UNIT 2.4 Production of Polyclonal Antisera

 

Introduction

Antibodies are serum immunoglobulins with binding specificity for particular antigens. Although antibodies can be identified in the serum of individuals or patients that have been exposed to particular pathogens, the usual methods for eliciting antibodies involve immunization with purified or partially purified antigen preparations. Antigens used are most commonly proteins or peptides, but carbohydrates, nucleic acids, small organic molecules (haptens) conjugated to appropriate protein carriers, cells, and cell and tissue extracts can also be employed.

The first consideration is usually whether polyclonal or monoclonal antibodies are needed. Polyclonal antibodies are particularly valuable for immunoprecipitation and immunoblotting (see units 8.3 & 8.10, respectively), whereas monoclonal antibodies can have exquisite specificity and can be derived for almost any purpose. Choice of the species of animal to be used for immunization is based in part on whether antibodies of great specificity are required—in which case genetically defined strains can be very helpful—or antibodies of wide cross-reactivity are needed.

The amount of antibody needed must also be evaluated. Clones of hybridomas (somatic cell hybrids of B cells from an immunized animal's spleen and myeloma tumors permissive for the production of monoclonal immunoglobulins) provide an essentially limitless supply of a constant reagent. Nevertheless, the initial investment in producing a monoclonal antibody is quite large, whereas relatively large amounts of a polyclonal antiserum can be obtained from a single rabbit or from several genetically identical rats or mice.

In this unit, the Basic Protocol and the Alternate Protocol describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. The Support Protocol presents a method for preparing serum from blood. Polyclonal antipeptide antisera can be produced by substituting carrier-conjugated peptides (see unit 9.2) for the purified protein antigens.

 

Strategic Planning

Production of good antisera depends in large part upon the quality, purity, and amount of available antigen, as well as on the specificity and sensitivity of the assay. For protein antigens, if possible, the material should be biochemically homogeneous and, depending on the intended use, should be in either a native or denatured conformation. Be aware that minor contaminants are often (unfortunately) more antigenic than the immunogen of interest, and antisera resulting from immunization may have more activity against the contaminants than against the protein of interest. Antisera to be used for screening bacterial expression cDNA libraries or for immunoblots are best made against denatured protein, whereas those to be used for screening cDNAs expressed in eukaryotic transfection systems or for immunoprecipitation of native-cell-synthesized structures might best be made against native protein.

Although the advances offered by the development of monoclonal antibody techniques have revolutionized the specificity, uniformity, and quantity of antibodies, there remain many circumstances in which polyclonal antibodies are more desirable than monoclonal antibodies. Production of polyclonal antisera takes less time and effort than production of monoclonal antibodies, requires relatively simple and readily available equipment, and produces reagents that can be used for immunoprecipitation, immunoblotting, and enzyme-linked immunosorbent assays (ELISAs). In addition, with the advent of antibody-based proteomic strategies for profiling protein expression and post-translational modification, a requirement for high-throughput antibody production has emerged. Multiplexing of peptide and protein immunizations within a single rabbit allows rapid generation of specific antibodies while minimizing the number of animals used (Larsson et al., 2006). This approach has obvious benefits in terms of animal welfare and cost efficiency.

Choice of animal for the production of antibodies depends upon the amount of antiserum desired, the evolutionary distance between the species from which the protein of interest has been derived and the species of the animal to be immunized, and prior experience with the immunogens. Rabbits are the usual animal of choice because they are genetically divergent from the human and mouse sources of the proteins most often studied. Rabbits provide as much as 25 ml of serum from each bleed without significant harmful effects. For smaller-scale experiments, or for those that rely on precisely defined antibody specificities, inbred mouse strains may be the system of choice. Because mice are smaller, the volume of antigen suspension used for immunization is significantly less and the amount of serum that can be obtained from a single bleed does not exceed 0.5 ml. Rats and hamsters may be used when larger amounts of serum are needed, or when the greater evolutionary distance is advantageous. With repeated bleeding, as much as 5 ml of serum can be obtained from these species. Additional discussion of the choice of species for the production of monoclonal antibodies can be found in unit 2.5.

The choice of adjuvant for in vivo animal use has become problematic in recent years. Freund's adjuvant has been reliably and widely used for over fifty years (Freund et al., 1937). However, there is a degree of distress and discomfort to the animal associated with its use, which requires that the responsible investigator explore alternatives (McWilliam and Niemi, 1988). In most studies that have compared available alternatives to Freund's adjuvant, the Basic Protocol given here, which uses a CFA/IFA immunization regimen, gives far superior antibody titers to the commonly used commercially available adjuvants (Johnston et al., 1991; Deeb et al., 1992; Smith et al., 1992). However, the TiterMax adjuvant has now emerged as an effective alternative to CFA/IFA (Bennett et al., 1992; also see the TiterMax Web site at http://www.titermax.com). TiterMax has two significant advantages over CFA/IFA in that it is less toxic and can deliver high, sustained titers after a single immunization. While the use of TiterMax is described in the Alternate Protocol, the researcher should seriously consider employing this adjuvant over CFA/IFA. It should be kept in mind that not all antigens are equal; therefore, one adjuvant may perform better than another for a given antigen.

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals. The ethical issues associated with the production of polyclonal antisera in animals are addressed on many University Web sites. For example, the Duke University and Medical Center Web site provides a comprehensive guide to the selection of animals and alternative adjuvant choices (http://vetmed.duhs.duke.edu/guidelines_for_adjuvant_use.htm). It is also recommended that, before embarking on the production of antiserum, the researcher should read the report and recommendations of ECVAM (European Center for the Validation of Alternative Methods) on the production of polyclonal antibodies in laboratory animals (http://altweb.jhsph.edu/publications/ECVAM/ecvam35.htm). It is also highly recommended that appropriately trained animal technicians within the institute animal facility carry out the immunization and bleeding of the animals.

 

Basic Protocol: Immunization to Produce Polyclonal Antibodies Using Freund's Adjuvant

In the presence of adjuvant, the protein antigen is injected intramuscularly (i.m.), intradermally (i.d.), or subcutaneously (s.c.) into an animal of the chosen species. Booster immunizations are started 4 to 8 weeks after the priming immunization and continued at 2- to 3-week intervals. Prior to the priming immunization and following the primary and each booster immunization, the animal is bled and serum prepared from whole blood (see Support Protocol).

Instructions on the different strategies for immunization (i.m., i.d., or s.c.) can be found in unit 1.6 and for bleeding (from marginal vein or artery of ear for rabbit; various other sites for mouse, rat, or hamster) in unit 1.7. Factors important in preparing specific high-titer antisera, procedures for modifying protein antigens to enhance their immunogenicity, and choice of host animal are discussed in the Commentary. Protocols for immunization prior to production of monoclonal antibodies (unit 2.5) should be reviewed for these purposes.

 Materials

• Rabbit, rat, mouse, or hamster of appropriate strain
• Complete Freund's adjuvant (CFA; Sigma)
• 1 to 2 mg/ml purified protein antigen in PBS (appendix 2A)
• Incomplete Freund's adjuvant (IFA; Sigma)

• 50-ml disposable polypropylene centrifuge tubes
• 3-ml glass syringes with 19-, 21-, and 22-G needles
• Double-ended locking hub connector (Luer-Lok, Becton Dickinson) or plastic 3-way stopcock
• 100-ml beaker

• Additional reagents and equipment for animal restraint (unit 1.3), parenteral injection (unit 1.6), and blood collection (unit 1.7)

CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced i.d. or into the eyes and may cause profound sloughing of skin or loss of sight. Self-injection can cause a positive TB test and lead to a granulomatous reaction. Use gloves and protective eyewear when handling CFA.

 1.

Bleed the animal prior to immunization and collect blood sample (unit 1.7) in a 50-ml centrifuge tube. Prepare serum from blood, then assay and store (see Support Protocol).

p type = annotation

This pre-immune bleed is critical as a control to ensure that the antibody activity detected in later bleeds is due to the immunization.

 2.

Shake CFA to disperse insoluble heat-killed Mycobacterium tuberculosis bacilli. Add 2 ml CFA to 2 ml of 0.25 to 0.5 mg/ml purified protein antigen in PBS at 4°C.

p type = annotation

These volumes produce immunogen sufficient to immunize four rabbits or up to 80 mice. Do not use Tris-based buffers for generating the emulsion.

p type = annotation

An effective and simple method for preparing purified protein antigen is by preparative SDS-PAGE (unit 8.4). As much as 2 mg of a homogeneous protein can be loaded across multiple precast gels (1.5-mm thick gel, 0.5-cm wide comb). Following electrophoresis, an edge can be sliced off with a razor blade, fixed and stained, and used to identify the region containing the protein band. The gel slice containing the protein may then be directly added to several milliliters of PBS (appendix 2A) and emulsified as described below with an equal volume of CFA. The acrylamide serves as an additional component for the protein depot provided by the adjuvant.

 3.

Draw up the CFA/antigen mixture into a 3-ml glass syringe with a 19-G needle. Remove needle, expel as much air as possible, and attach syringe to the double-ended locking hub connector or the plastic 3-way stopcock (see Fig. 2.4.1). Attach an empty 3-ml glass syringe at the other end and force the mixture back and forth from one syringe to the other repeatedly. When the mixture is homogeneous and white, disconnect the connector or stopcock, attach a 21-G needle, and test whether the emulsion is stable by extruding a small drop onto the surface of 50 ml cold water in a 100-ml beaker. A good oil-in-water emulsion should hold together as a droplet on the surface of the water. If the drop disperses, mix the antigen using the hub-connected syringes until it forms an emulsion.

p type = annotation

Heat will be generated by this procedure. Chill on a bed of ice from time to time to keep the mixture as close to 4°C as possible.

p type = annotation

Images of stable emulsions can be found in Koh et al. (2006).

Figure 2.4.1 Double-syringe device for preparation of antigen-adjuvant emulsions.

 4.

Transfer all of the adjuvant-antigen emulsion to one syringe and remove the connector or stopcock. Attach a 22-G needle to the syringe and remove air bubbles.

 5.

Restrain the animal (unit 1.3) and inject the adjuvant/antigen emulsion into multiple i.m., i.d., or s.c. sites.

p type = annotation

Discard the unused immunogen. For extremely valuable antigens, the emulsion may be stored at 4°C for several weeks and re-emulsified before use. However, denaturation of protein antigens may take place under these conditions. For immunization of small rodents (e.g., mice), it is often better to carry out injections intraperitoneally (i.p.; units 1.6 & 2.5).

 6.

Bleed the animal 10 to 14 days following the priming immunization and collect blood sample (unit 1.7). Prepare serum from blood (see Support Protocol).

 7.

Prepare antigen for booster immunizations, following steps 2 to 4. When CFA is the primary adjuvant, use IFA as the adjuvant for all subsequent immunizations.

 8.

Administer the first booster immunization 4 to 8 weeks after the priming immunization, bleed the animal 7 to 14 days later, and collect blood sample. Prepare serum from blood (see Support Protocol).

p type = annotation

Some investigators will administer the first booster immunization as early as 2 weeks after the primary immunization.

 9.

Administer further booster immunizations at 2- to 3-week intervals. Bleed animal 10 to 14 days after each boost and collect blood sample. Prepare serum from blood (see Support Protocol).

p type = annotation

Repeated i.d. immunization should be avoided as it can cause skin ulceration. Following primary i.d. or s.c. immunization, it is preferable to boost with i.m. injections for the rabbit. Some investigators prefer primary i.m. injections with boosters at other sites.

 

Alternate Protocol: Immunization to Produce Polyclonal Antiserum Using TiterMax

For highly immunogenic antigens the use of Freund's adjuvant can certainly be avoided. For other immunogens it may be necessary to test the different adjuvant systems.

 Additional Materials (also see Basic Protocol)

• TiterMax #R-1 (CytRx Corporation, Sigma, or TiterMax; store <24 months at 4°C)
• 1-ml plastic syringes

1.	Emulsify aqueous antigen with TiterMax adjuvant (see Basic Protocol; follow steps 1 through 5, except use 0.5 ml antigen and 0.5-ml vial TiterMax in step 2 and plastic syringe in step 3). p type = annotation TiterMax contains microparticulate silica coated with the non-ionic block copolymer, CRL-8941, and squalene. p type = annotation Although glass syringes are recommended for Freund's adjuvant emulsions, all-plastic syringes should be used with TiterMax. p type = annotation Each reconstituted 0.5-ml vial will immunize 20 mice or 10 rabbits. Unused antigen/adjuvant emulsion can be stored at 4°C, –20°C, or –70°C for as long as the antigen is stable. It may be necessary to re-emulsify before using.
2.	Transfer the antigen emulsion to a 1-ml syringe, attach a 22-G needle to the syringe, and remove air bubbles.
3.	Restrain the animal and inject the adjuvant/antigen emulsion. p type = annotation Rabbits should receive (i) 40 µl TiterMax/antigen emulsion i.m. in each thigh, or (ii) 4 s.c. injections of 100 µl each over shoulders and hind limbs, or (iii) 10 i.d. injections of 40 µl each along the back. High antibody titers have been obtained with 30 to 50 µg of antigen per rabbit.
4.	Bleed the animal and prepare antigen for booster immunization (see Basic Protocol, steps 6 and 7). p type = annotation Boosting with TiterMax may not be necessary for all antigens. If a second immunization is necessary, use soluble antigen in place of antigen/adjuvant emulsion at 4 weeks. If titers are still low after 10 to 14 days, a booster dose of antigen/TiterMax adjuvant can be given immediately. Increasing the amount of antigen may also help.
5.	Administer booster immunizations at 4, 8, and 12 weeks. Bleed the animal 10 to 14 days after each booster immunization. Prepare serum from blood (see Support Protocol) and cease immunization when high antigen-specific titers have been achieved.

 

Support Protocol: Preparation of Serum from Blood

Each blood sample is allowed to stand 4 hr at room temperature and overnight at 4°C until a clot forms. After removal of the clot and debris, the serum is assayed and stored at –20°C.

 Additional Materials (also see Basic Protocol)

• Blood samples (see Basic Protocol)
• Wooden applicator stick
• Sigma 4K15 centrifuge and 11150 rotor or equivalent

• Additional reagents and equipment for immunoblotting (unit 8.10), immunoprecipitation (unit 8.3), ELISA (unit 2.1), and double-immunodiffusion assay in agar (unit 2.3)

1.	Allow blood to stand in the 50-ml centrifuge tube 4 hr at room temperature to allow clot to form, then place overnight at 4°C to allow clot to retract.
2.	Gently loosen the clot from the sides of the tube with a wooden applicator stick (do not break up the clot), then remove the clot from the tube with the applicator. p type = annotation If a clot has not formed, initiate clotting by placing a wooden applicator stick into the tube containing the collected blood, then begin again at step 1.
3.	Transfer serum to a 50-ml centrifuge tube. Pellet any remaining blood cells and debris by centrifuging 10 min at 2700 × g, 4°C, and save supernatant.
4.	Assay antibody titer by the appropriate method: immunoprecipitation (unit 8.3), immunoblotting (unit 8.10), ELISA (unit 2.1), or double-immunodiffusion assay in agar (unit 2.3).
5.	Store serum in aliquots of suitable volume in screw-top tubes at –20°C. p type = annotation Some sera lose activity on repeated freezing/thawing; others are not stable at 4°C.

sect1 type = commentary

 

Commentary

 

Background Information

The kinetics of development of a specific antibody response upon immunization of a rabbit with antigen are illustrated in Figure 2.4.2. After the primary immunization, naive B cells are stimulated to differentiate into antibody-secreting plasma cells. For most soluble protein antigens, specific antibody begins to appear in the serum 5 to 7 days after the animal is injected. The antibody concentration (titer) continues to rise, and peaks around day 12, after which it decreases. Similar kinetics are observed with mice, rats, hamsters, and rabbits.

Figure 2.4.2 Kinetics of development of the specific antibody response. Arrows indicate when priming and boosting immunizations were administered. Actual amounts of specific antibody produced will vary considerably depending on immunogenicity of the protein.

In addition to differentiating into antibody-forming cells, the antigen-stimulated B cells proliferate to form a large population of memory B cells, which quickly become activated after the booster injection is administered. Thus, the lag period before the appearance of the specific antibody is much shorter after a booster injection than that observed for the initial immunization. In addition, a significantly higher titer of specific antibody is achieved and sustained for a longer period of time. The peak of antibody production occurs 7 to 14 days after boosting. As a consequence of the existence of the memory B cells, less antigen is required to stimulate a strong secondary response. Memory B cells are long-lived; therefore, a specific antibody response can be elicited as much as 6 months to a year after the last booster. Finally, the average affinity and degree of specificity of the antibody population for the antigen increase with repeated immunizations (Klinman and Press, 1975). A more in-depth discussion of the immunological concepts underlying the generation of the humoral immune response, including the potency of a variety of adjuvants, is given by Aguilar and Rodríguez (2007) and Liang et al. (2006).

Adjuvants greatly enhance the specific antibody titer, as they allow the antigen to be released slowly, thus ensuring the continual presence of antigen to stimulate the immune system. Freund's adjuvant has been used extensively in the preparation of antigen because it induces a high, long-lasting antibody titer that is often still measurable 25 weeks or more after boosting. The presence of killed mycobacteria in complete Freund's adjuvant (CFA) activates the T cell population, providing necessary lymphokines for B cell stimulation and maturation. CFA may cause granuloma and subsequent necrotic abscesses, so it should be used only for primary immunization. Thus, some countries are restricting the use of CFA in laboratory animals for this reason. Incomplete Freund's adjuvant (IFA) is adequate for booster injections. Wherever possible, to reduce animal discomfort, less noxious adjuvants should be used as described in the Alternate Protocol.

Over recent years many different adjuvant systems have been formulated for use in humans in the clinical context (reviewed in Liang et al., 2006; Aguilar and Rodríguez, 2007). While, in general, these adjuvants do not boost the immune response to the same extent as CFA/IFA or TiterMax, they are less toxic and therefore may be worth considering, especially when the antigen is expected to be highly immunogenic. Published comparisons of commercial adjuvants with the basic CFA/IFA protocol vary widely in their conclusions. In most cases, however, CFA/IFA produces higher titers of higher-affinity antibodies in a shorter time period (Johnston et al., 1991; Deeb et al., 1992; Smith et al., 1992; Liang et al., 2006; Aguilar and Rodríguez, 2007). For this reason CFA/IFA has always been favored for the generation of polyclonal antisera in animals for laboratory studies. Presently, the TiterMax adjuvant appears to be one of the only viable alternatives to CFA/IFA and should, therefore, be the adjuvant of choice due to its lower toxicity and ability to generate high antibody titers after a single immunization.

 

Critical Parameters

New Zealand red or white rabbits are generally the best source of specific antisera because 30 to 50 ml of whole blood can be obtained at each bleed. The life span of a rabbit is 5 to 6 years, so a continual source of specific antiserum can be provided over a period of time by one rabbit after booster injections. In this regard, the recommended times between booster injections are not critical; the animal may be rested for several months between subsequent boosters, after the primary and secondary booster injections. Blood collection, however, must take place 7 to 14 days after each booster to ensure a high titer.

Preimmune serum from the same animal is the preferred negative control. If additional control serum is required, either immune serum from animals immunized with totally unrelated antigens or pooled serum from naive animals will be adequate. Occasionally, spurious antibody activities from nonimmunized animals may mimic the activity of the immune serum.

Antibody specificity may vary widely between individual animals with respect to the dominant antigenic epitopes recognized on a given protein antigen. Therefore, antiserum from a single animal should be used throughout a study. If more than one animal must be used for particular antisera, the antisera should be pooled. Large-scale production of antisera can be carried out in goats, sheep, and horses with appropriate veterinary guidance. If serum is taken from inbred animal strains, the variability in antibody specificity, as observed in outbred rabbits, is less of a problem.

The most important factor in producing a highly specific polyclonal antiserum is the purity of the antigen preparation used for immunization. The immune system is very sensitive to the presence of foreign proteins. Any contaminating proteins in the antigen preparation can potentially induce a strong immune response when injected in the presence of adjuvant. When antisera are employed in sensitive techniques, such as immunoblotting or the screening of cDNA libraries, significant antibody titers to protein contaminants can be a major problem. Thus, the antigen preparation should contain no significant contaminating proteins. Ideally, there should be no visible contaminating bands when 10 to 20 µg are analyzed on an SDS-polyacrylamide gel stained with Coomassie brilliant blue (unit 8.9).

If the antiserum is to be used in functional assays, extra care must be taken to ensure that the immunizing antigen is in its native form, because antibodies directed to denatured forms of the protein antigen will interact weakly, if at all, with the antigen in its native conformation. On the other hand, antibodies used in immunoblots, immunoprecipitation of primary in vitro translation products, and immunoscreening of cDNA expression libraries may be most effective if generated against a denatured protein with reduced and carboxymethylated disulfide bonds. Similar considerations are also relevant to the use of antisera in proteomic applications.

 

Troubleshooting

Inability to attain high-titer antiserum after several booster injections may be due to a variety of factors as described below.

Use of inappropriate adjuvant. Some experimentation may be necessary to optimize the antigen/adjuvant ratio for different antigens. If the Alternate Protocol still fails to produce a good antibody titer after three immunizations, switch to the Basic Protocol. Further adjuvant systems are described in Liang et al. (2006) and Aguilar and Rodríguez (2007).

Inadequate antigen emulsification. If the emulsion fails the drop-on-water test described in the Basic Protocol (step 3; also see Koh et al., 2006), repeat the emulsification process. Be sure to use phosphate-buffered saline. Avoid plastic syringes and Tris-based buffers with CFA and IFA. Alternative emulsification techniques include homogenization, sonication, or vortexing (Koh et al., 2006; http://www.titermax.com).

The antigen is a poor immunogen. In general, the immunogenicity of a protein is related to the degree to which it differs from “self” proteins (Benjamin et al., 1984). Large bacterial or viral proteins, such as hemagglutinin or bacterial-coat proteins, are highly immunogenic, whereas proteins from mammalian sources, such as polypeptide hormones or cell-surface receptors, may be poorly immunogenic due to tolerance. Protein antigens can be made more immunogenic in two ways. First, they can be chemically linked to a carrier protein that is known to be a good immunogen. Common carrier proteins include keyhole limpet hemocyanin (KLH), fowl immunoglobulin, and bovine serum albumin (BSA). Coupling peptides to carrier proteins is described in unit 9.2; the same protocols can be used to couple the protein antigen of interest to the desired carrier. Second, the immunogenicity of an antigen may be enhanced by its polymerization into large aggregates via a cross-linking agent, such as glutaraldehyde. The protocol in unit 9.2 for the coupling of peptide antigens to a carrier protein with glutaraldehyde can also be used to polymerize any protein antigen. With both the coupling and polymerization procedures, any insoluble antigen complexes formed should be removed prior to immunization by centrifuging 10 min at 15,000 × g, 4°C.

Host animal's immune system may be compromised by bacterial or viral infection. Refer to units 1.1 & 1.2 for discussion of the consequences of poor animal husbandry. Utilize animals from reliable, pathogen-free sources and maintain them in appropriate infection-free facilities.

Only a few animals have been immunized. Because of the vagaries of immune-response genes in outbred animals such as rabbits, some antigens may not induce a good antibody response in a significant proportion of randomly selected animals. Thus, it is best to immunize several different animals and to screen the sera for the best responder. Obviously, this is less of a problem in homozygous inbred strains, but with a new antigen it is wise to test several strains for their antibody response.

An insufficient amount of antigen was used. Although recommended concentrations of antigen for rabbits are 0.25 to 0.5 mg/ml injected into multiple sites, for a total of 1 to 2 ml in the same animal, good results can be obtained with 1/10 to 1/20 of the concentration in the same volume. It is always tempting to use less of a precious antigen, but often too low a dose leads to too low a response.

 

Anticipated Results

For large or nonevolutionarily related proteins, a titer of 5 to 10 mg/ml of serum can be expected after repeated boosts (hyperimmunization). When immunizing with small or highly conserved protein species, a titer of 1 to 2 mg/ml of specific antibodies is more likely. Antibody titers and affinity for the antigen will be low after primary immunization and the first booster immunization, but both titer and affinity will increase with subsequent immunizations.

 

Time Considerations

Preparation of the immunogen and immunization will take ~3 hr on each occasion. Collection of antisera will take 1 to 2 hr, depending on the number and species of animals.

When using the CFA/IFA adjuvant system, collection of antiserum after the primary immunization will be at 10 to 14 days. This will be a low-titer, low-affinity serum. The first booster normally is given 4 to 8 weeks after the primary immunization but can be given as early as 2 weeks after the primary immunization if time is critical. Ideally, there should be at least 19 days between the primary and the secondary bleeds. A second booster is given at 6 weeks with a bleed on day 52 to 59. This will usually be the first high-titer bleed. If a titer of <1 mg/ml of specific antibody is obtained, subsequent boosting immunization will be necessary. When using TiterMax as the adjuvant, only a single immunization is required. In this case a low titer, equivalent to that seen after priming with CFA, is observed. Subsequently, the titer continues to rise without further boosting, allowing serum to be collected at 2 weekly intervals. If titers begin to decrease, the rabbit should be boosted as described in the Alternate Protocol.