A novel technique for producing antibody-coated microprobes using a thiol-terminal silane and a heterobifunctional crosslinker.
A novel technique for producing antibody-coated microprobes using a thiol-terminal silane and a heterobifunctional crosslinker.
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一种使用硫醇末端硅烷和异双功能交联剂生产抗体包被微探针的新技术。
DOI：
10.1016/s0165-0270(96)00138-0
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发表时间：
1997
影响因子：
3
通讯作者：
Helke,CJ
中科院分区：
文献类型：
--
作者：
Routh,VH;Helke,CJ
抗体包被的微探针用于测量中枢神经系统中神经肽的释放。虽然它们不是定量的，但它们提供了任何当前可用方法的体内释放位置的最精确的空间分辨率。以前的抗体微探针包被方法困难且耗时。此外，使用这些方法，我们不能产生均匀包被的抗体微探针。本文介绍了一种新的方法，用于生产抗体微探针使用硫醇末端硅烷和异双功能交联剂，4-（4-N-马来酰亚胺苯基）丁酸酰肼盐酸1/2二氧六环（MPBH）。在硅化后，将玻璃微量移液管与已缀合至MPBH的P物质（SP）抗体一起孵育。该方法导致抗体的致密、均匀包被，而不降低抗体的生物活性。此外，该方法比先前描述的方法花费显著更少的时间，而不牺牲抗体微探针作为微量移液器的使用。微探针对SP的灵敏度在皮摩尔范围内，SP浓度（M）的对数与B/B 0之间存在线性相关性（r2=0.98）。当在5°C下储存在含有50 mM NaCl（pH 7.4）的0.1 M磷酸钠缓冲液中时，微探针可稳定长达3周。最后，插入麻醉大鼠暴露的脊髓中15分钟，对抗体涂层没有产生损伤。
Antibody-coated microprobes are used to measure neuropeptide release in the central nervous system. Although they are not quantitative, they provide the most precise spatial resolution of the location of in vivo release of any currently available method. Previous methods of coating antibody microprobes are difficult and time-consuming. Moreover, using these methods we were unable to produce evenly coated antibody microprobes. This paper describes a novel method for the production of antibody microprobes using thiol-terminal silanes and the heterobifunctional crosslinker, 4-(4-N-maleimidophenyl)butyric acid hydrazide HCl 1/2 dioxane (MPBH). Following silation, glass micropipettes are incubated with antibody to substance P (SP) that has been conjugated to MPBH. This method results in a dense, even coating of antibody without decreasing the biological activity of the antibody. Additionally, this method takes considerably less time than previously described methods without sacrificing the use of antibody microprobes as micropipettes. The sensitivity of the microprobes for SP is in the picomolar range, and there is a linear correlation between the log of SP concentration (M) and B/B0(r2=0.98). The microprobes are stable for up to 3 weeks when stored in 0.1 M sodium phosphate buffer with 50 mM NaCl (pH 7.4) at 5°C. Finally, insertion into the exposed spinal cord of an anesthetized rat for 15 min produces no damage to the antibody coating.