请问关于 ELIspot的问题...

请问关于 ELIspot的问题

你好，我可以回答你的这个问题。我是深圳达科为生物技术公司专门负责Elispot技术的技术支持，在这里潜水查点些免疫学知识，很高兴能帮的到你。
一般能够做细胞培养的实验室就可以做Elispot实验的。但是实验结果需要自己用显微镜进行观察，可能就没有用自动分析仪器进行分析客观精确，但目前国内基本都是用显微镜观察或带到国外去检测，我们代理的德国BIOSYS公司的Elispot自动图像分析仪很快就将进入国内，相信随着该技术的发展，别的国家和公司的仪器包括国产的仪器都会逐步投入使用，所以检测就越来越不成为问题。
言归正传，简单的说Elispot的基本原理是包被在培养板上的包被抗体捕获淋巴细胞分泌出来的细胞因子，然后经过一系列的反应显色后在培养板底上形成斑点。因为比ELISA多了孵育细胞的过程，这样对你准备细胞、细胞入板后的刺激和培养就有比较高的要求，包括动物（或人）的免疫情况、细胞的活力，细胞分离的纯度（有无粒、红、巨噬细胞及贴壁细胞）、是否是单个细胞、孵育过程培养板的静置等，都很重要。
因为要注意的问题有很多，您如果需要，可以打我们公司的免费电话800 830 4366或发EMAIL给我tech@dakewe.com来详细交流（注：我可不是在这里卖东西哈，我只是希望论坛里的TX能真心互相帮助，知识越交流便越丰富）

ELISPOT听起来很神秘，主要是因为技术较新，价格是一般单位所担负不起的。其实跟做ELISA一样简单。关键是分析。如果要定量，最好有reader.另一问题是细胞密度，所以，要多设几个不同细胞密度的实验组。另外，要注意使细胞均匀的散步在孔底。

I am doing ELIspit now. If you have any questions, I will help you resolve ithem.
ELISPOT (Enzyme-linked ImmunoSPOT) technique was originally used to enumerate antibody secreting T cells,B cells and DC. In the current technique, cells are deposited onto a membrane coated with one antibody specific for a protein followed by an appropriate incubation period. Subsequently, the protein of interest is detected in the environment immediately surrounding the cell secreting it, with another antibody specific for a different epitope of the protein. The signal detected by the HRP enzyme/substrate results in a colorimetric footprint of the cells and can be quantitated by visual scoring or specialized plate-readers.

For cytokines, ELISPOT may in general be more sensitive than sandwich ELISA, since it takes advantage of the higher concentration of the secreted cytokine close to its source. In addition, ELISPOT allows quantitation of cytokine secretion on a per cell basis. However, the experimental conditions including the number of cells, the best combination of antibodies, and the stimulation and incubation times for optimal cytokine release have to be empirically determined.
Due to experimental variation, tweaking of the number of cells depositied per well, the proper stimulation and culture period for secretion of a specific cytokine may be required.