Biotinylated liposomes can be conjugated non-covalently with (strept)avidin through either direct interaction with the protein/antibody conjugated to (strept)avidin or by coupling with other biotinylated proteins using (strept)avidin as a bridging molecule. Both avidin and (strept)avidin form strong non-covalent bond with biotin. The high resistance to breakdown makes them very useful in bioconjugate chemistry. However, (strept)avidin has replaced avidin in most bioconjugation applications due to its enhanced properties. NeutrAvidin (ThermoFisher) is a modified avidin without negative properties. It performs much better than original avidin and sometimes (strept)avidin.
In order to exploit the high-affinity interaction of biotin with (strept)avidin, a two-step “sandwich” protocol (Method A) has been developed for the preparation of targeted immunoliposomes. In this methodology, (strept)avidin is first attached to biotinylated liposomes, then a biotin-modified protein/antibody is introduced into the biotinylated (strept)avidin-labeled liposomes. This non-covalent approach is rapid, extremely versatile and applicable to numerous targeting ligands of interest with respect to in vitro and in vivo applications. Alternatively, instead of forming a (strept)avidin bridge, (strept)avidin molecule can also be covalently conjugated to antibody or ligand (Method B) and non-covalently bound to liposomes containing biotin on surface in order to form immunoliposomes.
ImmunoFluor™-Biotinyl Cap is a Non-PEGylated product. For other reactive (PEGylated and non-PEGyalated products) biotinylated liposomes and also ImmunoFluor™ products suitable for other types conjugation methods see here.
ebiomall.com
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用Mabteck公司的ALP标记抗体吧，配合Millipore公司的Multiscreen-IP96孔板，所有的试剂都可以减半，照样可以出非常好的结果，4*96孔的试剂盒我一直做8*96孔，但BCIP/NBT显色液没试过减半，因为它很便宜。记得当年第一次做实验就出了很好的结果，经过不断摸索，做出的蓝紫色斑点确实非常漂亮，几乎没有非特异斑点，嗯，ELISPOT是我最拿手的实验之一。
关于PBMC:
拿到血液标本后，首先我要做的是尽快分离PBMC（一般2h内），国产的分离液里天津灏洋的比较好，然后用冻存液冻存PBMC。PBMC的冻存过程比一般的细胞株要更为严格，最好用冻存器。ELISPOT试验的第一天，在包被抗体的同时，将细胞复苏，完全培养液培养过夜，第二天收集细胞并计数即可。
尤其是获得SCI文献引用的产品，
绝对ok的
请问一般的实验室可以进行ELIspot吗？（我可以买到现成的试剂盒，资金这个应该没有问题。）ELIspot的难点在哪里？多谢大虾们的指点！这个对我很重要，多谢！！！
