Antibody Production (Immunogen Preparation)
Antibody production is conceptually simple. However, because it depends upon such a complex biological system (immunity of a living organism), results are not entirely predictable. Individual animals – even those that are genetically identical – will respond uniquely to the same immunization scheme, generating different suites of specific antibodies against an injected antigen. Even so, equipped with a basic understanding of how the immune system responds to injection of a foreign substance and a knowledge of available tools for preparing a sample for injection, researchers can greatly increase the probability of obtaining a useful antibody product.
For example, small compounds (drugs or peptides) are not sufficiently complex by themselves to induce an immune reponse or be processed in a manner that elicits production of specific antibodies. For antibody production to be successful with small antigens, they must be chemically conjugated with immunogenic carrier proteins such as keyhole limpet hemocyanin (KLH). Adjuvants can be mixed and injected with an immunogen to increase the intensity of the immune response.
Carrier protein conjugation, use of adjuvants and other issues relating to preparation of samples for injection are described in this section on antibody production. Procedures for generating, purifying and modifying antibodies for use as antigen-specific probes were developed during the 1970s and 1980s and have remained relatively unchanged since Harlow and Lane published their classic book "Antibodies: A Laboratory Manual" in 1988.
Antibody production and purification guide
The updated Antibody Production and Purification Technical Handbook is an essential resource for any laboratory working with antibodies. The handbook provides an overview of antibody structure and types, as well as technical information on the procedures, reagents and tools used to produce, purify, fragment and label antibodies.
The Immune System
The immune system is a surveillance system designed to provide protection to its host from foreign invaders. The surveillance is mediated by proteins and cells that circulate throughout the organism to identify and destroy foreign cells, viruses or macromolecules.
Immune protection is provided by a dual system consisting of the cellular immune response and the humoral immune response. The cellular immune response is mediated by T lymphocytes and cannot be transferred from one individual to another by transfusion of serum. Humoral immunity involves soluble proteins found in serum (antibodies) that can be transferred to a recipient when serum is transfused.
Every cell in a vertebrate organism expresses the class I Major Histocompatibility Complex (MHC I) on its plasma membrane. The MHC I presents endogenously derived peptide antigens to cytotoxic T lymphocytes (CTL). If the T cell receptor of a CTL binds to the MHC I/peptide antigen on a cell, the whole cell is destroyed. This is a general description of the cellular immune response, which targets intracellular pathogens such as viruses or bacteria (non-self) and cancer cells (altered self).
The humoral response targets extracellular antigens. B-lymphocytes use membrane IgM (mIgM) to bind antigen in its native form. Crosslinking of many mIgM and antigen molecules occurs (capping) and the complex is then taken into the cell by receptor mediated endocytosis. This endosome then fuses with a lysosome and the resulting endolysosome digests the antigen into small peptides. The endolysosome fuses with a vesicle containing class II Major Histocompatibility Complex (MHC II) molecules and the peptide antigens are bound by a cleft in the MHC II. This MHC II/antigen complex is then expressed on the plasma membrane of the B-lymphocyte. The T cell receptor of a T helper lymphocyte then binds the MHC II/antigen and the T cell secretes cytokines that signal the B-lymphocyte to divide, differentiate and secrete antibodies. Without T help, the humoral response shuts down; in fact, the cellular response shuts down as well, as in AIDS.
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Definitions of Antigen and Immunogen
Successful generation of antibodies depends upon B-lymphocytes to bind, process and present antigen to T helper lymphocytes, which signal the B cells to produce and secrete antibodies. An antigen is any molecule that is identified as non-self by components of the immune system. An immunogen is an antigen that is able to evoke an immune response, including production of antibody via the humoral response. All immunogens are antigens, but not all antigens are immunogens. It is important to distinguish between the terms "antigen" and "immunogen" because many compounds are not immunogenic, and successful production of antibodies against such antigens requires that they be made immunogenic by chemically attaching them to known immunogens before injection.
Properties Determining Immunogenicity
Immunogenicity is the ability of a molecule to solicit an immune response. There are three characteristics that a substance must have to be immunogenic: foreignness, high molecular weight and chemical complexity. Foreignness is required so that the immunized animal does not recognize and ignore the substance as "self." Generally, compounds from an organism are not immunogenic to that same individual and are only poorly immunogenic to others of the same or related species.
The second requirement for immunogenicity is high molecular weight. Small compounds (MW less than 1000), such as penicillin, progesterone and aspirin, as well as many moderately sized molecules (MW from 1000 to 6000), are not immunogenic. Most compounds with a molecular weight greater than 6000 are immunogenic. Compounds smaller than this can often be bound by mIgM on the surface of the B-lymphocyte, but they are not large enough to facilitate crosslinking of the mIgM molecules. This crosslinking is commonly called "capping" and is the signal for receptor mediated endocytosis of the antigen.
Finally, some degree of chemical complexity is required for a compound to be immunogenic. For example, even high molecular weight homopolymers of amino acids and simple polysaccharides seldom make good immunogens because they lack the chemical complexity necessary to generate an immune response.
Macromolecules as Immunogens
It is possible to make certain generalizations about immunogenicity of the four major classes of macromolecules: carbohydrates, lipids, nucleic acids and proteins. Carbohydrates are immunogenic only if they have a relatively complex polysaccharide structure or form part of more complex molecules, such as glycoproteins. Lipids usually are not immunogenic but can be made so by conjugation to a carrier protein. Likewise, nucleic acids are poor immunogens but can become immunogenic when coupled to a carrier protein.
Because of their structural complexity and size, proteins are generally strong immunogens. Given that most natural immunogens are macromolecules composed of protein, carbohydrate or a combination of the two, it is understandable that proteins are so broadly immunogenic. Peptides may have the complexity necessary to be antigenic, but their small size usually renders them ineffective as immunogens on their own. Peptides are most often conjugated to carrier proteins to insure that they induce an immune response and production of antibodies.
Haptens vs. Epitopes
Peptides and other small molecules that are used as antigens are referred to as haptens. They are able to act as recognition sites for production of specific antibodies but cannot by themselves stimulate the necessary immune response. Haptens can be made immunogenic by coupling them to a suitable carrier molecule.
An epitope is the specific site on an antigen to which an antibody binds. For very small antigens, practically the entire chemical structure may act as a single epitope. Depending on its complexity and size, an antigen may effect production of antibodies directed at numerous epitopes. Polyclonal antibodies are mixtures of serum immunoglobulins and collectively are likely to bind to multiple epitopes on the antigen. Monoclonal antibodies by definition contain only a single antibody clone and have binding specificity for one particular epitope.
Specific antibodies can be generated against nearly any sufficiently unique chemical structure, either natural or synthetic, as long as the compound is presented to the immune system in a form that is immunogenic. The resulting antibodies may bind to epitopes composed of entire molecules (e.g., small haptens), particular functional groups of a larger molecule, unique arrangements of several amino acid functional groups in the tertiary structure of proteins, or any other unique structure in lipoproteins, glycoproteins, RNA, DNA or polysaccharides. Epitopes may also be parts of cellular structures, bacteria, fungi or viruses.
Carrier Proteins for Immunogen Preparation
A carrier protein is any protein used for coupling with peptides or other haptens that are not sufficiently large or complex on their own to induce an immune response and produce antibodies. The carrier protein, because it is large and complex, confers immunogenicity to the conjugated hapten, resulting in antibodies being produced against epitopes on the hapten and carrier. Many proteins can be used as carriers and are chosen based on immunogenicity, solubility, and availability of useful functional groups through which conjugation with the hapten can be achieved. The two most commonly used carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
In a typical immune response, antibodies are produced by B-lymphocytes. In the majority of hapten-carrier systems, the B cells will produce antibodies that are specific for both the hapten and carrier. Because an antibody response will be directed against epitopes on both the carrier protein and hapten, it is important to plan carefully how hapten-specific antibodies will be identified and purified from the final immunized serum. To create the best immunogen, it may be beneficial to prepare the conjugates with several different carriers and with a range of hapten:carrier coupling ratios.
Keyhole Limpet Hemocyanin (KLH)
Keyhole limpet hemocyanin (KLH) is the most widely used carrier protein. The copper-containing polypeptide belongs to a group of non-heme proteins called hemocyanins, which are found in arthropods and mollusks. KLH is isolated from keyhole limpets (Megathura crenulata).
Because KLH is from a class of proteins and a group of organisms that are evolutionarily distant from mammals, it is very "foreign" to the animal systems typically used to produce antibodies. The protein is also highly immunogenic because of its very large size and complex structure. The molecule is composed of 350kDa and 390kDa subunits that associate to form aggregates ranging from 0.5 to 8 million daltons.
Each KLH protein molecule contains several hundred surface lysine groups that provide primary amines as targets for covalent attachment of haptens using a variety of crosslinking techniques. These features make KLH an extremely immunogenic and effective carrier protein for immunogen preparation. Although the large protein is sometimes difficult to work with because it has limited solubility, the commercial availability of stabilized and pre-activated formulations make it convenient to use.
Thermo Scientific Imject Mariculture Keyhole Limpet Hemocyanin (mcKLH) is purified and lyophilized in a stabilizing buffer. After reconstitution, the suspension-solution is an opalescent blue, which is characteristic of highly purified, nondenatured KLH.
Traditionally, KLH was obtained from giant keyhole limpets harvested directly from the natural environment. This method disturbs the sensitive shoreline ecosystems where these limpets live. Current methods to obtain KLH are much less threatening to the natural habitat and limpet species survival. Giant keyhole limpets are raised in tanks and harvested (marine culture or “mariculture”) where they are occasionally milked of some of their fluids, similar to humans donating blood.
Blue Carrier* Immunogenic Protein
Blue Carrier* Protein is a purified preparation of Concholepas concholepas hemocyanin (CCH). The large protein exhibits most of the same immunogenic properties as the popular carrier protein, keyhole limpet hemocyanin (KLH). However, its signficantly better solubility provides greater flexibility in immunogen preparation protocols by allowing a broader range of buffer and pH conditions for coupling peptides, proteins and other haptens using crosslinking methods.
Blue Carrier Protein is specially purified hemocyanin from the mollusk Concholepas concholepas. The CCH protein is composed of two very large polypeptide subunits (404 and 351kDa) that form an extremely stable heterodidecameric structure even in the absence of divalent cations. (By contrast, KLH has a less stable and soluble homodidecameric structure). The complex molecular arrangement of CCH subunits contains diverse repeated antigenic structures that elicit a strong immune reaction mediated by T and B lymphocytes.
Because of their large size and molecular complexity, KLH and CCH hemocyanins are carrier proteins of choice for use as immunogens to produce antibodies against haptens and peptides. Moreover, studies suggest that the strong DTH immune response elicited by hemocyanins in animals and in humans has beneficial therapeutic effects in certain types of cancer. New developments in the immunotherapy of cancer have taken advantage of the unique immunogenic properties of hemocyanins in the development of novel conjugate vaccines for treatment of emerging diseases.
Bovine Serum Albumin as a Carrier Protein
Bovine serum albumin (BSA; 67kDa) belongs to the class of serum proteins called albumins. Albumins constitute about half the protein content of plasma and are quite stable and soluble. BSA is much smaller than KLH but is nonetheless fully immunogenic. It is a popular carrier protein for weakly antigenic compounds. BSA exists as a single polypeptide with 59 lysine residues, 30 to 35 of which have primary amines that are capable of reacting with a conjugation reagent. Numerous carboxylate groups give BSA its net negative charge (pI 5.1). Thermo Scientific Imject BSA is a highly purified (i.e., Fraction V) bovine serum albumin that, once reconstituted, can be used for conjugation to haptens without dialysis or further purification.
BSA is commonly used in development of immunoassays because it is readily available, is fully soluble and has numerous functional groups useful for crosslinking to small molecules that otherwise would not coat efficiently in polystyrene microplates. Furthermore, BSA is the most popular standard for protein assays, well-characterized as a molecular weight marker in SDS-PAGE and widely used as a blocking agent. These same characteristics that make BSA easy to use in immunoassay development also make it simple to use for preparing and testing conjugation efficiency of carrier-hapten conjugates. However, such multiple uses for BSA also require that steps be taken to avoid undesired cross-reactivity with the carrier in antibody-screening procedures and final applications.
For this reason, BSA is often used as a non-relevant protein carrier for antibody screening and immunoassays after using KLH as the carrier protein to generate the immune response against the hapten. Only by using different carrier proteins in the immunization and screening/purification steps can one be assured of detecting hapten-specific rather than carrier-specific antibodies. Using BSA as the non-relevant carrier protein generally allows one to take greater advantage of its properties as standard, MW marker and blocking agent.
Cationized bovine serum albumin (cBSA) is prepared by modifying native BSA with excess ethylenediamine, essentially capping all negatively-charged carboxyl groups with positively-charged primary amines. The result is a highly positively-charged protein (pI > 11) that has significantly increased immunogenicity compared to native BSA. In addition, the increased number of primary amines provides for a greater number of antigen molecules to be conjugated with typical crosslinking methods.
A series of research articles by Muckerheide, Domen and Apple (1987, 1988) reported these researchers' studies of the effects of carrier protein cationization on the generation and regulation of immune responses. (See the linked product pages for complete references.) In their studies, using cBSA as the carrier protein resulted in immunogen that stimulated a much higher antibody response than the native form of BSA. In vivo, the antibody response increased and remained elevated for an extended period of time. In vitro, much less cBSA than native BSA was required to produce the same degree of T cell proliferation. Interestingly, the immune response enhancement caused by cBSA extended to haptens or other proteins to which it was conjugated. For example, when used to immunize mice, ovalbumin conjugated to cBSA elicited greater anti-ovalbumin antibody production than ovalbumin alone or ovalbumin-BSA conjugate.
Ovalbumin as a Carrier Protein
Ovalbumin (OVA; 45kDa) can be used as a carrier protein. Also known as egg albumin, ovalbumin constitutes 75% of protein in hen egg whites. OVA contains 20 lysine groups and is most often used as a secondary (screening) carrier rather than for immunization, although it is somewhat immunogenic. The protein also contains 14 aspartic acid and 33 glutamic acid residues that afford carboxyl groups. These groups can be used as targets for conjugation with haptens. Ovalbumin exists as a single polypeptide chain having many hydrophobic residues and an isoelectric point of 4.63. The protein denatures at temperatures above 56°C or when subject to electric current or vigorous shaking. OVA is unusual among proteins in being soluble in high concentrations of the organic solvent DMSO, enabling conjugation to haptens that are not easily soluble in aqueous buffers.
Methods of Hapten-Carrier Conjugation
Several approaches are available for conjugating haptens to carrier proteins. The choice of which conjugation chemistry to use depends on the functional groups available on the hapten, the required hapten orientation and distance from the carrier, and the possible effect of conjugation on biological and antigenic properties. For example, proteins and peptides have primary amines (the N-terminus and the side chain of lysine residues), carboxylic groups (C-terminus or the side chain of aspartic acid and glutamic acid), and sulfhydryls (side chain of cysteine residues) that can be targeted for conjugation. Generally, it is the many primary amines in a carrier protein that are used to couple haptens via a crosslinking reagent.
EDC Conjugation (carboxyl and amine crosslinking)
Because most proteins contain both exposed lysines and carboxyl groups, immunogen formation with the carbodiimide crosslinker EDC is often the simplest and most effective method for protein-carrier and peptide-carrier conjugations. EDC reacts with available carboxyl groups on either the protein carrier or peptide hapten to form an active o-acylisourea intermediate. This intermediate then reacts with a primary amine to form an amide bond and a soluble urea by-product. This efficient reaction produces a conjugated immunogen in less than two hours.
In general, conjugations mediated by EDC result in a certain amount of polymerization when polypeptide antigens and protein carriers are involved. This occurs because most peptides and antigens contain both primary amines and carboxylates (at least in their N- and C-termini, respectively). Some peptides will conjugate to themselves (end-to-end by their N- and C-termini or through side chains) as well as to the carrier protein. Likewise, the carrier protein will conjugate to itself.
Such polymerization is not necessarily a disadvantage for immunogenicity or the desired antigen-specific antibody production. Large polymers can decrease the solubility of the conjugate, making its subsequent handling and use more difficult. Some polymerized peptide on the surface of the carrier may actually enhance the immunogenicity of the peptide, effecting a greater antibody response. Peptides will become conjugated in a variety of orientations, ensuring that all parts of the molecule are presented and available as antigens within the entire population.
Maleimide Conjugation (sulfhydryl crosslinking)
A peptide synthesized with a terminal cysteine residue has a sulfhydryl group that provides a highly specific conjugation site for reacting with certain crosslinkers. For example, the heterobifunctional crosslinker Sulfo-SMCC contains a maleimide group that will react with free sulfhydryls, plus and a succinimidyl (NHS) ester that will react with primary amines. By reacting the reagent first to the carrier protein (with its numerous amines) and then to a peptide containing a reduced terminal cysteine, all peptide molecules can be conjugated with the same predictable orientation.
It is a two-step reaction strategy. The carrier protein is "activated" in isolation by reaction with a molar excess of Sulfo-SMCC. Numerous molecules of SMCC become attached to the carrier protein when the NHS-ester group is displaced by the abundant amino group of the carrier protein.The modified carrier protein is then purified by gel filtration (desalting) to remove excess crosslinker and byproducts. At this stage, the purified carrier possesses modifications generated by the crosslinker, resulting in a number of reactive maleimide groups projecting from its surface. Finally, cysteine-terminated peptide or other sulfhydryl hapten is added to the maleimide-activated carrier protein. The maleimide groups react with the peptide sulfhydryl (–SH) groups to form stable thioether bonds.
Any protein can be maleimide-activated in this manner to allow efficient conjugation of haptens via reduced thiols. However, KLH, BSA and other popular carrier proteins are available in pre-activated forms and convenient kits that are ready for immediate conjugation with sulfhydryl peptides. Purchasing quality-tested, stabilized, maleimide-activated carrier proteins ensures consistent performance and saves several steps.
Glutaraldehyde Conjugation (amine-to-amine crosslinking)
Glutaraldehyde can be used to crosslink peptides and carrier proteins via amines on the respective polypeptides. This approach randomly targets lysine residues or the N-terminus of a peptide and surface lysines of the carrier protein. Depending upon the peptide amino acid composition (i.e., whether it possesses more than one primary amine), the opportunity for variable antigen presentation (orientation) and high loading (polymerization) are not as great as with EDC conjugation. However, glutaraldehyde is an efficient crosslinker, if not particularly specific and predictable, and it is still commonly used by antibody production facilities.
Amine-to-amine crosslinking also can be accomplished with crosslinkers such as disuccinimidyl suberate (DSS) and its water-soluble analog, BS3. If longer spacer arms are desired, pegylated versions of BS3 are also available.
Adjuvants and Immunization
To enhance the immune response to an immunogen, various additives called adjuvants can be used. When mixed and injected with an immunogen, an adjuvant will enhance the immune response. An adjuvant is not a substitute for a carrier protein because it enhances the immune response to immunogens but cannot itself render haptens immunogenic. Adjuvants are nonspecific stimulators of the immune response, helping to deposit or sequester the injected material and causing a dramatic increase in the antibody response.
There are many popular adjuvants, including complete Freund's adjuvant (CFA or FCA). This reagent consists of a water-in-oil emulsion and killed Mycobacterium. The oil-and-water emulsion localizes the antigen for an extended period of time, and theMycobacterium attracts macrophages and other appropriate cells to the injection site. Complete Freund's adjuvant is used for the first injection (immunization). Subsequent boosts use immunogen in an emulsion incomplete Freund's adjuvant (IFA or FIA), which lacks the Mycobacterium. Freund's adjuvants are very effective, but they do pose risks to both animal and researcher because of the toxic mycobacterial components.
Solutions of aluminum hydroxide (alum) are convenient alternatives to Freund's adjuvants. Alum is considerably easier to mix with immunogens than Freund's adjuvants, as it does not require laborious emulsification. It is not as strong of a stimulator as complete Freund's adjuvant (CFA or FCA), as is less likely to elicit an immune response for a completely non-immunogenic compound. However, the vast majority of peptide-carrier protein conjugates are immunogenic, and alum provides significant stimulation for them. Alum is safer to use than Freund's adjuvants, as it is much less likely to cause tissue necrosis at the injection site.
The concentration of the immunogen before mixing with adjuvant will ultimately determine the amount of conjugate that will be administered per injection. The following protocols have been proven successful for injection and bleeding. The schedules can be customized for your convenience or when the condition of the animals warrants such consideration. In any case, injections should be discontinued whenever a severe reaction is observed in the animals, either locally or systemically. Only qualified and certified personnel should perform these animal procedures. Individuals not familiar with these techniques should consult an experienced investigator for training before attempting to immunize and bleed animals.
Immunization Schedule for Mice:
- Day 0: Collect pre-immune serum from the mouse to use as a blank when performing ELISA screening after immunization. Store frozen. Inject 50 to 100µg of immunogen (equal to 100 to 200µL of antigen-adjuvant mixture) per mouse. Typical routes of injection include intraperitoneal (i.p.) or subcutaneous (s.c.). One or two such injections may be made per animal.
- Day 14: Boost with an equivalent amount of immunogen in adjuvant.
- Day 21: Test bleed and assay antibody response by ELISA. (Typically, mice are bled under anesthesia through the tail vein or the retro-orbital plexis).
- Day 28: Boost again if necessary. Continue with a similar schedule of alternating boosts and test bleeds until a satisfactory response is observed. For monoclonal antibody production, inject either i.p. or intravenously (i.v.) 4 to 5 days before fusion with the immunogen dissolved in saline (no adjuvant).
Immunization Schedule for Rabbits:
- Day 0: Collect pre-immune serum from the rabbit to use as a blank when performing ELISA after immunization. Store frozen. Inject 100µg of immunogen (equal to about 200µL of the antigen-adjuvant mixture) into each of 8 to 10 subcutaneous sites on the back of the rabbit. Other routes of injection may also be used, but this is by far the easiest with the rabbit.
- Day 14: Boost with an equivalent amount of adjuvant.
- Day 21: Test bleed and assay antibody response by ELISA. (Typically, rabbits are bled through the ear vein without anesthetic). It is not difficult to collect 5 to 10mL of blood, which is more than adequate for measuring antibody response.
- Day 28: Boost again if necessary. Continue with a similar schedule of alternating boosts and test bleeds until a satisfactory response is observed.
Hapten-specific Antibody Screening and Purification
Antibody screening, titering and isotyping are important in-process and final antibody-testing steps in any custom antibody production project. These tests provide the information needed to decide which immunized animals or cell lines to select and continue, what dilutions and secondary reagents should be used in specific applications, and how the antibody can be effectively purified.
General purification of the total IgG component of serum or ascites is usually accomplished with Protein A, Protein G or other such affinity resin. For many applications, general IgG purification is adequate, even though the hapten-specific antibodies may account for only 2 to 5% of the total. Provided that the other immunoglobulin components do not cause off-target binding and background, their presence is not problematic.
Some assay systems and specific experiments require purification of hapten-specific antibodies. This can be accomplished by immobilizing the original hapten to a solid support in a form that does not contain the same carrier protein used to prepare the immunogen. A number of activated affinity resins are available for doing this.
General references about antibody production
- Benjamini, E., et al. (1991). Immunology, A Short Course, Second Ed. Wiley-Liss, New York, NY.
- Germain, R. (1986). The ins and outs of antigen processing and presentation.Nature 322: 687-689.
- Sell, S. (1987). Immunology, Immunopathology, and Immunity. Elsevier, New York, NY.
- Bartel, A. and Campbell, D. (1959). Some immunochemical differences between associated and dissociate hemocyanin. Arch. Biochem. Biophys. 82: 2332.
- Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Chapter 5 discusses the use of carrier proteins.
- Hermanson, G.T. (2008). Bioconjugate Techniques. 2nd edition, Academic Press, New York. (Part No. 20036). Chapter 19 discusses carrier protein uses and immunogen preparation.
- Oliva H., et al. (2002) Monoclonal antibodies to molluskan hemocyanin fromConcholepas concholepas demonstrate common and specific epitopes among subunits. Hybridoma and Hybridomics. 21: 365-373.
- Muckerheide, A., et al. (1987). Cationization of protein antigens. I. Alteration of immunogenic properties. J. Immunol. 138: 833-837.
- Muckerheide, A., et al. (1987). Cationization of protein antigens. II. Alteration of regulatory properties. J. Immunol. 138: 2800-2804.
- Domen, P.L., et al. (1987). Cationization of protein antigens. III. Abrogation of oral tolerance. J. Immunol. 139: 3195-3198.
- Apple, R.J., et al. (1988). Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells. J. Immunol. 140: 3290-3295.
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