iLite anti-HER2 ADCC Activity Set
iLite anti-HER2 ADCC Activity Set Developed and Manufactured by Svar Life Science
The Eagle Biosciences iLite® anti-HER2 ADCC Activity Set includes (components are also sold separately):
- iLite® ADCC Effector (V) Assay Ready Cells (BM5001)
- iLite® ADCC Target HER2 (+) Assay Ready Cells (BM5011)
- iLite® ADCC Target HER2 (-) Assay Ready Cells (BM5016)
For Research Use Only
Key benefits of iLite® ADCC Activity Assays
- Unparalleled sensitivity
- High serum tolerance
- Normalization read-out included
- Negative control available for screening of unspecific activity
- Easy to use – no culturing required on target or effector cells
Assay Principle
The iLite® ADCC Assay Ready Cells are engineered cells that enable antibody-dependent cell-mediated cytotoxicity (ADCC) to be examined through the specific expression of Firefly luciferase. When the antibodies of interest bind to the antigens on the surface of the target cell, the target-bound antibodies will be presented to the Fc receptors (FcγRIIIa) on the effector cell. When the Fc-portion of the target-bound antibodies binds to the receptor, multiple cross-linking of the two cell types occurs. This will initiate a signaling cascade which triggers the expression of Firefly luciferase (FL) in the effector cell. In this application note, we describe the use of an effector cell line (iLite® ADCC Effector (V) Assay Ready Cells) that over-express FcγRIIIa and contain the FL reporter gene that responds to the principal transcription factors that mediate signaling from the FcγRIIIa receptor, together with a positive target cell line which over-expresses the surface antigen HER2 (iLite® ADCC Target HER2 (+) Assay Ready Cells). iLite® ADCC Effector (V) Assay Ready Cells also contain the Nano Luciferase (NL) reporter gene, under the control of a constitutive promoter, that allows drug-induced FL activity to be normalized with respect to the constitutive expression of NL. This renders assay results independent of variations in cell number, serum matrix effects, or lysis of the effector cells by the target cells. In addition, we also describe the use of a negative control in the form of a target cell line depleted of HER2 expression (iLite® Target HER2 (-) Assay Ready Cells). The Firefly luciferase signal can be measured in a luminometer following addition and incubation of luciferase substrate. The Firefly luciferase signal is proportional to the functional activity of trastuzumab in the sample.
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尤其是获得SCI文献引用的产品，
绝对ok的
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。
我想用酶联免疫斑点（ELISPOT）技术测定NK细胞活性，但不知道哪里能买到试剂盒，特向大家求助，谢谢。
