What are Primary Antibodies?
Primary antibodies are immunoglobulins which bind to antigens from a protein or biomolecule of choice. They are a key research means of purifying, detecting, or measuring a protein in preclinical in vitro and in vivo research. Primary antibodies are developed in a range of species including rat, mouse, and rabbit.
Primary antibodies have two main types – monoclonal or polyclonal antibodies, each with their own advantages and disadvantages (which we’ve reviewed in an more detailed post).
Briefly, monoclonal antibodies are identical, developed from a single B cell clone and binding to a single epitope. This makes them homogeneous, and great for identical production and standardization experiments with little cross reactivity. They can, however, be inflexible and sensitive to assay changes.
Polyclonal antibodies are the reverse – developed from multiple B cell clones so providing a heterogenous mix, which binds to several epitopes and is great for amplifying signals from low expression targets. Drawbacks include lot variability and cross reactivity.
Monoclonal Antibodies and Isotype Controls
When using monoclonal antibodies across a range of experiments (including in vivo studies), it’s essential to have a good negative control. This allows you to optimize study performance and downstream data analysis.
Isotype controls match the primary antibody characteristics, but are raised against antigens not found in common preclinical species. This results in isotype controls that don’t have specificity for the target antigen.
Using isotype controls is important in drug development to mimic widespread FcR engagement effects, mimic FcR and protein staining for flow cytometry, and as negative controls in IHC experiments.
Choosing a Primary Antibody and Isotype Control
When you look to choose a primary antibody, one main point to consider is will this be for research use only/research grade or for in vivo studies.
Research Use Only/Research Grade Antibodies
Antibodies which aren’t for in vivo use (research use only) are available from many vendors. They are often supplied preserved, with unknown endotoxin levels, and in small quantities.
In Vivo Grade Primary Antibodies and Isotype Controls
For in vivo research, primary antibodies and isotype controls need to be higher purity monoclonal antibodies, without preservatives, carriers, or stabilizers, and with low endotoxin levels. Bulk quantities are often needed.