ELISA Assay

What Is It? Enzyme immunoassay (EIA) is a test used to detect and quantify specific antigen-eliciting molecules involved in biological processes, specifically processes related to cancer and autoimmune disorders. EIA can be used on most types of biological samples, such as plasma, serum, urine, and cell extracts. In the assay, a plate is coated with a primary antibody, which recognizes the antigen of the target molecule and bonds with it. The antigen-antibody complex is recognized by a secondary antibody that is joined to an enzyme that catalyzes the reaction mixture, yielding a specific color.

ELISA Enzyme Linked Immunosorbent Assay In this test, an enzyme is conjugated to an antibody and reacts with a colorless substrate to generate a colored reaction product. In the indirect ELISA the antigen is coated into the wells of a microtiter plate and is used to detect the presence of an antibody to the specific pathogen in the patient’s body fluid sample.

ELISA Serum or some other sample is added to the well of the plate and is allowed to react with the antigen that is coating the well. The presence of the antigen-antibody complex in the well is detected by adding an enzyme- conjugated secondary anti-isotope antibody (Ab 2 ) which binds to the primary antibody. A substrate for the enzyme is then added. If there has been a reaction between the antigen, antibody, and enzyme a colored reaction will take place, and a positive test result is indicated.

STEPS IN ELISA TESTING Step One: This is a dry well from a microtiter plate it has been coated with the antibody (primary antibody) for the nucleocapsid protein present in for influenza A Y Y Y Y This well is DRY

Step Two Step Two: You will add the patient sample to this well this will contain the virus for Influenza A if the patient has been exposed to the Influenza virus then it will be present in the saliva and the virus binds with the coated antibody (Y) on the plate. This captures the virus present in the patient’s sample. Y Y Y Y

Step Three Step Three: Add the anti influenza antibody (secondary antibody) that has been combined with horse radish peroxidase. This antibody then combines with the captured virus from the patient’s sample. This produces a sandwich complex with antibody on each side of a captured antigen

Step Four Step Four: Add the substrate (S) for the enzyme which would be hydrogen peroxide and tetramethylbenzidine (TMB). If the second antibody is present a color change will occur, and that will be proportional to the amount of virus present in the patient’s sample.