastname: Ma
authors.forename: Kaori
authors.initials: K
authors.lastname: Sasaki-Tabata
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
authors.forename: Lijiang
authors.initials: L
authors.lastname: Xuan
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage / Q000032:analysis / Q000276:immunology*; D001667:Binding, Competitive; D002138:Calibration; D002681:China; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005260:Female; D006241:Haptens / Q000008:administration & dosage / Q000276:immunology; D006825:Hybridomas; D007114:Immunization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017239:Paclitaxel / Q000008:administration & dosage / Q000032:analysis / Q000276:immunology*; D008517:Phytotherapy; D010946:Plants, Medicinal; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020946:Taxus / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D000972:Antineoplastic Agents, Phytogenic; D006241:Haptens; D017239:Paclitaxel