How to Choose a Secondary Antibody

The selection of a secondary antibody is a critical step in the experimental design of various immunodetection methods, including Western Blot, ELISA, immunohistochemistry (IHC), and immunofluorescence (IF). Secondary antibodies serve as crucial tools for the amplification of signal detection, enabling researchers to observe specific antigens with high sensitivity and specificity. This article provides a comprehensive guide on how to choose the right secondary antibody for your research, ensuring the success of your immunodetection assays.

Understanding Secondary Antibodies

Secondary antibodies are antibodies that bind to the primary antibodies, which are directly bound to the target antigen. They are typically conjugated with a detection marker, such as an enzyme or a fluorophore, which facilitates the visualization of the antigen-antibody complex. The choice of secondary antibody depends on several factors, including the host species of the primary antibody, the specificity of the secondary antibody, the type of conjugation, and the intended application.

Host Species and Cross-Reactivity

One of the first considerations in choosing a secondary antibody is the host species of the primary antibody. It is crucial to select a secondary antibody that is raised against the species of the primary antibody. For instance, if your primary antibody is raised in rabbits, you should choose an anti-rabbit secondary antibody.

Cross-reactivity is another important factor. Ideally, the secondary antibody should not cross-react with endogenous immunoglobulins from the species of the sample tissue or cells. This consideration is especially important in IHC and IF applications, where endogenous immunoglobulins can lead to non-specific staining.

Conjugation and Detection Methods

The choice of conjugation is dictated by the detection method. For colorimetric detection, enzymes like horseradish peroxidase (HRP) or alkaline phosphatase (AP) are commonly used. For fluorescence detection, fluorophores such as FITC, TRITC, or Alexa Fluor dyes are preferred. The selection depends on the available detection equipment, such as fluorescence microscopes or flow cytometers, and the need for multiplexing, which requires fluorophores with non-overlapping emission spectra.

Affinity and Specificity

The affinity and specificity of the secondary antibody are critical for achieving high signal-to-noise ratios. Affinity-purified secondary antibodies are preferred as they have higher specificity and lower cross-reactivity. Furthermore, some secondary antibodies are adsorbed against other species to reduce cross-reactivity further.

Applications and Experimental Conditions

The choice of secondary antibody is also influenced by the specific application and experimental conditions. For example, in Western Blot, the size of the secondary antibody can affect the migration of the antigen-antibody complex during electrophoresis. In IHC and IF, the tissue type and fixation method can influence the accessibility of the antigen and the choice of secondary antibody.

Best Practices for Selection

To ensure the optimal selection of a secondary antibody, consider the following best practices:

Review the literature to identify the secondary antibodies used in similar experiments.
Consult with antibody suppliers and utilize their expertise to select the most suitable secondary antibody.
Consider pre-adsorbed secondary antibodies for applications susceptible to cross-reactivity.
Opt for conjugated secondary antibodies that match your detection system.
Validate your choice by performing control experiments to assess specificity and sensitivity.

Conclusion

The careful selection of a secondary antibody is essential for the success of immunodetection assays. By considering factors such as the host species of the primary antibody, specificity, conjugation, and the intended application, researchers can select the most appropriate secondary antibody for their experiments. Following best practices and conducting validation experiments will further ensure the reliability and reproducibility of the results.

References

Green, N. M. (1990). Avidin and streptavidin. Methods in Enzymology, 184, 51-67.
Harlow, E., & Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
Hermanson, G. T. (2013). Bioconjugate Techniques (3rd ed.). Academic Press.
Atha, D. H., Manne, U., Grizzle, W. E., Wagner, P. D., Srivastava, S., & Reipa, V. (2010). Standards for immunohistochemical imaging: A protein reference device for biomarker quantitation. Journal of Histochemistry & Cytochemistry, 58(11), 1005-1014.
Nerenberg, S. T., & Peetoom, F. (1970). Use of Immunoelectrophoresis and Immunodiffusion in Clinical Medicine. CRC Critical Reviews in Clinical Laboratory Sciences, 1(2), 303-350.
Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press.
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350-4354.
Chang, C. J., Yang, Y. H., Liang, Y. C., Chiu, C. J., Chu, K. H., Chou, H. N., & Chiang, B. L. (2011). A novel phycobiliprotein alleviates allergic airway inflammation by modulating immune responses. American journal of respiratory and critical care medicine, 183(1), 15-25.