Abc method of ihc


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Abc method of ihc

  1. 1. ABC Method of IHC
  2. 2. Biotin • Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells and is critical for a number of biological processes. • The valeric acid side chain of the biotin molecule can be derivatized in order to incorporate various reactive groups that are used to attach biotin to other molecules. • In the context of IHC, biotin is conjugated to antibodies or to the enzyme reporters used to detect target antigens.
  3. 3. Avidin • The extraordinary affinity of avidin for biotin allows biotin-containing molecules in a complex mixture to be specifically bound to avidin. • Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibia. • It contains four identical subunits having a combined mass of 67 to 68kDa. Each subunit consists of 128 amino acids and binds one molecule of biotin; thus, a total of four biotin molecules can bind to a single avidin molecule. • Avidin has a basic isoelectric point (pI) of 10 to 10.5 and is stable over a wide range of pH and temperatures. Extensive chemical modification has little effect on the activity of avidin, making it especially useful for protein purification. • However, because of its carbohydrate content and basic pI, avidin exhibits relatively high nonspecific binding properties.
  4. 4. Principle Reporter intensity is a function of the localized enzyme activity, and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the target antigen. The multiple biotin binding sites in each tetravalent avidin molecule are ideal for achieving this amplification.
  5. 5. • The primary antibody is incubated with the tissue sample to allow binding to the target antigen. • A biotinylated secondary antibody, with specificity against the primary antibody, is incubated with the tissue sample to allow binding to the primary antibody. • A biotinylated enzyme (HRP or AP) is pre-incubated with free avidin to form large avidin-biotin-enzyme complexes. Typically, the avidin and biotinylated enzyme are mixed together in a specified ratio to prevent avidin saturation. • An aliquot of this solution is then added to the tissue sample, and any remaining biotin-binding sites on the avidin bind to the biotinylated antibody that is already bound to the tissue.
  6. 6. Important features of the Avidin-Biotin Complex (ABC) staining method. Advantages Disadvantages Increased enzyme reporter localized to the target antigen Some tissue may require endogenous biotin blocking to avoid nonspecific labeling Increased detection efficiency The ABC complex is large, which hinders tissue penetration in some applications Requires less primary antibody than direct methods of detection Reduced assay time compared to the PAP method
  7. 7. SUMMARY
  8. 8. • Direct method In this method a labelled antibody (e.g. with a substrate- chromogen) reacts directly with the antigen in the tissue. The advantage is that only one antibody is needed, hence the application is fast and produces little nonspecific binding. On the other hand, since only one antibody binds one epitope, the signal intensity is low. For applications with small amounts of antigen the signal may be too weak.
  9. 9. • Indirect method In order to upscale the low signal intensity of the direct method, the indirect method has been developed. The primary antibody binds to the antigen, followed by a second (labelled) antibody binding the primary. The signal is amplified due to the binding of multiple secondary antibodies to a single primary antibody. Another advantage is that only one labelled antibody needs to be employed for different targets, which may reduce costs. One disadvantage is that nonspecific binding occurs more frequently than with the direct method.
  10. 10. • PAP method Today, the peroxidase anti-peroxidase method is rarely used, but has been popular in pathology laboratories previously. It is an indirect method which depends on a third layer of antibodies, bound to peroxidase, that binds an unconjugated second layer antibody. The peroxidase is not chemically conjugated to the IgG but immunologically bound. That means that the third layer antibody is specific for peroxidase. This leads to a much higher activity of the peroxidase, in turn increasing the assay sensitivity by two to three orders of a magnitude.
  11. 11. • (Strept)Avidin-Biotin Complex (ABC) methods Nowadays, one of the most commonly used method for staining is based on the high affinity that avidin (chicken egg) and streptavidin (Streptomyces avidinii) have for the glycoprotein biotin. The basic principle is that avidin (or streptavidin) reacts with a biotinylated secondary antibody, followed by a horseradish peroxidase reaction.