–Uses: –In estimation of – hormones –Drugs –Enzymes –Detect Infectious microbial Ags or Abs e.g. HIV, Hepatitis A, B etc –To detect the markers of tumer
Enzyme-Linked ImmunoSorbent Assay (ELISA) Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a abiochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample..
Principle: in ELISA an unknown amount of antigen is affixed to a surface (solid phase), and then a specific antibody is added on the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to and give detectable color.
Types of ELIZA: 1-Indirect ELISA, "indirect," ELISA for detect antibodies in patient serum the steps as follow: 1.The antigen is fixed to the surface of microtiter to make it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. 2.The plate wells or other surface are then coated with serum samplesserum
3-The plate is washed to remove any unbound detection antibody.. 4-Secondary antibodies conjugated to the enzyme are added to the wells. 5- Wash the plate, 6- Apply a substrate which is converted by the enzyme to specific color 7-put stop solution 8-Read the result(color) using a spectrophotometer,
Used for testing the amount of antibody to an antigen in serum
ELISA Antibody Response Micro-plate reader 96-well micro-plate Positive result
2.Direct ELIZA(Sandwich ELISA ) To detect unknown Ags in patient serum Plate is coated with a capture antibody sample is added, and any antigen present binds to capture antibody detecting enzyme-linked antibody is added, and binds to antigen substrate is added, so it is converted by enzyme to detectable color. then stop solution also added to stop reaction
Add the solution containing antigen to be measured
3.Competitive ELISA For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in serum (if serum is positive for the antibody).
Competition occurs between the two antibodies for the same antigen. Sera to be tested are added to these wells and incubated at 37 degrees and then washed. If antibodies are present, antigen-antibody reaction occurs. Substrate is added but there is no enzyme to act on it, therefore, positive result shows no color change.
2- radioimmunoassay RIA Detected by X ray 3- immunofluoresecent IF Detected by fluorescent microscopy