The LISPOT method is derived from ELISA. Compared with the traditional ELISA method, it is a breakthrough. It is the extension and development of quantitative ELISA technology. Due to the different half-period of free circulating antibody or CK, it is continuously metabolized in body fluids or combined with target organs. Traditional enzyme-linked immunosorbent assay (ELISA) cannot accurately reflect the level of antibody and CK . In the 1980s, foreign researchers established the solid-phase enzyme-linked immunospot (ELISPOT) technique for detecting specific antibody-secreting cells and CK-secreting cells in vitro based on the basic principles of ELISA technology.
The origin of ELISPOT can be traced back to the hemolytic plaque forming cell assay (HPF) created by Jerne ELISA kit and others in 1963. This technology can be used to detect and count individual antibody-forming cells. With the maturation of monoclonal antibody technology, in 1983, Czerkinsky et al. Used this method to detect the number of cells secreting specific antibodies in spleen cells, and found that the method was highly sensitive and simple. Later, they used this method to quantitatively analyze the number of IFN2γ secreting cells in peripheral blood stimulated by mitogens. Subsequently, the ELISPOT method was used to detect the amount of other cytokines (such as IL-2, IL-4, IL-5, IL-10, TNFα and IL-6) secreted at the single cell level. Nordstrom et al. Used biotin-avidin biomagnification to improve the sensitivity of this method. Herr used a computer-assisted video image analysis (CVIA) to automatically analyze the size and number of TNF2α ELISA spots. Counting the number of antigen-specific CD8 + T cells in peripheral blood mononuclear cells (PBMC) after contact with target cells loaded with antigen peptides not only improves the resolution of background staining but also enables large-scale research. Vaquerano et al. Combined a digital camera and a computer to develop a similar speckle counting system. It is believed that this method is more objective, repeatable, and saves time when the number of speckles per hole is greater than 100.
With the continuous development of ELISPOT technology, its advantages are gradually shown. The following will distinguish between ELISPOT and ELISA methods.
ELISA uses a color reaction to measure absorbance on a microplate reader and compares it with the standard curve to obtain the total soluble protein.
ELISPOT also shows clear and discernible spots on the corresponding positions where the soluble protein is secreted by the cells through the color reaction. The spots can be counted directly under the microscope or by computer-aided analysis system. 1 spot represents 1 Cells to calculate the frequency of cells secreting the protein. (Some studies need to measure not only the amount of cytokine production, but also the frequency of cells that secrete this cytokine)
Because it is a single cell level test, ELISPOT is more sensitive than ELISA, and can detect one cell secreting the protein from 200,000 to 300,000 cells.
The capture antibody has high affinity, high specificity, and low endotoxin monoclonal antibodies. The ELISA kit does not affect the secretion of cytokines by activated cells when researchers activate cells with stimulants.
At present, the ELISA method still occupies the mainstream position in the research. The universal application of the ELISA kit makes the experiment more convenient, economical and accurate. The advantages of the ELISPOT technology also determine its development potential.
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