Antigens & Immunization

How much antigen is required for immunizations and screening?

In general, 2-5 mg of antigen for immunizations and 1-3 mg for screening. We can adjust project strategies if the antigen is difficult to isolate or not readily available.

Why is adjuvant used?

Adjuvants enhance the cellular immune response against the injected antigen. When mixed with antigen, they help deposit or sequester the injected material. The effect of adjuvants on antigen immunogenicity is uncertain.

How long does it take to produce an adequate immune response?

Mice typically receive 3-6 injections over 2 to 3 months. The first injection exposes the cells of the immune system to the antigen. Subsequent booster injections are needed to increase the number of Ig-secreting plasma cells.

What happens if no immune response is produced?

The immune response of any given animal is dependent on several variables in addition to the immunization time frame. Important variables include: the size of the antigen; the genetics of the mouse; the evolutionary distance between the species of the antigen and the immunized animal; how the antigen is processed by the immune system; the amount of antigen given in each immunization. Because of these variables, there may be little or no response to a given antigen. If this occurs we will work with you to outline more aggressive immunization protocols or suggest a change in your antigen. Alternative immunization protocols will be recommended if serum titers are unacceptable for fusion.

How do you determine immune response?

We define titer as the dilution of serum at 50% maximum signal using a solid-phase ELISA. If you require antibodies that function in other immunoassays, we will send you the sample sera for testing.

Should my antigen be coupled to a carrier?

Haptens, peptides, and weak or nonimmunogenic antigens may require conjugation to carrier proteins, such as keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin.

What protein sequence should I select if I want to make an antibody to a peptide?

Longer is better (greater than 12 residues) and lots of hydrophilic residues (Glu, Asp, Arg, Lys). Consider putting a Cys at one of the ends for coupling to a carrier. Your peptide should be soluble in aqueous solvents. Although there are a number of predictive algorithms for picking sequences, we know of no data saying that one is better than any other. Try the Hopp-Wood analysis. It’s been around for awhile and certainly biases toward charged residues.

Do you make peptides?

We don’t. But we can suggest some excellent companies who do. If you have a favorite synthesis company, ask if they will determine coupling efficiency. Not all peptides couple to carrier with high efficiency and this is a project stopper if peptide is not conjugated. Our recommendation is to use amino acid composition to determine coupling efficiency.

How pure does my antigen need to be?

As pure as you can make it. Remember, contaminants are usually as good at producing antibodies as your antigen of interest. There are strategies for using non-homogenous preparations of antigen but please talk to us first.

My expressed protein has GST (or His, or Flag). Will it matter if I don’t remove it?

Yes, it will make a difference. We’ve made lots of antibodies through the years to GST. Some even have specificity for the non-GST part but require GST for binding. Our recommendation is to remove the expression tags for screening. If this is a problem, call and we can discuss alternative strategies.

My antigen is hard to make (or purify). What if I only have 0.5 mg?

At the present time, our personal best is generating two specific monoclonal antibodies using 120 ug total protein. Talk to us but remember: if it doesn’t look like we can do it or that you won’t be happy with the length of time it will take us, it’s probably best to go back to the drawing board and redesign how and where the antigen will come from. Our procedures, processes, and technical staff are only as good as your antigen.

 

Return to Technical Support