Primary and secondary antibodies

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.

[2] 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] 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.