Overview:
ZAP70 is a non-receptor protein tyrosine kinase (part of the Syk/Zap70 family) that is involved in signaling by the T-cell antigen receptor (TCR). Ligation of the TCR/CD3 receptor in Jurkat T-cells induces phosphoprotein complexes which contain ZAP70 (1). TCR zeta chains are initially phosphorylated by p56Lck that lead to the recruitment of ZAP70 via its SH2 domain. ZAP70 in turn phosphorylates other proteins in the TCR-phosphoprotein complex. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex (2).
References:
1. Duplay, P. et al: p56lck interacts via its src homology 2 domain with the ZAP-70 kinase. J Exp Med. 1994 Apr 1;179(4):1163-72.2. Chan, A C. et al: Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling. J Immunol. 1994 May 15;152(10):4758-66.3. Salomon AR, et al. (2003) Proc Natl Acad Sci U S A; 100(2): 443-448 4. Ku GM, et al. (2001) EMBO J; 20(3): 457-465.5. Tang J, et al. (1999) Proc Natl Acad Sci U S A; 96(17): 9775-9780.6. Zhao Q, et al. (1996) Mol Cell Biol; 16(12): 6765-6774.7. Williams BL, et al. (1999) EMBO J; 18(7): 1832-1844.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
多谢!
不知道能不能直接买到大鼠抗豚鼠血管IgG的ELISA试剂盒。
如果买不到这种试剂盒,用空白板的话(就是反应孔中没有抗原或者抗体包被),把自制的豚鼠血管抗原加到反应孔中进行孵育,能不能使抗原附着在孔壁上?这样的话就可以按照elisa的操作步骤进行大鼠血清检测了。
目前的设想是自制抗原,粉碎豚鼠的血管,制成悬液,通过反复多次的冻融,离心后收集上清液作为抗原(即豚鼠血管抗原)。
然后将抗原加入到elisa的反应孔中(这种是特制的空白板,就是反应孔中没有抗原或抗体包被)进行孵育,使豚鼠血管抗原附着在孔壁上,就是让反应孔充当固相载体,形成固相抗原。倒掉多余的抗原。
再加入待检测的大鼠血清,这样血清中的特异性抗豚鼠血管IgG就可以跟固相的抗原结合,形成固相抗原抗体复合物。
加入酶标的兔抗鼠或者羊抗鼠IgG,形成酶标的抗原抗体复合物。
然后就是一些显色步骤。
现在关键的问题就是抗原加入空白板,能不能形成固相抗原,如果不能跟孔壁附着的话,在后面洗涤的过程中就被洗掉了,那就没办法完成检测了。
请各位大侠给指条明路啊!!!小弟在此多谢了!!!

