PREPARATION OF POLYCLONAL ANTIBODIES
 P r e p a r a t i o n o f P o l y c l o n a l A n t i b o d i e s
By BONNIE S. DUNBAR and ERIC D. SCHWOEBEL The term "polyclonal antibody" is defined as the total population of antibodies present in animal serum. This complex population contains different antibody subclasses including IgG, IgM, IgE, IgA, and IgD. Each antibody represents the secretory product from a single stimulated lymphocyte and its clonal progeny. A complex antigen such as a protein, glycolipid, etc., may contain many distinct antigenic determinants or epitopes, each of which is specifically recognized by antibodies from a single lymphocyte clone. These epitopes may be composed of amino acid sequences, conformational determinants, or molecular structures derived from posttranslational modification (e.g., glycosylation, phosphorylation, or acetylation). The term "immunogen" refers to any molecule that is capable of eliciting an immune response. Immunogenicity therefore refers to the level of immune response elicited. It is advised that basic texts on introductory immunology 1'2 be consulted prior to initiating studies utilizing antibodies. It is important to first decide whether the investigators' studies are amenable to the use of polyclonal antibodies. It is often desirable to develop banks of both monoclonal and polyclonal antisera such that the advantages of each can be exploited. The major advantages and disadvantages of polyclonal antisera are outlined in Table I. A complementary table describing monoclonal antibodies is given elsewhere in this text. 3 Antigen Purification and Preparation It is first necessary to obtain sufficient quantities of the highly purified immunogen that will be used to immunize the animal. In a protein preparation that contains as little as 1% contamination, the majority of antibodies may recognize that contaminant if it is highly immunogenic. Both the level of immune response (immunogenicity) and the type of epitopes recognized (conformational vs sequential) can be modulated by the method chosen for immunogen purification. Immunogens purified by nondenaturing bioB. Benacerraf and E. R. Unanue, " T e x t b o o k of I m m u n o l o g y . " Williams & Wilkins, Baltimore, Maryland, 1979. 2 W. R. Clark, " T h e Experimental Foundation of Modern I m m u n o l o g y . " Wiley, N e w York, 1986. 3 B. S. Dunbar and S. M. Skinner, this volume .
METHODSIN ENZYMOLOGY,VOL. 182
Copyright © 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
TABLE I ADVANTAGESAND DISADVANTAGESOF POLYCLONALANTIBODIES Major advantages Multiple subclass and high-affinity antibodies are present in antibody population Multiple specificities of antibodies are likely to recognize sequential as well as conformational antigenic determinants Antibodies should recognize multiple determinants specific for a protein (important if screening gene expression libraries !) Highly specific polyclonal antisera may easily be developed by immunization with proteins purified in a single step by 2D-PAGE Amenable to experiments involving immunoprecipitation Major disadvantages Immunogen must be highly purified to obtain desired specificity Individual domains of complex antigens are difficult to study because multiple antigenic determinants are recognized by the polyclonal antisera Quantities of antibodies are limited to the life of the immunized animal Different bleedings have to be characterized individually due to changes in antibody affinity, specificity, and subclass
chemical methods tend to elicit a strong response toward the conformational epitopes present in the molecule. Most of these methods do not, however, purify proteins to homogeneity. Procedures which denature the immunogen tend to yield antisera reactive toward sequential determinants. In many instances this is desirable since antibodies made against denatured proteins are usually best for immunoblotting of S D S - P A G E separated proteins. The use of two-dimensional gel electrophoresis has revolutionized the ability to easily isolate denatured proteins or peptides which are sufficiently purified to generate specific polyclonal antibodies.4'5 However, even though acrylamide may act as a "carrier" and help to enhance the immune response in some instances, denatured proteins are generally less immunogenic than are native proteins. It may therefore be necessary to use greater concentrations of denatured protein than the native protein or to enhance the immunogenicity of these proteins. Exceptions include the highly conserved proteins actin and tubulin, which are not immunogenic unless denatured. 6 Two other methods are also commonly used to increase the immune response to an antigen. The first method is to combine the immunogen with 4 B. S. Dunbar, H. Kinura, and T. M. Timmons, this volume . 5 B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. 6 S. L. Brenner, R. L. Pardue, L. Wible, L. Reading, and B. R. Brinkley, in "Laboratory Methods Manual for Hormone Action and Molecular Endocrinology" (W. T. Schrader and B. W. O'Malley, eds.), p. 12-1. Houston Biol. Assoc., Houston, Texas, 1987.
PREPARATION OF POLYCLONAL ANTIBODIES
an adjuvant, a broad term encompassing several substances that enhance the overall immune response. Some examples of adjuvants include alum, dextran sulfate, and large polymeric anions. 7 Two of the most effective adjuvants are the water and oil emulsions developed by Freund. 8 Immunogen is slowly released from the oil, provoking local inflammation and prolonging the immune response. The addition of bacterial antigens (Freund's complete adjuvant) further stimulates lymphocyte infiltration and response. A second method to enhance immune response is to modify the immunogen itself. The immunogen can simply be polymerized, or it may be conjugated to other carrier proteins or synthetic antigens. These modifications are particularly useful for highly conserved proteins or unusually small immunogens (e.g., steroids, prostaglandins, or cyclic nucleotides). 9,~° In addition to these methods, the immune response is also enhanced by choosing a species that is phylogenetically removed from the species that is the source of antigen. Other factors affecting choice of animal species are outlined in Table II. Antibody Preparation: Polyclonal Antibodies Because rabbits are the most commonly used animal for the production of polyclonal antibodies, this procedure is given in detail (similar methods are used for other animals). (Note: Before initiating animal experiments, it will be necessary to have protocols approved by the animal welfare committee of the institution.) Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. Immunization. A highly purified antigen (optimal total dosage 20-200 /zg) is emulsified using two 3-ml Luer-Lock syringes with 18-gauge needles connected by Tygon microbore tubing (0.3 × 0.09, Fisher, Pittsburgh, PA, catalog #14-170-15F). The first syringe contains 0.75 ml water or buffer and antigen (solubilized or minced gel pieces), and the second contains an equal volume of Freund's complete adjuvant (FCA) (see photographs of equipment set up in Ref. 5). Be sure to thoroughly vortex the FCA to resuspend the bacteria settled in the bottom of the vial. The immunogen 7 H. S. Warren, F. R. Vogel, and L. A. Chedid, Annu. Rev. lmmunol. 4, 369 (1986). 8 j. Freund, K. J. Thompson, H. B. Hough, H. E. Sommer, and T. M. Pisani, J. lmmunol. 60, 383 (1948). 9 B. F. Erlanger, this series, Vol. 70, p. 85. ~0M. Reichlin, this series, Vol. 70, p. 159.
TABLE II ANIMALSELECTIONFORANTIBODYPRODUCTION Species Rabbits
Mice and rats
Sheep and goats
Easy to maintain Quantities of antisera may be limYield large amounts of sera ited if very large amounts are (about 20 ml/week from a large required rabbit) Many rabbits are infected with Multiple subclasses of IgG b i n d pasteurellosis,which affects protein A immune response and limits life of animal (pathogen-free animals are available) Easy to maintain Only small amounts of antisera Multiple subclasses of IgG bind can be obtained protein A Blood usually is collected by carGenerally makes high-titer antidiac puncture, risking loss of bodies animal; can be collected from hind leg with vacuum apparatus Easy to maintain Small amounts of antisera obtained Antibody does not bind well to protein A, necessitating use of second antibody for detection Large quantities of antisera can Special facilities necessary for be obtained housing unless commercial proImmunogen can be shipped offducer employed site for commercial immunizaOnly one subclass of IgG binds to tion protein A, necessitating use of second antibody for detection
and adjuvant mixture is emulsified by passing the mixture back and forth between the syringes until the sample is thick. (Note: Some i m m u n o g e n adjuvant mixtures will be extremely viscous, while others will not be as thick.) Be sure that the tubing is fitted tightly over the needles and that L u e r - L o c k syringes are used, or the antigen may be lost if the tubing comes off from the pressure! The emulsified immunogen mixture is injected intradermally (not subcutaneously) into the layers of the skin of a shaved rabbit using a 22-gauge needle. The needle is quickly inserted just under the surface of the skin and enough emulsion is added to form small raised " p o c k e t s " in the skin. A total volume of approximately 0.5-1.0 ml should be injected into 10-12 sites along the upper sides (5-6 sites/side) of the rabbit. Be sure that the immunization sites are not too large or too close together to cause serious lesions. The remainder of the emulsion (0.5 ml) is also given subscapularly at this time. Because intradermal injections can be painful, it is preferable to use a light anesthetic prior to immunization
PREPARATION OF POLYCLONAL ANTIBODIES
(i.e., xylazine at a dosage of 5-10 mg/kg). A general schedule for boost immunization is given in Table III. Bleeding and Processing Serum. We have found that the best rabbit restrainer is one that does not trap the rabbit's neck since rabbits can easily be injured in cages where only the head is exposed. Pull the rabbit's ear through the bars and shave the hair along the outside margin of the ear with a single-edge razor blade. The ear should be rinsed with ethanol and dried. Apply a very small amount of xylene with a cotton swab on the center artery. Never apply on marginal vein. If too much xylene is used, the bleeding will be difficult to stop. Generously apply petroleum jelly on the marginal edge of the ear and on the region just under the ear. Do not apply where the cut is to be made. Once the ear is prepared, a razor blade is used to cut the vein using a firm, fiat stroke perpendicular to the vein. Be careful not to cut through the ear! For a few seconds the animal may not bleed; then a steady dripping of blood should start. Blood should be collected in glass tubes because clotting will be more efficient and more serum can be obtained with minimal hemolysis. The animal should stop bleeding immediately or very shortly after the ear is released. Wash ear thoroughly to remove xylene. As soon as the blood clots in the tube, rim the edges of the clot with a wooden applicator or glass pipet. (Note: This is a critical step for obtaining the maximum amount of serum from blood.) Allow the blood
T A B L E Ili GENERAL SCHEDULE FOR IMMUNIZATION OF RABBITS FOR PREPARATION OF POLYCLONAL ANTIBODIES
lmmunogen dose total
I (preimmune bleeding)
Carrier Freund's complete adjuvant: immunogen ratio 0.5 ml:0.5 ml PBS + immunogen Freund's incomplete adjuvant; 0.5 ml:
Site of injection Multiple sites: intradermal (>10); 1 site subscapular Two to four sites subcutaneous
0.5 ml PBS +
immunogen 31 (test bleed) I f antibody detected: 40
I f no or very low antibody titer detected: 10-100/~g
Freund's incomplete adjuvant
Two to four sites subcutaneous
Freund's complete adjuvant
Four to six sites intradermal Two sites subscapular
TABLE IV ADVANTAGESAND DISADVANTAGESOF ANTIBODYDETECTIONMETHODS Technique
"Western" blotting a,b
High sensitivity Determines directly which proteins are recognized by the antisera Antibody detection reagents commercially available
Yields detailed analysis of antibody specificity Complex antisera and antigen mixtures can be analyzed Usually performed under nondenaturing conditions High sensitivity Detects antibodies to either conformational or sequential determinants Assay can be used to quantify either antibody or antigen May be possible to use crude antigen Large numbers of assays can be easily processed Reagents commercially available High sensitivity Large numbers of assays can be easily processed Detects antibodies to either conformational or sequential determinants Reagents commercially available Detects immunological similarities between antigens Usually performed under nondenaturing conditions Simple to perform and inexpensive
Enzyme-linked immunoassay d
Ouchterlony double diffusion e
Disadvantages Generally detects only sequential determinants, thus not definitive proof of specificity Binding properties of different proteins to different papers must be optimized Large quantities of antibody and antigen usually necessary Specialized equipment required May require large quantities of antigen to coat microtiter plates Large number of assays requires specialized equipment Conditions for antigen adherance to microtiter plates must be optimized Yields little or no information on specificity Yields little or no information on specificity Binding properties of different proteins to different papers must be optimized
Salts or detergents can result in false positives Large amounts of antibody and antigen are usually required Not accurate for determining specificity of antibody Antigen-antibody ratio critical for immunoprecipitation; therefore, negative results are meaningless
a H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). b N. L. Anderson, S. L. Nance, T. W. Pearson, and N. G. Anderson, Electrophoresis 3, 135 (1982). c B. Weeke, Scand. J. Irnmunol., Suppl. No. 1, p. 15 (1973). a E. EngvaU and P. Perlman, Immunochemistry 8, 871 (1971). e O. Ouchterlony, in "Progress and Allergy" (P. Kallos, ed.), Vol. V, p. 1. Karger, Basel, 1958.
PREPARATION OF POLYCLONAL ANTIBODIES
to clot for 2-4 hr at room temperature, then decant serum and centrifuge at 1000 g for 10 min to remove blood cells. Some additional serum can be obtained by letting the blood clot overnight at 4° , but there is usually more hemolysis in this sample. (For photographic details of this procedure, refer to Ref. 5) Fractionation o f l g f r o m Serum. It is often desirable to partially purify antibody from antiserum prior to use. Two commonly used methods are ammonium sulfate fractionation and DEAE chromatography. Ammonium sulfate fractionation is a simple but crude method for antibody partial purification. Slowly add 40 ml saturated ammonium sulfate [77.7% (NH4)2SO4, pH 7.2-7.4] to 60 ml of serum while stirring. Continue to stir for 1 hr. Centrifuge at 5000 g for 20-30 min at 22°. Resuspend pellet in minimal volume of distilled H20. Dialyze sample extensively to remove ammonium sulfate. DEAE-Sephacel ion-exchange chromatography yields IgG purified from other immunoglobulin subclasses and most serum proteins. For chromatography dialyze the above sample extensively against phosphate equilibration buffer (made by the addition of 0.02 M NaHzPO4" H20 to 0.02 M Na2HPO4 until pH 7.4). Pour the resin as a slurry into the column (1.6 × 20 cm) and let settle. The top of the bed should be at least 2 cm from the top of the column. Equilibrate the bed with three column volumes of equilibration buffer. Apply the sample [approximately 100 mg of (NH4)2504 fraction in 5-20 ml equilibration buffer] to the column and begin collecting 8-ml fractions. The first IgG peak should come offin tubes 8-10. Continue to rinse the column until absorbance returns to background. Switch buffer to 0.02 M phosphate with 0.05 M NaC1. The second IgG peak should come off in another 8 to 10 fractions. IgM is removed with 0.1 M NaCI in TABLE V SUMMARY OF COMMON METHODS USED TO CHARACTERIZE ANTIBODIES
Antibody characteristics Specificity Quantitation Subclass determination Cytochemical localization
Methods Western blotting Immunoelectrophoresis Enzyme-linked immunoassay Dot-blot assays Immunoelectrophoresis Enzyme-linked immunoassay Ouchterlony double diffusion lmmunocytochemistry Organelle fractionation
phosphate buffer, and other serum proteins in 0.5-1.0 M NaCI. Regenerate the column by washing with three bed volumes of regeneration buffer (0.2 M phosphate buffer, pH 7.4, made as described for equilibration buffer) and then with equilibration buffer. Fractions can be tested for the presence of IgG by SDS-PAGE. In addition, new products have been designed to isolate IgG by affinity chromatography using Protein A attached to cellulose disks. These may prove to be useful for some applications. It is critical to properly characterize antiserum prior to use. Antibody titer, specificity, subclass, and affinity can greatly affect subsequent experimental protocols. Common methods for antibody detection and characterization are outlined in Tables IV and V and discussed in detail elsewhere. 3,5,11 11 T. M. Timmons and B. S. Dunbar, this volume .
 P r e p a r a t i o n o f M o n o c l o n a l A n t i b o d i e s
By BONNIE S. DUNBAR and SHERI M. SKINNER The report by Kohler and Milstein 1 that a cell hybrid made by fusing normal spleen cells with malignantly transformed antibody-secreting cells (plasmacytoma or myeloma cell line) can provide a continuous source of antibody of predefined specificity has led to the explosion of the use of "monoclonal antibodies." Because this technology has been so widely used, several excellent texts and reviews have been written which describe this technology in great detail. 2-5 The properties of these antibodies as well as the advantages and disadvantages of their use as compared to polyclonal antibodies are summarized in Table I. These guidelines should assist in the choice of determining which type of antibodies are optimal for use in designated experiments. G. Kohler and C. Milstein, Nature (London) 256, 495 (1975). 2 R. H. Kennett, T. J. McKearn, and K. B. Bechtol, "Monoclonal Antibodies." Plenum, New York, 1980. 3 G. Kohler, "Hybridoma Techniques." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1980. 4 j. W. Goding, "Monoclonal Antibodies: Principles and Practice," 2nd Ed. Academic Press, New York, 1986. 5 H. Zola and D. Brooks, in "Monoclonal Hybridoma Antibodies: Techniques and Applications" (J. Hurrell, ed.), p. 1. CRC Press, Boca Raton, Florida, 1981.
METHODSIN ENZYMOLOGY,VOL. 182
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.