Taxane-specific monoclonal antibodies: measurement of taxol, baccatin III, and "total taxanes" in Taxus brevifolia extracts by enzyme immunoassay.
Taxane-specific monoclonal antibodies: measurement of taxol, baccatin III, and "total taxanes" in Taxus brevifolia extracts by enzyme immunoassay.
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DOI：
10.1021/np50121a004
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发表时间：
1995-07
影响因子：
5.1
通讯作者：
P. Grothaus;Gary S. Bignami;Sean O'Malley;Kent E. Harada;Jilanne B. Byrnes;David F. Waller;T. J. G. Raybould;Mary T. McGuire;Belinda Alvarádo
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文献类型：
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作者：
P. Grothaus;Gary S. Bignami;Sean O'Malley;Kent E. Harada;Jilanne B. Byrnes;David F. Waller;T. J. G. Raybould;Mary T. McGuire;Belinda Alvarádo
制备了三种单克隆抗体，分别对紫杉醇或baccatin III具有特异性，或与几种常见紫杉烷具有交叉反应性，并用于开发敏感的竞争抑制酶免疫测定。通过将P3X63Ag8.653浆细胞瘤细胞与锁孔冠贝血青素-7-琥珀基紫杉醇或-7-琥珀基芽孢素III偶联物过度免疫小鼠的脾细胞融合，获得了产生这些单克隆抗体的杂交瘤。利用这些单克隆抗体和涂有互补半抗原偶联物的牛血清白蛋白微滴板，建立了直接和间接竞争性抑制酶免疫测定。直接竞争抑制酶免疫测定，在含有10% MeOH的缓冲液中，检测限为0.6 nM紫杉醇对3C6（抗紫杉醇）；1.1 nM baccatin III用于3H5 (anti-baccatin III)；0.6 nM紫杉醇或baccatin III用于8A10（抗紫杉烷）。免疫分析法准确检测了短叶红豆杉树皮MeOH粗提物和高效液相色谱萃取物中紫杉醇、baccatin III和“总紫杉烷”的含量。
Three monoclonal antibodies with either specificity to taxol or baccatin III, or cross-reactivity with several common taxanes have been prepared and used to develop sensitive competitive-inhibition enzyme immunoassays. The hybridomas producing these monoclonal antibodies were obtained by fusing P3X63Ag8.653 plasmacytoma cells and splenocytes from mice hyperimmunized with keyhole limpet hemocyanin-7-succinyltaxol or -7-succinylbaccatin III conjugates. Direct and indirect competitive inhibition enzyme immunoassays were developed with these monoclonal antibodies and microtiter plates coated with bovine serum albumin conjugates of the complementary hapten. Detection limits for the direct competitive inhibition enzyme immunoassays, conducted in buffer containing 10% MeOH, were 0.6 nM taxol for 3C6 (anti-taxol); 1.1 nM baccatin III for 3H5 (anti-baccatin III); and 0.6 nM taxol or baccatin III for 8A10 (anti-taxane). The immunoassays accurately detected taxol, baccatin III, and "total taxanes" in crude MeOH extracts of Taxus brevifolia bark and in hplc fractions of these extracts.