Epitope mapping of monoclonal antibodies to bovine prolactin.
Epitope mapping of monoclonal antibodies to bovine prolactin.
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牛催乳素单克隆抗体的表位作图。
DOI：
10.1152/ajpendo.1992.263.3.e520
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Smith,M
中科院分区：
文献类型：
--
作者：
Scammell,JG;Luck,DN;Valentine,DL;Smith,M
用免疫斑点法测定了三种抗羊催乳素单克隆抗体与14种氨基酸缺失或取代的牛催乳素突变体的交叉反应性，确定了它们所识别的表位。在整个分子中进行突变，包括破坏氨基末端、羧基末端和中心二硫环。缺乏免疫反应性被认为是突变位点是表位的一部分的指示。抗体6 F11与所有测试的牛催乳素突变体反应，除了羧基末端半胱氨酸（位置199）被丝氨酸取代的那些。抗体5G 2和4C 10与所有牛催乳素突变体反应，除了其中氨基末端半胱氨酸（位置4）被丝氨酸取代的那些。羊，松鼠猴，和大鼠催乳素与单克隆抗体的Western印迹分析显示，5G 2和4C 10是特异性的羊催乳素，而抗体6 F11与催乳素从所有三个物种的交叉反应。在存在抗体的情况下测定Nb 2生物测定中绵羊或大鼠催乳素的促有丝分裂活性，以确定表位是否为这些催乳素功能结构域的一部分。羊催乳素的生物活性（0.4 ng/ml）不受单克隆抗体[0.01-1 μ g免疫球蛋白G（IgG）/ml]的影响，而大鼠催乳素的生物活性（1.25 ng/ml）则被6 F11抑制，表观50%抑制浓度为0.25 μ g IgG/ml。这些结果表明单克隆抗体5G 2和4C 10与牛催乳素氨基末端二硫环的物种特异性区域交叉反应。（250字处删节）
The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)