Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. One of the most popular and commonly used methods is to covalently couple free carboxylic groups to primary amines through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). EDC, which is a so-called zero-length crosslinking agent, reacts with the carboxyl to form an amine reactive intermediate (O-acylisourea). The produced O-acylisourea can be easily displaced by nucleophilic attack from primary amino groups in the reaction mixture. However, this intermediate is unstable and hydrolyzed in aqueous solutions. In order to prevent the intermediate hydrolysis, sulfo-NHS (N-hydroxysulfosuccinimide) is added to EDC to produce a significantly more stable and more soluble active intermediate (NHS ester).
Consequently, the immunoliposomes are prepared by a two-step coupling procedure: first, activating the free carboxyl group on the antibody, peptide or protein with EDC and sulfo-NHS, and then covalently conjugating antibody to the lipids through displacement of sulfo-NHS groups by amine groups of the liposomes, as depicted below. EDC/sulfo-NHS coupling reactions are highly selective and highly efficient, and the biological activity of the protein or peptide is preserved.
ImmunoFluor™-Amine is a PEGylated product. For other amine reactive (PEGylated and non-PEGyalated products) 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！！！
