Primary and secondary antibodies
Primary and secondary antibodies are two groups of
Primary
A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of absorption, distribution, metabolism, and excretion (ADME) and multi-drug resistance (MDR) of therapeutic agents.
Secondary
Secondary antibodies provide signal detection and amplification along with extending the utility of an antibody through conjugation to proteins.
Whole Immunoglobulin molecule secondary antibodies are the most commonly used format, but these can be enzymatically processed to enable assay refinement. F(ab')2 fragments are generated by pepsin digestion to remove most of the Fc fragment, this avoids recognition by Fc receptors on live cells, or to Protein A or Protein G.[3] Papain digestion generates Fab fragments, which removes the entire Fc fragment including the hinge region, yielding two monovalent Fab moieties. They can be used to block endogenous immunoglobulins on cells, tissues or other surfaces, and to block the exposed immunoglobulins in multiple labeling experiments using primary antibodies from the same species.[4]
Applications
Secondary antibodies can be conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); or fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine derivatives, Alexa Fluor dyes; or other molecules to be used in various applications. Secondary antibodies are used in many biochemical assays [5] including:
References
- ^ https://ptglab.com/news/blog/secondary-antibody-selection/ Secondary antibody selection guide
- ^ "Secondary Antibodies as Probes". www.thermofisher.com. Retrieved 2017-05-31.
- ^ "F(ab')₂ Fragment Secondary Antibodies - Jackson ImmunoResearch". www.jacksonimmuno.com. Retrieved 2021-01-29.
- ^ "Fab Fragment Secondary Antibodies - Jackson ImmunoResearch". www.jacksonimmuno.com. Retrieved 2021-01-29.
- ^ "Secondary antibody review based on formal publications". secondary-antibody.com. Archived from the original on 2008-04-17. Retrieved 2008-04-16.