FAQ
General
Q. How should TiterMax® be stored?
A. TiterMax® contains the following:
- Block copolymer CRL-8941
- Microparticulate silica coated with CRL-8941
- Sorbitan Monooleate 80
- Squalene
Q. What is block copolymer CRL-8941?
A. CRL-8941 is a synthetic polymer composed of blocks or chains of hydrophobic polyoxypropylene (POP) and hydrophilic polyoxyethylene (POE). The adjuvant activity of block copolymers was described in 1981 by Hunter et al. CRL-8941 was selected for its adjuvant activity which is typical of other block copolymers, and its ability to form stable water-in-oil emulsions with squalene.
Q. How should TiterMax® be stored?
A. TiterMax® should be stored at 4° C.
Q. Can TiterMax® be stored and re-used after it has been emulsified with an antigen?
A. A 50:50 water-in-oil emulsion can usually be stored at room temperature, 4° C, -20° C, or -70° C, for as long as your antigen is stable. Upon storage, approximately 20% of the oil will disassociate from the emulsion. You may leave the emulsion in a syringe and simply re-emulsify it when you are ready to use it again for injecting. The stability of an emulsion will depend upon the inherent stability of the antigen.
Q. Is TiterMax® reliable?
A. TiterMax® has the reliability and reproducibility of FCA. TiterMax® can be used reliably with virtually any antigen because, like FCA, TiterMax® can entrap any antigen in a water-in-oil emulsion, which is critical for dependable adjuvant activity. In contrast, many new adjuvants simply bind antigens by adsorption. If the antigens do not adsorb, they seldom induce high antibody titers. TiterMax® combines the benefits of a water-in-oil emulsion with those of potent block copolymer adjuvants, yielding highly reproducible results.
Q. In what volumes may I purchase TiterMax®?
A. TiterMax® can be purchased in 0.5, 1.0, 5.0, and 10.0 ml vials to fit all of your research needs.
Efficacy
Q. What is TiterMax®’s efficacy in producing:
| Cell-Mediated Immune Response (CMI) | Some investigators report good responses |
|---|---|
| Delayed-Type Hypersensitivity (DTH) | Does produce a mild response |
| Autoimmune Disease | These have been produced only when immunizing with specific antigens. The adjuvant itself does not induce autoimmune disease. |
| Uveitis | These have been produced only when immunizing with specific antigens. The adjuvant itself does not induce Uveitis. |
| Adjuvant Arthritis | Does not cause |
| Cytotoxic T-cells | Some investigators report good responses |
There is no standard protocol for monoclonal antibody production; many different methods have been effective. Furthermore, monoclonal antibody protocols do not depend upon the adjuvant except insofar as the adjuvant is used to increase the number of antibody-producing cells. TiterMax® can be expected to work with any antigen that has been successfully used with immunizations with FCA.
Q. Does TiterMax® produce high antibody titers?
A. Optimal immunization schedules may vary depending on the antigen, species of animal, and needs of the researcher. TiterMax® produces antibody titers, which are acceptably high for most applications without boosting. However, it is safe to boost with TiterMax®. Since TiterMax® is a water-in-oil emulsion, we believe that it can be used in any protocol for inducing antibody titers that have proven successful with FCA. TiterMax® saves time and money by generating high titers rapidly, increasing the productivity of your research staff while enabling you to achieve the immune response you desire.
Emulsion
Q. How do I prepare TiterMax® for injection?
A. Emulsions of TiterMax® can be prepared using any technique that works with FCA. However, the volume per injection is measured in ul There are several methods from which you may choose, depending on the equipment you have available and the volume of emulsion you are going to prepare. The two methods we find to be the best in terms of simplicity and recovery of emulsion are the two syringe, double hub needle method, and the Kontes Pellet Pestle® homogenizer method. The latter method is especially suited to small volumes.
Q. What is the optimum emulsion ratio and antigen concentration range for TiterMax®?
A. Good immune responses have been achieved with ratios of water to TiterMax® of 50 to 90%, but the 50:50 water-in-oil emulsion is usually optimal. As you increase the water content of the water-in-oil emulsion, remember that you are decreasing the amount of the active ingredient in the oil phase. The investigator must carefully titrate the dose and emulsion formulation for each individual antigen. We have been successful in producing high antibody titers with an antigen concentration range of 15 to 125 ug/dose, depending on the antigen.
Q. What is the optimum emulsion ratio and antigen concentration range for TiterMax®?
A. Good immune responses have been achieved with ratios of water to TiterMax® of 50 to 90%, but the 50:50 water-in-oil emulsion is usually optimal. As you increase the water content of the water-in-oil emulsion, remember that you are decreasing the amount of the active ingredient in the oil phase. The investigator must carefully titrate the dose and emulsion formulation for each individual antigen. We have been successful in producing high antibody titers with an antigen concentration range of 15 to 125 ug/dose, depending on the antigen.
Q. I am trying to make an emulsion with TiterMax®, but it is still a liquid. My protein is formulated in Tris buffer. What can I do to make my emulsion the proper consistency?
A. First, ensure that you followed the emulsification directions correctly, specifically vortexing the TiterMax® prior to preparation. Several researchers who have used Tris buffer have noted they get a liquid consistency. Try adding small amounts of TiterMax® to your emulsion until it thickens to the proper consistency (similar to whipped cream). A droplet of the emulsion should float on water.
Q. My antigen contains 0.5% Triton X-100. Will this break the TiterMax® emulsion due to its detergent activity?
A. First, try 0.5% Triton without antigen with TiterMax® to see if the emulsion is stable. If the emulsion is stable, it should work fine with the antigen. In the past, 1% Triton with TiterMax® has been used successfully.
Q. Can I emulsify my antigen with TiterMax® if my antigen is contained in a band of an SDS-PAGE (polyacrylamide gradient electrophoresis) gel? Also, can I use glass syringes for the emulsification?
A. First, ensure that your IACUC will approve the injection of acrylamide. If this is approved, cut the band out of the gel. Do not worry if there is a residual amount of buffer remaining in the gel. Pull up the gel into the syringe, estimating the volume. Mix with the appropriate amount of TiterMax®, according to the emulsification method being used. Glass or plastic syringes can be used for the emulsification.
Toxicity
Q. Why is TiterMax® less toxic than Freund’s Complete Adjuvant (FCA)?
A. Toxicity of FCA is due to the combination of mineral oil with mycobacteria. TiterMax® contains no protein, peptide, or plant component. It consists of a metabolizable oil, squalene, which is a normal body component, and a newly developed block copolymer adjuvant. TiterMax® is formulated with emulsifying and stabilizing agents selected to provide an optimal balance between efficacy and toxicity for research applications. Local inflammatory reactions occurring with TiterMax® vary with the nature of the antigen used and with the dose. In our studies, confirmed by an independent testing laboratory, only mild and transient reactions have been observed. TiterMax® does not produce tuberculin hypersensitivity, granulomas, or adjuvant arthritis.
By incorporating both the antigen and adjuvant into a water-in-oil emulsion, TiterMax® is effectively delivered to the immune system for successful immunization while minimizing or eliminating the side effects of FCA.
Q. What is the maximum dose I could give a rabbit or mouse and not experience toxicity?
A. There should be no toxicity problems with injections of 50 ul or less per injection site. It is, however, dependent upon the route of administration and your antigen.
Immunization
Q. What are the preferred routes of injections?
A. Dosing regimens that have proven successful with other adjuvants may certainly be used with TiterMax®. Principle investigators have found that the subcutaneous route of injection frequently gives better antibody responses than the intraperitoneal route of injection.
Q. I have injected my animals with FCA on the initial injection. Can I boost with TiterMax®?
A. There should be no toxicity problems with injections of 50 ul or less per injection site. It is, however, dependent upon the route of administration and your antigen.
Q. How soon after injecting my mice initially can I begin bleeding? Can I bleed them weekly? Should I boost?
A. Wait 4-6 weeks after your initial immunization to begin bleeding. You can bleed the mice weekly as long as 0.25-0.30 ml of blood per week is not exceeded. If titers begin to decline, you may boost using either just your antigen or half of the immunizing dose. For best results, wait another 4 weeks after boosting to bleed.
Q. What volume per injection do you recommend when mice are immunized intraperitoneally with TiterMax®?
A. When injecting mice intraperitoneally with TiterMax®, I would suggest using a total emulsion of 0.1-0.5 ml. Most investigators have found optimum results with 0.5 ml. You may go up to 1.0 ml if necessary, but this is not recommended. If possible, we recommend using the subcutaneous route over the intraperitoneal route.
Q. I am going to be immunizing rabbits, should I boost? Several of my colleagues have seen Arthus’s reactions after boosting.
A. When injecting mice intraperitoneally with TiterMax®, I would suggest using a total emulsion of 0.1-0.5 ml. Most investigators have found optimum results with 0.5 ml. You may go up to 1.0 ml if necessary, but this is not recommended. If possible, we recommend using the subcutaneous route over the intraperitoneal route.
Antigens
Q. I store my antigen with sodium azide or thimerosal. Will it affect the shelf life of the TiterMax® emulsion?
A. Neither compound will affect the shelf life of the emulsion, although at high concentrations of either, the emulsion could become toxic.
Q. With what antigen does TiterMax® work best?
A. At this time, several investigators have reported success with the antigens listed below. TiterMax® appears to work particularly well with peptides and other poorly immunogenic materials. With both proteins and peptides, several investigators have reported that TiterMax® induces antibodies against epitopes with specificities that were not responding when using Freund’s. TiterMax® does not work effectively with pure carbohydrates without a protein component. Additional input is being received regularly, and data is being compiled. For specific questions regarding your antigen, call our technical service number: 1 (800) 345-2987.
- small peptide
- plant enzymes
- small peptide conjugated to KLH or BSA
- cellular antigens
- bacterial antigen (E. Coli)
- acrylamide gels
- thyroid receptors
- LPS
- elastase inhibitor proteins
- DNA (nuclear peptide or protein)
- membrane proteins
- Carbohydrate-protein conjugates
- whole tumor cell lines
Q. If my antigen contains surfactants, will it interfere with TiterMax® emulsion?
A. Immunogens that contain high concentrations of surfactants or other materials may interfere with emulsification. We have found that SDS, which may be present in acrylamide gels in concentrations greater than 1% or urea concentrations greater than 1.0M, significantly reduces the emulsifying capacity of TiterMax®.
Other similar materials are likely to have the same effect. Some surface active agents serve as demulsifying agents that break emulsions. The modern emulsifiers and microparticulate stabilizers of TiterMax® are able to overcome the effects of most such agents present in moderate quantities.
Please email us with any unanswered questions you may have.