In order to develop a rapid and straightforward coupling procedure at the PEG terminus, a method of direct coupling antibodies to the PEG terminus of liposomes was introduced by Bendas et al. In this methodology, antibodies are simply attached to the PEG terminus of liposomes, which had been endgroup-functionalized with cyanuric chloride, in mild basic conditions (pH 8.8) without prior antibody derivatizations. It has been shown that in order to obtain a stable attachment of proteins on liposome, the DSPE-PEG-cyanur was added into the liposomes to chemically conjugate with proteins to form a stable complex and minimize the denaturation of proteins.
Proteins can be covalently coupled to the liposomes via amine-reactive cyanur-groups, either directly to the vesicle surface using cyanuric chloride-activated DSPE (cyanur-DSPE) or to the distal ends of PEG-spacers using activated cyanur-PEG-PE (ammonium salt). Cyanuric chloride at the PEG terminus functions to link peptides, antibodies and other amine-containing biomolecules or nanoparticles via a nucleophilic substitution reaction under basic conditions. Antibodies or other proteins can be conjugated without any previous derivatization.
ImmunoFluor™-Cyanur is a PEGylated product. For other reactive (PEGylated and non-PEGyalated products) ImmunoFluor™ products suitable for other types conjugation methods see here.
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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！！！
