Issues with polyclonal antibody quality and signal-to-noise concerns can be addressed by performing affinity purification.
Contributed by Mike Browning
One of the key issues with polyclonal antibody quality is the purity of the antibody preparation.
Polyclonal antibodies are sold in many different preparations including “crude serum” and “purified”. Crude serum has an IgG concentration of about 10 mg/ml, but the concentration of any specific antibody in that pool of IgG is rarely more than 1% in even the best antibodies.
That creates a signal to noise problem of 100:1 when one uses crude serum for immunolabeling. Thus, if only 1% of the nonspecific IgG remains in the sample after washing that non-specific IgG could create a background signal equal to the signal from even the best antibody. For this reason, any user of crude serum must employ very rigorous washing to minimize the high background effect.
Antigen affinity purification of polyclonal antibodies is a much more effective method of reducing the signal to noise problem.
This solution separates the specific antibody away from the non-specific IgGs. Unfortunately, there is some confusion in the literature and in product data sheets regarding antibody purification. Two methods of “purification” that have little, if any, effect of the prevalence of non-specific IgG are known as Protein A purification and ammonium sulfate treatment. These two forms of purification do not purify the specific IgG, but rather just isolate the total pool of IgGs. Do not be misled. These two treatments do nothing to remove non-specific IgGs.
The only method to truly purify the specific antibody is affinity purification.
In this method, a column is prepared with the same antigen that was used to prepare the specific antibody. The sample containing the total IgG is then passed over the column and only those IgGs that bind to the antigen are retained on the column. After vigorous washing, the specific antibodies are eluted from the column. This typically results in a highly enriched preparation containing mostly the antigen-specific IgGs. So be attentive that the polyclonal antibody you use has been purified on an antigen column and not simply on a Protein A column or by using ammonium sulfate precipitation.