Perforin ELISpot Assay Kit

$350.00$1,550.00

This Perforin ELISpot Assay Kit is designed to determine the frequency of Perforin producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. The Eagle Biosciences Perforin ELISpot Assay Kit is for research use only and not to be used for diagnostic procedures.

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Perforin ELISpot Assay Kit

The Perforin ELISpot Assay Kit is for research use only.

Incubation Time: 18hrs – 23hrs
Sample Size: 100 µl


Assay Principle

The ELISpot is a highly specific immunoassay for the analysis of cytokine and other soluble molecule production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. The ELISpot assay constitutes an ideal tool in the investigation of Th1 / Th2 responses, vaccine development, viral infection monitoring and treatment, cancerology, infectious disease, autoimmune diseases and transplantation. Utilizing sandwich immuno-enzyme technology, ELISpot assays can detect both secreted cytokines and single cells that simultaneously produce multiple cytokines. Cell secreted cytokines or soluble molecules are captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured cytokines are revealed by tracer antibodies and appropriate conjugates. A capture antibody highly specific for the analyte of interest is coated to the wells of a PVDF bottomed 96 well microtiter plate either during kit manufacture or in the laboratory. The plate is then blocked to minimize any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any analytes produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured analyte. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing substrate is then applied to the wells resulting in colored spots which can be quantified using appropriate analysis software or manually using a microscope.


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Perforin ELISA Assay Kit

Additional Information

Assay Background


Perforin is involved in the killing function by CTLs and NKs. Upon degranulation, perforin inserts itself into the target cell’s plasma membrane, forming a pore. It leads to osmotic lysis of the target cells and subsequently enables granzymes to enter the target cells and activate apoptosis, the cell death program. Perforin has structural and functional similarities to complement component 6, 7, 8 and 9 (C6, C7, C8, C9). Like C9, this protein creates transmembrane tubules and is capable of lysing non-specifically a variety of target cells. This protein is one of the main cytolytic proteins of cytolytic granules, and it is known to be a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis. Perforin is a 70kDa protein containing 534 amino acid. Its role in the immune response against tumors and virus infection has been demonstrated.

ELISpot Assay Kit Procedure

Documents

Product Manual