Technical Information

Properties | Toxicity | Differences | Immunization | Hybridomas | Emulsification | Storage | Bibliography

• Block Copolymer CRL-8941
• Squalene, a Metabolizable Oil
• A Unique Microparticulate Stabilizer

Like FCA, TiterMax® can be used with a wide variety of antigens because it can entrap any antigen in a water-in-oil emulsion.

Since TiterMax® #R-1 contains no mycobacteria or mineral oil, one would not expect it to induce adjuvant arthritis or the severe systemic granulomatous reactions characteristic of FCA. In our studies, we have seen no evidence of such reactions.

This included many years of experience with block copolymer adjuvants and microparticulate stabilized emulsions in addition to work with the TiterMax® #R-1 formulation, including work done at an independent testing laboratory (Hazleton). TiterMax® has been optimized for the induction of IgG antibody and has little ability to stimulate delayed-type hypersensitivity reactions, which are responsible for the most serious complications of FCA. Most of the studies conducted in the development of the TiterMax® #R-1 formulation utilized subcutaneous foot pad injections in mice specifically to facilitate quantitative assessment of local reactions.

On several occasions, we have observed inflammatory reactions which begin at two to three weeks after injection, intensify for a week, and then gradually subside. They have the characteristics of local antigen antibody-complement (Arthus) reactions rather than the delayed-type hypersensitivity granulomas characteristic of FCA. With superficial subcutaneous injection, the reactions may resemble an abscess and drain. The most severe reactions were seen following the injection of a total volume of 1 ml of adjuvant distributed equally among four sites of a 400-gram animal. Only mild reactions were observed when the same antigen was injected in doses of 20 to 50 µl per site. Other severe local reactions were observed in animals following the injection of TiterMax® plus E. coli lipopolysaccharide (endotoxin). Consequently, TiterMax® does not counteract and may exacerbate the toxicity of some antigens.

While TiterMax® has been remarkably non-toxic in our hands, its production of high antibody titers may make the animals susceptible to localized Arthus reactions at the site of immunization, particularly when boosting. This can be minimized without compromising titers by injecting the antigen emulsion in multiple sites using small volumes. Many investigators find that TiterMax® produces highly acceptable titers without boosting. If boosting is desired, however, it is advisable to measure the antibody titer first. If it is high, then boosting should be done with soluble antigen without adjuvant and/or distributed among several sites.

TiterMax® contains no mineral oil and no protein, polysaccharide, or other microbial products. In order to achieve successful immunization, the antigen must be delivered to the lymph node together with the adjuvant. The most effective way to accomplish this is to incorporate both into a water-in-oil emulsion. TiterMax® has been specially formulated to maximize the desirable effects of water-in-oil emulsions while minimizing or eliminating the undesirable side effects of FCA. Table 1 summarizes the composition of several commercially available adjuvants.

*TiterMax® contains no mineral oil and no protein, polysaccharide, or other microbial products.

Adjuvants influence the titer, duration, isotype, and avidity of antibody in addition to their well-known effects on cell-mediated immunity. Recent investigations have shown that their influence extends even to inducing class I restricted CD-8 positive cytotoxic T lymphocytes and modulating the specificity of antibody among available epitopes on protein antigens. It has been known for many years that FCA preferentially induces antibody against epitopes on denatured proteins, while other adjuvants preferentially stimulate antibody against epitopes on native proteins. B epitope selection by adjuvants is seldom considered in experimental protocols even though it has been correlated with biological activity such as protection against simian immunodeficiency virus. TiterMax® was designed to maximize the production of high avidity antibody titer representing the spectrum of IgG subclasses while minimizing the undesirable induction of granulomatous delayed-type hypersensitivity.

Comparison Data

Procedures

Studies were conducted with various research adjuvants. Six to eight-week-old female C57BL/6 mice (10 per group) were immunized with a constant dose of 25 µg ovalbumin with each commercial adjuvant. Optimal dosing regimens for TiterMax® Gold were determined. Secondary immunizations were with antigen and adjuvant. All commercial adjuvants were prepared, injected, and boosted according to the manufacturers’ protocols. Serum samples were obtained at various times after immunization (days 28, 56, and 84), and IgG antibody titers against ovalbumin were determined using a microtier plate ELISA. Data is expressed as units/ml against an in-house standard.

Results

Effects of Injection Route on Efficacy of TiterMax®
Figure 6 shows the time course of anti-TNP antibody titers achieved using TiterMax® in mice immunized with a single dose of antigen using one of three routes: subcutaneous at the base of the tail, intraperitoneal, or intraplantar in one hind footpad. The data show that TiterMax® works well without boosting by several injection routes, with the intraplantar route superior. Four weeks after a single injection, titers of >100,000 were readily achieved. The subcutaneous route yielded a titer of >60,000, while the intraperitoneal route achieved titers >40,000. After eight weeks, the intraplantar route had produced titers >500,000, with the subcutaneous injection yielding titers >300,000 and the intraperitoneal route producing antibody titers >80,000. In general, during the first month after immunization, both the titers and the slope of their rise were comparable, regardless of route. However, the intraperitoneal route did not produce the persistent titers reproducibly, as seen with the other routes, probably because of the shortened survival of the antigen depot.

Figure 6. Effects of Injection Route on Efficacy of TiterMax® #R-1. Mice were immunized with a single dose of TNP-HEA (50 µl) in TiterMax® (total volume 40 µl) using one of three routes. Sera were collected at intervals, and antibody titers were measured using ELISA. Antibody titers are expressed as mean ± sem. Immunization with multiple small volumes of TiterMax®/antigen emulsion. 

FCA is frequently injected in multiple subcutaneous sites on the back and flank of animals in order to increase titers and reduce local inflammation. Although we saw essentially no signs of inflammation with TiterMax® in any of the experiments described here, we evaluated protocols using antigen with TiterMax® in multiple injection sites. These might be useful for investigators using more toxic antigens. In the experiment shown in Figure 7, animals injected with identical volumes of TNP-HEA distributed over four subcutaneous sites gave higher antibody titers than those in which the antigen was distributed between only two sites. Thus, distributing antigen among several sites may facilitate immunization with TiterMax®.

Figure 7. Effect of Number of Injection Sites on Antibody Titer. Mice were immunized with a single dose of TNPHEA (50 µg) in TiterMax® (total volume 40 µl). The antigen emulsion was injected subcutaneously into either two or four flanks. Sera were collected at intervals, and antibody titers (mean ± sem) were measured via ELISA.

IgG antibody subclass distribution

Figure 8 illustrates the IgG subclass distribution of anti-TNP antibody using TiterMax® in mice immunized with a single dose of antigen injected in one hind footpad. Sera were collected after 28 days, and antibody titers were measured. Unlike the IgG1 predominance seen with FCA, TiterMax® produced considerable proportions of the IgG2a and IgG2b subclass as well. Interestingly, in these studies with outbred mice, the range of IgG2a responses was broader than that of the other subclasses, suggesting a stronger genetic component to the regulation of IgG2a production. Protection against a variety of infectious agents appears to be subclass-dependent, with the IgG2 isotypes frequently implicated in effective immunity.

Figure 8: IgG Subclass Distribution Following TiterMax®/TNP-HEA Immunization. Mice were immunized with a single dose of antigen in TiterMax® injected in one hind footpad. Plasma samples were collected 28 days later, and antibody titers were measured via ELISA. Each point represents data from an individual animal.

Studies on delayed-type hypersensitivity

TiterMax® has not been extensively evaluated for induction of delayed-type hypersensitivity (DTH), although the nature of the adjuvant and preliminary data strongly suggest that it preferentially stimulates humoral immunity. In one experiment to evaluate DTH, three Hartley guinea pigs were immunized with 100 µg TNP-HEA in TiterMax® (80 µl intramuscularly, divided between the hind flanks). Animals were boosted on day 28 with 100 µl soluble TNP-HEA (intramuscularly, divided between the hind flanks). Skin tests were performed by injection TNp-HEA (50 µg in 50 µl intradermally) six days and, into different sites, 14 days later. After 24 hrs, no site injected on day six showed erythema or induration, while those injected on day 14 showed only about 1 cm. of erythema. After 48 hrs, all sites injected on day six remained negative, no induration. Antibody titers on day 14 were 45,299±3,261 (mean ± sem). In other experiments, we have failed to induce adjuvant arthritis in Lewis rats under conditions where all of the control FCA-treated rats succumb.

TiterMax® can produce high titers without boosting

The duration of an antibody response has been related to the persistence of antigen. One would predict that an adjuvant such as TiterMax®, which forms stable depots of water-in-oil emulsion in tissue, might produce long-lasting responses. In addition, it should facilitate the generation of an antibody response with only a single injection of antigen. This was found to be the case with TiterMax® (Figure 4). Whether mice were injected with only a single dose of TNP-HEA in TiterMax® or boosted after 28 days, the antibody titers reached high levels by six weeks. In unboosted animals, the titers began to decline after a few months, but they rose again after seven months and, at one year, were comparable to levels in boosted animals.

Figure 4. High, Long-Lasting Titers Can Be Achieved with TiterMax® without boosting. Mice were immunized with 100 μg of TNP-HEA in TiterMax®(80 μl, split between two hind footpads). One group was boosted with 100 μl of TNP-HEA in TiterMax®(80 μl, split between two hind footpads), while the other group was not. Antibody titers (mean ± sem) were measured via ELISA.

Figure 5. High, Long-Lasting Titers Can Be Achieved with TiterMax® When Boosted with Soluble Antigen. Mice were immunized with 50 μg of TNP-HEA in TiterMax®(40 μl, split among the four footpads). On day 35, one group was boosted with 25 μg of TNP-HEA in TiterMax®(20 μl, split between two hind footpads), while the other group was boosted with 25 μg of TNP-HEA in saline (20 μl, split between two hind footpads). Antibody titers (mean ± sem) were measured via ELISA.

Boosting may be accomplished with soluble antigen 

We evaluated the relative efficacy of boosting with soluble antigen vs. boosting with antigen in TiterMax® in the following experiment. Mice were injected with TNP-HEA, and after 35 days, they were boosted with either antigen in saline or antigen in TiterMax® emulsion. Interestingly, antibody titers showed a transient depression after boosting with antigen in TiterMax®. However, both groups showed strong titers, which persisted throughout the six months of the experiment (Figure 5).

Antibodies to TNP

The efficacy of TiterMax® in various species was evaluated by measuring antibody titers 14 and 28 days following a single immunization. Groups of mice received 50 μg of TNP-HEA emulsified with 20 μl of TiterMax® in a total volume of 40 μl injected subcutaneously at the base of the tail. Rats, guinea pigs, and rabbits each received 100 μg of antigen emulsified with 40 μl of TiterMax® in a total volume of 80 μl equally distributed between two sites (except for the intraperitoneal route). Rabbits and guinea pigs received intramuscular flank injections, while rats received intraplantar, subcutaneous, or intraperitoneal injections. The results are summarized in Table 3. All four species produced significant antibody titers, although the magnitude and duration of the response varies with species and route of immunization. The antibody titers of both rabbits and mice increased 5 to 6-fold between days 14 and 28 but were essentially unchanged in rats and decreased in guinea pigs. In rats, the intraplantar route was superior to either the subcutaneous or intraperitoneal routes. Thus, TiterMax® was effective in four different species of experimental animals. Immunization protocols should be tailored to each experimental system.

Table 3. Efficacy of TiterMax® in Various Species

• Number of animals per group: 5 mice, 4 rats per route, 2 guinea pigs, 3 rabbits
• Mice received a total volume of 40 µl; all other groups received 80 µl
• Antibody titers (mean ± sem) were measured using an ELISA

Antibodies to BSA

Studies performed by Hazleton Laboratories evaluated TiterMax® against several other commercially available adjuvants in rabbits, mice, and goats immunized with LHRH-BSA. IgG antibody titers against BSA are shown in Table 4. Eight weeks after immunization, mice who received TiterMax® had 1.5-fold higher antibody titers than those who received FCA, while rabbits had 3.5-fold higher titers. Goats showed the highest antibody titers with FCA, but in all three species, TiterMax® induced at least 4.5-fold higher titers than either Adjuvax(TM) or RAS.

Table 4. Anti-BSA Titers in Different Species

Groups of experimental animals were immunized with a total of 50 µl LHRH-BSA in several adjuvants according to the manufacturers’ protocols. All groups were injected on day 1 and boosted on day 28 (TiterMax® and FCA groups), day 21 (RAS group), or day 21 and 35 (Adjuvax(tm) group). The second injection in the FCA group was with incomplete Freund’s adjuvant. Results from week 8 are shown. IgG antibody titers (mean ± sem) against the carrier BSA were measured using an ELISA.

Antibodies to peptide

Antibody activity against the LHRH peptide was measured using a radioimmunoassay with labeled peptide. Table 5 shows that immunization with TiterMax® induced anti-peptide titers comparable to FCA in all three species.

Table 5. Antibody to LHRH Peptide in Different Species

Groups of experimental animals were immunized, as described in Table 4. Results from week 8 using sera are shown. Antibody activity against the LHRH peptide was measured using a radioimmunoassay with labeled peptide. Data (mean ± sem) for the percent peptide bound using a serum dilution (final concentration 1:200 for rabbits; 1:100 for mice; 1:5 for goats) in the linear portion of the dose-response curves are shown. TiterMax® induced anti-peptide titers were comparable to FCA in all three species.

Many investigators have asked us for a TiterMax® immunization protocol suitable for priming spleen cells for infusion with myeloma cells to produce monoclonal antibodies. There is no standard protocol for monoclonal antibody production; many different methods have been effective. Furthermore, monoclonal antibody protocols do not depend on 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 in immunizations with FCA. For weak antigens or antigens available in minute amounts, the immunization protocols must be adapted so that the animals are producing detectable antibodies prior to fusion, a prerequisite for success. While no protocol can be written to anticipate every immunogen of interest, certain principles related to immunization can be considered in fine-tuning a general protocol for a specific immunogen.

Factors to consider when choosing the immunogen include the degree of purity, whether denaturation will impact the desired application, and the need to chemically aggregate small molecules or attach them to carriers to enhance their immunogenicity. These are beyond the scope of this document, but detailed information can be obtained from review articles, monoclonal antibody directories, or specific publications about monoclonal antibodies against immunogens similar to the one of interest.

The choice of animal species and strain as immune spleen cell donor for fusion depends on the myeloma cell line available and the origin of the immunogen. Mice are most commonly used, with the BALB/c strain preferred since most murine myelomas competent for fusion were derived from that strain. If a low response to a particular antigen in BALB/c mice requires immunization of other inbred strains, the hybridomas can be propagated in the appropriate F1 hybrid. Rats (LOU/C or LOU/M strain) are used when monoclonal antibodies against murine antigens are desired or to produce large amounts of ascites. Hamsters have been used to produce monoclonal antibodies against mouse antigens to which rats do not respond. In specialized cases, protocols have been designed to eliminate unwanted monoclonal antibodies against particularly immunogenic epitopes by treating the animals with cytotoxic agents immediately after the initial immunization.

Immunization procedures used by various investigators to produce monoclonal antibodies differ widely. Immunization is started by injecting 1-125 µg antigen per mouse or rat (in groups of 5 to 10) either intraperitoneally or subcutaneously. Soluble antigens have usually been administered with an adjuvant such as FCA. The animals are boosted after 10 days to three weeks, generally with the same dose as used in the original immunization. Multiple booster injections (as many as seven times weekly or every other week) have been used with weak antigens. Most protocols boost with antigen in incomplete Freund’s adjuvant, although some investigators have used multiple injections with FCA. This will result in severe inflammatory reactions and is to be discouraged.

Five to seven days following a booster immunization, serum may be tested for antibodies. This guides the investigator in deciding how long to pursue the immunization. It also ensures that the screening assay which will be used to screen the hybridoma supernatants is perfected.

Animals that show the highest antibody titers are selected for further immunization. At least three to four weeks after the last boost and three to four days before fusion with the myeloma cells, the animals are challenged with intraperitoneal or intravenous injection of antigen in aqueous solution to stimulate dividing plasma cell blasts in the spleen. Some investigators use two intraperitoneal injections at day -4 and -3, followed by intravenous injection on day -2; others barrage with 1/5 to 1/10 the original dose of immunogen on each of the four days before fusion. Most investigators administer a single intravenous antigen dose on day -3 or an intraperitoneal injection on day -4, followed by an intravenous injection on day -3. In some situations, the adoptive transfer may be used to enrich antibody cells before the fusion. In that case, spleen cells from immunized mice are transferred into lethally irradiated (750 R) recipients together with antigen. The donor spleen cells repopulate the recipient spleen, and four to six days after transfer, the recipient spleen contains a high proportion of antigen-specific antibody-forming cells suitable for fusion with the myeloma cell line.

Do not perform with myeloma cells until a satisfactory antibody titer has been achieved.

1. Prepare emulsion of equal volumes of antigen (300 ug to 2.5 mg/ml for protein antigens) and TiterMax®#R-1 Research Adjuvant according to the TiterMax® instruction brochure.
2. Day 0. Immunize a group of 5 to 10 two-month-old BALB/c mice by injecting 50 ul of the antigen TiterMax® emulsion containing 15-125 ug antigen subcutaneously in two sites at the base of the tail (25 ul into each) using a 27 gauge hypodermic needle. Alternatively, the antigen emulsion may be injected intradermally into each hind footpad (25 ul into each).

   For relatively strong immunogens, a single immunization with TiterMax® will result in high titers of high avidity antibody 30 to 40 days after a single immunization. With weaker antigens, variable extents of boosting may be necessary.

3. Day 14. (Optional depending on antigen). Repeat immunization with antigen-TiterMax® emulsion.
4. Seven days after a boost or 30-40 days after a single immunization. Collect samples of serum from each animal. Do not pool them. Assay each antibody titer to the relevant antigen using the same assay that will be used for screening the hybridoma supernatants. If antibody titers are low (<1:100 by most assay procedures), repeat immunization with antigen in TiterMax® emulsion. Collect serum 7 days later and assay for antibody titer.

   If fusing lymph node cells with the myeloma cells, lymph nodes may be harvested whenever an adequate antibody titer has been achieved.

   If fusing spleen cells (more common), the animals must be boosted with soluble antigen to increase the proportion of antibody-producing cells in the spleen as described in step 5.

5. 3 to 4 weeks after last boost and 5 days before fusion with myeloma cells. Select 2 to 3 animals with the highest antibody titers. Inject intraperitoneally with 20 to 100 µg of soluble antigen on day 1, inject intravenously with the same amount of antigen on day 2, and harvest spleens on day 5. Prepare cells for fusion and proceed according to desired fusion, screening, cloning, and propagation protocols. Production of high-affinity antibodies may be enhanced by prolonging the period between the last boost and pre-fusion challenge and by challenging with lower doses (2-10 µg of antigen).

TiterMax® Research Adjuvant combines the benefits of a potent synthetic adjuvant (copolymer CRL-8941) with those of a microparticulate-stabilized, water-in-oil emulsion containing a metabolizable non-toxic oil, squalene. Emulsions of TiterMax® can be prepared using any technique that works with Freund’s Complete Adjuvant (FCA). There are several methods from which you may choose to emulsify your antigen, 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 emulsifying needle method and the Kontes Pellet Pestle® homogenizer method. The latter is especially suited to small volumes. A number of other methods suggested by recent TiterMax® users are also described. Each of these methods will produce stable water-in-oil emulsions in 1 to 5 minutes.

Stable water-in-oil emulsions are notoriously difficult to make. The TiterMax® formulation was developed to produce a very stable emulsion. TiterMax® was developed to meet the specific needs of investigators for an immunoadjuvant that is at least as effective as Freund’s Complete Adjuvant (FCA) and safer and easier to use. Like FCA, TiterMax® can be used with a wide variety of antigens. Please follow the step-by-step instructions carefully for the method you choose.

Method 2: Two-Syringe, 3-Way Stopcock

This method is suitable for emulsion volumes between 1 ml and 10 mls. Available 3-way stopcocks have larger bores than the 18 gauge double hub needles (Method 1), so that emulsification takes longer, and the syringes connected to 3-way stopcocks are often more difficult to hold. Recovery of emulsion is approximately 70 to 80%.

Materials:

1. TiterMax® Research Adjuvant
2. Two 3.0 ml all plastic or siliconized glass syringes (preferably lock tip).
3. One 18 gauge needle for withdrawing TiterMax® from the vial or syringe without needle or positive. Displacement precision pipette if you open the entire vial.
4. One 3-way plastic disposable or stainless steel reusable stopcock.
5. Antigen in saline or other suitable fluid (typical dose range in mice is 15 to 125 µg/ mouse).

Method 3: One Syringe, Blunt Needle

This method is useful for volumes less than 0.5 ml and has been used with volumes as low as 0.05 ml final emulsion volume. Recovery of emulsion is approximately 50 to 75%.

Materials:

1. TiterMax® Research Adjuvant
2. 1 ml all-plastic syringe
3. 18-gauge x 1.5-inch blunt needle for emulsifying
4. 1.5 ml conical bottom plastic centrifuge tube
5. One 18-gauge needle for withdrawing TiterMax® from the vial or syringe without a needle or positive displacement precision pipette if you open the entire vial
6. Antigen in saline or other suitable fluid (typical dose range in mice is 15 to 125 µg/ mouse)

Method 4: Emulsifying in Syringe Using Sonication

This method is suitable for emulsion volumes between 0.5 ml and 2 ml. Recovery of emulsion is approximately 80 to 90%.

Materials:

1. TiterMax® Research Adjuvant
2. One 18 gauge needle for withdrawing TiterMax® from the vial or syringe without a needle or positive displacement precision pipette if you open the entire vial
3. One syringe for preparation of emulsion and immunization; piston removed; tip sealed with Parafilm®
4. Parafilm® for sealing the tip of the syringe
5. Antigen in saline or other suitable fluid (typical dose range in mice is 15 to 125 µg/ mouse)
6. Sonic dismembrator with microtip

Q. How should TiterMax® be stored?

A. TiterMax® should be stored at 4° C.

Q. What is the shelf life of TiterMax®?

A. TiterMax® has a shelf life of 24 months.

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 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. If 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.

Selected articles that reference TiterMax®:

1. Roberge, F.G., Xu, D. and Chan, C.C. A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis. Current Eye Research 11:371-376, 1992.
2. Bennett, B., Check, I.J., Olsen, M.R. and Hunter, R.L. A comparison of commercially available adjuvants for use in research. Journal of Immunological Methods 153:31-40, 1992.
3. Smith, D.E., O’Brien, M.E., Palmer, V.J. and Sadowski, J.A. The selection of an adjuvant for polyclonal antibody production using a low-molecular-weight antigen in rabbits. Laboratory Animal Science 42:599-601, 1992.
4. Kast, W.M., Brandt, R.M.P. and Melief, C.J.M. Strict peptide length is not required for the induction of cytotoxic T lymphocyte-mediated antiviral protection by peptide vaccination. European Journal of Immunology 23:1189- 1192, 1993.
5. Roberge, F.G., Xu, D., Chan, C.C., de Smet, M.D., Nussenblatt, R.B. and Chen, H. Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocyte growth factor signal transduction. Current Eye Research 12:197-203, 1993.
6. Broekhuyse, R.M., Kuhlmann, E.D. and Winkens, H.J. Experimental autoimmune anterior uveitis (EAAU). III. Induction by immunization with purified uveal and skin melanins. Experimental Eye Research 56:575-583, 1993.
7. Shenoy, M. and Christadoss, P. Induction of experimental autoimmune myasthenia gravis with acetylcholine receptors using a nonionic block copolymer as adjuvant. Immunological Investigations 22:267-282, 1993.
8. Urbanski, M. and Cone, R.E. T cell-derived antigen-specific humoral immune response. II. Further characterization of the response and the antigen binding T cell immunoproteins. Cellular Immunology 153:131-141, 1994.
9. Leenaars, P.P., Hendriksen, C.F., Angulo, A.F., Koedam, M.A. and Claassen, E. Evaluation of several adjuvants as alternatives to the use of Freund’s adjuvant in rabbits. Veterinary Immunology and Immunopathology 40:225-241, 1994.
10. Robinson, K., Bellaby, T. and Wakelin, D. Vaccination against the nematode Trichinella spiralis in high- and low-responder mice. Effects of different adjuvants upon protective immunity and immune responsiveness. Immunology 82:261-267, 1994.
11. McClimon, L.B., Glick, B. and Dick, J.W. Effect of three commercially available adjuvants on the production of antibodies to Pasteurella multocida in broilers. Avian Diseases 38:354-357, 1994.
12. Hunter, R.L., Olsen, M.R. and Bennett, B. Copolymer Adjuvants and TiterMax®. In: The Theory and Practical Applications of Adjuvants. D.E.S. Stewart-Tull, editor. John Wiley & Sons, New York, pp. 51-94, 1995.
13. Soong, C.J., Torian, B.E., Abd-Alla, M.D., Jackson, T.F., Gatharim, V. and Ravdin, J.I. Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen. Infection and Immunity 63:472-477, 1995.
14. Soong, C.J., Kain, K.C., Abd-Alla, M.D., Jackson, T.F. and Ravdin, J.I. A recombinant cysteine-rich section of the Entamoeba histolyticagalactose-inhibitable lectin is efficacious as a subunit vaccine in the gerbil model of amebic liver abscess. Journal of Infectious Diseases 171:645-651, 1995.
15. Dyall, R., Vasovic, L.V., Molano, A. and Nikolic-Zugic, J. CD4-independent in vivopriming of murine CTL by optimal MHC class 1-restricted peptides derived from intracellular pathogens. International Immunology 7:1205-1212, 1995.
16. Robuccio, J.A., Griffith, J.W., Chroscinski, E.A., Cross, P.J., Light, T.E., and Lang, C.M. Comparison of the Effects of Five Adjuvants on the Antibody Response to Influenza Virus Antigen in Guinea Pigs. Laboratory Animal Science 45:420-425, 1995.
17. Ke, Y., Hunter, R.L., Kapp, J.A. Induction of Humoral and Cytolytic Responses by Ovalbumin in TiterMax® and a New Synthetic Copolymer Adjuvant. Vaccine Research 4:29-45, 1995.
18. Wang, R., Charoenvit, Y., Corradin, G., Porrozzi, R., Hunter, R.L., Glenn, G., Alving, C.R., Church, P., and Hoffman, S.L. Induction of Protective Polyclonal Antibodies by Immunization with a Plasmodium yoeli Circumsporozoite Protein Multiple Antigen Peptide Vaccine. The Journal of Immunology 154:2784-2793, 1995.
19. Jennings, Veronica M. Review of Selected Adjuvants Used in Antibody Production. ILAR Journal 37:119-124, 1995.
20. Keohane, E.M., Takvorian, P.M., Cali, A., Tanowitz, H.B., Wittner, M., And Weiss, L.M. Identification of a microsporidian polar tube protein reactive monoclonal antibody. Journal of Eukaryotic Microbiology 43 (1):26-31, 1996 Jan – Feb.
21. Sjolander A., Lovgren Bengtsson K., Johansson M., and Morein B. Kinetics, localization and isotype profile of antibody response to immune stimulating complexes(iscoms) containing human influenza virus envelope glycoproteins. Scandinavian Journal of Immunology 43(2): 164-72, Feb 1996.
22. Wang, R., Charoenvit, Y., Corradin, G., De La Vega, P., Franke, E.D., and Hoffman, S. Protection Against Malaria by Plasmodium yoelii Sporozoite Surface Protein 2 Linear Peptide Induction of CD4+ T Cell- and IFN-g-Dependent Elimination of Infected Hepatocytes. Journal of Immunology 157(9): 4061-4067, Nov 1, 1996.
23. Guex-Crosier, Y., Raber, J., Chan, C-C., Kriete, M.S., Benichou, J., Pilson, R.S., Kerwin, J.A., Waldmann, T.A., Hakimi, J., and Roberge, F. Humanized Antibodies Against the a-Chain of the IL-2 Receptor and Against the bChain Shared by the IL-2 and IL-15 Receptors in a Monkey Uveitis Model of Autoimmune Diseases. Journal of Immunology 158 (1):452-458, January 1, 1997.