Which component is colored in ELISA (Enzyme-linked Immunosorbent Assay)?
Which component is colored in ELISA (Enzyme-linked Immunosorbent Assay)?
Last Updated on Thursday, 24 June 2010 01:11 Written by Administrator Thursday, 24 June 2010 01:11
Question by heyheyhey: Which component is colored in ELISA (Enzyme-linked Immunosorbent Assay)?
also, In an ELISA used to detect antibody, which component is linked to the enzyme?
Best answer:
Answer by spasmodeus
The color comes from the chemical alteration of an enzyme substrate, such as 3,3-diaminobenzidine (DAB). Conversion of DAB by an enzyme like horseradish peroxidase (HRP) produces a brown color which can be detected using a spectrophotometer.
The way it typically works is a multiwell assay plate is coated with an antibody against the desired antigen for detection. Samples are loaded into the wells, and the antigen allowed to bind to the antibody coating the plate. Unbound material is washed away, and a secondary antibody conjugated to an enzyme (like HRP) is loaded into the well. This antibody binds to the antigen already immobilized in the well by the coating antibody, forming a so-called “sandwich”. Finally, after unbound secondary antibody is washed away, the enzyme substrate (such as DAB) is introduced. The amount of enzyme still present in the well is directly proportional to the amount of antigen captured in the well and cross-linked to the enzyme by the secondary antibody. The amount of colored enzyme product is also proportional to the amount of enzyme. So, one can compare the amount of product produced from well to well over time, and from this value quantitate the amount of antigen present in a particular sample.
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Tags: assay, colored, component, elisa, EnzymeLinked, Immunosorbent