The Choice of Secondary Antibody is one of the Most Overlooked Issues in Immunochemistry.

After the tissue of interest has been incubated with the primary antibody, the next critical step involves incubating the tissue in a labeled secondary antibody to detect the presence of the primary antibody. The choice of the secondary antibody has to satisfy the same requirements of specificity and reproducibility, as did the primary antibody used. Unfortunately many researchers consider specificity and reproducibility only for primary antibodies. Given that any signal seen in the assay is due to the secondary antibody, it is clear that a non-specific or irreproducible secondary antibody will yield nonspecific or irreproducible signals.

There are two principal reasons why the choice of secondary antibody is critical.

  1. The secondary antibody can cross-react with endogenous serum IgGs that are present in the tissue being examined and thus give a high background.
  2. The secondary antibody can vary greatly in signal strength from bleed to bleed (lot-to-lot) unless the manufacturer has pooled all bleeds of the secondary antibody to ensure consistency in all subsequent lots produced.

Let’s examine each of these crucial points in detail:

  1. The secondary antibody can react with residual IgGs that are left in the tissue being examined.

Let’s first consider the lack of specificity in the secondary antibody. It is important to realize that the sequences of many mammalian IgGs are highly homologous. So if one prepares an antibody against rabbit IgG, one will often obtain an antibody that recognizes IgGs of other mammalian species. The most common problem here is cross-reaction with the IgG of the species being studied. Suppose that a manufacturer prepares a secondary anti-IgG by injecting purified rabbit IgG into a goat. The goat makes anti-rabbit IgG antibodies that are then isolated, labeled and then sold as HRP or FITC-labeled Goat anti-rabbit IgG. This secondary antibody is then used to label the primary rabbit antibody used for example on rat tissue.

However, if the Goat anti-rabbit IgG also recognizes rat IgG imagine what will happen to the signal generated in rat tissue. The signal will reflect the presence of the primary rabbit IgG AND the presence of any residual rat IgG in the rat tissue. Even with extensive perfusion it is extremely difficult to eliminate all residual rat serum proteins from the tissue and therefore there will likely be a high non-specific background staining in the tissue.

There is a straightforward solution to this problem. This involves pre-absorbing the Goat anti-rabbit IgG against rat serum proteins to remove any secondary antibody that would bind to endogenous rat serum proteins. This simple step is highly recommended, and all secondary antibodies used for immunostaining should undergo this step. Unfortunately this procedure does not eliminate the very high background staining one obtains when using a mouse monoclonal to stain mouse tissue. Elimination of the high background in such situations is particularly problematic. One solution to this problem was suggested by my colleague Dr. Tom Finger from the University of Colorado School of Medicine. “To use a monoclonal (mouse) antibody on mouse tissue, we first treat the sections with an unlabeled anti-mouse Fab which covers up much of the endogenous, the native mouse Ig molecules. Then we put on the primary (monoclonal anti-X) then after washing we apply a labeled anti-mouse Fab secondary. Since the majority of endogenous Ig is already covered by the unlabeled Fab, the only uncovered mouse IgG present is the primary one you applied. Since the Fab is monovalent, there is no sticking of your primary to the already applied Fab.”

  1. The secondary antibody can vary greatly in signal strength from bleed to bleed.

This issue is the same issue that all polyclonal antibodies face, namely each bleed for a polyclonal antibody will have slightly or, in some cases, greatly divergent properties. Thus one lot of antibody purified from bleed X may have a very strong level of antibody present while a later lot from bleed Y may have a much weaker level. This will inevitably lead to irreproducible results. This fact has led some to argue that we should spend billions of dollars and convert all antibodies to recombinant antibodies. Fortunately there is a simple solution to this problem that has virtually zero cost. The solution involves pooling all antibody bleeds into a single large lot before the antibodies are sold. This simple step virtually eliminates all lot-to-lot variability. So be sure to check that your secondary antibodies all come from pooled serum, otherwise when you want to recheck your results 6 months from now you may experience irreproducibility within your own research.

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