* cheapest and simplest technique
* an analytical method used in Clinical Toxicology
* commonly used in screening blood (serum) for TDM (Therapeutic Drug Monitoring) and as a primary screening procedure of abused drugs and their metabolines in the urine. * it is the first homogenous assay to be widely used.
* the separation using antibody specificity (antigen binding). * measurement of enzyme-substrate reactions using visible spectroscopy, and standard curve. * produces reliable results
* the test utilizes antibodies that are enzyme linked and react only with the specific substance in the sample(urine or blood) is positive with the particular drug being tested.
1) Combine a sample containing an unknown concentration of antigen (Ag) & a solution containing a known concentration of antibody against the Ag (Ab). 2) Allow binding of Ag & Ab (incubation #1).
3) Add a known concentration of prepared Ag-enzyme conjugate. 4) Allow binding of Ag-enzyme conjugate with any remaining unbound Ab in solution (incubation #2). The conjugate is constitutionally active; binding to any unbound Ab will render it inactive. 5) Add enzyme substrate.
6) Measure enzyme activity.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to...