Thursday, 7 January 2016

ELISA

ELISA


  1. There are a number of different ways to determine whether an antibody has bound to its target antigen.
  2.  The enzyme-linked immuno sorbent assay (ELISA) is one method, and it is frequently used for diagnostic detection.
  3. The ELISA procedure may be either indirect or direct  
  4. A generalized indirect ELISA protocol  has the following steps.
  5. 1. Bind the sample being tested for the presence of a specific molecule or organism to a solid support, such as a plastic micro titer plate, which usually contains 96 sample wells. Wash the support to remove unbound molecules.
  6. 2. Add a marker-specific antibody (primary antibody directed against the target antigen) to the bound material, and then wash the support to remove unbound primary antibody.
  7. 3. Add a second antibody (secondary antibody) that binds specifically to the primary antibody and not to the target molecule. Bound (conjugated) to the secondary antibody is an enzyme, such as alkaline phosphatase, peroxidase, or urease, that can catalyze a reaction that converts a colorless substrate into a colored product.  Wash the mixture to remove any unbound secondary antibody–enzyme conjugate.
  8. 4. Add the colorless substrate.
  9. 5. Observe or measure the amount of colored product. If the primary antibody does not bind to a target site in the sample, the second washing step removes it. Consequently, the secondary antibody– enzyme conjugate has nothing to bind to and is removed during the third washing step, and the final mixture remains colorless. Conversely, if the target site is present in the sample, then the primary antibody binds to it,
  10. the secondary antibody binds to the primary antibody, and the attached enzyme catalyzes the reaction to form an easily detected colored product. Since secondary antibodies that are complexed with an enzyme are available commercially, each new diagnostic test requires only a unique primary antibody.
  11.  In addition, several secondary antibody molecules, each with
  12. several enzyme molecules attached, bind to one primary antibody molecule, thereby amplifying the intensity of the signal.
  13. With a direct ELISA protocol (Fig. 9.1B), a monoclonal antibody specific for the target antigen is first bound to the surface of the microtiter plate. 
  14. Generalized ELISA protocol for detecting a target antigen. The primary antibody is often obtained from rabbits that have been immunized with the target antigen, while the secondary antibody is from goats immunized with rabbit antibodies.
  15. The enzyme (E) is conjugated to the secondary antibody. (A) Indirect ELISA; (B) direct ELISA.
  16. assess the amount of a particular antigen in a sample, the sample is added to the well of the microtiter plate and allowed to interact with the bound antibody. 
  17. This is followed by a wash to remove any unbound molecules.
  18. Then, the primary antibody and the secondary antibody conjugated to an enzyme are added, as described above, before the presence of bound antigen is visualized.
  19. The principal feature of an ELISA system is the specific binding of the primary antibody to the target site. If the target molecule is, for example, a protein, then a purified preparation of this protein is generally used to generate the antibodies that will be used to detect the target. 
  20. The resulting antibody mixture, which is found in the serum (antiserum) of an inoculated animal, usually a rabbit, contains a number of different antibodies that would each bind to a different antigenic determinant (epitope) on the target molecule.
  21.  Such a mixture of antibodies is called a polyclonal preparation.
  22. For some diagnostic assays, the use of polyclonal antibodies has two drawbacks:
  23. (1) the amounts of the different antibodies within a polyclonal preparation may vary from one batch to the next, and (2) polyclonal antibodies cannot be used to distinguish between two similar targets, e.g., when the difference between the pathogenic form (target) and the nonpathogenic one (nontarget) is a single determinant. 
  24. However, these problems can be overcome,because it is now possible to generate an antibody preparation that is directed against a single antigenic determinant, namely, a monoclonal antibody.
  25. Also, despite these drawbacks, diagnostic assays employing polyclonal antibodies are widely used for a variety of purposes.