1. There is insufficient antigen present and an
   amplification protocol may be needed.
   
   
2. Change from a more sensitive detection
   method to a less sensitive detection method.
   
   
3. The primary antibody is diluted too far.
   
   
4. Enzyme activity is inhibited.
   
   
5. The secondary antibody does not recognize
   certain primary antibodies well.
   
   
6. The secondary antibody cross-reacts with
   immunoglobulins in the diluent.
   

There is insufficient antigen present and an amplification protocol may be needed.

Signal may be increased by changing from an enzyme- or fluorophore-conjugated antibody to a biotinylated secondary antibody followed by enzyme- or fluorophore-conjugated streptavidin. Some fluorophores are brighter than others, so changing from an FITC conjugate to DyLight 488 also will increase signal.

Change from a more sensitive detection method to a less sensitive detection method.     top of page

For example, switching the detection method from immunoperoxidase to immunofluorescence results in a 10 to 100 fold decrease in sensitivity. Sometimes, using a higher concentration of the primary antibody may solve the problem. Sometimes, an amplification protocol is required to obtain desired signal.

The primary antibody is diluted too far.     top of page

Titrating the primary antibody is recommended for each protocol. The working dilution of a primary antibody may be known for a particular protocol, but may need to be modified if a new detection system is used.

Enzyme activity is inhibited.     top of page

This is especially a concern for peroxidase. Sodium azide is a potent inhibitor of peroxidase activity. It may be present in commercially prepared buffers and stabilizers (or diluent). Sodium azide may be used as long as it is washed out thoroughly from the experimental system prior to incubation with the peroxidase conjugate. The water used for reconstitution and the glycerol used to prolong the shelf life also may contain an unknown peroxidase inhibitor(s).

The secondary antibody does not recognize certain primary antibodies well.
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Our secondary antibodies are raised against and purified on the solid phase column of immunoglobulins isolated from "normal serum". Therefore, they may not recognize certain less dominant isotypes of immunoglobulins well. For example, our anti-mouse (or rat) IgG (Fcγ) does not recognize the heavy chains of all subclasses equally well.

This is usually not a problem for polyclonal primary antibodies (made in rabbits, goats, guinea pigs etc.) since polyclonal antibodies usually contain more than one isotype.

A secondary antibody that has been adsorbed against closely related species, such as anti-mouse adsorbed against rat, only recognizes a few epitopes on mouse IgG that are different from rat. Therefore, if a certain monoclonal mouse IgG primary antibody does not bear a lot of those unique epitopes, it may not be recognized by the labeled secondary antibody well.

The secondary antibody cross-reacts with immunoglobulins in the diluent.
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For example, anti-goat IgG, diluted with buffer containing BSA or dry milk (contaminated with bovine IgG), may lose some of its activity due to cross-reactivity of the antibody with bovine IgG. The cross-reactivity also may result in higher background due to the formation of sticky immune complexes. To avoid these problems, dilute antibodies in buffer without any carrier proteins.