Weak Signals from Labelled Secondary Antibodies


What are the possible causes of weak signals from labelled secondary antibodies assuming that antigens are present and primary antibodies are working?

(First determine that the primary antibodies do not contribute to the background)
If there is not sufficient antigen present. An amplification protocol might be needed.

Moving from a more sensitive detection method to a less sensitive detection method.

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.

For example, a working concentration of the primary antibody has been determined previously by immunoperoxidase to be 0.1 μg/ml. For various reasons, subsequently, immunofluorescence is tried as another method of detection on the same antigen using the same concentration of the primary antibody. No signal could be detected although the fluorophore-labelled secondary antibody was used at the recommended working dilutions (or at a final working concentration of approximately 2 – 30 g/ml). Using a 20 to 300 fold molar excess of the secondary antibody won’t help if there is not enough primary antibody to begin with.

The Enzyme activity is inhibited.

This is especially a concern for peroxidase. Azide is a potent inhibitor of peroxidase.
Sometimes, azide may be present in certain commercially prepared buffers and stabilisers (or diluent). Azide may be used as long as it is washed out thoroughly prior to the incubation with the peroxidase conjugate. Bottled distilled water used for reconstitution or glycerol used to prolong the shelf life may also contain unknown peroxidase inhibitor(s).

The secondary antibody does not recognise certain primary antibodies well.Jackson ImmunoResearch’s secondary antibodies are raised against, and purified on the solid phase column of immunoglobulins isolated from “normal serum”. Therefore, they may not recognise certain less dominant isotypes of immunoglobulins well. For example, our anti-mouse (or rat) IgG (Fc ) does not recognise the heavy chains of all subclasses equally well. This is not usually 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 recognises 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 recognised by the labelled secondary antibody well.

The secondary antibody cross-reacts with immunoglobulins in the diluent.

For example, an anti-goat IgG, diluted in a diluent containing horse serum or BSA contaminated with bovine IgG, may lose some of its activity due to the crossreactivity of the antibody with bovine or horse IgG. The cross-reactivity may also result in higher background due to the formation of sticky immune complexes.


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