monoclonal antibody protocols

CHAPTER Methods of Immunization to Enhance the Immune Response to Specific Antigens InVivo in Preparation for Fusions Yielding Monoclonal Antibodies Jon A Rudbach, John L Cantrell, and J II Ulrich Introduction The first step in preparing useful monoclonal antibodies (MAbs) is to immunize an animal with an appropriate “vaccine.” Animal and vaccine are both emphasized in the preceding sentence because this chapter describes how to generate satisfactory MAbs by maximizing interactions between the two The term vaccine was used purposefully to connote that not only antigens of interest may be contained in the immunizing product, but carriers and adjuvants may also be included These latter components can influence greatly the success of obtaining useful hybridomas, which produce antibodies of the desired specificity and quality Immunization protocols for obtaining only murine MAbs are covered herein Although cross-species hybridizations can be made, they usually involve very specialized techniques Moreover, the lessons that can be derived from mouse immunization protocols can, in general, be extrapolated to other species as well When considering which mouse strains to immunize, even though some initial advantage may be obtained by selecting a strain other than a Balb/c, there is an overriding consideration that must be taken into From Methods m Molecular Bology, Vol 45’ Monoclonal Antrbody Protocols Edlted by W C Davis Humana Press Inc., Totowa, NJ Rudbach, Cantrell, and Ulrich account The usual mouse myeloma used for fusion IS a HAT-sensitive variant of the Balbk-derived MOPC-2 myeloma The fusion product of the myeloma with the antibody-producing spleen cells will express both Balbk antigens (from the MOPC-21) and those of the donor strain that provided the spleen cells Therefore, any production of ascites as a source of MAb must be performed in a histocompatible mouse strain This is easiest if the spleen cell donor is a syngeneic Balbk mouse If the spleen cell donor is an inbred strain other than Balbk, then the F, progeny of a Balbk-“spleen cell donor” cross, which contains both sets of histocompatibility antigens, must be used to grow the hybridoma for ascites production With these genetic restrictions, hybridomas generated from spleen cells donatedby outbred mice would be allogeneic and precluded from growth m any recipient One way around this problem would be to generateMAbs only from cell-culture fluids, thus avoiding the histocompatibility problem However, this usually resultsin lower yields of antibodies Therefore, most investigators find it easier to manipulate the immunological responses of Balbk mice with adjuvants and/or carriers rather than reverting to the use of other inbred mouse strains for immunization An antigen is a molecule that, when introduced into an appropriate animal, will stimulate an immunological (antibody) response in that animal In basic terms, an epitope is the minimal chemical configuration in an antigen that can be immunologically recognized as uniquely specific by the immune system Inasmuch as a controlling reason for generating MAbs, instead of polyclonal antiserum, is to obtain a high degree of specificity, immunization with an epitopically restricted antigenic material is usually preferable to the use of a crude antigen Such antigens with restricted diversity can be obtained by blocking nondesired epitopes, by chemically conjugating purified chemical groupings to a carrier, or by synthesizing/cloning epitopically pure antigens However, regardless of the antigenic material used in the vaccine, the final selection of specificity will be made during screening of the hybridoma supernatant fluids for antibody The nature of the ligand attached to the solid support is of prime importance at this point There is a paradigm in immunology that the first antibody developed after immunization is usually more specific than that produced later in the immune response On the other hand, a later antibody may have more of the desired properties of affinity, class, and subclass (1) These considerations, as well as the desirability of generating sufficient numbers Immunization to Enhance Immune Response of antibody-secreting cells to yield a quantitatively satisfactory fusion run, require a well-designed immunization protocol It is thought that high-affinity antibody-producing cells can be selected by using minimal (suboptimal) amounts of antigen (I) In order to use this approach and not compromise some of the practical aspects of the procedure, immunological adjuvants can be employed Appropriate adjuvants can be selected that will increase the number of antibody-forming cells and also can direct the response to yield a qualitatively desirable antibody Antigens, which are weakly immunogenic because they are functional molecules, related to tissue antigens of the mice, denatured or lack appropriate physicochemical properties, or too small, can have their immunogenicity increased through the use of adjuvants Furthermore, conjugation of antigens to carriers, with or without coadministration of adjuvants, can turn marginally immunogenic materials into useful antigens (2) Use of carriers and procedures for conjugating them have been described elsewhere (3), and are not covered in this chapter Adjuvants are materials that are not (usually) themselves immunogenic, but which can be used in conjunction with antigens to alter an immune response quantitatively and/or qualitatively (4) Although many types of adjuvants are available, only those with proven utility for generating cells useful for MAb production in mice are covered These are commercially available and not require extensive preparation or manipulation Complete Freund’s Adjuvant (CFA) is a potent adjuvant that has been used successfully for decades It can be used with weakly antigenic materials and has a reputation for stimulating the production of large amounts of high-quality antibody (5) CFA, however, suffers from its toxicity It has a history of inducing necrotic lesions in animals even after a single use Moreover, many animal care committees have banned the use of CFA in their facilities An alternative to CFA is the Ribi Adjuvant System (RAS), which has gained wide acceptance both by immunologists and animal care committees (6) RAS is a ready-to-use product, two forms of which are recommended for use in mice to generate cells suitable for fusions leading to MAb production One of these contains synthetic trehalose dicorynomycolate (S-TDCM) in a form that can be readily formulated into an oil-in-water emulsion The second form contains monophosphoryl lipid A (MLA) as a second immunostimulant, in addition to the S-TDCM The choice of which one to use is somewhat empirical, but can be Rudbach, Cantrell, and Ulrich directed by the nature of the antigen and the quality of the antibodies desired Our experience has shown that the use of S-TDCM only as the adjuvant produces predominantly IgGr isotype MAbs The use of S-TDCM + MLA increases the probability of a fusion yielding MAbs of the IgG2 isotype Materials 2.1 Preparing Vaccine 1, Antigen: Prepareor obtain antigen of choice Adjuvant RAS (see Section 3.2 for details) Phosphate-buffered salme (PBS): 0.15M NaCl and O.OlM NaH2P04-Na2HP04, pH 7.4 Mouse: Use female mice (see Note 1) 1, 2.2 Collecting Blood and Serum Dry ice or a CO2 tank and regulator Cotton, 500~mL beaker (or other container that can be covered) Solution of sodium heparin (1500 U/mL) in saline Pasteur pipets Razor blade (single-edged) mL Tuberculin syringes and 1/2-m., 27-gage needles 70% Alcohol Microcentrifuge and tubes Methods 3.1 Antigen Preparation Antigens soluble in PBS: Solubilize selected antigen in sterile PBS, ideally at a concentration per milliliter of about 50 times the amount to be administered For example, tf the antigen dose per injection for a mouse is 100 pg, then a stock solution of (50 x 100 or) 5000 pg/mL is desirable, Because a mouse dose will be contained in 0.2 mL, this solution will be a lo-fold concentrate Store the PBS-soluble antigen preparation under conditions deemed appropriate for the material (-7O”C, 4OC, and so forth) This recommendation for preparation of an antigen solution is ideal, but is not absolutely necessary Antigens soluble m detergent: Sometimes detergents are necessary to solubilize very hydrophobic proteins When possible, solubrlize antigen in detergentat a concentrationsuch that when the solution is diluted to an antigen concentration of five times a dose expected to be given to a mouse, the detergent concentration should be 0.2% or less Immunization to Enhance Immune Response Immobilized antigen: Another type of antigen preparation that is frequently encountered is a band cut from a polyacrylamide gel electrophoresis (PAGE) gel The slice of gel should be reduced to the smallest particles practical by suspendmg it in a small amount of salme and expressing it repeatedly through successively smaller hypodermic needles, beginning with an 18-gage and finishing with a 27-gage needle This suspension can be treated as an antigen solution and prepared with the adjuvant as described in Section 3.2 It is recommended that the antigen under consideration be incorporated into the emulsion at a concentration range of 50-250 pg/mL of saline However, weak immunogens can be used at concentrations of up to 1.0 mg/rnL If the amount of antigen available is very limited, the lower limit is the amount recommended In these latter cases, experience has shown that it is better to give multiple doses of small amounts of antigen rather than to administer all of it in a single dose; this should be considered when deciding on formulations of a precious antigen 3.2 Vaccine Preparation with RAS The RAS is available as an oil concentrate, which only requires reconstitution with a solution of antigen Vaccines are formulated with RAS adjuvants as follows: Each vial of lyophilized adjuvant emulsion contains 0.5 mg of each immunostimulant, 40 pL of oil (Squalene) and uL of Tween-80 Vials should be stored at 2-8°C until used Prior to reconstituting the emulsion, place the vial in a water bath at 40-45”C for 5-10 mm (alternatively, the vial can be warmed in a beaker of hot tap water for 5-10 min) Reconstitute each vial with 2.0 mL of sterile PBS containing the desired amount of antigen as follows: a Inject the antigen-PBS solution (2 mL) directly into the veal through the rubber stopper, using a syringe fitted with a 20- or 21-gage needle (leave the cap seal in place) b Vortex the vial vigorously for 2-3 mm to form emulsion, with rubber stopper in place The final vaccine will contain 50 ug of each adjuvant/O.:! mL (a mouse dose) The final emulsion also contains 2% oil (Squalene) and 0.2% Tween-80 If the entire contents of the vial will not be used initially, reconstitute to mL with saline, and mix aliquots 1: with antigen in saline imrnedi- Rudbach, Cantrell, and Ulrich ately before use Unused emulsion can be stored at 4°C (for up to 60 d) or lyophilized Do not store frozen Prior to animal inoculation, warm the vial to 37”C, and vortex briefly 3.3 Immunization Protocol When using a vaccine prepared with a RAS emulsion, it is recommended to inject mice with 0.2 rrL ip or SC(0.1 mL in each of two SC sites) Our experience suggests the SCroute is the preferred route A minimum protocol for immunizing mice to generate cells for preparing hybridomas is as follows: immunize on d 0, boost on d 21, take a trial bleeding on d 26; if the antibody titers are satisfactory, boost on d 35 with antigen only, intravenously, and remove the spleen to obtain cells for fusion on d 38 (see Notes and 4) 3.4 Collecting Sera When screening mice for antibody responses during an immunization regimen, in anticipation of deciding when to take the spleen for fusion, it is preferable to test the serum of the actual potential spleen donor, rather than that of a companion animal immunized in parallel This necessitates repeated bleedings of a single mouse Repeated bleedings are possible, owing to the very small volumes of sera needed for assay (see Chapter lo), if care is taken with handling of the mice Some institutional animal care committees stipulate that mouse bleedings be performed under carbon dioxide anesthesia as outlined: Either place a small piece of dry ice beneath cotton m a beaker or fill the covered beaker with CO, gas from a tank Place mouse in beaker until It is anesthetized Remove the mouse, and rapidly bleed by one of the followmg techniques: a Retro-orbital: Insert the tip of a Pasteur pipet, which has been “wetted” with the heparin solution, into the retro-orbltal space, anterior to the eye Rotate gently to disrupt the vascular plexus, and collect by capillary action about 100 FL of blood b Cardiac: With the mouse on its back, wet the chest with alcohol, and insert a 27-gage needle into the heart, between the ribs or under the sternum, through the diaphragm, Collect 100 PL of blood mto the “heparm-wetted” syringe c Tall vein: With the corner of a new, alcohol-wiped razor blade, nick a lateral vein, longitudinally, near the tip of the tad Collect, by capdlary action, 100 pL of blood into a “heparm-wetted” Pasteur pipet Compress Immunization to Enhance Immune Response with dry cotton to stop the blood flow Warming mice under a heat lamp for a few minutes immediately before bleeding will increase blood flow through the veins and speed the process of blood collection Express the blood into a nncrocentrifuge tube that contains 10 pL of the heparm solution in its tip, vortex well, and centrifuge to separatethe plasma Remove the plasma to a second microcentrifuge tube, seal, and store m a freezer, if not tested immediately Notes It is recommended that female mice be used for immunization Male Balb/c mice fight; many times the tails are so damaged that inJections and bleedings are impaired If sufficient antigen is available, mice should be immuruzed to prepare a pool of polyvalent antiserum The enzyme immunoassay (EIA) assay should be optimized with this antiserum pool (see Chapter 10) The short time between successful screenmg of the culture supernatant fluids for antibody after the fusion and reculturmg for clonmg or expansion generally is not sufficient for optimizmg the EIA assay With most antigens, a good antibody titer can be achieved after a single booster injection If, however, the serum antibody titer is too low, a second booster injection, with adJuvant, should be given, and another test bleeding taken to determine if satisfactory titers have been obtained In order to increase the chances of obtainmg a hybridoma that will yield the desired quality of antibody, the immunization protocol should be designed to yield the maxtmum number of antibody-forming cells from the spleen Experience has shown that a mouse with a higher serum antibody response yields splenic cells that result in proportionately greater numbers of specific MAb-producing hybridomas Therefore, the immunization protocol should be designed to maximize serum antrbody titers References Davis, B D., Dulbecco, R , Eisen, H N., Ginsberg, H S., Wood, W B., and McCarty, M (1973) Antibody formation, in Mzcrobiology, 2nd ed , Harper & Row, New York, pp 484,485 Benjamini, E and Leskowitz, S (1991) Immunology A Short Course, 2nd ed., Wiley-Liss, New York, pp 38-40 Kabat, E A and Mayer, M M (1961) Experimental Immunochemistry, 2nd ed , Thomas, Spnngfield, IL, pp 446-450,798-802,8 13-8 15 Hui, G S N., Chang, S P , Gibson, H., Hashimoto, A., Hashno, C , Barr, P J., and Kotam, S (1991) Influence of adJuvants on the antibody specificity to the Plasmodmm falciparum maJor merozoite surface protein, gp195 J Immunol 147, 39353941 Rudbach, Cantrell, and Ulrich Kabat, E A and Mayer, M M (1961) Experimental Immunochemuty, 2nd ed., Thomas, Springfield, IL, pp 309-310, 872 Rudbach, J A., Johnson, D A., and Ulrich, J T (1995) Ribi adJuvants: chemistry, biology and utility in vaccines for human and veterinary medicine, in Adjuvants Theory and Practical Applications (Stewart-Tull, D E S , ed.), Wiley, New York, pp 287-313 CHAPTER Methods of Immunization to Enhance the Immune Response to Specific Antigens In Vitro Margaret E SchelZing Introduction In vitro immunization involves the exposure of spleen cells to antigen in tissue culture rather than the antigenic stimulation of spleen cells via immunization of mice The production of monoclonal antibodies (MAbs) to highly conserved molecules, such as enzymes (1,2), is possible using in vitro immunization MAbs to such “self’-antigens often are not possible to make using traditional in vivo methods owing to immune suppression or tolerance Utilizing in vitro immunization, it is possible to elicit the formation of MAbs in response to picogram quantities of antigen (3-6) Although certain protocols (I, 7) indicate a minimum requirement of from 30-100 yg antigen for in vitro immunization, we have found that the nanogram or picogram quantities of antigen available from blotted polyacrylamide gels provide sufficient antigen for the preparation of MAbs by in vitro immunization (3,.5) Additional advantages of in vitro immunization include shortening the immunization procedure from the or wk required for in vivo immunization to d, allowing defined antigen concentrations, and controlling antigen degradation (3) In vitro immunization is modulated by regulating the activation and maturation of antigen-specific B-lymphocytes using growth/differentiation factors An extensive literature describes interactions between the various lymphokines and cell types involved in the regulation of B-cell proliferation and differentiation, but these interactions are not sufficiently From Methods m Molecular Brology, Vol 45 Monoclonal Antibody Protocols Edtted by W C Davis Humana Press Inc , Totowa, NJ Schelling defined to provide a complete overview Factors that appear to increase the immune response to specific antigens in vitro include interleukin-2 (IL-2) When IL-2 was included in the in vitro immunization, Pollock and d’Apice (8) found that cultures produced a higher yield of hybridomas producing MAbs of the desired specificity Additionally, muramyl dipeptide (MDP) has been reported to increase the yield of specific antibodies in in vitro systems (8-10) The MDP effect on lymphocytes is attributed (8) to the ability of MDP to stimulate interleukin- (IL- 1) production by monocytes/macrophages, which activates helper T-cells, and its adjuvant effect on immunizations MDP is not a polyclonal activator of human lymphocytes, which may be important in limiting the number of activated but irrelevant lymphocytes available for fusion following antigen stimulation The IL-2 effect is also possibly the result of its effect on helper T-cells Jacot-Guillarmod (II) reported the use of 10% conditioned medium as a source of B-cell growth and differentiation factors This conditioned medium consisted of a 2-d-old supernatant from human spleen cells cultured in the presence of pokeweed mitogen The activity of the conditioned medium was replaced by 20 p,g/niL MDP and 200 UlmL IL-2 (I I) &helling (3) reported the addition of dextran sulfate to thymocyte-conditioned medium (TCM) (12,13) for increased specific MAb formation for viral proteins Martin et al (14), however, reported that the addition of specific and nonspecific cell activators such as Staphylococcus aweus Cowan I strain cells, lipopolysaccharide, or dextran sulfate, to the immunizing medium did not increase the in vitro secretion of specific human antibodies to Haemophilus influenzae type B The number of specific MAbs produced is higher when no more than 2% fetal bovine serum (FBS) is used in the in vitro unmunization system (3) Additionally, the number of specific MAbs produced is greater when the addition of FBS is delayed until 24 h following the addition of antigen to the in vitro immunization system, thus avoiding a competition of the antigen with components of the FBS in the in vitro immunization system for the production of MAbs In vitro immunization of B-lymphocytes frequently results in the production of IgM MAbs If IgG MAbs are preferred, it is possible to inject mice prior to harvest of the spleen cells for in vitro immunization according to in vivo technique immunization schedules It has been reported that sequential in vitro immunizations are possible, but given the short-lived existence of spleen cells in culture, it is difficult to 246 Vaccaro and Markinac Materials Cell-culture medium with antibiotics: The researcher should use the medium that best preserves the health of the cells during the sorting process A base medium, such as RPMI-1640, with 5-10% fetal bovine serum and appropriate levels of antibiotics, 1srecommended Some laboratories omit antibiotics after the cells are in culture Complete cell-culture medium can be stored at 4°C prior to use for no longer than d Magnetic particles: Magnetic beads or particles can be obtained from several commercial sources: coupled directly with the MAb of interest, polyclonal anti-immunoglobulm (IgG and IgM or isotype-specific), or as free particles prepared for couplmg by the investigator Magnetic particles from PerSeptive Diagnostics (Cambridge, MA; formerly Advanced Magnetics) are stable for at least mo when stored at 4°C a Directly coupled magnetic particles available commercially: Magnetic anti-CD10 particles are available from PerSeptive Diagnostics (Cambridge, MA) At the time of use, magnetically wash the particles three times in culture medium with antibiotics Particles must not be frozen or centrifuged Resuspend the particles in culture medium such that only a small volume is added to the cells b Magnetic particles prepared with the antibody of interest by the investtgator: MAbs (and others) can be purchased from commercial sources and covalently attached to amme-termmated BioMag magnetic particles available from PerSeptive Diagnostics through a glutaraldehyde crosslinking of the amines 111 MAb to the ammes on the magnetic parthe ticle If using this procedure, activate the magnetic particles according to the instructions provided (see Note 1) The overnight coupling procedure requires electron microscopy (EM)-grade glutaraldehyde, 15 mg of MAb (purified or unpurified), and 100 mg of a carrier protein, such as bovine serum albumin (BSA), per 500 mg of amme-terminated BioMag c Magnetic particles coupled with anti-tmmunoglobulin: A two-step procedure involvmg the use of magnetic partrcles coupled with antiimmunoglobulin can also be used Magnetic particles coupled with polyclonal antrmouse immunoglobulin (IgG and IgM or rsotype-specific) can be purchased commercially Depending on the source of antibody and the manufacturer’s recommendations, anti-Ig-coupled magnetic particles require 5-20 l.rg of MAb/million cells for the twostep procedure The MAbs need not be purified, but should be sterile (see Note 2) Equipment: Sterile tubes, bottles, and flasks as needed Cell preparation: Cells to be separated may be partially purified prior to use Separation out of whole blood should not be attempted unless the cells Negative Selection are diluted in medium Cells should be washed in medrum prtor to separation to reduce the amount of immunoglobulin in the medium and attached to cells Magnetic separator: Magnetic separator appropriate for the tube or flask to be used Magnetic separationunits are available from PerSeptive Diagnostics that accommodate 12 x 75 mm test tubes, microcentrifuge tubes, tissueculture flasks, 96-well tissue-culture plates, and 15- and 50-mL conical tubes Method Negative cell selection is best carried out in sterile tissue-culture tubes or flasks The following is a one-step protocol that uses BioMag magnetic particles to which CD10 MAb is covalently attached (see Notes and 2) The target population is assumed to be 10% of the total (see Note 3) All steps are performed at 4°C (see Note 4) Deliver mL containing approx x lo6 cells in RPM1 with 5% FBS and 1% penicillin-streptomycin into an appropriate tube (see Notes and 6) Wash the cells three times with mL of sterile medium by centrifuging the cells at 15Og for 10 Gently, but thoroughly, resuspend the cellular pellet (see Note 7) Wash 0.2 mL of magnetic anti-CD10 parttcles three times in 0.2 mL of sterile medium Use a magnet to pull the magnetic particles to the side of the tube, and shake vigorously to resupend the magnetic particles during washing Add 0.2 mL of washed magnetic anti-CD10 particles to the resupended washed cells (The particle-to-cell ratio in this example is 50 particles/cell based on the total cell population; see Note 8.) Incubate the cells with the magnetic particles for 30 Swirl the cell/particle suspension once every 10 to promote attachment (see Note 9) Magnetically separate for 10 twice (see Note 10) and save the supernatant (see Note 11) Centrifuge and resuspend the cells in fresh medium, and use the cells as desired Following separation, react an aliquot of the purged cell preparation with fluorescein isothiocyanate (FITC)-labeled CD10 antibody, and examine by flow cytometry to determine the efficiency of removal of CDlO+ cells Depending on FACS results, a second or third purging may be necessary in some cases Notes Amine-terminated BioMag is a l+m magnetic particle with an iron oxide core, a silane coatmg, and a functionalized amme surface MAbs may be covalently attached to amine-terminated BioMag through a glutaralde- 248 Vaccaro and Markinac hyde crosslinking of the ammes on the particle to the amines in the monoclonal The magnetic particles are activated with glutaraldehyde for h After magnetically separating the activated magnetic particles and washing excess glutaraldehyde away with a pyridme buffer at pH 6, 15 mg of MAb (purified or unpurified) and 100 mg of BSA are added to the actrvated particles for an overnight incubation After the overnight mcubatron, the particles are washed magnetically and incubated with 1M glycme for 30 mm to quench any activated amines that may not have bound protein The monoclonal coupled magnetic particles are then suspended in sterile medium and are ready to use A two-step cell-separation procedure can be used In the two-step procedure, an MAb is incubated with magnetic goat antimouse IgG for 20 at 4°C The magnetic goat antimouse IgG-MAb complex is washed to remove excess antibody and then incubated with cells for 20 at 4OC After magnetic separation, the supernatant containing the purtfied cells is removed, and the cells are resuspended in fresh medium Other variations of the two-step procedure include the use of biotinylated or fluoresceinated MAbs with either magnetic streptavidin or magnetic sheep antifluorescein particles Although magnetic protein A and magnetic protein G preparations are commercially available, they are not recommended in cell-sorting applications, because protein A and G will bind all immunoglobulins, including those introduced into media by FBS or other serum proteins Negatrve selection mvolving populations of cells >50% of the total may be difficult to work with Unless the investigator is willing to accept a low yield, the technology is not yet specific enough to gave an appropriate yield It is recommended that positive selection be used in those cases The protocol is usually performed at 4°C The reason for this is to minimize patching, capping, and phagocytosis.Also, cell viability may be bestpreserved by keeping the cells on ice However, room temperature or even 37°C may be optimal for certain cell types If the investigator is having problems with low yield and/or viabihty, other temperatures should be mvestigated The maximum cell concentration should be about mllhon cells/ml The denser the mixture of cells, the more likely there will be nonspecific binding, clumping, and trapping of cells The investigator should work with the most dilute cellular suspension reasonable with the goals of the experiment If poor depletions are being obtamed, try drlutmg the mixture lo-fold, and repeat the experiment It is important to have protem in the culture medium during cell sorting to minimize nonspecific bindmg Too much protein in the medium may inhibit the bindmg of particles to cells Thus, the investigator may have to titrate the protein concentration to optimize the particle binding Negative Selection 249 If the cells are clumped at this stage, it may be because of DNA release by dead cells These clumps can be easily broken up with the use of 0.1% DNase in the medium Depending on antigen availability and size of the target cell population, cell-sorting applications may require 20-80 magnetic particles/cell based on the total cell population, If the purity of the final cell population is not sufficient, then higher particle-to-cell ratios can be used Also, the investigator can try lengthening the incubation time or multiple incubations of particles with cells One-micron or smaller particles, such as those supplied by PerSeptive Diagnostics, can be used in the method in Section Magnetic beads 4-pm or larger, such as those supplied by Dynal (01~0, Norway), require continuous rotation to maintain the suspension Overswirling will damage the cells The investigator will have to monitor cell viability with the particular cell type and magnetic particle in use 10 Magnetic separation must be performed with the pellet formed on the side of the flask or tube This is so that the unselected cells will not contaminate the pellet owing to gravity One-micron particles will take up to 10 to separate, depending on the volume of medium Larger particles will separate faster, but it still may be appropriate to wait the same amount of time 11 Aspirate the flask or tube very carefully after the magnetic separation The magnetic field must be applied during aspiration It is very easy to disturb the pellet and to pipet particles inadvertently because of the gentle packing of the pellet It is recommended that a propipet rather than vacuum aspiration or decanting be used Additional magnetic separations can be performed to reduce further the amount of contaminating magnetic particles in the supernatant Because the particles are black, it is very easy to see if they have not been removed References Adkins, B., Ghanei, A., and Hamilton, K (1993) Development of IL-4, IL-2, and IFN-y production by murine peripheral T lymphocytes J Immunol 151, 66 17-6626 Berman,J F and Center,D M (1987) Chemotacticactivity of porcine insulin for human T lymphocytes in vitro J Zmmunol 138,2100-2103 Bieva, C J., Vanderbrugghen, F J., and Stryckmans, P A (1989) Malignant cell 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Lagoo-Deenadayalan, S., Lorenz, H M., Byrne, J H., Barber, W H., and Hardy, K J (1994) IL-2, IL-4, and IFN-y gene expression versus secretion m superantigen-activated T cells J Immunol 152, 1641-1652 Negative Selection 251 19 Lederman, S., Yellin, M J., Cleary, A M., Pernis, A., Inghirami, G , Cohn, L E., et al (1994) T-BAM/CD40-L on helper T lymphocytes augments lymphokineinduced B cell Ig rsotype switch recombination and rescues B cells from programmed cell death J Immunol 152,2163-2171 20 Lee, W T and Vitteta, E S (1991) The differentral expression of homing and adhesion molecules on virgin and memory T cells in the mouse Cell Zmmunol 132,2 15-222 21 Levin, D., Constant, S., Pasqualini, T., Flavell, R., and Bottomly, K (1993) Role of dendritic cells in priming of CD4 T lymphocytes to peptide antigen in uivo J Immunol 151,6142-6750 22 Linette, G P., Hartzman, R J , Ledbetter, J A., and June, C H (1988) HIV-linfected cells show a selective signaling defect after perturbation of CD3-antigen receptor Science 241,573-576 23 Loeffler, C M., Platt, J L., Anderson, P M., Katsanis, E., Ochoa, J B., Urba, W J., et al, (1991) Antitumor effects of interleukin liposomes and anti-Cd3stimulated T cells against murine MCA-38 hepatic metasis Cancer Res 51, 2127-2132 24 Nagata, M., Santamaria, P., Kawamura, T., Utsugi, T , and Yoon, J (1994) Evidence for the role of CD8+ cytotoxic T cells in the destruction of pancreatic p-cells in nonobese diabetic mice J Immunol 152,2042-2050 25 Nestle, F., Zheng, X., Thompson, C , Turka, L., and Nickoloff, B (1993) Characterizatron of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets J Zmmunol 1X,6535-6545 26 Padmanabhan, R , Corsrco, C D., Howard, T H., Holter, W., Fordis, C., Willingham, M., and Howard, B (1989) Purification of transiently transfected cells by magnetic affinity cell sorting Anal Biochem 170,341-348 27 Padmanabhan, R , Corsica, C D., Holter, W , Howard, T., and Howard, B H (1989) Purification of transiently transfected cells by magnetic-affinity cell sorting J Immunogenetics 116,91-102 28 Pricop, L., Rabinowich, H., Morel, P A., Sulica, A., Whiteside, T L., and Herberman, R B (1993) Characterization of the Fcp receptor on human natural killer cells J Zmmunol 151,3018-3029 29 Rabinowich, H., Sedlmayr, P., Herberman, R., and Whiteside, T (1992) Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2 activated natural killer cells J Zmmunol 149, 340-349 30 Rasmussen, R., Counts, S., Daley, J., and Schlossman, S (1994) Isolation and characterization of CD6- T-cells from peripheral blood J Zmmunol 152,527-536 31 Smyth, M J., Strobl, S L., Young, H A., Ortaldo, J R., and Ochoa, A C (1991) Regulation of lymphokine-activated killer activity and pore forming protein gene expression in human peripheral blood CD8+ T lymphocytes J Immunol 146, 3289-3297 32 Spangude, G J and Scollay, R (1990) A simplified method for enrichment of mouse hematopoietic stem cells Exp Hematol 18,920-926 252 Vaccaro and Markinac 33 Spangude, G J., Heimfeld, S., and Weissman, I L (1988) Purtfication and characterization of mouse hematopoietic stem cells Science 241,58-62 34 Trickett, A E., Ford, D J., Lam-PO-Tang, P R L., and Vowels, M R (1990) Comparison of magnetic particles for immunomagnetic bone marrow purging using an acute lymphoblastic leukemia model Transplant Proc 22,2177-2178 35 Vaccaro, D E (1990) Application of magnetic separation: cell sorting Am Biotech Lab April, 30-35 CHAPTER26 Use of Monoclonal Antibodies with Magnetic Particles to Separate Cell Subpopulations by Positive Selection Dennis E Vaccaro and Joan E Markinac Introduction The use of magnetic particles in combination with monoclonal antibodies (MAbs) has greatly simplified sorting heterogeneouspopulations of cells into specific subpopulations (l-35) MAbs which recognize particular cell surface markers provide the specificity necessaryto distinguish one cellular population from another.Magnetic particles provide an effective, yet gentle, physical force to move the target population away from the remaining cells Two forms of selection have been used by researchers: positive and negative selection Positive selection is the isolation of the desired population from the cellular mixture and does not imply the complete removal of a target cell population Negative selection is the elimination of an undesired population from the cellular mixture The methods for negative selection were outlined in Chapter 25 In this chapter, we focus on the methods used in positive selection (9,28) As with negative-selection techniques, there are a variety of methods in use We outline one general method and discuss variations in methodology in Section of this chapter Materials Cell-culture medium with antibiotics: The researcher should use the medium that best preserves the health of the cells during the sorting process.A base medium, such as RPMI-1640, with 5-10% fetal bovine serum From- Methods fn Molecular Brology, Vol 45 Monoclonal Ant/body Protocols Edlted by: W C Davs Humana Press Inc., Totowa, NJ 253 Vaccaro and Markinac (FBS) and appropriate levels of antibiotics is recommended Some laboratories omit antibiotics after the cells are in culture Complete cellculture medium can be stored at 4OCprior to use for no longer than d Magnetic particles: Magnetic beads or particles can be obtained from several commercral sources coupled directly with the MAb of Interest, polyclonal anti-immunoglobulin (IgG and IgM or isotype-specific), or as free particles prepared for coupling by the investigator Magnetic particles from PerSeptive Diagnostics (Cambridge, MA; formerly Advanced Magnetics) are stable for at least mo when stored at 4°C a Directly coupled magnetic particles available commercially: Magnetic particles are available from PerSeptive Diagnostics At the time of use, magnetically wash the particles three times in culture medium with antibiotics Particles must not be frozen or centrifuged Resuspendthe particles in culture medium such that only a small volume is addedto the cells b Magnetic particles prepared with the antibody of interest by the investigator: MAbs can be purchased from commercial sources and covalently attached to amine-terminated BioMag magnetic particles available from PerSeptive Diagnostics through a glutaraldehyde crosslinking of the amines m the MAb to the ammes on the magnetic particle If usmg this source, activate the magnetic particles according to the instructions provided (see Chapter 25, Note 1) The overnight couplmg procedure requires election microscopy (EM)-grade glutaraldehyde, 15 mg of MAb (purified or unpurified), and 100 mg of a carrier protein, such as BSA, per 500 mg of amine-terminated BioMag c Magnetic particles coupled with anti-immunoglobulin: A two-step procedure involving the use of magnetic particles coupled with antiimmunoglobulin can also be used Magnetic particles coupled with polyclonal antimouse immunoglobulm (IgG and IgM or isotype-specific) can be purchased commercially Depending on the source of antibody and the manufacturer’s recommendations, anti-Ig-coupled particles are used with 5-20 ug of MAb/million cells, which is typically needed for the two-step procedure The MAbs need not be purified, but should be sterile (see Chapter 25, Note 2) Equipment: Sterile tubes, bottles, and flasks as needed Cell preparation: Cells to be separated may be partially purified prior to use Separation out of whole blood should not be attempted unless the cells are diluted m medium Cells should be washed in medmm prior to separation to reduce the amount of immunoglobulm in the medium and attached to cells Magnetic separator: magnetic separator appropriate for the tube or flask to be used Magnetic separation units are available from PerSepttve Positive Selection 255 Diagnostics that accommodate 12 x 75 mm test tubes, microcentrifuge tubes, tissue-culture flasks, 96-well tissue-culture plates, and 15 and 50-mL conical tubes Method Use sterile tissue-culture tubes or flasks The following is a one-step protocol The target population is assumed to be 10% of the total (see Note 1) All steps are done at 4°C (see Note 2) Place approx lo7 lymphocytes in a tube in 10 mL of RPM1 with 5% FBS and antibiotics (see Notes and 4) Wash 0.4 mL of the appropriate magnetic anti-CD (or anticell membrane molecule of interest) antibody three times in sterile medium, and then add to the cells, (The particle-to-cell ratio in this example is 20 magnetic particles/cell based on the total cell population; see Note 5.) Incubate the cells with magnetic particles for 30 to h at 4°C (see Note 6) Swirl the cell/particle suspension once every 10 to promote attachment (see Note 7) Magnetically separate for 10 min, and discard the supernatant or keep for further evaluation (see Notes and 9) Methods for detaching magnetic particles from cells after separation include: a Culture the cells bound to the magnetic particles for 48 h During this time, the magnetic particles should separate from the cells owing to the turnover of cell-surface molecules Remove the freed particles magnetically b Use a protease, such as chymopapain, to break the antigen-antibody bond, and then remove the particles magnetically (see Note 10) Notes Approx 70-90% of the total target cell population can be positively selected This protocol is usually performed at 4°C The reason for this is to minimize patching, capping, and phagocytosis Also, cell viability may be best preserved by keeping the cells on ice However, room temperature or even 37°C may be optimal for certain cell types Other temperatures should be investigated if the investigator is having problems with low yield and/or viability The maximum cell concentration should be about x lo6 cells/ml The denser the mixture of cells, the more likely there will be nonspecific binding, clumping, and trapping of cells The investigator should work with the most dilute cellular suspension reasonable with the goals of the experiment If poor yields are being obtained, try diluting the mixture lo-fold, and repeat the experiment 256 Vaccaro and Markinac It is important to have protein m the culture medium durmg cell sortmg to minimize nonspecific binding Too much protein in the medium may inhibit the binding of particles to cells The mvestigator may have to titrate the protein concentration to optimize the particle bmding and cell separation Depending on antigen availability and the size of the target cell population, cell sortmg applications may require 20-80 magnetic particles/cell based on the total cell population Increasing the incubation time beyond h may be necessary to obtain the desired yield of cells If the cells are clumped at this stage, it may be because of DNA release by dead cells These clumps can be easily broken up with the use of 0.1% DNase in the medium Magnetic separation must be performed with the pellet formed on the side of the flask or tube This is so that the unselected cells will not contaminate the pellet owing to gravity One-micron particles will take up to 10 to separate depending on the volume of medium Larger particles will separate faster However, it still may be appropriate to wait the same amount of time Aspirate the flask or tube very carefully after the magnetic separation The magnetic field should be maintained during aspiration It is very easy to disturb the pellet and to pipet particles inadvertently because of the gentle packing of the pellet It is recommended that a propipet rather than vacuum aspiration or pouring off be used Additional magnetic separations can be performed to reduce further the amount of contaminating magnetic particles in the supernatant Because the particles are black, it is very easy to see if they have not been removed 10 Each of theseprocedures haslimitations Not all particles may detach from cells during culturing, and the use of a protease may damage cells Depending on the application, it may not be necessaryto remove the cells from the BioMag particles BioMag particles are only pm m sizeand can be successfully used in flow cytometry equipment, because they not jam the machine and are distingmshable from cells If considerable problems are encountered when attempting to select a population of cells positively, negative selection should be considered (see Chapter 25) References Adkins, B., Ghanei, A., and Hanulton, K (1993) Development of IL-4, IL-2, and JFN-y production by murine peripheral T lymphocytes J Zmmunol 151,6617-6626 Berman, J F and Center, D M (1987) Chemotactic activity of porcine insulin for human T lymphocytes in vitro J Zmmunol 138,2100-2103 Positive Selection Bieva, C J., Vanderbrugghen, F J., and Stryckmans, P A (1989) Malignant cell separation by iron colloidal immunomagnetic adsorption Exp Hematol 17, 14-920 Brissette-Storkus, C., Kaufman, D L., Pasewicz, L., Worsey, H M., Lakomy, R., Ildstad, S T., and Chambers, W H (1994) Characterization and function of the NKR-Pldlm/T-cell receptor-@+ subset of rat T-cells J Zmmunol 152, 388-396 Chervenak, R., Dempsey, D., Soloff, R., Wolcott, M., and Jennings, S (1993) The expression of CD4 by T cell precursors resident in both the thymus and the bone marrow J Zmmunol 151,4486-4493 Chilton, P and Fernandez-Botran, R (1993) Production of soluble IL-4 receptors by murine spleen cells is regulated by T cell activation and IL-4 J Zmmunol 151, 5907-5917 Cruickshank, W W., Berman, J S., Theodore, A C., Bernado, J., and Center, D M (1987) Lymphokine activation of T4 and T lymphocytes and monocytes J Zmmunol 138,3817-3823 Ettinger, R., Wang, J K M., BOSSU,P., Papas, K., Sidman, C L., Abbas, A K., and Marshak-Rothstein, A (1994) Functional distinctions between MRL-lpr and MRL-gld lymphocytes J Zmmunol 152,1557-1568 Ford, J R and Terzaghi-Howe, M (1992) Characteristics of magnetically separated rat tracheal epithelial cells populations Am J Physiol 263, L568-L574 10 Fowler, D., Kurasawa, K., Husebekk, A., Cohen, P., and Gress, R (1994) Cells of Th2 cytokine phenotype prevent LPS-induced lethality during murine graft-versus-host reaction J Zmmunol 152, 1004-1013 11 Freedman, M S., Blain, M., and Antel, J (1991) Differential responses of CD4+CD45RA+ and CD4+CD29+ subsets to activated CD8+ cells: enhanced stimulation of the CD4+CD45RA+ subset by cells from patients with multiple sclerosis Cell Zmmunol 133,306-3 16 12 Gribben, J G., Saporito, L., Barber, M., Blake, K W., Edwards, R M., Griffin, J D., et al (1992) Bone marrows of non-Hodgkin’s lymphoma patients with a bcl-2 translocation can be purged of polymerase chain reaction-detectable lymphoma cells using monoclonal antibodies and immunomagnetic bead depletion Blood 80, 1083-1089 13 Gryllis, C., Wainberg, M A., Gornitsky, M., and Brenner, B (1990) Diminution of inducible lymphokine-activated killer cell activity in individuals with AIDS-related disorders AIDS 4, 1205-1212 14 Haregewoin, A S., Soman, G., Horn, R C., and Finberg, R W (1989) Human T cells respond to mycobacterial heat shock protein Nature 340,309-312 15 Hermentin, P., Doenges, R., Franssen, U., Bieva, C., Vander Brugghen, F J., Stryckmans, P., et al (1990) Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion Bioconjugate Chem 1,411418 16 June, C H., Ledbetter, J A., Gillespie, M M., Lindsten, T., and Thompson, C B (1987) T cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin-2 gene expression Mol Cell Biol 7,4472-4480 258 Vaccaro and Markinac 17 Kornfield, H., Crulckshank, W W., Pyle, S W , Berman, J S., and Center, D M (1988) Lymphocyte activation by HIV-l envelope glycoprotein Nature 335, 445-448 18 Lagoo, A., Lagoo-Deenadayalan, S., Lorenz, H M., Byrne, J H., Barber, W H., and Hardy, K J (1994) IL-2, IL-4, and IFN-)I gene expression versus secretion in superantigen-activated T cells J Zmmunol 152, 1641-1652 19 Lederman, S , Yellm, M J , Cleary, A M., Pernis, A., Inghiraml, G , Cohn, L E , et al (1994) T-BAM/CD40-L on helper T lymphocytes augments lymphokineinduced B cell Ig isotype switch recombination and rescues B cells from programmed cell death J Zmmunol 152,2163-2171 20 Lee, W T and Vitteta, E S (1991) The differential expression of homing and adhesion molecules on virgin and memory T cells m the mouse Cell Zmmunol 132,2 15-222 21 Levm, D., Constant, S , Pasqualml, T., Flavell, R., and Bottomly, K (1993) Role of dendritic cells in priming of CD4 T lymphocytes to peptide antigen in vzvo Immunol 151,6742-6750 22 Linette, G P , Hartzman, R J., Ledbetter, J A., and June, C H (1988) HIV-l- 23 24 25 26 27 28 29 30 infected cells show a selective signaling defect after perturbation of CD3-antigen receptor Science 241,573-576 Loeffler, C M., Platt, J L., Anderson, P M., Katsanis, E., Ochoa, J B., Urba, W J., et al (1991) Antitumor effects of interleukin liposomes and anti-Cd3stimulated T cells against murine MCA-38 hepatic metasis Cancer Res 51, 2127-2132 Nagata, M , Santamaria, P., Kawamura, T., Utsugi, T , and Yoon, J (1994) Evidence for the role of CD8+ cytotoxlc T cells m the destruction of pancreatic p-cells in nonobese diabetic mice J Immunol 152,2042-2050 Nestle, F., Zheng, X., Thompson, C , Turka, L., and Nickoloff, B (1993) Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets J Immunol 151,6535-6545 Padmanabhan, R., Corsica, C D , Howard, T H , Holter, W., Fordis, C., Willingham, M., and Howard, B (1989) Purification of transiently transfected cells by magnetic affinity cell sorting Anal Biochem 170,341-348 Padmanabhan, R., Corsica, C D , Holter, W., Howard, T , and Howard, B H (1989) Purification of transiently transfected cells by magnetic-affinity cell sorting J Immunogenetics 116,91-102 Pricop, L., Rabinowich, H., Morel, P A , Sulica, A., Whiteside, T L., and Herberman, R B (1993) Characterization of the Fcp receptor on human natural killer cells J Zmmunol 151,3018-3029 Rabmowich, H., Sedlmayr, P., Herberman, R., and Whiteslde, T (1992) Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2 activated natural luller cells J Zmmunol 149, 340-349 Rasmussen, R., Counts, S., Daley, J , and Schlossman, S (1994) Isolation and characterization of CD6- T-cells from peripheral blood J ImmunoZ 152,527-536 Positive Selection 259 Smyth, M J , Strobl, S L., Young, H A., Ortaldo, J R., and Ochoa, A C (1991) Regulation of lymphokine-activated killer activity and pore forming protein gene expression in human peripheral blood CD8+ T lymphocytes J ZmmunoE 146, 3289-3297 32 Spangude, G J and Scollay, R (1990) A simplified method for enrichment of mouse hematopoietrc stem cells Exp Hematol l&920-926 33 Spangude, G J., Heimfeld, S., and Weissman, I L (1988) Purification and characterization of mouse hematopoietlc stem cells Science 241,58-62 34 Trickett, A E., Ford, D J , Lam-PO-Tang, P R L., and Vowels, M R (1990) Comparison of magnetic particles for immunomagnetic bone marrow purging using an acute lymphoblastic leukemia model Transplant Proc 22,2177-2178 35 Vaccaro, D E (1990) Application of magnetic separation* cell sorting Am Biotech Lab April, 30-35 ... antibody References Teng, N N H., Reyes, G R., Bieber, M., Fry, K E., Lam, K S., and Herbert, J M (198.5) Strategies for stable human monoclonal antibody production, in Human Hybridomas and Monoclonal. .. but these interactions are not sufficiently From Methods m Molecular Brology, Vol 45 Monoclonal Antibody Protocols Edtted by W C Davis Humana Press Inc , Totowa, NJ Schelling defined to provide... and rat (cross-species) spleen cells with From* Methods m Molecular B/ology, Vol 45’ Monoclonal Antibody Protocols Edlted by: W C Davts Humana 17 Press Inc , Totowa, NJ 18 Hamilton and Davis