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Peptide competition assay
Important note:
Each antibody-antigen pair will require careful optimisation of neutralisation assay conditions and
initial trial might not give an ultimate answer. Titration of both, antibody and free peptide needs
to be done, to obtain best restult.
Absorption of antibodies with antigen can be of importance during characterisation of
unknown target proteins by antibodies. In some cases there is
unclear if the band which is seen during Western Blot detection or the staining
pattern of the tissue is the effect of a specific binding of the antibody to
the target protein or is due to non-specific interactions. In this case procedure
given below might be helpful.
Procedure for absorption of antibodies with antigen:
- Dilution of the antibody used in the assay have to be determined. Usually
sub-optimal dilution of the antibody should be used. In other words this dilution
of the antibody should give not maximal but consistent result (staining of the tissue
or signal on the Western Blot). There will be a difference in how much of the antibody/peptide has
to be used if working on total antibody pool (e.g. serum or total IgY) or if using affinity purified antibodies
- Consider use of a pure immunogen (protein or peptide). If the conjugate
peptide-carrier protein has been used to rise antibodies, using a conjugate in this
assay might give false results. Anti-carrier antibodies might be responsible for
the obtained staining pattern or signals on a Western Blot.
For help how to work with peptides check at: How to dissolve a peptide? - Recommended amount of the antigen: starting from 0,1:1 molar ratio between peptide and
antibodies. To our experience for some peptides 50-100 fold of excess of peptide is needed to
compete out the band. Titration of peptide is needed to find the right signal inhibition conditions.
Otherwise the assay will not work.
- Reaction of the antibodies with the antigen can be done at room temperature
for 2 hours or at 4°C over night. Afterwards, for background reduction, solution
containing antibody/antigen complexes should be centrifugated for 15 min. at
full speed. Resulting supernatant should be removed carefully (while leaving
some liquid at the bottom of the tube) and used
in the staining or Western Blot.
- In case of hydrophobic peptides, which will precipitate in solution, spotting them on nitrocellulose membrane before incubation with the antibody, might be helpful.
Last changed: 2009-04-02 by WebMaster