Detecting the concentration of IFN-γ in samples by double antibody sandwich ELISA



Introduction to IFN-γ,



IFN-y, known as type 1 I interferon, is composed of 143 residues. There are glycoproteins of 20 and 25kDa subtypes, which exist in the form of homologous glycoproteins connected head to tail.



IFN-γ is produced by T lymphocytes and NK cells to resist viruses and interfere with proliferation. In addition, IFN-γ has several functions related to immune regulation, including regulating cell proliferation and programmed death, and stimulating or inhibiting different genes. expression.



IFN-γ can resist the infection of Toxoplasma gondii and Chlamydia by inducing the production of indoleamine 2,3-dioxygenase. IFN-y is a potent mononuclear phagocyte enhancer. IFN-y enhances pinocytosis and phagocytosis by enhancing the expression of Mac-1 in mononuclear phagocytes. IFN-y enhances the effect of killing tumor cells by increasing the release of oxygen free radicals and TNF-a from macrophages. IFN-γ selectively increases the release of IgG2a from B cells including LPS-stimulated B cells and lgG3 from B cells due to antigen presentation by type 2 T cells. It has been reported that IFN-V can increase the autocrine IFN-y effect of cells.



Detection principle,



A double antibody sandwich ELISA method was used to detect the concentration of IFN-γ in the sample. TheIFN-γ capture antibody has been pre-coated on the ELISA plate. When the sample or reference material is added, the IFN-γ in it will be combined with the capture antibody, and other free components are removed through the washing process. When the biotinylated anti-human IFN-γ antibody is added, the anti-human IFN-γ antibody is combined with IFN-γ to form a sandwich immune complex, and other free components are removed through the washing process. Then add horseradish peroxidase labeled avidin. Biotin binds specifically to avidin, and the enzyme linked to avidin is connected to the sandwich immune complex; other free components are removed through the washing process. Finally, the color developer is added. If IFN-γ is present in the sample, an immune complex will be formed. Horseradish peroxidase will catalyze the oxidation of the colorless color reagent into a blue substance, which turns yellow after adding the stop solution. Detected by a microplate reader, read the OD value at 450nm, the IFN-γ concentration is proportional to the OD4so value, draw a standard curve with a reference product, and compare the OD value in the unknown sample to calculate the IFN-γ concentration in the specimen.



Specimen collection,

 

 



To collect specimens, please follow the following procedures:


After the cell supernatant sample is centrifuged to remove the suspended matter;


Serum samples should be spontaneously coagulated, and the supernatant should be taken to avoid clotting blood in the refrigerator; C. Plasma samples are recommended to be collected by EDTA. If the samples to be tested cannot be tested in time; D. After collection, please aliquot and freeze Store at -20°C, avoid repeated freezing and thawing.


Serum samples should not be added with any preservatives or anticoagulants;


The specimen should be clear and transparent. Any suspended matter in the sample should be removed by centrifugation before testing;
Do not use hemolytic, hyperlipidemia or contaminated specimens for testing, otherwise the results will be inaccurate.


Note: For normal human serum or plasma samples, please use the sample buffer to do multiple dilutions before testing.



Precautions,



Please store the kit at 2~8°C.


Concentrated washing liquid may crystallize at low temperatures. Please heat in a water bath to completely dissolve the crystals before preparing the working liquid. 3. If the standard product is reconstituted, please use it up within three days.


Do not contact the substrate with oxidants and metals.


When adding samples, please change the tip in time to avoid cross contamination.


It is strictly forbidden to mix kit components with different batch numbers.


Full mixing is very important to ensure the accuracy of the reaction results. After adding liquid, tap the edge to ensure mixing. 8. For room temperature reaction, please strictly control it at 25~28C.


The washing process is very important. Insufficient washing will reduce the accuracy and cause larger errors in the results. 10. It is recommended to make double multiple holes when testing standards and samples in the test.


Avoid air bubbles during sample loading.


In the detection of serum and plasma samples, the incubation time of the detection antibody should be appropriately extended.


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