| Kohler and Milstein, both biomolecue sciensts obtained hybridomas secreting specific antibody by using immuned mouse spleen cells to fuse with bone marrow tumor cells in1975, marking the birth of the monoclonal antibody technology. The so-called monoclonal antibody (MAb) is secreted by a single B-cell cloning and can identify the only specific epitope.Preparation of monoclonal antibody technology is divided into hybridoma technology and genetic engineering techniques. Hybridoma technology, the initial way of preparing of monoclonal antibodies, is currently the most widely used means for antibody preparation. The technology mainly include three parts:animal immunization, cell fusion and hybridoma screening. The MAbs prepared by this technology are widely used in the detection, separation of substances because they possess advantages such as high-affinity, high sensitivity. The technology of genetic engineering is of many kinds, incluing chimeric antibody technology, reconstruction of antibody technology, phage display technology, ribosome display technology, covalent display technology, et al.With the development of science and technology with monoclonal antibodies has been applied in many fields. It shows an unique advantage in lymphocyte identification, pathogen identification, parasite detection, cancer diagnosis and classification, and hormone detection in the clinical diagnosis. Humanized MAbs are widely used in the treatment of autoimmune disease, cardiovascular disease, cancer and other diseases. In addition, MAbs can also be applied to the detection of harmful substances in food as well as the detection, separation, purification of small molecule active substance and the neutralization of toxic substances in the crude drug research.Immunosorbent assay is a very important field of application of MAbs. It is based on the specificity response of antigen and antibody, while taking antigen or antibody labeled with enzymes or other colored or light-emitting substance, to conduct qualitative or quantitative detection. Its specificity and sensitivity is much higher than the general physical and chemical methods. At present, the application of immunoassay enzyme-linked immunosorbent assay (ELISA) occupies a large proportion of the immunoassay, which contains three commonly used methods: double-antibody sandwich method, indirect method, competition method. It is suitable for promotion and application because of its advantages such as high specificity, high sensitivity, simple sample pre-treatment, low cost, efficient, no environmental pollution, etc.The active substances of crude drugs are generally the hapten, which require to coupling to specific macromolecules as a carrier in order to constitute a complete antigen to stimulate the body’s specific immune response to produce specific antibody. Generally speaking, hapten-conjugated carrier are proteins, such as bovine serum albumin (BSA), rabbit serum albumin (RSA), chicken egg albumin (OVA), human serum albumin (HSA), keyhole limpet hemocyanin (KLH). it may also be other macromolecules, such as polylysine (the PLL).The common chemical method of coupling the hapten to carrier Include the periodate oxidation method, glutaraldehyde method, carbodiimide method, succinic anhydride method, re-ammoniation method and mixed anhydride method.Paclitaxel is a tricyclic diterpenoids separated from plant of Taxus, which has a unique anti-tumor mechanism that induced tubulin polymerization to inhibit cell division. Paclitaxel was approved as a new drugs against advanced cancer by the Food and Drug Administration (FAD) of U.S.A in1992. So far, it has been still a clinical first-line drug for treatment of breast cancer, uterine cancer, ovarian cancer and other cancers. The value of of paclitaxel has great upside potential because of two reasons. On one hand, the market demand for paclitaxel is incresing, on the other hand, its main raw material yew is of the scarcity of resources, coupled with its content in plants is very low.Accurate and efficient detection method is essential in the paclitaxel study. Until now, analysis methods for the determination of paclitaxel investigated include thin layer chromatography, capillary electrophoresis, high performance liquid chromatography, mass spectrometry, biochemical assay. The most widely used method is the high performance liquid chromatography. Although some of these methods has certain advantages, all of them suffer from some limitations such as high cost, complex operation, time consuming or low recovery rates. Immunoassay had been improved to be effective in quality control of natural medicines and had showed advantages in high sensitivity, low cost, and convenient operation.In present study, we report the preparation of highly specific monoclonal antibody (MAb) against paclitaxel, and the establishment of an indirect competitive enzyme-linked immunosorbent assay (icELISA) that would be convenient for quality control and appropriate use of paclitaxel, with some advantages over previously reported ones.Because paclitaxel is a hapten resulting from its small molecular weight (Mr= 853.93) as well as paclitaxel molecule has no functional groups combined with the carrier protein,7-xylosyltaxol was conjugated to the carrier protein bovine serum albumin (BSA) to construct the immunogen. The ratio of hapten in XylTax-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization of mice with this conjugate, hybridomas secreting MAb against paclitaxel were obtained by fusing the splenocytes with mouse myeloma cell line SP2/0. After screening by the limiting dilution method, hybridomas producing MAb reactive to paclitaxel were inoculated intraperitoneally to mice to get mass production of monoclonal antibody. Then, MAb against paclitaxel was purified from ascites by salting out method combined with proteinG Column chromatography. The anti-paclitaxel MAb3A3we obtained was identified as IgGl with κ light chains and 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-hydroxybacctin I,13-acetyl-9-dihydrobaccatinⅢ, and1-acetoxyl-5-deacety-baccatin I,<0.11%). Using the MAb3A3, we established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for paclitaxel determination with a detection range of0.0098-0.3125μg/mL. Determination of paclitaxel contents in various Yew tree samples with the established icELISA resulted in recovery rates ranging from92%to94.8%, and intra-and inter-assay variations of3.6%and4.7%, respectively. This icELISA provides a valuable means for paclitaxel determination for different purposes.Compared with HPLC, The icELISA we established shows greater practical value in that it does not utilize sophisticated equipments and can be carried out in routine laboratory conditions. This assay shows advantages in the effectiveness, precision, simplicity, low cost, and environment friendliness as a result of Simultaneous detection of multiple samples, no complex sample pre-treatment and using little organic solvents.The approach we adopted for preparing the hapten is more convenient than the method reported previously. The synthesis of the antigen conjugates is easier using7-xylosyltaxol because it is a commercialization natural derivate, which eliminate the need for synthesis procedure of taxol derivatives.The reactivity pattern with taxanes suggests that the epitope recognized by the MAb3A3probably resides in the distal portion of the C-13side chain. The MAb3A3has no cross-reaction with the taxanes lack of the C-13side chain, which present a solution in some extent to the defects in the existing paclitaxel immunoassay.The assay we established can be applied in rapid analysis of a large number of samples, and well serves the purposes of batch quality inspection of Taxus materials, screening of Taxus and other new resources yielding paclitaxel, and determining paclitaxel at extremely low concentrations below the detection limit of HPLC. |
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