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[ 16] Design and Production of Bispecific Monoclonal Antibodies by Hybrid Hybridomas for Use in
Irnmunoassay B y MIYoKO TAKAHASHI, STF.VEN A. FULLER, and SCOTT WINSTON
Introduction The production of novel antibody reagents has become possible through advances in hybridoma techniques, recombinant DNA manipulation, and gene transfection technology. One such novel reagent is the bispecific antibody, a hybrid immunoglobulin molecule that is formed of nonidentical heavy- and light-chain pairs expressing different antigen specificities. The ability of bispecific antibodies to cross-link two different a n t i gens has led to applications in immunohistochemistry, 1 cell targeting, 2,3 complement-mediated cytotoxicity,4 and immunoassays. 5,6 In conventional use, monoclonal antibodies (MAb) are covalently coupled to various reagents (e.g., enzymes, fluorochromes, radioisotopes, dyes, and toxins). Enzyme immunoassays (EIA) utilize MAb cross-linked to any of several marker enzymes. Such chemical modification of both antibody and enzyme has several limitations, including (1) an a n t i b o d y - e n z y m e mixture that includes both active and inactive species in varying proportions, (2) a preparation that contains both antibody and enzyme coupled to contaminants as well as to each other, (3) loss of antibody function, and (4) a n t i b o d y - e n z y m e conjugates that are often less stable than the unmodified components. Bispecific monoclonal antibodies (bsMAb), developed originally by Milstein and Cuello (1983), 1 can circumvent these problems by avoiding entirely the chemical modification. Antibodies with bifunctional specificities have been generated by a number of methods: (1) cross-linking of heterologous MAb, 7,s (2) reassociation of monovalent antibody fragments, 9- H and (3) cell fusion of hybrid1C. Milsteinand A. C. Cuello,Nature (London) 305, 537 (1983). 2 U. D. Staerz and M. J. Bevan, Proc. Natl. Acad. Sci. U.S.A. 83, 1453 (1986). 3A. Lanzavecchiaand D. Seheidegger,Eur. J. Immunol. 17, 105 (1987). 4 j. T. Wong and R. B. Colvin,J. Immunol. 139, 1369 (1987). 5M. R. Suresh, A. C. Cuello,and C. Milstein,Proc. Natl. Acad. Sci. U.S.A. 83, 7989 (1986). 6 M. Takahashiand S. A. Fuller, Clin. Chem. 34, 1693 (1988). 7U. D. Staerz, O. Kanagawa,and M. J. Bevan,Nature (London) 314, 628 (1985). s p. Perez, R. W. Hoffman,S. Shaw,J. A. Bluestone,and D. M. Segal,Nature (London) 316, 354 (1985). 9A. Nisonoffand W. J. Mandy, Nature (London) 194, 355 (1962). 1oV. Raso and T. Griffin,CancerRes. 41, 2073 (1981). H M. Brennan, P. F. Davison, and H. Paulus, Science 229, 81 (1985). METHODS IN ENZYMOLOGY, VOL. 203
~ t © 1991 by Academlc Pre~ Inc. All rlghts of ~ u c f i o n in any form re~rved.
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omas or sensitized B lymphocytes.~ The cell fusion approach has several advantages. Hybrid hybridomas form a high percentage of hybrid antibody molecules. The ease of the procedure allows any experienced cell culturist to perform the work. In addition, bsMAbs that are biologically produced by hybrid hybridomas often exhibit no observable changes in specificity or physical characteristics after prolonged storage at 4" (M. Takahashi, S. A. Fuller, and S. Winston, unpublished data, 1988). Our research interests lie in the development of EIAs for a wide variety of analytes. In this chapter we describe methods we have utilized or developed to prepare a panel of fusion partners that simplifies production of bsMAbs, the preparation of hybrid hybridomas, and the production and use of bsMAbs. While the focus of our work is urease-based EIAs, these methods should be readily applicable to other enzyme/detector systems. A detailed report concerning the production of bsMAbs by hybrid hybridomas (theory, materials, and methods) was published in an earlier volume in this series.~2
Hybrid Hybridoma Production In our attempts to utilize bsMAbs in urease-based EIAs, we wished to set up a simple and flexible system that might be useful for any analyte or MAb of interest. Consequently, we have prepared a panel of anti-urease fusion partners that represent the major murine IgG subclasses and that possess two selectable markers. This panel has the following advantages: I. bsMAbs of most homologous or heterologous heavy-chain subclasses can be readily prepared, which provides flexibility in stability, function, or purpose. 2. The hybridoma to be fused need not be modified, since the selectable markers of the anti-urease fusion panner allow both parental lines to be eliminated. Figure 1 is a schematic representation of the processes involved in producing hybrid hybridomas. Each of these is described in the methods that follow. Isolation of Hybridoma Lines for Use as Fusion Partners A panel of anti-urease-producing hybridoma lines was established from a 42% (w/v) polyethylene glycol (PEG) (Mr 4000; Merck, Darmstadt, Germany) fusion of Sp2/0 cells and splenocytes isolated from BALB/c ~2M. R, Suresh, A. C. Cuello, and C. Milstein, this series, Vol. 121, p. 210.
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Anti-Urease Hybridoma 8-Azaguanine Ouabain Anti-Ureose Hybridoma
(HAT Sensitive) (Ouabain Resistant)
+
Anti-hCG Hybridoma (HAT Resistant) (Ouabain Sensitive) Fusion Selection (HAT, Ouabain) Cloning
Hybrid Hybridoma (Anti-Urease and Anti-hCG) FIG. 1. How diagram for preparation of hybrid hybridomas. HAT, Hypoxanthine/aminopterin/thymidine medium; hCG, human chorionic gonadotropin.
mice immunized with jackbean urease.6 In order to use these anti-ureaseproducing hybridoma lines as universal fusion partners with either immunocytes (lymphocytes) or other hybridomas, double-mutant fines resistant to both 8-azaguanine and ouabain were isolated. Procedure to Isolate 8-Azaguanine-Resistant Hybridomas. In hybridoma technology, the most common selection method is the use of a parental cell line that is sensitive to hypoxanthine/aminopterin/thymidine (HAT) medium. Is,14 Aminopterin blocks de nova purine and pyrimidine synthesis by inhibition of the enzyme dihydrofolate reductase. However, if present, the enzymes hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thymidine kinase can utilize hypoxanthine and thymidine, respectively, via salvage pathways. Animal cell lines deficient for HGPRT (HAT sensitive) can be isolated by adaptive growth and selection in 8-azaguanine (8-AG). This antimetabolite is cytotoxic to normal cells and only HGPRT- cells can survive. Selection of 8-AG-resistant variants involves adaptive growth in successively increasing concentrations of the drug (to 20/~g/ml). 1. Prepare a 2 mg/ml stock solution of 8-AG. Weigh 200 mg of the purine analog and add to 90 ml distilled water. Add 1 N NaOH dropwise ,3 S. A. Fuller, M. Takahashi, and J. G. R. HurreU, in "Current Protocols in Molecular Biology" (F. Ausubel, B. Brent, R. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, ¢¢ls.),Unit 11. Greene Publishing, New York, 1987. 14G. Galfr~ and C. Milstein, this series, Vol. 73, p. 3.
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until dissolved. Add distilled water to 100 ml. Filter sterilize the solution and then store frozen at - 20 ° in convenient aliquots. 2. Prepare six wells (six-well cluster dish; Costar, Cambridge, MA) containing actively growing hybridomas (3 X l05 cells/ml, anti-urease cell lines in this case) in 10 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS). Add the 8-AG stock solution to a final concentration of 1, 2, and 4 ]tg/ml, using two wells for each of the concentrations (we have found that different murine hybridoma lines show varied sensitivity to the drug). Grow cells in an incubator under 8O/oCO2. 3. Make a 50% medium change every 2 days using growth medium containing appropriate amounts of 8-AG. 4. Select the culture growing at the highest drug concentration. Double the drug concentration and continue to culture for several generations. 5. Repeat step 4 until a viable cell population growing in the presence of 20/~g/ml 8-AG is obtained. 6. When cells are adapted to the 20/tg/ml drug concentration (approximately 3 weeks), clone by the limiting dilution method ~3 using medium containing 20 #g/ml 8-AG. 7. When the clones are confluent, select 10 wells containing a single colony and expand them in duplicate in a 24-well plate (Costar). Culture one set in HAT selection medium 13,14and discard the clones that survive. 8. Screen the remaining clones that are 8-AG resistant and HAT sensitive for antibody production (anti-urease). 9. Select clones with good growth and antibody production. Freeze aliquots for safe keeping. The remaining cells are subjected to another round of adaptive growth in the presence of ouabain.
Procedurefor Isolation of Ouabain-Resistant Hybridomas. Ouabain is a plant glycoside that inhibits membrane-bound Na+,K+ - ATPase. Ouabain resistance is an additional property useful for the selection of desirable fusion products. An 8-AG-resistant hybridoma line that is also ouabain resistant can be fused with any hybridoma line. The fusion products are selectively cultured in medium containing HAT and ouabain. In this medium, the double-mutant parental cells do not survive because of their deficiency for HGPRT. The other parental hybridoma will die due to its lack of resistance to ouabain, and only the hybrid hybridomas will survive. The procedure used to isolate ouabain-resistant hybridomas is similar to that described in the previous section for azaguanine. 1. Prepare 50 m M ouabain (Sigma, St. Louis, MO) stock solution in distilled water. Dissolve by heating to 70 ° . Mix with an equal
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2.
3.
4.
5.
6. 7.
8. 9. 10.
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volume of 2× DMEM (room temperature), filter sterilize, and store frozen at - 2 0 ° in convenient aliquots. In a 75-cm2 tissue culture flask, grow the 8-AG-resistant hybridoma line to a cell density of approximately 4 × 105 cells/ml in 30 ml medium with 15% FCS. Add the ouabain stock solution to a final concentration of 0.1 mM. Set up a small control flask and grow the cells in the same medium without the drug, in order to ensure that the cell growth retardation is due to the drug. Make a 50% medium change by removing the spent medium and supplementing with fresh medium or by merely doubling the medium as required. Allow cells to grow for several generations in medium containing 0.1 m M ouabain, then double the culture volume with fresh medium containing 0.2 m M ouabain. When the culture has reached a cell density of 4 - 6 X 105 cells/ml, make an appropriate split with medium containing 0.2 m M ouabain. Repeat step 4, by doubling the culture volume with medium containing 0.4 m M ouabain. It is very important to increase the ouabain level gradually. In comparison to human cell lines, rodent cell lines have been reported to be far more resistant to ouabain. All of the nonadapted murine hybridoma lines are very sensitive to the drug at around 0.2-0.4 mM. Continue the process and gradually increase the ouabain level up to lmM. When the cells are well adapted to 1 m M ouabain, add 20 #g/ml 8-AG to the medium supplemented with 15% (v/v) FCS. Allow them to grow for several generations in the presence of the two drugs. Clone by the limiting dilution method ~ in medium containing 1 m M ouabain and 20 gg/ml 8-AG, supplemented with 20% FCS. Screen only those wells with a single colony for antibody activity. Choose the clone(s) with the best growth characteristics and the best production of antibody.
Using the procedures described above, we have isolated numerous 8-AG/ouabain-resistant hybridoma lines that produce anti-urease of IgM or IgG (subtypes 1, 2a, and 2b) class. Each of these lines were continuously cultured for at least 8 weeks in DMEM containing 10% FCS, after which all lines still demonstrated resistance to 20/tg/ml 8-AG and 1 m M ouabain and were sensitive to HAT medium. Production of anti-urease in the variant lines was measured by enzyme-linked immunosorbent assay (ELISA) following continuous culture. Despite the stability of drug resist-
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ance characteristics, some hybridomas expressed reduced antibody production relative to wild type. Cell doubling times of the variant lines in the presence and absence of azaguanine and ouabain were as reported previously.6 Cell Fusions and Cloning Two cell fusion procedures used to obtain hybrid hybridomas are described in the following sections. Most reagents and procedures are as for conventional hybridoma workJ TM Differences exist in the use of selection medium, the fusion cell ratio for hybridoma × hybridoma fusions, and screening for bispecific antibody activity. The latter is discussed separately. Hybridoma × Splenocyte Cell Fusions. Fusions between the 8-AGresistant hybridomas that produce anti-urease and splenocytes obtained from an immunized mouse, or splenocytes immunized in vitro, are basically as for Sp2/0 × splenocyte fusions: The ratio of splenocytes to hybridoma cells is 5 : 1. The polyethylene glycol (PEG)-mediated fusion products are resuspended in HAT selection medium and incubated for 1 - 3 hr in a CO2 incubator. The cells are then plated out at 1- 2 × 105 cells/well on 96-well Costar plates that were preseeded with mouse peritoneal exudate cells (PEC) as feeders in HAT selection medium. Hybrid hybridomas are generally screened 2 - 3 weeks postfusion. Selected positive cultures are then subjected to cloning by the limiting dilution method. We have successfully established hybrid hybridomas that produce bsMAb by fusions of anti-urease producing hybridomas with splenocytes sensitized in vitro with human luteinizing hormone as well as with splenocytes obtained from BALB/c mice immunized with various immunogens, including human chorionicgonadotropin (hCG). The limitation of this approach, i.e., fusions of hybridoma× splenocytes, is that the isolation of hybrid hybridomas that produce bsMAbs of desired specificity and affinity depends on chance. Hybridoma × Hybridoma Cell Fusions. We describe here the protocol used for the fusion experiments between anti-urease hybridomas and hybridomas secreting anti-hCG (schematically shown in Fig. 1). 1. From each of the two hybridoma lines to be fused, harvest 4 × 107 cells from a logarithmically growing culture. 2. Centrifuge both hybridoma tines. Pool the cell pellets, and wash three times in warm DMEM. 3. Subject the final cell pellet (which is a 1 : 1 cell mixture of the two parental hybridomas) to PEG cell fusion. '3 4. Resuspend the fused cells in 40 ml DMEM supplemented with 20%
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6.
7.
8. 9. 10.
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FCS, 2× HAT, and 2 m M ouabain. Incubate for 30 min in a CO2 incubator prior to plating out at 2 X 105 cells/0.1 ml into each well on 96-well Costar plates. The plates should be preseeded with 0.1 ml/well mouse PEC in DMEM with 20% FCS, and without HAT/ ouabain. Prepare control cultures as in the experimental plates for both parental hybridomas using 10 wells each. PEC feeder cells are preseeded and each hybridoma line is plated at 2 X 105 cells/well. In the HAT/ouabain selection medium, the control anti-urease hybridomas should not survive because of their sensitivity to HAT, while anti-hCG hybridomas are killed due to ouabain sensitivity. Leave plates undisturbed in the CO2 incubator for 1 week, except for a brief check for microbial contamination a few days after the fusion. One week postfusion, examine the control cultures carefully. When both parental cells are dead in the HAT/ouabain medium, start feeding the experimental plates with DMEM supplemented with 20% FCS and HT (HAT medium from which aminopterin is omitted) and ouabain. Feed cultures as required until the hybrid hybridomas become confluent and the culture medium becomes acidic (yellow in appearance). Screen the culture supernatants for parental and bispecific antibody activities, as described in the following sections. Select cultures indicating bispecific antibody activity, and clone by the limiting dilution method. When the hybrid hybridomas are established and cloned, freeze a number of vials and confirm a good recovery from the liquid nitrogen freezer.
The procedure described above to prepare bispecific antibody-producing hybridomas by the fusion between two hybridoma lines has the advantage that the characteristics of antibody specificity and affinity as well as the heavy-chain isotypes of immunoglobulins produced by each of the parental hybridomas are already known. This allows for an easy selection of clones that meet the specific criteria desired. Our initial bsMAb work6 indicated that fusion ofIgM, IgG~, IgG2~, and IgG2b anti-urease hybridomas with an IgG~ anti-hCG hybridoma resulted in efficient production of bispeeific monoclonal antibodies only for the homologous IgG1 X IgG~ fusions. Based on this observation, we have performed extensive fusion experiments to evaluate the outcome of homologous as well as heterologous heavy-chain combinations using our expanded panel of anti-urease variant hybridoma cell lines. Table I presents the
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TABLE I PRODUCTION OF BISPECIFIC ANTIBODY FROM FUSIONS BETWEEN HYBRIDOMAS PRODUCING HOMOLOGOUS AS WELL AS HETEROLOGOUS IMMUNOGLOBULIN SUBCLASSESOF IGG Percentage of positive well~ Parental antibodies
Parental hybridoma fines*
Anti-urease
Anti-hCG
3U66 r X 2G131 7U114 r × 6G94 7U215 • X IG151
96 100 100
57 17 52
100 97 100
53 99 37
bsMAb
Number of wells screened
Homologous fusions IgGl-IgGi IgG~.- IgG2~ IgG2b- IgG2b Heterlogous fusions
IgGl-IgGz~ IgGl-IgG2b IgGi-IgGz,
3U66 r × 6G94 2G131 × 6U334 r 3U66 r X IGl51
57 17 49 6.9 2.2 5.5
130 180 180 160 180 110
a Superscript r designates the anti-urease-producing variant hybridoma fines. b Calculated as (number of positive wells/number of wells screened) 100.
results obtained from cell fusions between anti-urease and anti-hCG hybridomas of homologous or heterologous heavy-chain subclass. In fusion experiments between homologous heavy chains of IgGl, IgG~, and IgG2b subclasses, bispecific antibody-positive wells were identified in 17-57% of wells screened. Compared to the homologous fusion results, the fusions between heterologous subclasses of IgG yielded only 2.2-6.9% positive wells for bispecific antibody activity. Moreover, use of IgM or IgG3 hybridomas yielded no or only a few positive wells regardless of homologous or heterologous fusions. It is important to note that isolation of bsMAbs composed of heterologous heavy chains is possible despite the lower efficiency compared to homologous fusions.
Antibody Screening of Culture Supernatants of Hybrid Hybridomas Enzyme immunoassays using 96-well microtiter plates are routinely used in our laboratory for the screening of culture supernatants for bsMAb activity as well as monospecific MAb activities of parental cells.
Materials Polyvinyl or polystyrene 96-well microtiter plates Coating buffer: 0.05 M carbonate buffer, pH 9.6 TEN buffer: 0.05 M Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.15 MNaC1, pH 7.3 Wash buffer (WB): Phosphate-buffered saline (PBS), pH 7.2, with 0.05% Tween 20
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Diluting buffer (DB): PBS, pH 7.2, with 0.05% Tween 20 and 0.25% bovine serum albumin (BSA) hCG (Sigma) Urease and urease substrate solution (ADI Diagnostics, Inc., Rexdale, Canada) o-Phenylenediamine (OPD) (Sigma) Peroxidase-conjugated goat anti-mouse IgG (heavy and light chain) (Cappel Worthington, Malvern, PA) Urease-conjugated rabbit anti-mouse F(ab')2 ofIgG (ADI Diagnostics)
Methods Anti-hCG Screening 1. Coat 96-well polyvinyl microtiter plates (Dynatech, Chantilly, VA) with 100/tl/well of hCG (5 IU/ml) in 0.05 M carbonate buffer. Seal the plates and store at 4" for at least 24 hr before use. 2. Remove antigen solution and fill all wells with DB to block additional protein binding sites for 1 hr at room temperature. 3. Wash the plates three times in WB. 4. Add hybrid hybridoma culture supernatants (100/d/well) into the plates and incubate for 60-90 rain at 37 °. 5. Wash plates three times in WB. 6. Add 100 /d/well of urease-conjugated rabbit anti-mouse F(ab')z fragment of IgG. Seal the plates and incubate for 30 rain at 37*. 7. Wash the plates three times with WB, then three times with 0.85% NaC1. 8. Add 100/~l/well urease substrate solution, and after a 10- to 20-rain incubation at room temperature, read the plates at 590 nm.
Anti-Urease Screening. The procedure is the same as for anti-hCG screening with minor modifications. The marker enzyme conjugated to the detector antibody must be one other than urease. 1. Coat polyvinyl plates with urease (10/tg/ml) in TEN buffer. Seal the plates and store at 4* until use. 2. Block plates for 1 hr at room temperature. 3. Wash three times in WB. 4. Add culture supernatants to the plate, and incubate for 60-90 min at 37 ° . 5. Wash plates three times. 6. Add peroxidase-conjugated goat anti-mouse IgG (heavy and light chains) and incubate for 30 min at 37 °. 7. Wash plates four times with WB.
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8. Add OPD in phosphate-citrate buffer (2.43 ml 0.1 M citric acid, 2.57 ml 0.2 M phosphate, 5 ml H20 , and 0.015 ml 30% H202). Incubate for 30 rain in the dark at room temperature. 9. Stop the reaction with 2 M H2SO4,and read the results at 492 nm.
Anti-Urease-Anti-hCG Screening (bsMAb Activity). The bispecific antibody activities can be screened either by direct assay involving the binding of hCG directly to microtiter wells, or by sandwich assay, in which hCG is attached to the solid phase by capturing it with an antibody that is bound to the microtiter well. In general, we find that the sandwich assay works better for bsMAb screening. The anti-urease-anti-hCG bispecific monoclonal antibody activities in culture supernatants of hybrid hybridomas were assayed by simultaneous sandwich immunoassay using 96-well plates coated with affinity purified capture antibody. I. Coat polyvinyl plates with capture antibody (10 ltg/ml, either polyclonal or monoclonal antibody specific for the antigen, hCG) in 0.05 M carbonate buffer, pH 9.6. When monoclonal capture antibodies are used, they should not compete with the bsMAbs for the binding site of analyte. 2. Prior to use, block the plate with DB for 1 hr at room temperature. 3. Wash plates three times in WB. 4. Add an appropriate amount of antigen (50 mIU hCG) to each well in a 20-gl volume. Mix the plate by gently tapping the side of plate. Then add culture supernatants (70/zl/well) and 400 ng urease in 10 gl PBS. 5. Incubate the plates for 30 min at 37 °. 6. Wash the plates three times with WB, followed by three times with 0.85% NaC1. 7. Add 100 gl/well urease substrate solution and incubate for 30 min at room temperature prior to reading the reaction at 620 nm. 8. Repeat the assay on positive wells, and run control assays in parallel in which only culture supernatants and urease are added to the assay wells. If the sample contains functional bsMAb, the experimental wells should remain positive, while the control assays should be negative.
Production and Purification of Bispecific Monoclonal Antibodies
Production of bsMabs In Vitro Production. Large-scale culture of hybridomas has been described in detail in this series. ~4For the production of murine bsMAbs, it is more practical and economical to use an in vivo system. However, if the derivation of bsMAbs utilizes species other than mouse, in vitro large-scale
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production of bsMAbs might be the method of choice. Culture in vitro has the advantage of obtaining purer antibody preparations. Contamination of the harvested end product is further reduced by the use of serum-free medium for short cultures of 24-48 hr using a high cell density of 1-2 × 106 cells/ml. In Vivo Production. Procedures for the production of bsMAbs from ascitic tumors are similar to conventional hybridoma work? TM It is important to remember that the animals used, e.g., mice or rats, for ascites production must be histocompatible with the parent cells. If, for example, two strains of mice are involved, an F~ hybrid of the two strains should be used for in vivo propagation. Use of irradiated mice at 300-400 rad or nude mice may be considered for nonhistocompatible situations. However, such mice should be maintained in a germ-free environment. Processing Antibodies Produced. Antibodies obtained from in vitro propagation are very dilute. They can be concentrated by precipitation at 40-50% saturation with ammonium sulfate, dissolving the precipitates in a smaller volume, and dialyzing against PBS. Antibodies in ascitic fluid are already concentrated, along with contaminating proteins such as albumin. Albumin levels can be greatly reduced by (NH4)2SO 4 precipitation at 50% saturation at 4 °. Antibodies sensitive to (NH4)2SO 4 precipitation can be concentrated by dialysis against hygroscopic materials (e.g., PEG powder), by vacuum dialysis, or by filtration through selective Amicon (Danvers, MA) membranes. Partially purified or concentrated antibodies as described above are stored at 4 ° with preservatives such as NaN3 at 0.1%, or frozen at - 2 0 ° if a long period of storage is required prior to further purification by column chromatography. Affinity chromatography using various immobilized staphylococcal protein A matrices is a common method used to prepare purified antibody. Procedures have been described elsewhere ~3or are available from manufacturers [e.g., Pharmacia (Piscataway, NJ) and Bio-Rad (Richmond, CA)]. Protein A chromatography of ascites fluid or culture supernatant will provide a preparation containing both bsMAbs and parental MAbs free of contaminants. Antibody should be concentrated and dialyzed against appropriate buffer prior to use in immunoassay or further purification.
Purification of Bispecific Monoclonal Antibodies Functional bsMAbs may be purified by a number of techniques. Anion-exchange chromatography has been used successfully to prepare bsMAbs substantially free of contamination by parental antibodies when two different heavy-chain subclasses are present in the hybrid molecule.5
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This technique has been discussed earlier in this series. 12Others have used ion-exchange and hydroxylapatite chromatography with mixed results. ~5,16 Affinity chromatography may be used to purify bsMAbs when sufficient quantities of antigen are available to prepare affinity matrices. 1. Prepare antigen affinity columns. ~a 2. Ascitic fluid containing bsMAbs is diluted in 3 vol phosphate-buffered saline (PBS; 10 m M sodium phosphate, 150 m M sodium chloride, pH 7.2) and applied to the first antigen affinity column. 3. Wash the column with PBS and elute antibody with glycine-HC1 buffer (50 m M glycine, 150 m M sodium chloride, pH 2.3). 4. Concentrate eluate by ultrafiltration and dialyze against PBS. 5. Apply dialyzed eluate to the second antigen affinity column and repeat steps 3 and 4. Only bsMAbs can bind to both columns. Parental antibodies will bind only one column. When bsMAb is to be used to detect or quantitate an analyte by enzyme or other detector [e.g., detection of human chorionicgonadotropin (hCG) in an EIA by anti-hCG-anti-urease bsMAb], it may be necessary to eliminate only the anti-analyte parental antibody. Purification on only one antigen affinity column (that of the detector) will yield both bsMAb and one parental antibody (e.g., anti-urease), which should not interfere in detection of the analyte. In the case of urease-based EIAs, the size of the urease molecule (Mr 540,000) can be utilized to separate bsMAb by size exclusion chromatography. 1. Antibody from bsMAb-containing ascitic fluid is purified by protein A-chromatography. 13 2. Antibody (250/zg) is mixed with 800/zg urease in a total volume of 200/zl PBS for 10 min at room temperature. 3. This mixture is then loaded onto a 0.75 × 30 cm TSK G3000SW HPLC column (Varian, Lexington, MA). The sample is eluted at a flow rate of 1 ml/min using a mobile phase of 67 m M sodium phosphate, 300 m M sodium chloride, pH 7.0. 4. Fractions of 0.5 ml are collected and assayed for bsMAb activity as described earlier. Figure 2 shows a set of elution profiles of antibody plus urease, urease 15M. R. Clark and H. Waldman, J. Natl. Cancerlnst. 79, 1393 (1987). 16L. Karawajew, O. Behrsing, G. Kaiser, and B. Michel, J. Immunol. Methods 111, 95 (1988).
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[ 16] Design and Production of Bispecific Monoclonal Antibodies by Hybrid Hybridomas for Use in
Irnmunoassay B y MIYoKO TAKAHASHI, STF.VEN A. FULLER, and SCOTT WINSTON
Introduction The production of novel antibody reagents has become possible through advances in hybridoma techniques, recombinant DNA manipulation, and gene transfection technology. One such novel reagent is the bispecific antibody, a hybrid immunoglobulin molecule that is formed of nonidentical heavy- and light-chain pairs expressing different antigen specificities. The ability of bispecific antibodies to cross-link two different a n t i gens has led to applications in immunohistochemistry, 1 cell targeting, 2,3 complement-mediated cytotoxicity,4 and immunoassays. 5,6 In conventional use, monoclonal antibodies (MAb) are covalently coupled to various reagents (e.g., enzymes, fluorochromes, radioisotopes, dyes, and toxins). Enzyme immunoassays (EIA) utilize MAb cross-linked to any of several marker enzymes. Such chemical modification of both antibody and enzyme has several limitations, including (1) an a n t i b o d y - e n z y m e mixture that includes both active and inactive species in varying proportions, (2) a preparation that contains both antibody and enzyme coupled to contaminants as well as to each other, (3) loss of antibody function, and (4) a n t i b o d y - e n z y m e conjugates that are often less stable than the unmodified components. Bispecific monoclonal antibodies (bsMAb), developed originally by Milstein and Cuello (1983), 1 can circumvent these problems by avoiding entirely the chemical modification. Antibodies with bifunctional specificities have been generated by a number of methods: (1) cross-linking of heterologous MAb, 7,s (2) reassociation of monovalent antibody fragments, 9- H and (3) cell fusion of hybrid1C. Milsteinand A. C. Cuello,Nature (London) 305, 537 (1983). 2 U. D. Staerz and M. J. Bevan, Proc. Natl. Acad. Sci. U.S.A. 83, 1453 (1986). 3A. Lanzavecchiaand D. Seheidegger,Eur. J. Immunol. 17, 105 (1987). 4 j. T. Wong and R. B. Colvin,J. Immunol. 139, 1369 (1987). 5M. R. Suresh, A. C. Cuello,and C. Milstein,Proc. Natl. Acad. Sci. U.S.A. 83, 7989 (1986). 6 M. Takahashiand S. A. Fuller, Clin. Chem. 34, 1693 (1988). 7U. D. Staerz, O. Kanagawa,and M. J. Bevan,Nature (London) 314, 628 (1985). s p. Perez, R. W. Hoffman,S. Shaw,J. A. Bluestone,and D. M. Segal,Nature (London) 316, 354 (1985). 9A. Nisonoffand W. J. Mandy, Nature (London) 194, 355 (1962). 1oV. Raso and T. Griffin,CancerRes. 41, 2073 (1981). H M. Brennan, P. F. Davison, and H. Paulus, Science 229, 81 (1985). METHODS IN ENZYMOLOGY, VOL. 203
~ t © 1991 by Academlc Pre~ Inc. All rlghts of ~ u c f i o n in any form re~rved.
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omas or sensitized B lymphocytes.~ The cell fusion approach has several advantages. Hybrid hybridomas form a high percentage of hybrid antibody molecules. The ease of the procedure allows any experienced cell culturist to perform the work. In addition, bsMAbs that are biologically produced by hybrid hybridomas often exhibit no observable changes in specificity or physical characteristics after prolonged storage at 4" (M. Takahashi, S. A. Fuller, and S. Winston, unpublished data, 1988). Our research interests lie in the development of EIAs for a wide variety of analytes. In this chapter we describe methods we have utilized or developed to prepare a panel of fusion partners that simplifies production of bsMAbs, the preparation of hybrid hybridomas, and the production and use of bsMAbs. While the focus of our work is urease-based EIAs, these methods should be readily applicable to other enzyme/detector systems. A detailed report concerning the production of bsMAbs by hybrid hybridomas (theory, materials, and methods) was published in an earlier volume in this series.~2
Hybrid Hybridoma Production In our attempts to utilize bsMAbs in urease-based EIAs, we wished to set up a simple and flexible system that might be useful for any analyte or MAb of interest. Consequently, we have prepared a panel of anti-urease fusion partners that represent the major murine IgG subclasses and that possess two selectable markers. This panel has the following advantages: I. bsMAbs of most homologous or heterologous heavy-chain subclasses can be readily prepared, which provides flexibility in stability, function, or purpose. 2. The hybridoma to be fused need not be modified, since the selectable markers of the anti-urease fusion panner allow both parental lines to be eliminated. Figure 1 is a schematic representation of the processes involved in producing hybrid hybridomas. Each of these is described in the methods that follow. Isolation of Hybridoma Lines for Use as Fusion Partners A panel of anti-urease-producing hybridoma lines was established from a 42% (w/v) polyethylene glycol (PEG) (Mr 4000; Merck, Darmstadt, Germany) fusion of Sp2/0 cells and splenocytes isolated from BALB/c ~2M. R, Suresh, A. C. Cuello, and C. Milstein, this series, Vol. 121, p. 210.
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Anti-Urease Hybridoma 8-Azaguanine Ouabain Anti-Ureose Hybridoma
(HAT Sensitive) (Ouabain Resistant)
+
Anti-hCG Hybridoma (HAT Resistant) (Ouabain Sensitive) Fusion Selection (HAT, Ouabain) Cloning
Hybrid Hybridoma (Anti-Urease and Anti-hCG) FIG. 1. How diagram for preparation of hybrid hybridomas. HAT, Hypoxanthine/aminopterin/thymidine medium; hCG, human chorionic gonadotropin.
mice immunized with jackbean urease.6 In order to use these anti-ureaseproducing hybridoma lines as universal fusion partners with either immunocytes (lymphocytes) or other hybridomas, double-mutant fines resistant to both 8-azaguanine and ouabain were isolated. Procedure to Isolate 8-Azaguanine-Resistant Hybridomas. In hybridoma technology, the most common selection method is the use of a parental cell line that is sensitive to hypoxanthine/aminopterin/thymidine (HAT) medium. Is,14 Aminopterin blocks de nova purine and pyrimidine synthesis by inhibition of the enzyme dihydrofolate reductase. However, if present, the enzymes hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thymidine kinase can utilize hypoxanthine and thymidine, respectively, via salvage pathways. Animal cell lines deficient for HGPRT (HAT sensitive) can be isolated by adaptive growth and selection in 8-azaguanine (8-AG). This antimetabolite is cytotoxic to normal cells and only HGPRT- cells can survive. Selection of 8-AG-resistant variants involves adaptive growth in successively increasing concentrations of the drug (to 20/~g/ml). 1. Prepare a 2 mg/ml stock solution of 8-AG. Weigh 200 mg of the purine analog and add to 90 ml distilled water. Add 1 N NaOH dropwise ,3 S. A. Fuller, M. Takahashi, and J. G. R. HurreU, in "Current Protocols in Molecular Biology" (F. Ausubel, B. Brent, R. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, ¢¢ls.),Unit 11. Greene Publishing, New York, 1987. 14G. Galfr~ and C. Milstein, this series, Vol. 73, p. 3.
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until dissolved. Add distilled water to 100 ml. Filter sterilize the solution and then store frozen at - 20 ° in convenient aliquots. 2. Prepare six wells (six-well cluster dish; Costar, Cambridge, MA) containing actively growing hybridomas (3 X l05 cells/ml, anti-urease cell lines in this case) in 10 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS). Add the 8-AG stock solution to a final concentration of 1, 2, and 4 ]tg/ml, using two wells for each of the concentrations (we have found that different murine hybridoma lines show varied sensitivity to the drug). Grow cells in an incubator under 8O/oCO2. 3. Make a 50% medium change every 2 days using growth medium containing appropriate amounts of 8-AG. 4. Select the culture growing at the highest drug concentration. Double the drug concentration and continue to culture for several generations. 5. Repeat step 4 until a viable cell population growing in the presence of 20/~g/ml 8-AG is obtained. 6. When cells are adapted to the 20/tg/ml drug concentration (approximately 3 weeks), clone by the limiting dilution method ~3 using medium containing 20 #g/ml 8-AG. 7. When the clones are confluent, select 10 wells containing a single colony and expand them in duplicate in a 24-well plate (Costar). Culture one set in HAT selection medium 13,14and discard the clones that survive. 8. Screen the remaining clones that are 8-AG resistant and HAT sensitive for antibody production (anti-urease). 9. Select clones with good growth and antibody production. Freeze aliquots for safe keeping. The remaining cells are subjected to another round of adaptive growth in the presence of ouabain.
Procedurefor Isolation of Ouabain-Resistant Hybridomas. Ouabain is a plant glycoside that inhibits membrane-bound Na+,K+ - ATPase. Ouabain resistance is an additional property useful for the selection of desirable fusion products. An 8-AG-resistant hybridoma line that is also ouabain resistant can be fused with any hybridoma line. The fusion products are selectively cultured in medium containing HAT and ouabain. In this medium, the double-mutant parental cells do not survive because of their deficiency for HGPRT. The other parental hybridoma will die due to its lack of resistance to ouabain, and only the hybrid hybridomas will survive. The procedure used to isolate ouabain-resistant hybridomas is similar to that described in the previous section for azaguanine. 1. Prepare 50 m M ouabain (Sigma, St. Louis, MO) stock solution in distilled water. Dissolve by heating to 70 ° . Mix with an equal
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2.
3.
4.
5.
6. 7.
8. 9. 10.
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volume of 2× DMEM (room temperature), filter sterilize, and store frozen at - 2 0 ° in convenient aliquots. In a 75-cm2 tissue culture flask, grow the 8-AG-resistant hybridoma line to a cell density of approximately 4 × 105 cells/ml in 30 ml medium with 15% FCS. Add the ouabain stock solution to a final concentration of 0.1 mM. Set up a small control flask and grow the cells in the same medium without the drug, in order to ensure that the cell growth retardation is due to the drug. Make a 50% medium change by removing the spent medium and supplementing with fresh medium or by merely doubling the medium as required. Allow cells to grow for several generations in medium containing 0.1 m M ouabain, then double the culture volume with fresh medium containing 0.2 m M ouabain. When the culture has reached a cell density of 4 - 6 X 105 cells/ml, make an appropriate split with medium containing 0.2 m M ouabain. Repeat step 4, by doubling the culture volume with medium containing 0.4 m M ouabain. It is very important to increase the ouabain level gradually. In comparison to human cell lines, rodent cell lines have been reported to be far more resistant to ouabain. All of the nonadapted murine hybridoma lines are very sensitive to the drug at around 0.2-0.4 mM. Continue the process and gradually increase the ouabain level up to lmM. When the cells are well adapted to 1 m M ouabain, add 20 #g/ml 8-AG to the medium supplemented with 15% (v/v) FCS. Allow them to grow for several generations in the presence of the two drugs. Clone by the limiting dilution method ~ in medium containing 1 m M ouabain and 20 gg/ml 8-AG, supplemented with 20% FCS. Screen only those wells with a single colony for antibody activity. Choose the clone(s) with the best growth characteristics and the best production of antibody.
Using the procedures described above, we have isolated numerous 8-AG/ouabain-resistant hybridoma lines that produce anti-urease of IgM or IgG (subtypes 1, 2a, and 2b) class. Each of these lines were continuously cultured for at least 8 weeks in DMEM containing 10% FCS, after which all lines still demonstrated resistance to 20/tg/ml 8-AG and 1 m M ouabain and were sensitive to HAT medium. Production of anti-urease in the variant lines was measured by enzyme-linked immunosorbent assay (ELISA) following continuous culture. Despite the stability of drug resist-
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ance characteristics, some hybridomas expressed reduced antibody production relative to wild type. Cell doubling times of the variant lines in the presence and absence of azaguanine and ouabain were as reported previously.6 Cell Fusions and Cloning Two cell fusion procedures used to obtain hybrid hybridomas are described in the following sections. Most reagents and procedures are as for conventional hybridoma workJ TM Differences exist in the use of selection medium, the fusion cell ratio for hybridoma × hybridoma fusions, and screening for bispecific antibody activity. The latter is discussed separately. Hybridoma × Splenocyte Cell Fusions. Fusions between the 8-AGresistant hybridomas that produce anti-urease and splenocytes obtained from an immunized mouse, or splenocytes immunized in vitro, are basically as for Sp2/0 × splenocyte fusions: The ratio of splenocytes to hybridoma cells is 5 : 1. The polyethylene glycol (PEG)-mediated fusion products are resuspended in HAT selection medium and incubated for 1 - 3 hr in a CO2 incubator. The cells are then plated out at 1- 2 × 105 cells/well on 96-well Costar plates that were preseeded with mouse peritoneal exudate cells (PEC) as feeders in HAT selection medium. Hybrid hybridomas are generally screened 2 - 3 weeks postfusion. Selected positive cultures are then subjected to cloning by the limiting dilution method. We have successfully established hybrid hybridomas that produce bsMAb by fusions of anti-urease producing hybridomas with splenocytes sensitized in vitro with human luteinizing hormone as well as with splenocytes obtained from BALB/c mice immunized with various immunogens, including human chorionicgonadotropin (hCG). The limitation of this approach, i.e., fusions of hybridoma× splenocytes, is that the isolation of hybrid hybridomas that produce bsMAbs of desired specificity and affinity depends on chance. Hybridoma × Hybridoma Cell Fusions. We describe here the protocol used for the fusion experiments between anti-urease hybridomas and hybridomas secreting anti-hCG (schematically shown in Fig. 1). 1. From each of the two hybridoma lines to be fused, harvest 4 × 107 cells from a logarithmically growing culture. 2. Centrifuge both hybridoma tines. Pool the cell pellets, and wash three times in warm DMEM. 3. Subject the final cell pellet (which is a 1 : 1 cell mixture of the two parental hybridomas) to PEG cell fusion. '3 4. Resuspend the fused cells in 40 ml DMEM supplemented with 20%
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5.
6.
7.
8. 9. 10.
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FCS, 2× HAT, and 2 m M ouabain. Incubate for 30 min in a CO2 incubator prior to plating out at 2 X 105 cells/0.1 ml into each well on 96-well Costar plates. The plates should be preseeded with 0.1 ml/well mouse PEC in DMEM with 20% FCS, and without HAT/ ouabain. Prepare control cultures as in the experimental plates for both parental hybridomas using 10 wells each. PEC feeder cells are preseeded and each hybridoma line is plated at 2 X 105 cells/well. In the HAT/ouabain selection medium, the control anti-urease hybridomas should not survive because of their sensitivity to HAT, while anti-hCG hybridomas are killed due to ouabain sensitivity. Leave plates undisturbed in the CO2 incubator for 1 week, except for a brief check for microbial contamination a few days after the fusion. One week postfusion, examine the control cultures carefully. When both parental cells are dead in the HAT/ouabain medium, start feeding the experimental plates with DMEM supplemented with 20% FCS and HT (HAT medium from which aminopterin is omitted) and ouabain. Feed cultures as required until the hybrid hybridomas become confluent and the culture medium becomes acidic (yellow in appearance). Screen the culture supernatants for parental and bispecific antibody activities, as described in the following sections. Select cultures indicating bispecific antibody activity, and clone by the limiting dilution method. When the hybrid hybridomas are established and cloned, freeze a number of vials and confirm a good recovery from the liquid nitrogen freezer.
The procedure described above to prepare bispecific antibody-producing hybridomas by the fusion between two hybridoma lines has the advantage that the characteristics of antibody specificity and affinity as well as the heavy-chain isotypes of immunoglobulins produced by each of the parental hybridomas are already known. This allows for an easy selection of clones that meet the specific criteria desired. Our initial bsMAb work6 indicated that fusion ofIgM, IgG~, IgG2~, and IgG2b anti-urease hybridomas with an IgG~ anti-hCG hybridoma resulted in efficient production of bispeeific monoclonal antibodies only for the homologous IgG1 X IgG~ fusions. Based on this observation, we have performed extensive fusion experiments to evaluate the outcome of homologous as well as heterologous heavy-chain combinations using our expanded panel of anti-urease variant hybridoma cell lines. Table I presents the
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TABLE I PRODUCTION OF BISPECIFIC ANTIBODY FROM FUSIONS BETWEEN HYBRIDOMAS PRODUCING HOMOLOGOUS AS WELL AS HETEROLOGOUS IMMUNOGLOBULIN SUBCLASSESOF IGG Percentage of positive well~ Parental antibodies
Parental hybridoma fines*
Anti-urease
Anti-hCG
3U66 r X 2G131 7U114 r × 6G94 7U215 • X IG151
96 100 100
57 17 52
100 97 100
53 99 37
bsMAb
Number of wells screened
Homologous fusions IgGl-IgGi IgG~.- IgG2~ IgG2b- IgG2b Heterlogous fusions
IgGl-IgGz~ IgGl-IgG2b IgGi-IgGz,
3U66 r × 6G94 2G131 × 6U334 r 3U66 r X IGl51
57 17 49 6.9 2.2 5.5
130 180 180 160 180 110
a Superscript r designates the anti-urease-producing variant hybridoma fines. b Calculated as (number of positive wells/number of wells screened) 100.
results obtained from cell fusions between anti-urease and anti-hCG hybridomas of homologous or heterologous heavy-chain subclass. In fusion experiments between homologous heavy chains of IgGl, IgG~, and IgG2b subclasses, bispecific antibody-positive wells were identified in 17-57% of wells screened. Compared to the homologous fusion results, the fusions between heterologous subclasses of IgG yielded only 2.2-6.9% positive wells for bispecific antibody activity. Moreover, use of IgM or IgG3 hybridomas yielded no or only a few positive wells regardless of homologous or heterologous fusions. It is important to note that isolation of bsMAbs composed of heterologous heavy chains is possible despite the lower efficiency compared to homologous fusions.
Antibody Screening of Culture Supernatants of Hybrid Hybridomas Enzyme immunoassays using 96-well microtiter plates are routinely used in our laboratory for the screening of culture supernatants for bsMAb activity as well as monospecific MAb activities of parental cells.
Materials Polyvinyl or polystyrene 96-well microtiter plates Coating buffer: 0.05 M carbonate buffer, pH 9.6 TEN buffer: 0.05 M Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.15 MNaC1, pH 7.3 Wash buffer (WB): Phosphate-buffered saline (PBS), pH 7.2, with 0.05% Tween 20
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Diluting buffer (DB): PBS, pH 7.2, with 0.05% Tween 20 and 0.25% bovine serum albumin (BSA) hCG (Sigma) Urease and urease substrate solution (ADI Diagnostics, Inc., Rexdale, Canada) o-Phenylenediamine (OPD) (Sigma) Peroxidase-conjugated goat anti-mouse IgG (heavy and light chain) (Cappel Worthington, Malvern, PA) Urease-conjugated rabbit anti-mouse F(ab')2 ofIgG (ADI Diagnostics)
Methods Anti-hCG Screening 1. Coat 96-well polyvinyl microtiter plates (Dynatech, Chantilly, VA) with 100/tl/well of hCG (5 IU/ml) in 0.05 M carbonate buffer. Seal the plates and store at 4" for at least 24 hr before use. 2. Remove antigen solution and fill all wells with DB to block additional protein binding sites for 1 hr at room temperature. 3. Wash the plates three times in WB. 4. Add hybrid hybridoma culture supernatants (100/d/well) into the plates and incubate for 60-90 rain at 37 °. 5. Wash plates three times in WB. 6. Add 100 /d/well of urease-conjugated rabbit anti-mouse F(ab')z fragment of IgG. Seal the plates and incubate for 30 rain at 37*. 7. Wash the plates three times with WB, then three times with 0.85% NaC1. 8. Add 100/~l/well urease substrate solution, and after a 10- to 20-rain incubation at room temperature, read the plates at 590 nm.
Anti-Urease Screening. The procedure is the same as for anti-hCG screening with minor modifications. The marker enzyme conjugated to the detector antibody must be one other than urease. 1. Coat polyvinyl plates with urease (10/tg/ml) in TEN buffer. Seal the plates and store at 4* until use. 2. Block plates for 1 hr at room temperature. 3. Wash three times in WB. 4. Add culture supernatants to the plate, and incubate for 60-90 min at 37 ° . 5. Wash plates three times. 6. Add peroxidase-conjugated goat anti-mouse IgG (heavy and light chains) and incubate for 30 min at 37 °. 7. Wash plates four times with WB.
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8. Add OPD in phosphate-citrate buffer (2.43 ml 0.1 M citric acid, 2.57 ml 0.2 M phosphate, 5 ml H20 , and 0.015 ml 30% H202). Incubate for 30 rain in the dark at room temperature. 9. Stop the reaction with 2 M H2SO4,and read the results at 492 nm.
Anti-Urease-Anti-hCG Screening (bsMAb Activity). The bispecific antibody activities can be screened either by direct assay involving the binding of hCG directly to microtiter wells, or by sandwich assay, in which hCG is attached to the solid phase by capturing it with an antibody that is bound to the microtiter well. In general, we find that the sandwich assay works better for bsMAb screening. The anti-urease-anti-hCG bispecific monoclonal antibody activities in culture supernatants of hybrid hybridomas were assayed by simultaneous sandwich immunoassay using 96-well plates coated with affinity purified capture antibody. I. Coat polyvinyl plates with capture antibody (10 ltg/ml, either polyclonal or monoclonal antibody specific for the antigen, hCG) in 0.05 M carbonate buffer, pH 9.6. When monoclonal capture antibodies are used, they should not compete with the bsMAbs for the binding site of analyte. 2. Prior to use, block the plate with DB for 1 hr at room temperature. 3. Wash plates three times in WB. 4. Add an appropriate amount of antigen (50 mIU hCG) to each well in a 20-gl volume. Mix the plate by gently tapping the side of plate. Then add culture supernatants (70/zl/well) and 400 ng urease in 10 gl PBS. 5. Incubate the plates for 30 min at 37 °. 6. Wash the plates three times with WB, followed by three times with 0.85% NaC1. 7. Add 100 gl/well urease substrate solution and incubate for 30 min at room temperature prior to reading the reaction at 620 nm. 8. Repeat the assay on positive wells, and run control assays in parallel in which only culture supernatants and urease are added to the assay wells. If the sample contains functional bsMAb, the experimental wells should remain positive, while the control assays should be negative.
Production and Purification of Bispecific Monoclonal Antibodies
Production of bsMabs In Vitro Production. Large-scale culture of hybridomas has been described in detail in this series. ~4For the production of murine bsMAbs, it is more practical and economical to use an in vivo system. However, if the derivation of bsMAbs utilizes species other than mouse, in vitro large-scale
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production of bsMAbs might be the method of choice. Culture in vitro has the advantage of obtaining purer antibody preparations. Contamination of the harvested end product is further reduced by the use of serum-free medium for short cultures of 24-48 hr using a high cell density of 1-2 × 106 cells/ml. In Vivo Production. Procedures for the production of bsMAbs from ascitic tumors are similar to conventional hybridoma work? TM It is important to remember that the animals used, e.g., mice or rats, for ascites production must be histocompatible with the parent cells. If, for example, two strains of mice are involved, an F~ hybrid of the two strains should be used for in vivo propagation. Use of irradiated mice at 300-400 rad or nude mice may be considered for nonhistocompatible situations. However, such mice should be maintained in a germ-free environment. Processing Antibodies Produced. Antibodies obtained from in vitro propagation are very dilute. They can be concentrated by precipitation at 40-50% saturation with ammonium sulfate, dissolving the precipitates in a smaller volume, and dialyzing against PBS. Antibodies in ascitic fluid are already concentrated, along with contaminating proteins such as albumin. Albumin levels can be greatly reduced by (NH4)2SO 4 precipitation at 50% saturation at 4 °. Antibodies sensitive to (NH4)2SO 4 precipitation can be concentrated by dialysis against hygroscopic materials (e.g., PEG powder), by vacuum dialysis, or by filtration through selective Amicon (Danvers, MA) membranes. Partially purified or concentrated antibodies as described above are stored at 4 ° with preservatives such as NaN3 at 0.1%, or frozen at - 2 0 ° if a long period of storage is required prior to further purification by column chromatography. Affinity chromatography using various immobilized staphylococcal protein A matrices is a common method used to prepare purified antibody. Procedures have been described elsewhere ~3or are available from manufacturers [e.g., Pharmacia (Piscataway, NJ) and Bio-Rad (Richmond, CA)]. Protein A chromatography of ascites fluid or culture supernatant will provide a preparation containing both bsMAbs and parental MAbs free of contaminants. Antibody should be concentrated and dialyzed against appropriate buffer prior to use in immunoassay or further purification.
Purification of Bispecific Monoclonal Antibodies Functional bsMAbs may be purified by a number of techniques. Anion-exchange chromatography has been used successfully to prepare bsMAbs substantially free of contamination by parental antibodies when two different heavy-chain subclasses are present in the hybrid molecule.5
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This technique has been discussed earlier in this series. 12Others have used ion-exchange and hydroxylapatite chromatography with mixed results. ~5,16 Affinity chromatography may be used to purify bsMAbs when sufficient quantities of antigen are available to prepare affinity matrices. 1. Prepare antigen affinity columns. ~a 2. Ascitic fluid containing bsMAbs is diluted in 3 vol phosphate-buffered saline (PBS; 10 m M sodium phosphate, 150 m M sodium chloride, pH 7.2) and applied to the first antigen affinity column. 3. Wash the column with PBS and elute antibody with glycine-HC1 buffer (50 m M glycine, 150 m M sodium chloride, pH 2.3). 4. Concentrate eluate by ultrafiltration and dialyze against PBS. 5. Apply dialyzed eluate to the second antigen affinity column and repeat steps 3 and 4. Only bsMAbs can bind to both columns. Parental antibodies will bind only one column. When bsMAb is to be used to detect or quantitate an analyte by enzyme or other detector [e.g., detection of human chorionicgonadotropin (hCG) in an EIA by anti-hCG-anti-urease bsMAb], it may be necessary to eliminate only the anti-analyte parental antibody. Purification on only one antigen affinity column (that of the detector) will yield both bsMAb and one parental antibody (e.g., anti-urease), which should not interfere in detection of the analyte. In the case of urease-based EIAs, the size of the urease molecule (Mr 540,000) can be utilized to separate bsMAb by size exclusion chromatography. 1. Antibody from bsMAb-containing ascitic fluid is purified by protein A-chromatography. 13 2. Antibody (250/zg) is mixed with 800/zg urease in a total volume of 200/zl PBS for 10 min at room temperature. 3. This mixture is then loaded onto a 0.75 × 30 cm TSK G3000SW HPLC column (Varian, Lexington, MA). The sample is eluted at a flow rate of 1 ml/min using a mobile phase of 67 m M sodium phosphate, 300 m M sodium chloride, pH 7.0. 4. Fractions of 0.5 ml are collected and assayed for bsMAb activity as described earlier. Figure 2 shows a set of elution profiles of antibody plus urease, urease 15M. R. Clark and H. Waldman, J. Natl. Cancerlnst. 79, 1393 (1987). 16L. Karawajew, O. Behrsing, G. Kaiser, and B. Michel, J. Immunol. Methods 111, 95 (1988).
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[ 1 6]
O
,
