What is Immunohistochemistry?

Immunohistochemistry, also known as immunocytochemistry, refers to the specific antibody with a chromogenic reagent labeled in the tissue cell in situ through antigen-antibody reaction and histochemical coloration reaction, and the corresponding antigen is qualitatively, localized, and quantitatively determined. A new technology.

immunochemistry

Immunohistochemistry (IHC) is based on the principle of specific binding of antigens and antibodies, and uses a chemical reaction to develop a labeled antibody (fluorescein, enzyme, metal ion, isotope) to determine the antigen (polypeptide) in the tissue cell. And proteins), which are localized, qualitative, and quantitative, are called immunohistochemistry.

The whole process of immunohistochemistry includes:

Immunohistochemical staining has a very broad role in biomedical research. and

Antibodies for immunohistochemistry

The antibodies commonly used in immunohistochemistry are

Cause of cross reaction

Refers to antibodies that react specifically with other antigens in addition to their specific antigens. The causes are as follows:

1. Antigen specificity means that the antigenic substances used to immunize animals contain a variety of antigen molecules, which cause animals to produce corresponding antibodies specific to a variety of antigen molecules. Any other substance, as long as it contains one or more of the same antigen molecules as above, will cross-react with the above-mentioned multispecific antiserum.

2. The common determinant is the same epitope in both antigen molecules.

3. The determinants are similar. If the structures of two different antigenic determinants are approximately the same, due to the spatial conformation, the corresponding antibodies of a certain determinant can cross-react with the approximately the same determinant. Of course, the binding force when the antigen-antibody conformation is similar is smaller than the binding force when the anastomosis is performed.

Comparison of polyclonal and monoclonal antibodies

1. Uniformity. In a monoclonal antibody, the chemical structure and amino acid sequence of each antibody are the same, and there is only one Ig subclass. That is, the monoclonal antibody is a homogeneous antibody with high purity. Polyclonal antibodies obtained from different animals and at different times have different chemical compositions. Polyclonal antibodies are a mixture of multiple species and subclasses of Ig.

2. Stability. Monoclonal antibodies are less stable, sensitive to changes in pH, unstable to heat, and volatile during purification, while polyclonal antibodies have better stability.

3. Specificity. Monoclonal antibodies are directed against a certain determinant of the antigen, so when using it for serological reactions, they have strong specificity and high sensitivity, and generally do not cross-react. Polyclonal antibodies can bind to multiple epitopes on the antigen, so they have poor specificity and are more likely to cause cross-reactivity.

4. Repeatability. Monoclonal antibodies have good reproducibility, while multiple antibodies differ from batch to batch.

5. Precipitation reaction. Polyclonal antibodies easily form network-like precipitates due to their multivalent binding to antigens, while monoclonal antibodies only bind to antigens to produce dimers, and cannot directly form antigen-antibody precipitates.

Antibody storage

Antibody storage containers should be made of materials that do not adsorb proteins. Commonly used are polypropylene, polycarbonate, and borosilicate glass. If the protein concentration in the stored antibody is very low (10-100mg / L), an additional isolation protein should be added to reduce the adsorption of the antibody protein by the container. The isolation protein is usually 0.1% -1.0% bovine serum albumin. Most of the diluted antibodies should be stored at 4 -8 to avoid the harmful effects of freeze-thaw on antibody proteins.

The antibody stock solution and the separated immunoglobulin components should be stored at -20 ° C and avoid repeated freeze-thaw cycles. The frozen antibody solution should be thawed slowly at room temperature, and rapid thawing at high temperature should be absolutely avoided. Antibodies contaminated with bacteria often have false positive results, and contaminated antibody solutions and other reagents should be discarded. To prevent bacterial contamination, 0.01% sodium azide can be added to the antibody solution.

Antibodies can be stored under -20 ° C for 3-5 years after being freeze-dried under vacuum. The diluted monoclonal antibody should be added with 0.1% sodium azide concentration. Most diluted antibodies can be stored frozen, and a few antibodies may lose antigen activity. Most monoclonal antibodies, as long as the protein concentration is appropriate, can be stored at 4 ° C for several months.