The phrase "enzyme-linked immunosorbent assay," or "ELISA" for short, was first used in 1971 to refer to an enzyme-based immunoassay technique useful in measuring antigen concentrations. Enzyme-linked immunosorbent Assay (ELISA) kits are utilized in research to detect and quantifyparticular protein sequences and other targets inside a sample. Using the correct ELISA kits provides precise, quantifiable data that confirms and supports your study. You must select the appropriate kit type (direct, indirect, sandwich, or competitive ELISA) for your project. A high-quality kit should tolerate some technique variation and be thoughtfully designed to help you get the best results.
Applications Of ELISA Kits For Cytokine Detection
The ELISA technique includes a variety of immunoassays with few differences in their procedures. Several parameters, such as the antigen being detected, the monoclonal antibody available for a specific antigen, and the necessary test sensitivity, determine which version of ELISA to employ. Sandwich and indirect ELISA are the two most commonly used in cytokine detection.
1. Sandwich ELISA
Cytokine sandwich ELISAs are sensitive enzyme immunoassays that can preciselyidentify and measure the amount of soluble chemokine and cytokine proteins. Highly pure anti-cytokine antibodies (capture antibodies) are used in the basic cytokine sandwich ELISA method. These antibodies are noncovalently adsorbed onto plastic microwell plates. The immobilized antibodies work to selectively bind soluble cytokine proteins found in the samples that were added to the plate after platewashings.
Following the removal of unbound material, biotin-conjugated anti-cytokine antibodies (detection antibodies) are used to identify the cytokine proteins that have been collected. An enzyme-labeled avidin or streptavidin step comes after them.
An ELISA reader may be used to simply quantify the amount of colored product created by the bound, enzyme-linked detection reagents at an acceptable optical density (OD) spectrophotometrically after the addition of a chromogenic substrate. Data storage and reanalysis become easier when the plate reader is linked to a computer.
The ELISA sandwich method is the preferred option for high accuracy in measuring analytes. This format can reveal the specificity and sensitivity of analytes, starting with a capture antibody that immobilizes the analyte. When adding a detection antibody with an enzyme label, the sandwich is assembled like building blocks. It completes the complex sandwich structure by identifying an additional epitope on the analyte.
2. Indirect ELISA
An indirect ELISA is one in which a secondary conjugated antibody identifies the primary antigen-specific antibody. Antibody sequencing coordinates sensitivity and signal amplification in Indirect Elisa. Two antibodies work together in this configuration to defeat the analyte. With high specificity, the primary antibody seeks for the analyte and forms an irreversible association.
The secondary antibody then uses an enzyme label to identify and attach itself to the original antibody. Collaboratively, the two antibodies enhance the signal and lead to increased sensitivity, highlighting even the most minuscule amounts of analyte.
Sensitivity is high with indirect ELISA (https://www.biomatik.com/blog/unlocking-the-secrets-of-elisa-kits-an-introduction/) when used in cytokine detection. This is mainly because the primary antibody can attach to many secondary antibodies conjugated with enzymes. Additionally, one enzyme-conjugated secondary antibody mayidentify various primary antibodies. This provides the user with the option to employ the same enzyme-conjugated secondary antibody in several ELISA as needed (independent of the antigen being detected).