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Peptide competition assay

Important note:
Each antibody-antigen pair will require careful optimisation of neutralization 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 result. 

Absorption of antibodies with antigen can be of importance during characterisation of unknown target proteins by antibodies. In some cases it 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. 

Usually a peptide has too low molecular weight to allow separation on SDS gel. It can be used for a dot blot

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. In some cases increased overall background signal after incubation of antibodies with peptide/protein can be observed.

Estimation of the ratio between antibodies and a peptide – an example.
Assume that IgG concentration in the serum is 10mg/ml. Using a molecular weight of
75000 (for 1 antigen binding site), this comes to 1.33E-7 mol/ml in stock.
If using 50 000X dilution, the concentration is 2.67E-12 mol/ml on the blot. Multiply the antibody concentration by 100 to arrive at the peptide concentration that will give you a 100X excess of peptide. This is 2.67E-10 mol/ml. Peptide molecular weight needs to be known to complete the calculation in units of mol/ml, since you are working with an antibody concentration in mol/ml. (Peptide in mol/ml is g/ml divided by g/mol )

Calculation example: PsaC peptide is 1413 g/mol. Assume the concentration is 1mg/ml, which is therefore 7.08 E-7 mol/ml.
The dilution required to go from 7.08 E-7 down to 2.67E-10. This is a 2654X dilution of peptide. Then, 1/2654l = x/10000 l = 3.77l in the incubation mixture.
Note, again, that 10000 µl (10ml) is the final volume, and this is used for calculation, while the incubation is done in 5ml.

Neutralization (competition) assay
Pre-incubation of antibody and peptide is done for two hours in RT in TBST (without Blocking reagent - what is very important), with occasional mixing. The volume of this solution is ½ of that which is required for the blot (eg, if you intend to do a 10 ml incubation with antibody, do the competition in 5ml).

The concentration of antibody in pre-incubation mixture is 2X the final concentration. The peptide to antibody molar ratio in this mixture should be 100:1. A control solution - TBST with just antibody at 2X final concentration - should be done. Prepare 4 % blocking solution in TBST (5 ml as in this example). At the end of the pre-incubation, add4% blocking solution to the peptide/antibody mixture. Mix briefly and add to the blot. The final concentration of blocking reagent is now 2% and the antibody concentration is
1X (he antibody dilution that is normally used).
Following incubation of excess of peptide and antibody, solution needs to be shortly centrifugated and supernatant is used for western blot. 

Result:

100 X molar excess of the peptide has bee used in this experiment (right panel, + peptide) and completely competed out the signal obtained from a target protein.

example of peptide neutralisation experiment

5 µg of total protein from (1) Arabidopsis thaliana leaf extracted, (2) Spinacia oleracea leaf extracted with PEB, (3) Hordeum vulgare , (4) Zea mays, extracted with Protein Extration Buffer, PEB (AS08 300) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according to the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.

Conclusion: obtained band is a result of immunization with a peptide used to elicit this antibody as it is completely depleted by antibody-peptide neutralisation.



Guideline for working with peptides

When working with dissolving peptides following points may help:

* All such attempts should be carried out on very small samples rather than the bulk material.
* Most peptides will dissolve in aqueous media with the addition of a little acid (acetic acid) or a little base (dilute ammonia) depending on their overall charge.
* Before dissolving a peptide short analysis of its sequence might help. If hydrophobic amino acids like A, C, F, I, L, M, P, V, W, Y are consisting more than 50 % of a peptide, dissolve a peptide first in a small amount of either dimethyl sulphoxide (DMSO) or N, N-dimethylformamide (DMF) PRIOR to the addition of any aqueous component.
* Gentle heating or use of ultrasonication may also aid peptide dissolution.


Some general comments:

* Peptide should be slowly added to larger volumes of water/buffer, not opposite.
* Problems with peptide solubility can also result from oxidation and the formation of the disulphide bridges. Especially if a peptide has not been stored properly or is rather old. Check this by using Elman's reagent (Pierce).

* 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.


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