Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V37(3) 1995
Adjuvants and Antibody Production

Institutional animal care and use committees (IACUCs) are responsible for providing oversight in assuring the welfare and humane care and use of animals. As a component of fulfilling this obligation, many institutions have developed written policies and guidelines that describe acceptable techniques for conducting various experimental procedures. These guidelines generally combine currently available literature and experience to guide decision-making efforts so that justified scientific requirements are balanced with the ethical responsibilities for conducting in vivo procedures. Updating these guidelines with new information on procedural refinements and alternatives is a critical component of ensuring that these procedures are performed in a manner that avoids or minimizes pain or distress.

In the case of adjuvants and antibody production, published guidelines available to IACUCs include the National Institutes of Health (NIH) Intramural Recommendations for the Research Use of Complete Freund's Adjuvant (Grumpstrup-Scott and Greenhouse, 1988), the Canadian Council on Animal Care (CCAC) Guidelines on Acceptable Immunologic Procedures (CCAC, 1991) and the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia (Workman et al., 1988). While these guidelines provide a solid foundation for constructing acceptable procedural policies, none are sufficiently comprehensive to address all aspects of antibody production procedures.

In an attempt to define the currently acceptable policies and guidelines for adjuvant use and antibody production, we asked 55 academic and industrial institutions across the United States to provide us with their current recommendations. Written policies and guidelines were received from 30 of these institutions. This information as well as published data were used as resources to compile recommendations based on sound scientific rationale. We believe the guidelines presented below to be representative of current guidelines for adjuvant use and antibody production.

The information presented in this manuscript is intended to provide points for IACUCs to consider when developing or revising recommendations for these procedures. A select general reference list compiled from institutional guidelines and a literature search is also included.

INSTITUTIONAL POLICIES

Documents for antibody production can be written as guidelines or in specified instances as standard operating procedures. Guidelines, in general, are provided to assist investigators in appropriately designing experimental protocols for adjuvant use and antibody production. The guidelines may be included as part of the protocol form. The guidelines are often adopted as institutional policies and any departures from established procedures require justification by the investigator when completing the animal protocol form and subsequent review by the IACUC.

Background information should be made available and should include the objectives and sequential procedures conducted in the production of monoclonal and polyclonal antibodies. Descriptions of how adjuvants work and their potential to cause pain and distress when used in animals (particularly when used improperly) are not only helpful and informative, but provide scientific rationale for the guidelines per se. The potential for pain and distress associated with other aspects of antibody production such as tumor growth, abdominal distention and paracentesis in mice with ascites, and blood collection techniques should also be addressed.

Investigators should be encouraged to develop refinements in antibody production techniques, alternative adjuvant protocols, and specific alternatives to the use of Freund's complete adjuvant (FCA), as well as to consider in vitro alternatives in order to advance the field of antibody production. A general list of references and specific citations for individual recommendations will also benefit both the investigator and the IACUC.

MONOCLONAL ANTIBODY PRODUCTION

Selection of Animals

Different mouse strains may be used for immunization as some strains respond better than others to certain antigens based on MHC haplotype, genes encoding B- and T-cell receptors, and expression of various proteins involved in immunoregulatory mechanisms (Kuby, 1992). Other rodents, such as rats or hamsters, are infrequently used for immunization.

As most hybridomas have been derived from the fusion of BALB/c plasmacytomas and BALB/c spleen cells, hybridoma recipient mice must be histocompatible, or if not, must be immunodeficient or immunosuppressed by exogenous means such as irradiation. The UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia (Workman et al., 1988) suggest the use of retired female breeders. The females' abdominal musculature is presumed to be more distensible, which allows larger ascites volumes to be tolerated without discomfort. The utility of retired breeders is enhanced by their availability, large size, and tendency not to fight (Chandler, 1987). The use of BALB/c-derived crossbred F1 hybrids is another consideration given reports that cite greater ascites volumes and antibody production in F1 hybrids (Brodeur and Tsang, 1986; Stewart et al., 1989). However, others reported no significant difference between F1 hybrids and parental BALB/c mice (Chandler, 1987).

To the authors' knowledge, there is no definitive information in the literature to suggest that either sex is necessarily better for antibody production. In one study, ascites production and antibody production were greater in male mice (Brodeur et al., 1984) while results of another study suggested that there was no predictable difference between male and female mice with respect to ascites production parameters. It was suggested, however, that some cell lines may grow slightly better in one sex of mouse than the other, and for some cell lines, solid tumors without ascites production tend to form in one sex while ascites tumors tend to form in the opposite sex (Chandler, 1987). BALB/c male mice are aggressive and often require individual housing because of their tendency to fight, which has been a deterrent to their use.

Immunization Protocols

The immunization schedule and the selection of, or need for, adjuvants should be determined based on the nature of the antigen (Harlow and Lane, 1988; Kenney et al., 1989). For example, intravenous inoculation can be used for particulate antigens without adjuvant and for booster immunizations of soluble antigens in saline. Subcutaneous and intraperitoneal routes of injection are commonly used for primary immunization of mice with an antigen-adjuvant preparation. Because of limited muscle mass, intramuscular injections in mice are frequently discouraged. Intradermal injections are usually limited to a total volume of 0.05 ml. Volumes of 0.05 to 0.2 ml may be administered subcutaneously, and volumes < 0.5 ml may be administered intraperitoneally. Some institutions limit the total volume to 0.1 ml if FCA is used, and Freund's incomplete adjuvant (FIA) is recommended for subsequent boosts. Mice are generally boosted 14 to 28 days following primary immunization. Blood is collected to measure antibody titer approximately 10 to 24 days following the boost. Animals with sufficiently high titer are rested for 3-4 weeks, then given a final boost with aqueous antigen alone intravenously in < 0.2 ml total volume or intraperitoneally. Three days later, the mice are euthanized and the spleens are harvested for cell fusion. Animals that do not have a high titer following the first boost may be re-boosted multiple times and titers may be repeated.

In vitro immunization of lymphocytes, first described by Hengartner et al. (1978), may be considered as an alternative to in vivo immunization (Van Ness et al., 1984; Ma et al., 1984). Advantages cited for in vitro immunization include that the procedure may be performed in days rather than weeks, only very small amounts of antigen are required, and it is possible to generate antibodies that are not produced in vivo due to suppression or tolerance (Guzman et al., 1995). A more detailed description of in vitro immunization is presented in the manuscript by Grimaldi and French (p. 130 of this issue).

Priming Agents

In mice that do not receive a priming agent prior to hybridoma cell inoculation, solid tumors may form, but little or no ascites is produced. Pristane is the ascitogenic agent most frequently used to "prime" the peritoneal cavity for successful growth of hybridomas as ascitic tumors. The reported biologic effects of pristane include the induction of granulomatous inflammation in the peritoneum (Cranco and Potter, 1976; Leak et al., 1985), immunosuppression (Kripke and Weiss, 1970; Freund and Blair, 1982), induction of growth factors (Cranco and Potter, 1976; Platica et al., 1982), lymphatic obstruction (Leak et al., 1985), and reduced clearance of particulate materials and cells from the peritoneal cavity (Moore and Rajan, 1994). The recommended dose volume for pristane varies from 0.1 to 0.5 ml. Many guidelines recommended volumes of 0.1 to 0.3 ml, with the majority of these recommending 0.2 ml. One study demonstrated no significant difference in ascites volumes in animals receiving 0. 1 vs. 0.5 ml pristane (Hoogenraad and Wraight, 1986), and researchers have reported success using 0.2 ml pristane [(Kwan et al., 1980; Colwell et al., 1986). Other authors suggest that smaller volumes of pristane may reduce the potential for distress associated with its irritant properties (Amyx, 1987; McGuill and Rowan, 1989). In a study by Brodeur et al. (1984), pristane volumes ranging from 0.2 to 2.0 ml were investigated, and volumes of 0.5 ml were found to result in the largest volumes of ascites with high antibody titer.

Alternatives to the use of pristane include complete and incomplete Freund's adjuvant. Two guidelines in our survey stated that FCA was not acceptable for intraperitoneal administration. A study conducted to compare pristane to FCA, FIA, proteose-peptone, thioglycollate, corn oil, and mineral oil as alternative priming agents, demonstrated that FIA was as good or better than pristane based on number of taps per mouse, ascites volume, and antibody concentration (Gillette, 1987). A 0.25 ml volume of all agents was administered 7 days prior to hybridoma cell inoculation. In a subsequent study, using 0.5 ml FIA, results indicated that the time interval between priming and hybridoma inoculation could be reduced to 1 day, and as few as 1 x 10⁵ hybridoma cells could be inoculated without significant negative impact on ascites production (Mueller et al., 1986). Results of this study also demonstrated development of ascites tumors in control mice that received only FIA, without hybridoma cells. Another study demonstrated significantly greater ascites volumes with no significant difference in antibody concentration in FIA-primed mice as compared with pristane-primed mice (Jones et al., 1990), and the time interval over which the animals were tapped was also shorter for FIA-primed mice. The agents were administered in a volume of 0.1 ml.

Inoculation of Hybridoma Cells

The recommended time interval between pristane priming and inoculation of hybridoma cells varied from 5 days to 4 weeks. These recommendations are consistent with reports that intervals ranging from 3 days to 3-4 weeks have been successful for hybridoma growth, with optimum times reported to be 1 0 days (Hoogenraad et al., 1983; Hoogenraad and Wraight, 1986), 14 days (Brodeur et al., 1984), 3-4 weeks (Chandler, 1987), and 30 days (Cranco and Potter , 1976). In the study reported by Chandler, priming periods of 5 to 54 days were examined with two cell lines in large groups of mice (N=200-1000/group). The author suggested that the longer optimal time period between pristane priming and hybridoma inoculation (3-4 weeks) may be required for peritoneal inflammatory cells to establish an ideal microenvironment for hybridoma growth.

Guidelines for the number of cells in the hybridoma inoculum range from 10⁵ to 10⁷ cells in basal cell culture media or PBS, inoculated intraperitoneally in a total volume from 0.1 to 0.5 ml. One report indicated the optimum hybridoma inoculum to be 5 x 10⁵ cells. Inocula of 2 x 10⁶ cells were associated with significant mortality and with doses of 1 x 10⁵ cells, fewer mice developed ascites and the volume yield was significantly lower (Chandler, 1987). Differences in the number of cells inoculated in these experiments did not significantly alter antibody concentration. An inverse relationship between the number of cells inoculated and duration of time between injection of cells and onset of ascites production as well as duration of survival of the mice has been noted (Brodeur et al., 1984). Ascites formed sooner with larger cell inocula, but shortened survival times could limit the number of taps possible. Based on ascites volumes and antibody concentration, optimal cell dosage was between 0.6 and 3.2 x 10⁶ cells (Brodeur et al., 1984). Another reference recommended 10⁷ cells (Zola, 1995). Optimal cell number for inoculation may be dependent on the biologic behavior of the cell line and thus it may be appropriate to consider conducting pilot experiments with novel cell lines to investigate parameters such as number of cells for inoculation and thus permit optimization prior to using large numbers of animals.

Hybridomas should be tested for the presence of adventitious viral and mycoplasma agents prior to introduction into an animal host to prevent the potential transmission of infectious agents from contaminated cell lines into facility mouse colonies (Loew and Fox, 1983). Viral contamination was found in 69% of 465 murine leukemia and transplantable tumor specimens tested (Collins and Parker, 1972). The most common viral "passengers" were lactic dehydrogenase-elevating virus and minute virus of mice, but polyoma, mouse hepatitis virus, Sendai, lymphocytic choriomeningitis virus, and reovirus-3 were also encountered. One of these viruses, lymphocytic choriomeningitis, has been isolated in tumor cell lines by several laboratories. This virus is capable of producing illness in humans, and indeed investigators have been infected when handling tissue or animals harboring this virus Baum et al., 1966; Biggar et al., 1976; Bhatt et al., 1986; Dykewicz et al., 1992).

Abdominal Paracentesis

Institutional guidelines recommend that ascites pressure be relieved by abdominal paracentesis when visible abdominal distention becomes evident, and prior to the development of marked abdominal distention with associated clinical signs of pain or distress. The UKCCCR guidelines (Workman et al., 1988), recommend that ascitic fluid volumes not exceed 20% of the baseline body weight prior to performing abdominal paracentesis.

The use of anesthesia for abdominal paracentesis was required by one institution and was recommended in many guidelines although manual restraint was also generally acceptable. Anesthesia may be particularly helpful for training students and new personnel until the paracentesis technique is perfected. Methoxyflurane was commonly recommended for this procedure. Individuals with experience in clinical assessment of ascites mice, making determinations of proper time intervals between taps, and performing paracentesis procedures should provide hands-on training for inexperienced personnel.

To minimize bacterial contamination, preparation of the paracentesis site with an antiseptic is recommended. An 18 to 20 gauge needle is commonly used because the proteinaceous ascites fluid is frequently viscous. Use of a large gauge needle also permits more rapid collection of ascites which reduces the duration of anesthesia or manual restraint required for the collection procedure. To help prevent shock, which may result from rapid fluid loss especially when large volumes of ascites are collected, 2-3 ml of warm saline or lactated ringers solution may be administered subcutaneously to the mouse at the time of paracentesis. Intervals of 1-3 days (Galfre and Milstein, 1981) or 2-3 days (Epstein and Epstein, 1986) between taps are recommended in the literature.

The number of abdominal taps may vary (Tung et al., 1976). Institutional guidelines vary in their recommendations regarding number of times paracentesis is permitted to be performed--from once to unlimited times--provided that ascites fluid continues to accumulate, the animals continue to maintain good body condition without evidence of debilitation or complications from the procedure, and animals exhibit no signs of pain or distress. Euthanasia of animals prior to the initial collection of ascitic fluid is seldom recommended. Rather, the majority of institutions surveyed permitted a maximum of 2 or 3 taps and recommended that euthanasia precede the final abdominal paracentesis. If a maximum of 3 taps per animal is permissible, a requirement that animals be observed at least once daily, 7 days a week, with assurance that the animals would be euthanized if they appeared distressed, seems appropriate.

Clinical Observations

Clinical observation of the animals should be performed at least once daily. A number of institutions qualified this by stating that daily observations were to include weekends and holidays. Clinical observations every other day may be adequate until ascites develops. Daily clinical observations allow the degree of abdominal distention to be frequently assessed so that abdominal paracentesis can be performed as needed for each animal. Animals that exhibit severe clinical abnormalities or distress and warrant humane euthanasia can be identified so that animal pain and distress can be minimized.

Potential clinical and pathologic sequelae to hybridoma growth and ascites production procedures include granulomatous peritoneal inflammation induced by priming agents; solid tumor growth within the abdominal cavity; progressive ascites fluid distention of the abdomen, which may be associated with difficulty in ambulation and elevation of the diaphragm leading to respiratory compromise; and complications resulting from injection or paracentesis procedures such as development of hypovolemia following rapid removal of ascites by paracentesis. Any or all of these pathologic sequelae may cause pain or distress in the animal (McGuill and Rowan, 1989; Jackson, 1994; Jackson et al., 1996). In addition to daily observation of animals to assure that abdominal paracentesis is performed before marked abdominal distention or discomfort becomes evident, investigators should be instructed to monitor animals for hunched posture, roughened haircoat, anorexia, dehydration, weight loss, loss of body condition, inactivity, difficulty in ambulation, tachypnea, and dyspnea. Excessive abdominal distention not relieved by paracentesis should prompt abdominal palpation to diagnose solid tumor growth in the abdomen. Mice should also be monitored for pale eyes, ears, and mucous membranes, which may be indicative of anemia or shock. In one study, clinical signs that included roughened haircoat, hunched posture, inactivity, pallor most evident on the ears and muzzle, tachypnea, and dyspnea were observed in some animals shortly after abdominal paracentesis, and were considered compatible with signs of circulatory shock (Jackson, 1994; Jackson et al., 1996). While these signs were often transient, in some animals they were persistent and severe, leading to euthanasia or death within approximately 30 minutes from the time of tap (Jackson, 1994; Jackson et al., 1996). We therefore recommend that animals be monitored for at least 30 minutes following abdominal tap so that animals with severe or persistent clinical abnormalities that warrant consideration for fluid replacement therapy or euthanasia may be identified.

Animals showing significant abnormalities should be monitored closely to ascertain if signs of pain or distress develop; if animals exhibit severe clinical abnormalities or become moribund, they should be euthanized. Death is not considered an acceptable endpoint to the experiment. To easily identify these mice and to facilitate their monitoring while undergoing ascites production procedures, restricted housing of mice used for ascites production to designated rooms may in some cases be warranted. Proper supervision and training of personnel will undoubtedly facilitate the proper conduct of these procedures in mice.

Alternatives

Many alternatives are available for laboratory scale growth of hybridoma cells in vitro including stationary cultures in T-flasks and suspension cultures in roller bottles and spinner flasks (Kuhlmann et al., 1985; Boyd and James, 1989; Reuveny and Lazar, 1989). Other techniques include growth of cells in dialysis tubing within a culture bottle (Sjörgren-Jansson and Jeanson, 1990), a roller bottle-like apparatus (Falkenberg et al., 1993), or tumbling chamber (Jaspert et al., 1995); and the use of oscillating bubble dialysis chambers (Pannell and Milstein, 1992). Laboratory scale stirred tank reactors (Reuveny et al., 1986a; Shevitz et al., 1989), fermentors (Reuveny et al., 1986b; Falkenberg et al., 1995), ceramic-matrix bioreactors (Kurkela et al., 1993), packed-bed bioreactors (Moro et al., 1994), and hollow fiber bioreactors (Hopkinson, 1985; Altshuler et al., 1986; Kurkela et al., 1993; Jackson et al., in press) are also in use.

There are many advantages of monoclonal antibody production in mice against which in vitro technologies must compete. Production is rapid in mice, mice can support the growth of most cell lines, minimal materials and technical expertise are required, and the antibody in ascites is highly concentrated. Requirements for technical expertise in cell culture, availability of cell culture laboratory facilities and supplies, capital costs for purchase of equipment, and in general, production of less concentrated antibody than that obtained in ascites, has hindered widespread use of many of these methodologies on a laboratory scale.

Promoting availability of alternative in vitro technology for monoclonal antibody production is worthwhile. For example, some institutions have established cell culture core facilities that are operated as a service to investigators. Many contract antibody production companies also offer in vitro production services that may be used by investigators. Ongoing refinements in cell culture methodologies will likely offer more and improved alternatives for the future. Recombinant antibody technology also provides alternatives to the use of animals for monoclonal antibody production (Karu, 1993) and is discussed in detail in the manuscript by Karu, Bell, and Chin (p. 132 of this issue).

POLYCLONAL ANTIBODY PRODUCTION

Selection of Animals

Selecting a species for polyclonal antibody production is described in detail in the article by Hanly, Artwohl, and Bennett (p. 98 of this issue). The species most frequently used for polyclonal antibody production are mice, hamsters, rats, guinea pigs, rabbits, goats, sheep, and chickens. Rabbits are the most commonly used laboratory animal species. Because of the inherent value of these experiments and the animals in which they are conducted, combined with the stress associated with these immunization protocols, we recommend the use of specific-pathogen free rabbits. Preference of sex of the rabbit may vary, but often investigators prefer females because they tend to be more docile. Animals are usually over 2 kg in weight before the immunization protocol commences. These animals are readily available commercially and their use dramatically reduces morbidity and mortality documented frequently in rabbits infected with microbial pathogens, particularly Pasteurella multocida.

Antigen Preparation

The antigen preparation should be free of extraneous microbial contamination; sterilization by filtration through a 0.22 micron filter is routinely performed. The presence of byproducts, such as polyacrylamide gel, should be avoided because of their inflammatory properties (Stills, 1994). Antigen prepared by gel electrophoresis should be eluted, lyophilized, ground to a fine powder, and resuspended in sterile saline (Harlow and Lane, 1988). Alternatively, the antigen is transferred to nitrocellulose paper, trimmed, and cut into the smallest pieces possible (Knudsen, 1985; Larsson and Nilsson, 1988). If these materials are incorporated into the antigen preparation, care should be exercised when administered with FCA. Extremes of pH, particulate matter and contamination with chemicals such as SDS, urea, acetic acid, or other solvents or potentially toxic agents should also be avoided. Special precautions may be necessary if the antigen itself is a viable microbe.

Antigen-Adjuvant Emulsions

A description of procedural techniques to properly mix antigen-adjuvant emulsions should be provided to ensure proper preparation of the material (Herbert, 1978; Harlow and Lane, 1988). If FCA is used, the mycobacteria should first be resuspended by vortexing or shaking. One part or less of Freund's adjuvant to one part antigen (v/v) is recommended. Two sterile luer-lock syringes, one containing Freund's adjuvant and one containing the antigen, preferably in sterile saline, are used for these purposes. Glass syringes are often preferred because the oil will react with the rubber plunger on plastic disposable syringes (Stills and Bailey, 1991). The aqueous antigen solution is injected through a 3-way stopcock or mixing cannula into the adjuvant and then the emulsion is prepared by pushing the solution back and forth between syringes for several minutes. An emulsion is properly prepared when it becomes thick, is difficult to inject back and forth through the cannula, and will not separate on standing; a droplet placed into a saline solution should not disperse. Emulsification is enhanced by the use of cold (4°C) adjuvant. Failure to emulsify the preparation may be due to contamination of the antigen with SDS or organic solvents (Herbert, 1978). For antigen-adjuvant preparations using adjuvants other than Freund's, manufacturer's instructions should be followed.

Freund's Adjuvant and Alternatives

FCA (Freund and McDermott, 1942), first used in the late 1930s, still remains the most commonly used adjuvant for polyclonal antibody production. The adjuvant contains killed mycobacteria, paraffin oil, and mannide monoosleate. The use of killed mycobacteria elicits a delayed hypersensitivity reaction. As a part of this reaction, there is augmented recruitment of antigen-presenting macrophages and other antigen-processing cells, which helps T-cell dependent humoral antibody responses. In addition, adjuvants in general contain substances that have a depot effect. This results in slow degradation of the antigen with a prolonged period for antigen stimulation of the immune system (Osebold, 1982). Lesions associated with the use of Freund's adjuvant have been described and include granulomatous inflammation, focal necrosis, ulceration of skin, fistulous tracts, muscle atrophy, self-induced trauma, hypersensitivity reactions, and weight loss (Amyx, 1987; [Broderson, 1989; Toth et al., 1989; Stills and Bailey, 1991).

FCA should be used only once, usually for the initial immunization, in all species. Severe hypersensitivity reactions may result following re-exposure of the host to mycobacteria. For example, in rabbits, death with or without preceding acute respiratory distress was observed within 6 to 48 hours following booster inoculations with FCA given 3-6 weeks following primary immunization (Broderson, 1989). Severe pain was present in 5 of 9 people accidentally injected with FCA. All 5 of these individuals tested positive for tuberculosis prior to the accidental FCA inoculation (Chapel and August, 1975). Also, painful ulcers and abscesses in patients treated with autologous tumor extract in FCA occurred only following the second or subsequent immunization with FCA (Hughes et al., 1970). FIA, without killed mycobacteria, is commonly recommended for booster immunizations to avoid hypersensitivity reactions resulting from re-exposure of the host to mycobacteria.

Formulations of FCA not exceeding 0.1 mg dry mycobacterial cell mass/ml have been recommended to avoid proportionally increased inflammation and necrosis observed with higher concentrations of mycobacteria (Broderson, 1989). The use of FCA is specifically discouraged for use in food animals and nonhuman primates because of subsequent interference with routine tuberculin skin tests used for diagnostic purposes. Nonhuman primates inoculated with FCA may be tattooed to prevent misinterpretation of subsequent positive tuberculin test results (Pierce and Dukelow, 1988). Some institutions suggested that Freund's adjuvant should be limited to use with antigens known to be weak immunogens or when only very small amounts of antigen are available (Harlow and Lane, 1988).

Less inflammatory alternatives to Freund's adjuvant are available and should be considered (Allison and Byars, 1986; Allison and Byars, 1991; Altman and Dixon, 1989; Bennett et al., 1992; Croft et al., 1991; Lipman et al., 1992; Niemi et al., 1985; Smith et al., 1992; Woodward, 1989; Yarkoni and Rapp, 1979). Guidelines should cite references for alternatives to the use of Freund's adjuvant, and additional information or referral to individuals with experience in the use of alternative adjuvants is helpful for investigators (Rowsell, 1989). Ribi Adjuvant System® and TiterMax® are commonly cited as appropriate alternatives (Bennett et al., 1992; Check et al., 1990; Hunter et al., 1989; Lipman et al., 1992; Meyer et al., 1974; Ribi et al., 1984). Noninflammatory adsorptive adjuvants such as alum and aluminum hydroxide gel may also be considered (Bomford, 1988; Harlow and Lane, 1988). A more detailed description and comparison of various research adjuvants is presented in the manuscripts by Jennings (p.119 of this issue) and Hanly (p. 112 of this issue). Providing detailed recommendations outlining the specific rationale for selection of species and adjuvants for use with particular antigens and applications based on considerations for the amount of antibody required, the type of response required, and the nature of the antigen may sometimes be helpful (Warren et al., 1986).

Specific recommendations for the interval between primary and booster immunizations are usually not cited. It is important to remember that the primary antibody response continues as long as the antigen is still being released (Stills, 1994). This statement must be considered, however, in the context that immunoglobulin class switching is a dynamic process occurring during the prolonged immunologic response to the initial immunization with antigen and FCA or similar adjuvants. Sera analysis to determine that antibody levels have dropped sufficiently to prevent rapid clearance of antigen can be undertaken before administration of subsequent immunizations. Two to 3 weeks is generally considered the minimum time period between the initial and subsequent immunizations. Booster immunizations are sometimes delayed if significant inflammatory reactions are still present from the initial immunization. For adjuvant-antigen mixtures that cause excessive inflammatory reactions, due consideration should be given to using lesser concentrations, smaller volumes per injection site, or both for subsequent immunizations.

Injection Site Selection and Preparation

The selection of appropriate injection sites, irrespective of the adjuvant used with the antigen preparation, is important. Anatomic sites used for grasping, handling, or restraint such as the dorsal cervical/scapular areas and rump in rabbits and dorsal cervical/scapular areas and tail base in rodents should be avoided when possible. Extension of granulomatous inflammation into the spinal cord following inadvertent injection of a FCA-antigen mixture into the paraspinal musculature has been associated with posterior paresis in guinea pigs (Kleinman et al., 1993). Care therefore should be taken when making injections near the dorsal spinal column. Granulomas can also be noted in other organs after injections with FCA (Schiefer and Stunzi, 1979). Avoid sites that may be prone to self-mutilation, and avoid sites that may interfere with ambulation such as footpads. "Footpad" injections in rabbits are prohibited by most, if not all, institutions. The lack of anatomically defined footpads, plus the weight bearing function of the rabbit's feet, preclude this site for immunization. Furthermore, significant antibody titers are routinely produced by immunization at other sites (Leskowitz and Waksman, 1960; Amyx, 1987). Where scientific justification is provided, footpad injections may be permitted in rodents, but only in one hind foot, and with the animals housed on soft bedding. Suggested maximum injection volumes can range from 0.01 to 0.05 ml for mice and 0.10 ml for rats. Alternative strategies for tracing the fate of injected antigens in local lymph nodes includes intramuscular or subcutaneous injection in an area drained by a particular node such as the axillary or inguinal nodes, or injection in the hairless skin at the base of the tail. Sometimes direct inoculation into lymph nodes, such as the popliteal lymph node, is used (Sigel et al., 1983). With practice these nodes often can be palpated and the injection performed percutaneously, thus obviating the need to perform a surgical incision and dissection to locate the lymph node for injection. Visualization of nodes may be facilitated by prior injection of a vital dye (Harlow and Lane, 1988).

Hair should be clipped from intradermal and subcutaneous injection sites, and the site should be aseptically prepared with betadine or nolvasan scrub followed by alcohol or other appropriate antiseptics. Clipping of hair and aseptic preparation of the injection sites not only reduces the potential for the development of infection and abscess formation, but also facilitates visualization of the injection sites to permit appropriate treatment of lesions if they develop. The use of sterile needles and syringes is mandatory to minimize microbial contamination of injected tissue. The area where the needle penetrates the skin for intramuscular injections should be prepared with appropriate antiseptics.

Routes, Volumes, and Sites of Administration

The range of recommendations for routes and sites of administration of antigen-adjuvant preparations, volumes per site, and number of sites per animal for different species vary in the literature and institutional guidelines. Particularly with the use of Freund's adjuvant, it is important to note that the severity of potentially painful inflammatory reactions may be minimized by injection of a small volume of inoculum per site and the use of multiple injection sites when appropriate. Injection sites must be sufficiently separated to prohibit coalescing of the inflammatory lesions, which may result in disruption of blood supply to the area with subsequent formation of draining abscesses or occasional tissue sloughs. Using multiple sites for immunization also provides more foci for antigen presentation and the involvement of more lymph nodes, which ultimately improves antibody production (Amyx, 1987; Grumpstrup-Scott and Greenhouse, 1988; Stills and Bailey, 1991). Booster injection sites should be distanced from previous injection sites.

Intradermal and subcutaneous routes are commonly used to take advantage of antigen-processing dendritic cells present within the dermis. The Langerhans cells have class II major histocompatibility antigens, and Fc receptors that interact with and activate T-helper lymphocytes (Harlow and Lane, 1988; Stills, 1994). The following recommendations apply primarily to antigen solutions in FCA or FIA. Volumes ranging from 0.05 ml per site (Amyx, 1987; CCAC, 1991; Grumpstrup-Scott and Greenhouse, 1988) to 0.10 ml per site (Harlow and Lane, 1988; Johnston et al., 1991) have been recommended for intradermal injections in rabbits. Injections are most commonly placed on the back and can be performed using a small 25 to 27 gauge needle. A total of five intradermal sites has been recommended by Johnston et al. (1991), however, a larger number of sites is recommended in immunology texts (Harlow and Lane, 1988). Smaller granulomas (averaging 16 mm vs. 25 mm diameter at 5 weeks post-injection) are produced following FCA inoculation with 0.05 vs. 0.10 ml in rabbits (Stills and Bailey, 1991). Because intradermal sites ulcerate with FCA (Broderson, 1989; Stills and Bailey, 1991), sterile inocula must be used and the site must be properly disinfected to prevent secondary bacterial infection. Sites that are secondarily infected may be tender on palpation and show evidence of self-mutilation (scratching and chewing). Sites without secondary complications, however, usually don't appear to be painful or tender on palpation (Johnston et al., 1991; Stills, 1994). It is important to separate multiple intradermal and subcutaneous injection sites, which are often located along the back, to prevent coalescing of the inflammatory lesions. Intradermal injection in rodents is not recommended by the CCAC, however, intradermal injection of volumes ranging from 0.01 to 0.05 ml per site is recommended in a number of institutional guidelines.

Subcutaneous injection volumes in the rabbit vary from recommendations of 0.10 ml (Amyx, 1987) to 0.25 ml (CCAC, 1991; Johnston et al., 1991) to 0.40 ml per site (Harlow and Lane, 1988). Number of sites recommended varies from four (CCAC, 1991; Johnston et al., 1991) to 10 (Harlow and Lane, 1988). It must be cautioned, however, that with subcutaneous injection, the granulomatous inflammatory response can extend to adjacent sites and fistulous tracts may be formed (Stills and Bailey, 1991). Subcutaneous injection volumes in rodents have been recommended at 0.10 ml per site (Grumpstrup-Scott and Greenhouse, 1988; CCAC, 1991).

Intramuscular injections, usually made in the biceps femoris or quadriceps muscle mass, generally are larger volumes of 0.25 ml (Johnston et al., 1991) to 0.20-0.40 ml (Harlow and Lane, 1988) to < 0.50 ml per site (CCAC, 1991). Care must be exercised to avoid adjacent nerves and blood vessels as well as fascial planes when injecting into a muscle bundle. Intramuscular injection has been recommended for large animal species with volumes of 0.50 ml (Grumpstrup-Scott and Greenhouse, 1988) to < 1.0 ml (CCAC, 1991) recommended. As with subcutaneous administration, the inoculum can migrate to more ventral and superficial sites and form fistulous tracts and cutaneous ulcerations at distant sites (Broderson, 1989). Disagreement exists as to the appropriateness of intramuscular injection of Freund's adjuvant. The intramuscular route of injection was recommended in some institutional guidelines and specifically discouraged in other guidelines. While effective, lesions are difficult to evaluate unless loss of limb function or muscle atrophy occurs, which is considered to be a disadvantage (Amyx, 1987; Stills and Bailey, 1991). Intramuscular injection is generally not recommended in rodents because of limited muscle mass. Broderson (1989) concludes that more than 0.10 to 0.30 ml of FCA or FIA per site in rabbits and owl monkeys, regardless of route of administration, markedly increases the severity of lesions.

The intraperitoneal route of administration is only recommended in rodents. The CCAC recommends volumes < 0.10 ml. Clinical and pathological effects associated with intraperitoneal administration of FCA in mice have been described (Toth et al., 1989; Lipman et al., 1992).

Intravenous use of FCA or FIA should not be permitted (Harlow and Lane, 1988; CCAC, 1991). Following intravenous injection of FCA or FIA in rabbits adjuvant emboli were identified in the subpleural regions of the lung and multiple granulomas were formed. There was also hyperplasia of Type II alveolar epithelial cells and the alveolar septae became thickened (Brooks et al., 1978). Occasionally intravenous routes can be used for particulate antigens and for antigens soluble in physiological saline (Stills, 1994).

For adjuvants other than Freund's adjuvants, such as Ribi Adjuvant System® and TiterMax®, the manufacturer provides detailed information on preparation of antigen-adjuvant emulsions, recommended routes and sites, total volume and volume per site for injection in different species, and recommended immunization schedules. For TiterMax®, intradermal, subcutaneous, and intramuscular routes are recommended with volumes per injection site ranging from 0.01 to 0.25 ml in small and large animals. For Ribi, intradermal, subcutaneous, intramuscular, and intraperitoneal routes are recommended with volumes per injection site ranging from 0.05 to 0.50 ml in small and large animals. For these and other alternative adjuvants, it is recommended that investigators follow manufacturers' recommendations.

Post-injection Observations

Investigators and veterinary staff should observe animals for evidence of pain or distress, and for evidence of lesions such as swelling, abscess or fistula formation, and infection or ulceration at the immunization sites. CCAC Guidelines recommend that observations should be made at least 3 times weekly for a period of 4 weeks following immunization, or until all lesions have healed. An immunization clinical incidence form which includes the agent, route, site or sites, volume, date of injection and the body weight of the animal on the injection date may be helpful in maintaining careful data on individual animals. Veterinary follow-up, including clinical observations and palpations of the injected sites, and determination of the necessity for any supportive therapy is strongly recommended. All guidelines should instruct investigators to contact the veterinary staff if injection site lesions or evidence of pain or distress are identified in any animals. This will permit timely and appropriate assessment and institution of therapy when required. Supportive therapy may include topical cleansing, antibiotic administration, analgesic administration, or all three. Fluid replacement or nutritional supplements may also be required if the animals have sustained anorexia or decreased fluid intake.

Although lesser in severity and frequency by comparison to Freund's adjuvant, inflammatory lesions can occasionally be observed following immunization with alternative adjuvants such as TiterMax® or Ribi adjuvants. Inflammatory reactions, which include draining abscesses, have been observed 2-3 weeks following immunization with TiterMax® adjuvant (Check et al., 1990). These lesions generally persisted for approximately 1 week and then gradually subsided. It would therefore seem prudent to recommend similar post-injection observations for animals receiving alternative adjuvants.

Blood Collection

Recommendations for acceptable methods of blood collection from different species that include maximum permissible volumes and frequencies of collection is an indispensable part of the guidelines. One standard recommendation is that the amount of blood collected every two weeks should not exceed 15% of the total blood volume of the animal. Given that 5-7% of the animal's body weight is blood, simple calculations regarding blood volume withdrawal can be used. Withdrawal of 15% of total blood volume equates to approximately 1% of body weight. Investigators proposing to exceed these recommendations, or other IACUC guidelines, should be required to perform additional monitoring procedures such as periodically evaluating hematocrit and total protein or providing appropriate fluid or blood replacement therapy (Nerenberg et al., 1978). Plasmapheresis may also be considered.

Survival bleeding is most commonly recommended via tail vein or retro-orbital sinus from rodents under anesthesia and via the marginal ear vein or central ear artery in rabbits with appropriate sedation or tranquilization. Acepromazine and droperidol-fentanyl are commonly recommended for this purpose in rabbits (Tillman and Norman, 1983). Methoxyflurane is commonly recommended for anesthesia in rodents although other anesthetics are also commonly used, including tribromoethanol or ketamine and xylazine. Blood is generally collected from large animal species via jugular venipuncture with or without tranquilization.

Blood collection from rabbit ears by transsecting the vein with a razor cut is strongly discouraged and in many cases prohibited. The use of xylene for vasodilation is discouraged because of its irritant properties. If used, xylene should be removed rapidly with alcohol and then soap and water, followed by an application of a soothing lotion. Use of heat lamps, acepromazine, or droperidol-fentanyl are often recommended as preferred methods of enhancing vasodilation to facilitate blood collection. Because of the risk of cardiac tamponade, pulmonary hemorrhage, and pneumothorax, intracardiac blood collection is limited to terminal procedures and is performed under general anesthesia in both rodents and rabbits. Survival intracardiac blood collection was not recommended for any species in the guidelines in our survey.

Restraint

Proper methods for restraint during immunization and blood collection procedures must be used to avoid injuries to the animal as well as personnel. It is helpful to acclimate animals to handling and restraint procedures prior to the initiation of immunization or other experimental procedures. It is extremely important to train personnel to perform manual restraint techniques and to use commercially available restrainers properly. Sedation of rabbits with acepromazine or droperidol-fentanyl during immunizations to reduce stress, enhance vasodilation, and prevent injury to rabbits and personnel is often recommended (Tillman and Norman, 1983). Other species may also be sedated for immunization procedures.

Alternative Techniques

Antibody production in chickens is an alternative in vivo technique for production of polyclonal antibodies. Indications and procedural methods for this technique are described in the manuscript by Hanly, Artwohl, and Bennett (p. 93 of this issue). Antibody production in chickens offers the advantage of providing a non-invasive means to obtain antibody that is recovered from the egg yolk.

Polyclonal antibody can also be recovered from ascites induced in immunized mice via intraperitoneal inoculation of a sarcoma cell line. The host's B-lymphocytes secrete antibody into the ascitic fluid (Karu, 1993; Ou et al., 1993). Other methods have also been described for polyclonal antibody production in mice (Lacy and Voss, 1986; Mahana and Paraf, 1993).

Another alternative method for production of polyclonal antibodies is via placement of a subcutaneous whiffle ball chamber, which has been described in rabbits and chickens (Hillam et al., 1974; Clemons et al., 1992; Ermeling et al., 1992). Immunizations are made directly into the chamber and antibody-rich fluid is harvested from the chamber. Advantages cited for this technique include greater flexibility in preparation of the immunogen, minimal discomfort and minimal tissue reaction in the animal, ease of immunization and collection of fluid from the chamber, and recovery of large volumes of antibody-rich fluid with low cellularity and absence of lipids (Clemons et al., 1992). This procedure does require surgical placement of the chamber.

INSTITUTIONAL RESOURCES

Institutional guidelines should include information sources for investigators seeking training or assistance in performing technical procedures and veterinary assessment and treatment for animals. Guidelines should also indicate availability of reference materials. Investigators conducting their own monoclonal or polyclonal antibody production procedures will benefit from videotapes illustrating currently acceptable immunization, blood sampling, and ascites harvesting techniques. Operation of centralized facilities for in vivo or in vitro monoclonal antibody production or in vivo polyclonal antibody production can be useful, particularly for institutions where large scale antibody production is performed.

PERSONNEL SAFETY

Personnel safety associated with the materials and procedures for antibody production are an important component of these types of guidelines. Recommendations should urge caution when using FCA. Accidental needle sticks in humans have been associated with chronic, painful inflammatory lesions and abscesses, particularly in individuals that were already sensitized to mycobacteria. Also, in previously unsensitized individuals, subsequent positive turberculin tests have been documented (Chapel and August, 1975). Protective clothing, including lab coat, safety glasses, and gloves are recommended to be worn. The use of luer-lock syringes in mixing antigen-adjuvant emulsions; avoidance of re-capping needles; and appropriate restraint, sedation, or anesthesia of animals to prevent injury to animals or personnel during immunizations should be emphasized.

Pristane can cause severe irritation if it comes into contact with the eyes, mucous membranes, skin, or respiratory or gastrointestinal tract. It is classified as a tumorigenic agent; therefore, appropriate precautions when handling this agent are required. As mentioned above, xylene's use should be discouraged; xylene is also an irritant, a mutagen, and a suspect carcinogen and the same precautions should be used when handling this agent.

DISCUSSION

Generally, there is agreement among recommendations made in institutional guidelines, published guidelines, and reports in the literature regarding the appropriate conduct of procedures for adjuvant use and antibody production. There was, however, considerable variability in recommendations for the number of permissible abdominal taps in mice used for ascites production. Restricting the number of taps to one may provide greater assurance that pain and distress are minimized, but at the same time, a much larger number of animals would likely be needed to produce the required amount of antibody. This conflict reflects the inherent difficulties in making these kinds of balanced decisions.

While guidelines are, by definition, intended to serve as general recommendations, it should be emphasized that each animal must be treated on an individual basis and professional judgment must be exercised. For example, there is considerable variation in the biological behavior of different hybridoma cell lines. Some cell lines are very aggressive and cause rapid morbidity and mortality whereas other cell lines are more slowly progressive and cause minimal morbidity. Fewer taps may be appropriate for cell lines known to cause rapid morbidity, whereas multiple taps may be appropriate for cell lines causing more slowly progressive signs. Even within a given cell line there may be considerable variation in response between individual animals. For these reasons it is important to emphasize that each animal must be clinically evaluated when making determinations for the number of abdominal taps and the time for euthanasia. Likewise in polyclonal antibody production, individual animals may respond very differently to the same treatment. Sound professional judgment on the part of investigators and veterinarians is required to assure that the recommendations established in institutional guidelines are rationally executed.

The finding that institutional guidelines and the literature vary with respect to recommendations for routes and sites of administration, volumes per site, and number of sites per animal for antigen-adjuvant preparations using Freund's adjuvant in different species suggests that additional work in this area is necessary to better define acceptable parameters for these procedures in different species, which balance scientific requirements with humane care and use of animals.

In reviewing institutional guidelines, it is clear that while there are many commercially available alternatives to the use of Freund's adjuvant, Freund's adjuvant remains the most commonly used preparation. Vigorous attempts should be made by institutions to disseminate information about alternative adjuvants, investigators should be encouraged to investigate alternatives to the use of Freund's adjuvant, and efforts should be made to determine the inflammatory sequelae of alternative adjuvants being used in animals (Mallon et al., 1991). Development and careful analysis of adjuvants with less severe inflammatory reactions, which provide adequate antibody response, should promote wider use of these products in biomedical settings.

The establishment of guidelines that describe acceptable techniques for adjuvant use and antibody production are extremely helpful. These guidelines provide investigators with the information needed to design protocols and perform experiments in a humane fashion with due consideration for the effects of these procedures on animals (Toth and Olson, 1990). We therefore encourage all institutions to develop such guidelines for these and other in vivo procedures.

REFERENCES

Allison, A. C., and N. E. Byars. 1986. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and of cell-mediated immunity. J. Immunol. Methods 95:157-168.

Allison, A.C., and N.E. Byars. 1991. Immunologic adjuvants: Desirable properties and side-effects. Mol. Immunol. 28(3):279-284.

Altman, A. C., and F. J. Dixon. 1989. Immunomodifiers in vaccines. Adv. Vet. Sci. Comp. Med. 33:301-343.

Altshuler, G. L., D. M. Dziewulski, J. A. Sowek, and G. Belfort. 1986. Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators. Biotechnol. Bioeng. 28:646-658.

Amyx, H. L. 1987. Control of animal pain and distress in antibody production and infectious disease studies. J. Am. Vet. Med. Assoc. 191(10): 1287-1289.

Baum, S. G., A. M. Lewis, W. P. Rowe, and R. J. Huebner. 1966. Epidemic nonmeningitic lymphocytic-choriomeningitis-virus infectionAn outbreak in a population of laboratory personnel. N. Eng. J. Med. 274(17): 934-936.

Bennett, B., I. J. Check, M. R. Olsen, and R. L. Hunter. 1992. A comparison of commercially available adjuvants for use in research. J. Immunol. Methods 153(1-2):31-40.

Bhatt, P. N., R. O. Jacoby, and S. W. Barthold. 1986. Contamination of transplantable murine tumors with lymphocytic choriomeningitis virus. Lab. Anim. Sci. 36(2):136-139.

Biggar, R. J., R. Deibel, and J. P. Woodall. 1976. Implications, monitoring, and control of accidental transmission of lymphocytic choriomeningitis virus within hamster tumor cell lines. Cancer Res. 36:551-553.

Bomford, R. 1988. Aluminum salts: Perspectives in their use as adjuvants. Pp. 35-41 in Immunological Adjuvants and Vaccines, G. Gregoriadis, A. Allison, and G. Poste, eds. New York: Plenum Press.

Boyd, J. E., and K. James. 1989. Human monoclonal antibodies: Their potential, problems, and prospects. Pp. 1-43 in Monoclonal Antibodies: Production and Application, A. Mizrahi, ed. New York: Alan R Liss.

Broderson, J. R. 1989. A retrospective review of lesions associated with the use of Freund's adjuvant. Lab. Anim. Sci. 39:400-405.

Brodeur, B. R., P. Tsang, and Y. Larose. 1984. Parameters affecting ascites tumour formation in mice and monoclonal antibody production. J. Immunol. Methods 71(2):265-272.

Brodeur, B. R., P. Tsang, et al. 1987. Production of monoclonal antibodies in mouse ascites. Pp. 99-111 in Monoclonal Antibody Production Techniques and Applications, L.B. Schook, ed. New York: Marcel Dekker.

Brodeur, B. R., and P. S. Tsang. 1986. High yield monoclonal antibody production in ascites. J. Immunol. Methods 86:239-241.

Brooks, R. E., R. D. Betz, and R. D. Moore. 1978. Injury and repair of the lung: Response to intravenous Freund's adjuvant. J. Pathol. 124:205-217.

Canadian Council on Animal Care (CCAC). 1991. CCAC Guidelines on acceptable immunological procedures. Ottawa, Ontario, Canada: CCAC (Available from CCAC, Constitution Square, Tower II, 315-350 Albert, Ottawa ON K1R 1B1. Tel: (613) 238-4031; Fax: (613) 238-2837; E-mail: ccac@carleton.ca.)

Chandler, J. P. 1987. Factors influencing monoclonal antibody production in mouse ascites fluid. Pp. 75-92 in Commercial Production of Monoclonal Antibodies: A Guide for Scale-Up, S. S. Seaver, ed. New York: Marcel Dekker.

Chapel, H. M., and P. J. August. 1975. Report of nine cases of accidental injury to man with Freund's complete adjuvant. Clin. Exp. Immunol. 24:538-541.

Check, I. J., B. Bennett, et al. 1990. Hunter's TiterMax® #R-1 Adjuvant: Technical Background. Norcross, Ga.: Vaxcel Corporation.

Clemons, D. J., C. Besch-Williford, E. K. Steffen, L. K. Riley, and D. H. Moore. 1992. Evaluation of a subcutaneously implanted chamber for antibody production in rabbits. Lab. Anim. Sci. 42(3):307-311.

Collins, M. J., and J. C. Parker. 1972. Murine virus contaminants of leukemia viruses and transplantable tumors. J. Nat. Cancer Inst. 49:1137-1143.

Colwell, D. E., S. M. Michalek, and J. R. McGhee. 1986. Method for generating a high frequency of hybridomas producing monoclonal IgA antibodies. Methods Enzymol. 121:42-51.

Cranco, M., and M. Potter. 1976. The requirement of an adherent cell substratum for the growth of developing plasmacytoma cells in vivo. J. Exp. Med. 144:1554-1567.

Croft, S., J. Walsh, W. Lloyd, and G. J. Russell-Jones. 1991. TraT: A powerful carrier molecule for the stimulation of immune responses to protein and peptide antigens. J. Immunol. 146:793-798.

Dykewicz, C. A., V. M. Dato, S. P. Fisher-Hoch, M. V. Howarth, G. I. Perez-Oronoz, S. M. Ostroff, H. Gary, L. B. Schonberger, and J. B. McCormick. 1992. Lymphocytic choriomeningitis outbreak associated with nude mice in a research institute. J. Am. Med. Assoc. 267:1349-1353.

Epstein, N., and M. Epstein. 1986. The Hybridoma Technology: I. Production of Monoclonal Antibodies. Adv. Biotechnol. Processes 6:179-218.

Ermeling, B. L., E. K. Steffen, R. E. Fish, and R. R. Hook, Jr. 1992. Evaluation of subcutaneous chambers as an alternative to conventional methods of antibody production in chickens. Lab. Anim. Sci. 42(4):402-407.

Falkenberg, F. W., H. Weichert, M. Krane, I. Bartels, M. Palme, H. -O. Nagels, and H. Fiebig. 1995. In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter. J. Immunol. Methods 179:13-29.

Falkenberg, F. W., T. Hengelage, M. Krane, I. Bartels, A. Albrecht, N. Holtmeier, and M. Wüthrich. 1993. A simple and inexpensive high density dialysis tubing cell culture system for the in vitro production of monoclonal antibodies in high concentration. J. Immunol. Methods 165:193-206.

Freund, J., and D. McDermott. 1942. Sensitization to horse serum by means of adjuvants. Proc. Soc. Exp. Biol. Med. 49:548-553.

Freund, Y. R., and P. B. Blair. 1982. Depression of natural killer activity and mitogen responsiveness in mice treated with pristane. J. Immunol. 129:2826-2830.

Galfrè, G., and C. Milstein. 1981. Preparation of monoclonal antibodies: Strategies and procedures. Methods Enzymol. 73:1-46.

Gillette, R. W. 1987. Alternatives to pristane priming for ascitic fluid and monoclonal antibody production. J. Immunol. Methods 99:21-23.

Grumpstrup-Scott, J., and D. D. Greenhouse. 1988. NIH intramural recommendations for the research use of complete Freund's adjuvant. ILAR News 30(2):9.

Guzman, J., K. Frei, and D. Nadal. 1995. In vitro immunization: Generation of neutralizing monoclonal antibodies to human interleukin-10. J. Immunol. Methods 179-265-268.

[Harlow, E., and D. Lane. 1988. Adjuvants. Pp. 96-124 in Antibodies: A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.

Hengartner, H., A. L. Luzzati, and M. Schreier. 1978. Fusion of in vitro immunized lymphoid cells with X63Ag8. Curr. Top. Microbiol. Immunol. 81:92-96.

Herbert, W. J. 1978. Mineral oil adjuvants and the immunization of laboratory animals. Pp. A2.3-A3.15 in Handbook of Experimental Immunology, D. M. Weir, ed. Oxford: Blackwell Scientific Publications.

Hillam, R. P., R. P. Tengerdy, and G. L. Brown. 1974. Local antibody production against the murine toxin of Yersinia pestis in a golf-ball induced granuloma. Infect. Immun. 10:458-463.

Hoogenraad, N., T. Helman, and J. Hoogenraad. 1983. The effect of pre-injection of mice with pristane on ascites tumour formation and monoclonal antibody production. J. Immunol. Methods 61:317-320.

Hoogenraad, N. J., and C. J. Wraight. 1986. The effect of pristane on ascites tumor formation and monoclonal antibody production. Methods Enzymol. 121:375-381.

Hopkinson, J. 1985. Hollow fiber cell culture systems for economical cell-product manufacturing. Bio/Technology 3:225-230.

Hughes, L. E., R. Kearney, and M. Tully. 1970. A study in clinical cancer immunotherapy. Cancer 26:269-278.

Hunter, R. L., B. Bennett, D. Howerton, S. Buynitzky, and I. J. Check. 1989. Nonionic block copolymer surfactants as immunological adjuvants: Mechanisms of action and novel formulations. Pp. 133-144 in Immunological Adjuvants and Vaccines, G. Gregoriadis, A. C. Allison, and G. Poste, eds. New York: Plenum Press.

Jackson, L- R. 1994. Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production. Massachusetts Institute of Technology, Division of Toxicology, Masters Thesis.

Jackson, L. R., L. J. Trudel, J. G. Fox, and N. S. Lipman. In press. Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production. J. Immunol. Methods

Jackson, L. R., L. J. Trudel, J. G. Fox, and N. S. Lipman. 1996. Clinicopathologic features and production parameters for monoclonal antibody production in murine ascites. Lab. Anim. Sci. (Manuscript in progress.)

Jaspert, R., T. Geske, A. Teichmann, Y.-M. Kaßner, K. Kretzschmar, and J. L'age-Stehr. 1995. Laboratory scale production of monoclonal antibodies in a tumbling chamber. J. Immunol. Methods 178:77-87.

Johnston, B. A., H. Eisen, and D. Fry. 1991. An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits. Lab. Anim. Sci. 41:15-21.

Jones, S. L., J. C. Cox, and J. E. Pearson. 1990. Increased monoclonal antibody ascites production in mice primed with Freund's incomplete adjuvant. J. Immunol. Methods 129:227-231.

Karu, A. E. 1993. Monoclonal antibodies and their use in detection of hazardous substances. Pp. 205-321 in Hazard Assessment of Chemicals-Current Developments, J. Saxena, ed. Washington, D.C.: Taylor & Francis Intl Publishers.

Kenney, J. S., B. W. Hughes, M. P. Masada, and A. C. Allison. 1989. Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J. Immunol. Methods 121:157-166.

Kleinman, N. R., A. B. Kier, E. Diaconu, and J. H. Lass. 1993. Posterior paresis induced by Freund's adjuvant in guinea pigs. Lab. Anim. Sci. 43:364-366.

Knudsen, K. 1985. Proteins transferred to nitrocellulose for use as immunogens. Analytical Biochemistry 147: 285-288.

Kripke, M.C., and D. W. Weiss. 1970. Studies on the immune responses of BALB/c mice during tumor induction by mineral oil. Int. J. Cancer 6:422-430.

Kuby, J. 1992. Antigens. Pp. 75-78 in Immunology, J. Kuby, ed. New York: W.H. Freeman and Company.

Kuhlmann, I., W. Kurth, and I. Ruhdel. 1985. Monoclonal antibodies: In vitro and in vivo production on a laboratory scale, with consideration of the legal aspects of animal protection. ATLA 17:73-82.

Kurkela, R., E. Fraune, and P. Vihko. 1993. Pilot-scale production of murine monoclonal antibodies in agitated, ceramic-matrix, or hollow-fiber cell culture systems. Biotechniques 15(4):674-683.

Kwan, S. P., D. E. Yelton, and M. D. Scharff. 1980. Production of monoclonal antibodies. Genet. Eng. 2:31-46.

Lacy, M. J., and E. W. Voss. 1986. A modified method to induce immune polyclonal ascites fluid in Balb/c mice using Sp2/0-Ag14 cells. J. Immunol. Methods. 97:169-177.

Larsson, A., and B. O. Nilsson. 1988. Immunization with nanogram quantities of nitrocellulose-bound antigen, electroblotted from sodium dodecyl sulphate-polyacrylamide gels. Scand. J. Immunol. 27:305-309.

Leak, L. V., M. Potter, and W. J. Mayfield. 1985. Response of the peritoneal mesothelium to the mineral oil pristane. Curr. Top. Microbiol. Immunol. 122:221-233.

Leskowitz, S., and B. Waksman. 1960. Studies in immunization: I. The effect of route of injection of bovine serum albumin in Freund's adjuvant on production of circulating antibody and delayed hypersensitivity. J. Immunol. 84:58-72.

Lipman, N.S., L. J. Trudel, J. C. Murphy, and Y. Sahali. 1992. Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and Ribi adjuvants in mice. Lab. Anim. Sci. 42:193-197.

Loew, F. M., and J. G. Fox. 1983. Animal health surveillance and health delivery systems. Pp. 69-81 in The Mouse in Biomedical Research, H. Foster, J. D. Small, and J. G. Fox, eds. New York: Academic Press.

Ma, M., S. -J. Wu, M. Howard, and A. B. Borkovec. 1984. Enhanced production of mouse hybridomas to picomoles of antigen using EL-4 conditioned media with an in vitro immunization protocol. In Vitro 20(9):739-741.

Mahana, W., and A. Paraf. 1993. Mice ascites as a source of polyclonal and monoclonal antibodies. J. Immunol. Methods 167:187-192.

Mallon, F. M., M. E. Graichen, B. R. Conway, M. S. Landi, and H. C. Hughes. 1991. Comparison of antibody response by use of synthetic adjuvant system and Freund's complete adjuvant in rabbits. Am. J. Vet. Res. 52:1503-1506.

McGuill, M. W., and A. N. Rowan. 1989. Refinement of monoclonal antibody production and animal well-being. ILAR News 31(l):7-11.

Meyer, T. J., E. E. Ribi, I. Azuma, and B. Zbar. 1974. Biologically active components from mycobacterial cell walls. II. Suppression and regression of strain-2 guinea pig hepatoma. J. Nat. Cancer Inst. 52:103-111.

Moore, J. M., and T. V. Rajan. 1994. Pristane retards clearance of particulate materials from the peritoneal cavity of laboratory mice. J. Immunol. Methods 173:273-278.

Moro, A. M., M. T. A Rodrigues, M. N. Gouvea, M. L. Z. Silvestri, J. E. Kalil, and I. Raw. 1994. Multiparametric analyses of hybridoma growth on cylinders in a packed-bed bioreactor system with internal areation. Serum-supplementation and serum-free media comparison for Mab production. J. Immunol. Methods 176:67-77.

Mueller, U. W., C. S. Hawes, and W. R. Jones. 1986. Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice. J. Immunol. Methods 87:193-196.

Nerenberg, S. T., P. Zedler, R. Prasad, N. S. Biskup, and L. Pedersen. 1978. Hematological response of rabbits to chronic, repetitive, severe bleedings for the production of antisera. J. Immunol. Methods 24:19-24.

Niemi, S. M., J. G. Fox, L. R. Brown, and R. Langer. 1985. Evaluation of ethylene-vinyl acetate copolymer as a noninflammatory alternative to Freund's complete adjuvant in rabbits. Lab. Anim. Sci. 35:609-612.

Osebold, J. W. 1982. Mechanisms of action by immunological adjuvants. J. Am. Vet. Med. Assoc. 181:983-987.

Ou, S. K-W., J. M.-C. Hwang, and P. H. Patterson. 1993. A modified method for obtaining large amounts of high-titer polyclonal ascites fluid. J. Immunol. Methods 165:75-80.

Pannell, R., and C. Milstein. 1992. An oscillating bubble chamber for laboratory scale production of monoclonal antibodies as an alternative to ascitic tumours. J. Immunol. Methods 146:43-48.

Pierce, D. L., and W. R. Dukelow. 1988. Misleading positive tuberculin reactions in a squirrel monkey colony. Lab. Anim. Sci. 38(6)729-730.

Platica, M., C. Bojko, R. Clincea, and V. Hollander. 1982. Lipopolysaccharide and pristane-induced peritoneal exudate requirements for plasma tumor cell colony formation. J. Nat. Cancer Inst. 68:325-328.

Reuveny, S., and A. Lazar. 1989. Equipment and procedures for production of monoclonal antibodies in culture. Pp. 45-80 in Monoclonal Antibodies: Production and Application, A. Mizrahi, ed. New York: Alan R. Liss.

Reuveny, S., D. Velez, J. D. Macmillan, and L. Miller. 1986a. Factors affecting cell growth and monoclonal antibody production in stirred reactors. J. Immunol. Methods 86:53-59.

Reuveny, S., D. Velez, L. Miller, and J. D. Macmillan. 1986b. Comparison of cell propagation methods for their effect on monoclonal antibody yield in fermenters. J. Immunol. Methods 86:61-69.

Ribi, E., J. L. Cantrell, K. Takayama, N. Qureshi, J. Peterson, and H. O. Ribi. 1984. Lipid A and immunotherapy. Rev. Infect. Dis. 6:567-572.

Rowsell, H. C. 1989. Guidelines on immunological procedures. SCAW Newsletter 11(3):2-3, 6-7. (Available from Scientists Center for Animal Welfare, Golden Triangle Building One, 7833 Walker Drive, Ste. 340, Greenbelt, MD 20770. Tel: (301) 345-3500; Fax: (301) 345-3503.)

Schiefer, B., and H. Stunzi. 1979. Pulmonary lesions in guinea pigs and rats after subcutaneous injection of complete Freund's adjuvant or homologous pulmonary tissue. Zbl. Vet. Med. A. 26:1-10.

Shevitz, J., S. Reuveny, T. L. LaPorte, and G. H. Cho. 1989. Stirred tank perfusion reactors for cell propagation and monoclonal antibody production. Adv. Biotechnol. Processes 11:81-106.

Sigel, M. B., Y. N. Sinha, and W. P. VanderLaan. 1983. Production of antibodies by inoculation into lymph nodes. Methods Enzymol. 93:3-12.

Smith, D. E., M. E. O'Brien, V. J. Palmer, and J. A. Sadowski. 1992. The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular weight antigen in rabbits. Lab. Anim. Sci. 42(6):599-601.

Sjörgren-Jansson, E., and S. Jeanson. 1990. Growing hybridomas in dialysis tubing, optimization of technique. Pp. 1-41 in Laboratory Methods in Immunology, E. Zola, ed. Boca Raton, Fla.: CRC Press.

Stewart, F., A. Callander, and D. J. Garwes. 1989. Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice. J. Immunol. Methods 119:269-275.

Stills, H. F. 1994. Polyclonal antibody production. Pp. 435-448 in The Biology of the Laboratory Rabbit, 2nd Edition, P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds. San Diego, Ca: Academic Press.

Stills, H. F., and M. Q. Bailey. 1991. Use of Freund's complete adjuvant. Lab. Anim. 20(4):25-30.

Tillman, P., and C. Norman. 1983. Droperidol-fentanyl as an aid to blood collection in rabbits. Lab. Anim. Sci. 33(2):181-182.

Toth, L. A., A. W. Dunlap, G. A. Olson, and J. R. Hessler. 1989. An evaluation of distress following intraperitoneal immunization with Freund's Adjuvant in mice. Lab. Anim. Sci. 39(2):122-126.

Toth, L. A., and G. A. Olson. 1990. Strategies for minimizing pain and distress in laboratory animals. Lab. Anim. 20(4):33-39.

Tung, A. S., S. T. Ju, S. Sato, and A. Nisonoff. 1976. Production of large amounts of antibodies in individual mice. J. Immunol. 116:676-681.

Van Ness, J., U. K. Laemmli, and D. E. Pettijohn. 1984. Immunization in vitro and production of monclonal antibodies specific to insoluble and weakly immunogenic proteins. Proc. Natl. Acad. Sci. USA 81:7897-7901.

Warren, H. S., F. R. Vogel, and L. A. Chedid. 1986. Current status of immunological adjuvants. Ann. Rev. Immunol. 4:369-388.

Woodward, L. F. 1989. Adjuvant activity of water-insoluble surfactants. Lab. Anim. Sci. 39(3):222-225.

Workman, J. P., A. Balmain, J. A. Hickman, N. J. McNally, A. M. Rohas, N. A. Mitchison, C. G. Pierrepoint, R. Raymond, C. Rowlatt, T.C. Stephens et al. 1988. UKCCCR guidelines for the welfare of animals in experimental neoplasia. Lab. Anim. 22:195-201.

Yarkoni, E., and H. J. Rapp. 1979. Tumor regression after intralesional injection of mycobacterial components emulsified in 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene (squalene), 2,6,10,15,19, 23-hexamethyltetracosane (squalane), peanut oil, or mineral oil. Cancer Res. 39:1518-1520.

Zola, H. 1995. Conventional mouse monoclonal antibodies. P. 29 in Monoclonal Antibodies: The Second Generation, H. Zola, ed. Oxford: BIOS.

Copyright © 2008. National Academy of Sciences.
All rights reserved.
500 Fifth St. N.W., Washington, D.C. 20001.
Terms of Use and Privacy Statement