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Protocols > Peptide neutralization/competition assay

Important note

Each antibody-antigen pair will require careful optimization of neutralization assay conditions and initial trials might not give an ultimate answer. Titration of both, antibody and free peptide needs to be done, to obtain the best result.

Absorption of antibodies with the antigen can be of importance during characterization of unknown target proteins by antibodies. In some cases, it is unclear if the band 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 due to non-specific interactions.

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

Procedure

Absorption of antibodies with antigen

Dilution of the antibody used in the assay must be determined. Usually, sub-optimal dilution of the antibody should be used. In other words, this dilution of the antibody should not give maximal, but consistent, results (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.

Example

Estimation of the ratio between antibodies and a peptide

Assume that IgG concentration in the serum is 10 mg/ml. Using a molecular weight of 75000 (for 1 antigen binding site), the resulting concentration will be 1.33E-7 mol/ml in stock. When using a 50 000x dilution, the concentration is 2.67E-12 mol/ml on the blot. Multiplying the antibody concentration by 100 will give you the peptide concentration that will give you a 100x excess of peptide, namely 2.67E-10 mol/ml. The molecular weight of the peptide 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 1 mg/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, 1/2654 l = x/10000 l = 3.77 l in the incubation mixture.
Note: 10000 µl (10 ml) 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, with occasional mixing. It is very important that this is done without a blocking reagent. The volume of this solution is half of that which is required for the blot (e.g., if you intend to do a 10 ml incubation with antibody, do the competition in 5 ml).

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, add 4% 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 the antibody dilution that is normally used.
Following incubation of excess of peptide and antibody, the solution needs to be shortly centrifugated, and the resulting supernatant is used for western blot.

Results

100x molar excess of the peptide was used in the below presented experiment (right panel, + peptide), which completely competed out the signal obtained from the 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 Extraction 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 1 h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 for 1 h 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 1 h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according to the manufacturer's 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

The obtained band is a result of immunization with a peptide used to elicit this antibody, as it is completely depleted by antibody-peptide neutralization.

Guideline for working with peptides

When working with dissolving peptides, the 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, a 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 sulfoxide (DMSO) or N, N-dimethylformamide (DMF) prior to the addition of any aqueous components.
Gentle heating, or the use of ultrasonication may also aid peptide dissolution.

General comments

Peptide should be slowly added to larger volumes of water/buffer, not the other way around.
Problems with peptide solubility can also result from oxidation and the formation of disulphide bridges, especially if a peptide has not been stored properly or is old. Check this by using Elman's reagent (Pierce).
Recommended amount of the antigen starts from 0.1:1 molar ratio between peptide and antibodies. For some peptides, a 50-100-fold of excess of peptide is needed to compete out the band. Titration of the peptide is needed to find the right signal inhibition conditions, or 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. Subsequently, 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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