How To Prepare Your Antigen For Custom Antibody Production

There are a couple of general rules to follow having to do with protein size, protein concentration, vehicles (i.e., buffers), other chemicals in the vehicles, and form of the protein:

1. Protein Size: In general, the immune system doesn’t recognize small molecules, so there is a minimum size for a protein to become “immunogenic.” Although the exact cutoff depends on the protein, we recommend that the proteins under 20 kDa in size (about 200 amino acids) be coupled to something bigger in order to make them maximally immunogenic. This can be done by treating the protein with a bifunctional cross-linking agent (such as glutaraldehyde or paraformaldehyde) or by conjugating the protein to keyhole limpet hemocyanin (KLH).

2. Protein Concentration: It’s difficult to predict a priori what concentration of protein is necessary to inject, but we recommend using at least 2 mg of protein in a total volume of 5000 ul of buffer. This is probably a generous amount for highly immunogenic proteins, but with poorly immunogenic proteins, it’s probably necessary. In any cse, there doesn’t seem to be a maximal limit, so “the more, the better” is our motto.

3. Vehicles Buffers: The ideal buffer for injections is phosphate-buffered (10 mM, pH 7.2) isotonic saline (also called “PBS”). It’s “ideal,” because this is the buffer than Freund used so many years ago when he was perfecting his adjuvants. It’s also good because it doesn’t contain primary amine groups, which will interfere with conjugations using aldehydes or NHS-groups, in case the protein needs to be coupled to make it bigger. Tris buffered saline (“TBS”) is fine for injections, too, but it DOES contain a primary amine group, so it can’t be used when conjugations are required – yet another reason to stick with PBS!

4. Other Chemicals in the Vehicle: This issue is tricky, and if you have any questions, it’s best to call us for our opinions on your particular vehicle. But the rule of thumb is that if a chemical is toxic to the hens, or even if the chemical is an irritant, it should NOT be present in the vehicle. The reason is simple – dead chickens don’t lay eggs, and even hens that have been irritated a little bit, often go into a molt where they don’t lay eggs.

Chemicals that are commonly found in some preparations, and that are fine for including in vehicles include tris and imidazole (anything below 5 mM),

Detergents are problematic for another reason entirely – they disrupt the emulsification that is necessary to create a good adjuvant. Emulsification is the process by which miscelles of aqueous buffer (containing the protein) become surrounded by oil. This process is important, because it allows the protein to be released slowly into the hen’s connective tissue, which is the preferred time for delivery for antibody production. Consequently, we strongly recommend dialyzing away detergents – both ionic detergents (e.g., SDS) and non-ionic detergents (e.g., triton X-100, tween-20, etc.). Again, they’re not toxic to the hens per se, they just interfere with the emulsification.

The worry that some clients have, though, is that their protein will come out of solution. These clients will, however, be pleased to hear that insoluble proteins often make the best preparations for antibody production! Although the reason isn’t entirely clear, it is likely that insoluble proteins remain at the site of injection for a longer period, allowing them to interact with the antigen-presenting cells. So, if your protein comes out of solution while you’re making your injection mixture, celebrate!

5. Form of the Protein. We can inject proteins in solution or proteins in polyacrylamide gels.

5A. Proteins in Solution. The easiest and best way to prepare your protein is simply to put it into PBS at a concentration of 2 mg in 5000 ul (400 ug per ml). Do NOT put in anti-microbials such as sodium azide or anything else that will harm (or irritate) the hens.

5B. Proteins in Polyacrylamide Gels. If your protein is contaminated with other proteins, you might want to run an SDS-polyacrylamide gel to separate the protein of interest from the contaminants. If you do, run a thick gel (at least 1 mm in width) and then use a stain that doesn’t fix the protein inside the gel. The product we recommend is one from Thermo called Pierce Zinc Reversible Stain Kit. Once the gel is stained, the staining solution should be removed, allowing the protein to diffuse from the gel and stimulate the immune system. Do not use Coomassie stains, as these end up fixing the protein inside the gel, and the protein can’t get out.

An important trick with gels is to minimize the volume of the gel itself. Be sure to cut out the band WITHOUT extra bits of gel around. It is best to keep the gel volume under 1.0 ml (in a total volume of 5.0 mls PBS).