1. Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel.
- Author
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Chao Z, Tan M, Paudel MK, Sakamoto S, Ma L, Sasaki-Tabata K, Tanaka H, Shoyama Y, Xuan L, and Morimoto S
- Subjects
- Animals, Antibodies, Monoclonal biosynthesis, Antibody Specificity, Antineoplastic Agents, Phytogenic administration & dosage, Antineoplastic Agents, Phytogenic analysis, Binding, Competitive, Calibration, China, Cross Reactions, Enzyme-Linked Immunosorbent Assay standards, Female, Haptens administration & dosage, Haptens immunology, Hybridomas, Immunization, Male, Mice, Mice, Inbred BALB C, Paclitaxel administration & dosage, Paclitaxel analysis, Phytotherapy, Plants, Medicinal, Quality Control, Reference Standards, Reproducibility of Results, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Antibodies, Monoclonal immunology, Antineoplastic Agents, Phytogenic immunology, Enzyme-Linked Immunosorbent Assay methods, Paclitaxel immunology, Taxus chemistry
- 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.
- Published
- 2013
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