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.