IHC Primary Antibody Selection Guidance

Abstract:      Good immunohistochemistry test results take a great of time and effort. Here, IHC troubleshooting for choosing primary antibody is specified, including antibody type, applications, species and hosts.
Keywords:   IHC Primary Antibody, Immunohistochemistry, IHC Troubleshooting

1. Monoclonal or Polyclonal Antibody

Antigen determinant (also called antigenic epitope) is present in the antigen surface and determines antigen specificity as special chemical groups. An antigen has single or multiple different antigenic epitopes. Each epitope only has an antigen specificity.

Using the antigen including multiple antigen determinants to immunize animals can stimulate multiple B cells of the organism to produce the polyclonal antibody (pAb) against multiple antigenic epitopes. Clone of single B cell produces the monoclonal antibody (mAb).

In antigen-antibody reaction, monoclonal antibody usually has a higher specificity, lower affinity and sensitivity for antigen test. Background signal produced by sections and cell staining is less. Polyclonal antibody has a lower specificity and higher affinity. Independent of single epitope, it has a strong inclusion for minor antigenic changes (e.g. polymorphism, heterogeneity of glycosylation or slight degeneration). The sensitivity is higher. However, non-specific staining occurs easily and can be avoided by sealing.

2. Antibody Applications

Some primary antibodies can be applied in western blotting, immunohistochemistry, immunofluorescence or immunoprecipitation. Then, the antibody for immunohistochemistry is required.

3. Antibody Species

It's important to choose the right antibody which can detect the target species. The antibody between different species is varied.

4. Antibody Host

Rabbit polyclonal or monoclonal antibody; mouse monoclonal antibody. Secondary antibody should be chosen according to primary antibody host.

5. Recommended Antibodies

REFERENCES

Primary Antibody Selection and Blocking Techniques for Immunohistochemistry, PMID: 34859400.

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