A time- and cost-saving method of producing rat polyclonal antibodies
A time- and cost-saving method of producing rat polyclonal antibodies
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DOI：
10.1267/ahc.06003
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发表时间：
2006-01-01
影响因子：
2.4
通讯作者：
Iseki, Shoichi
中科院分区：
文献类型：
--
作者：
Wakayama, Tomohiko;Kato, Yukio;Iseki, Shoichi
产生抗体通常需要三个多月的时间。在本研究中，我们介绍了一种快速生产多克隆抗体的方法，基于制备的重组寡肽作为抗原，然后免疫大鼠。使用这种方法，我们产生了针对两种小鼠蛋白：ERGIC-53和c-Kit的抗血清。将编码该蛋白的一对互补的人工合成的寡脱氧核苷酸连接到表达载体中，并将与载体蛋白谷胱甘肽-S-转移酶融合的重组寡肽导入细菌中，纯化。Wistar大鼠通过将乳化抗原皮下注射到后足垫中进行免疫，然后在2周后进行加强注射。加强免疫后1周，收集血清，通过免疫组织化学检测抗体滴度。两种抗原均获得了最大效价为1600倍的抗血清，并通过Western印迹法确认了其特异性。抗ERGIC-53抗血清识别舌下腺中的腺泡细胞，抗c-Kit抗血清识别睾丸中的生精细胞和Leydig细胞。这些抗血清适用于与小鼠单克隆抗体或兔多克隆抗体的荧光双重免疫染色。因此，这种方法使我们能够在不到一个月的时间内以相对较低的成本生产出可用于免疫组织化学的特异性大鼠多克隆抗血清。
Producing antibodies usually takes more than three months. In the present study, we introduce a faster way of producing polyclonal antibodies based on preparation of the recombinant oligopeptide as antigen followed by immunization of rats. Using this method, we produced antisera against two mouse proteins: ERGIC-53 and c-Kit. An expression vector ligated with a pair of complementary synthetic oligodeoxyribonucleotides encoding the protein was introduced into bacteria, and the recombinant oligopeptide fused with the carrier protein glutathione-S-transferase was purified. Wistar rats were immunized by injecting the emulsified antigen subcutaneously into the hind footpads, followed by a booster injection after 2 weeks. One week after the booster, the sera were collected and examined for the antibody titer by immunohistochemistry. Antisera with 1600-fold titer at the maximum were obtained for both antigens and confirmed for their specificity by Western blotting. Anti-ERGIC-53 antisera recognized acinar cells in the sublingual gland, and anti-c-Kit antisera recognized spermatogenic and Leydig cells in the testis. These antisera were applicable to fluorescent double immunostaining with mouse monoclonal or rabbit polyclonal antibodies. Consequently, this method enabled us to produce specific rat polyclonal antisera available for immunohistochemistry in less than one month at a relatively low cost.