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surface of tight epithelia is recognized by the anti-a subunit in the distal colon and the urinary bladder, but not in jejunal cells. In contrast, the sodium pump is expressed on the basolateral cell surface of all three types of epithelia. The sodium channel and the a subunit of the sodium pump share an epitope that is recognized by the polyclonal antibody) 2 Conclusion Using the streptavidin-biotin bridge system, the number of steps required for the purification of rare cellular proteins is reduced and their tissue localization is improved. Most reagents are now commercially available, which greatly facilitates the use of the system. Acknowledgments We wish to thank Dr. Michel Aubert from the Department of Pediatrics of the University of Geneva who performed the mathematical analysis of the binding studies. We are grateful to Dr. Jean Wilson who kindly revised the manuscript and to Mine. Cesco who typed it. This work was supported by grants from the Swiss National Science Foundation (3.398-0.86) and from Amersham International, plc, Amersham, England.

[76] F a c i l i t a t e d C e l l F u s i o n for H y b r i d o m a P r o d u c t i o n

By MARY K. CONRAD and MATHEW M. S. Lo For many years hybridomas have been produced for a variety of purposes by fusion of two cell types in the presence of viruses I or polyethylene glycol (PEG). 2-3 There are, however, disadvantages to the method of chemical fusion. One is the fact that the fusion between cells is a random event, causing problems when cell populations cannot be specifically selected in advance. As a result, large numbers of growing hybridoma colonies need to be screened in order to find the usually rare colonies of interest arising from the fusion. More recently, attention has been given to the use of intense, shortduration electric pulses to achieve cell-cell fusion. *-6 Cell fusion using i G. Kohler and C. Milstein, Nature (London) 256, 495 (1975). 2 G. Galfre, S. C. Howe, and C. Milstein, Nature (London) 266, 550 (1977). 3 G. Galfr~ and C. Milstein, this series, Vol. 73, p. 3. 4 E. Neumann, G. Gerisch, and K. Opatz, Naturwissenschafien 67, 414 (1980). T. Y. Tsong, Biosci, Rep. 3, 487 (1983). 6 U. Zimmermann, J. Vienken, J. Halfmann, and C. C. Emeis, Adv. Biotechnol. Processes 4, 79 (1985).

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.

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this m e t h o d is usually 10-100 times more efficient than PEG-mediated fusion. 7 Also of advantage is the fact that exogenous chemicals need not be introduced into the cell suspensions to be fused. The key to successful fusion is cell-cell contact 8 upon application of the electric field. However, the same disadvantages that are found in P E G fusions also apply to this method and are, in fact, multiplied many times over because of the enhanced fusion efficiency. Therefore, unless specific cells of interest can be stringently isolated in advance, potentially millions of randomly fused hybridomas would need to be screened for the rare one of interest. When cell populations can be isolated in advance, there are a number of techniques that can be used to bring cells into contact prior to electrofusion. 6,9-~2 In order to benefit from the advantages of electrofusion, a method is required for selectively achieving contact between cells of interest within a bulk suspension of mixed cell types. Previously it has been reported that fusion between B cells and antigen-coated myeloma cells can be p r o m o t e d by such targeting techniques alone, without the use of f u s o g e n s ) 3 This method, however, has never been in widespread use, suggesting difficulties in its practical application. The technique illustrates the fact, though, that B cells possess convenient surface antibodies directed against one specific antigen, the use of which can facilitate targeted preselection in cell suspensions. Targeting We have used the high-affinity interaction between antigen and surface immunoglobulin receptors on B cells in conjunction with the tight binding b e t w e e n avidin and biotin to achieve specific preselected cell-cell contact in suspensions. The procedures we developed result in high-affinity monoclonal antibody production from hybrids formed during electrofusion.74 To accomplish this, myeloma cells are coated with either biotin or avidin, and the antigen of interest is conjugated to the affinity counterpart (Fig. 1). A mixture of these with spleefi cells, followed by electrofu7 u. Karsten, (3. Papsdorf, G. Roloff, P. Stolley, H. Abel, I. Walther, and H. Weiss, Eur. J. Cancer Clin. Oncol. 21, 733 (1985). 8 A. Sowers, J. Cell Biol~ 102, 1358 (1986). 9 H. A. Pohl, "Dielectrophoresis." Cambridge Univ. Press, Cambridge, 1978. 10j. Vienken and U. Zimmermann, FEBS Lett. 13/, II (1982). i1 R. Bischoff, R. M. Eisert, I. Schedel, J. Vienken, and U. Zimmermann, FEBS Lett. 147, 64 (1982). t2 M. K. Conrad, M. M. S. Lo, T. Y. Tsong, and S. H. Snyder, in "Cell Fusion" (A. Sowers, ed.), p. 427. Plenum, New York, 1987. t3 R. B. Bankert, C. DesSoye, and L. Power, Transplant. Proc. 12, 443 (1980). t4 M. M. S. Lo, T. Y. Tsong, M. K. Conrad, S. M. Strittmatter, L. H. Hester, and S. H. Snyder, Nature (London) 310, 792 (1984).

[76]

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FIG. 1. Basic protocol for bioselective cell-cell contact and electrofusion to form monoclonal antibody-secreting hybridomas. Myeloma and spleen cells are prepared as described in the text. Following coincubation, electrofusion is performed on the bulk cell suspension containing adherent pairs of interest. Resulting hybridomas are screened, and those producing the desired antibodies are cloned. See text for details. M, Myeloma cell; B, B-type spleen cell; H, hybridoma cell; Av-Ag, avidin-antigen conjugate. (From Ref. 12.)

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4000-

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FIG. 2. Radioimmunoassays for (A) initial screening for antiangiotensin-converting enzyme (ACE) antibody and (B) affinity determinations for two monoclonals. (A) Two fusions using spleen cells of mice immunized with ACE resulted in 31 and 11 growing colonies, respectively. Supernatants from the colonies were screened with a radioimmunoassay.14 All colonies produced anti-ACE antibody titers. Bars a-d are 3,000-, 10,000-, 30,000-, and 100,000-fold dilutions of a goat polyclonal antiserum raised against ACE. Bar g is a mouse IgG2a monoclonal antibody raised against Russell viper venom. (B) Known quantities of monoclonal antibodies were serially diluted for incubation with 125I-labeled ACE in the radioimmunoassay. Results for two monoclonals are shown. The affinity is equal to the concentration of antibody that binds 50% of added antigen. Affinities (Ko) ranged between 1 and 0.1 nM. (Figure 2A was originally published in Ref. 14.)

sion, r e s u l t s in r e l a t i v e l y f e w g r o w i n g c o l o n i e s , a v e r y high p r o p o r t i o n o f which secrete high-affinity antibodies directed against the antigen used (Fig. 2). U n i n t e r e s t i n g h y b r i d o m a s a r e r a r e l y p r o d u c e d , t h u s s a v i n g res o u r c e s in t h e s c r e e n i n g s t e p s .

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Construction of Avidin-Antigen Conjugates The chemical conjugation of antigen to avidin is complex, and a variety of chemical cross-linking procedures (similar to those used to prepare enzyme-antibody or enzyme-macromolecule conjugates) may be employed, j5 Conjugation to avidin is preferred over conjugation to biotin because of the relative ease of characterizing the avidinylated conjugate and the greater efficiency of biotinylating myeloma cells. The essential steps in preparing avidin-antigen conjugates consist of chemical activation of avidin (or antigen) and subsequent reaction with antigen (or avidin), followed by purification of the conjugate from other reaction products by chromatographic separation techniques based on either size or charge differences. Cross-linking reactions may be performed in solution or in the solid phase by immobilization of avidin onto iminobiotin-Sepharose. We have previously described the use of a small homobifunctional cross-linker 1,5-difluoro-2,4-dinitrobenzene ( D F D N B ) 16'17 in a solid-phase procedure. ~4 In this procedure, avidin, bound to iminobiotin-Sepharose, is reacted with a large excess of DFDNB, and the resin is washed to remove unreacted reagent. As a consequence, the immobilized avidin is chemically activated, and subsequent addition of proteins or peptides results in their covalent conjugation to avidin through free amino groups. Preparation oflminobiotin-Sepharose. Ethylenediamine is combined with CNBr-activated Sepharose CL-4B, j8 and the product is allowed to react subsequently with the N-hydroxysuccinimide derivative of iminobiotin.19.20 The free (unreacted) amines of the iminobiotin-Sepharose preparation are blocked by reaction with Sanger's reagent (2,4-dinitrofluorobenzene). Typically, the iminobiotin concentration is about 10-30 /zmol/ml of packed Sepharose resin. Preparation of Avidin-Conjugated Antigen. Avidin (70 /zg) is incubated with iminobiotin-Sepharose (0.1 ml packed resin) in a solution (0.5 ml) consisting of 0.1 M sodium borate buffer (pH 10.5). The incubation and subsequent reaction are carried out in a small 1-ml column. After a 1hr incubation period at 23°, the resin is washed with 2 ml of sodium borate buffer and reacted for 30 min at 23° with a solution (0.5 ml) containing 2 t5 p. Tijssen, "Practice and Theory of Enzyme Immunoassays," Vol. 26. Elsevier, New York, 1985. 16 H. S. Tager, Anal. Biochem. 71, 367 (1976). 17 E, E. Golds and P. E. Braun, J. Biol. Chem. 253, 8162 (1978). ~8 p. Cuatrecasas and I. Parikh, Biochemistry 11, 2291 (1972). ~9 K. Hoffmann, S. W. Wood, C. C. Brinton, J. M. Montibeller, and F. M. Finn, Proc. Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 20 G. A. Orr, J. Biol. Chem. 256, 761 (1981); See also B. F. Goldin and G, A. Orr, this volume [17].

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m M DFDNB in 0.1 M sodium borate buffer (pH 8.5). The resin is washed with 5 ml of sodium borate buffer (pH 10.5) to remove unreacted reagent. Antigen (0. I nmol) is dissolved in sodium borate buffer (pH 10.5) and added to the resin now containing the chemically activated avidin. These are reacted for 18 hr at 23 °. The resin is washed with 2 ml of sodium borate buffer (pH 10.5) and 1 ml sodium borate buffer containing 1 m M lysine or glycine to block unreacted DFDNB groups. The avidin-antigen conjugate is eluted with 0.1 M citrate buffer (pH 3.5). A carrier protein such as bovine serum albumin (BSA) is usually added to the conjugate, which is then desalted by gel filtration on a Sephadex G-25 column and eluted with phosphate-buffered saline (PBS). Comments. This method of conjugation is particularly effective with large proteins where modification of basic residues does not drastically affect the antigenicity of the protein of interest. However, smaller peptides or acidic proteins could become insoluble when conjugated in this manner. Smaller proteins or peptides can be derivatized with DFDNB and conjugated to avidin in solution.16 The molar ratio of the peptide to avidin should be reduced to less than 5 mol of peptide per mole of avidin to prevent formation of insoluble conjugate. The coupling efficiency of this method is very high. However, any amino groups present on the peptide will react with DFDNB and may form very large conjugated complexes, severely altering the antigenicity of the peptide. We have also used a heterobifunctional cross-linker, N-maleimidobenzoyl-N-hydroxysuccinimide (MBS). 15,21,22The advantage of using MBS is that conjugation occurs through sulfhydryl groups on the antigen. We have found this to be less detrimental to the antigenicity of proteins and peptides. Also, MBS reacts with lysine residues of avidin, which causes a beneficial reduction in the isoelectric point of avidin-MBS to a more neutral pH value. In contrast, native avidin is very basic and usually binds nonspecifically even mildly acidic proteins, such as those on cell surface membranes. Avidin is first reacted with MBS at a 5-fold molar excess of reagent for 1 hr at 23 °. Free reagent is removed by gel filtration on a Sephadex G-25 column. The concentration of avidin-MBS is calculated from the absorbance of the avidin-MBS at 282 nm. The ratio of avidin to MBS is usually between 3 : 1 and 5 : 1. Antigen is reduced with 10 m M dithiothreitol (DTT) prior to reaction with avidin-MBS for 20 hr. Maleimide groups react with free sulfhydryl groups on cysteines or with those produced from reduction of cystine residues in proteins. In most cases, conjugates may be used without further purification. However, 21 F, T. Lui, M. Finnecker, T. Hamaoka, and D. Katz, Biochemistry 18, 690 (1979). 2z R. J. Youle and D. M. Neville, Jr., Proc. Natl. Acad. Sci. U.S.A. 77, 5483 (1980).

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purification is easily accomplished by ion-exchange chromatography or by gel filtration. Cross-linkers may not be suitable for conjugating haptens or carbohydrates to avidin. Many haptens or carbohydrates can be chemically activated either by synthesizing the appropriate N-hydroxysuccinimide derivative 15 or by periodate oxidation, and the activated substances are in turn conjugated to avidin. These reactions are usually very efficient, and the conjugate can be easily purified by ion-exchange chromatography, since reaction with amino groups on avidin reduces its isoelectric point. For such conjugations, avidin from egg white (which contains many lysines) is more useful than, and therefore preferred over, streptavidin from

Streptomyces avidinii. 23 Construction of Biotin-Antigen Conjugates In some cases where shotgun experiments are conducted to raise antibodies to unknown antigens, biotinylation may offer advantages over avidinylation. For this purpose, biotinylation reactions are carried out with derivatives of biotin or iminobiotin. N-Hydroxysuccinimidyl (NHS) esters, reactive with amines on proteins, have been most commonly employed. The reactions are simple, quantitative, and are less likely to modify biochemical and antigenic properties. Other biotin-containing reagents that react with proteins and glycoconjugates may also be used. 24

Testing of Conjugate Before relying on the conjugate to bring about selective cell-cell cross-linking, the functionality of both the avidin (or biotin) and the antigen moieties must be ascertained. In vitro assays, including enzyme linked immunoassay, radioimmunoassay, and fluorescent immunoassay, are used for this purpose. The same assays can also provide some idea as to the amount of conjugate to use in the actual fusion procedure. Any of the assays requires a control antiserum to the antigen being used. Often one can use serum from the animals immunized for fusion purposes, in which detectable titers can often be found with enzyme or radioimmunoassays using native antigen. 15.25Commercially available polyclonal or other monoclonal antibody preparations can also be used. Once an appropriate standard antiserum is available, the binding activity of the conjugate is measured by incubating together biotinylated 23 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 24 M. Wilchek and E. A. Bayer, this volume [13]; E. A. Bayer and M. Wilchek, this volume

[14]. 2~ B. B. Mishell and S. M. Shiigi, "Selected Methods in Cellular Immunology." Freeman, San Francisco, California 1980.

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050 I° A b ~

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FIG. 3. Characterization of activity of antigen-avidin conjugates. An antigen-avidin conjugate was prepared by cross-linking with DFDNB to avidin. 16The conjugate was analyzed by adsorption to biotinylated microtiter plates. The plates (Dynatek) were coated with biotinylated bovine serum albumin, conjugate was added and incubated for 40 min at 37°, and the wells were washed 5 times with PBS containing 0.05% (v/v) Tween 20. A polyclonal antiserum, raised against the antibody, was added, and the plates were incubated for 1 hr at 37°. Following another wash cycle, the plates were treated with a second antibody covalently attached to horseradish peroxidase; after incubation and washing, the plates were developed with a suitable substrate solution. The curve shows the amount of conjugate bound as measured by enzymatic conversion of substrate to a colored product at 410nm. Conjugate activity can be detected to at least a 1 : 200 dilution of the original conjugate preparation. (From Ref. 12.) m y e l o m a cells, the a v i d i n - a n t i g e n conjugate, a p r e d e t e r m i n e d dilution o f the a n t i s e r u m , and a g o a t a n t i - m o u s e a n t i b o d y that has b e e n either radiolabeled o r c o n j u g a t e d to p e r o x i d a s e or fluorescent m i c r o s p h e r e s . Cells are t h e n w a s h e d , and the d e g r e e o f s e c o n d a n t i b o d y binding is quantified. N e g a t i v e c o n t r o l s s h o u l d include n o r m a l (unbiotinylated) m y e l o m a cells, n o r m a l m o u s e s e r u m ( u n i m m u n i z e d ) , and c o n j u g a t e r e p l a c e m e n t with native avidin o r streptavidin. Titrating the c o n j u g a t e or inhibiting with k n o w n c o n c e n t r a t i o n s o f free antigen p r o v i d e s quantification o f the degree o f binding. A n e x a m p l e o f the t y p e o f result that m a y be o b t a i n e d b y e n z y m e i m m u n o a s s a y is s h o w n in Fig. 3. P r e p a r a t i o n of Cells for Fusion

Myeloma Cells Biotinylation. B i o t i n y l - N - h y d r o x y s u c c i n i m i d e ester ( B N H S ) is used to biotinylate m y e l o m a cells. L o g - p h a s e ceils are h a r v e s t e d and w a s h e d

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with Dulbecco's PBS, which contains no divalent cations. BNHS (0.1 M) is freshly dissolved in dimethylformamide (a minimal volume is used as this solvent lyses cells at high concentrations) and diluted with PBS. Cells are resuspended in 100/zM BNHS, incubated for 15 min at 23 ° without further agitation, and centrifuged through 10 ml of serum at 200 g for 8 min. Cells are washed twice in Dulbecco's modified Eagle's medium (DME), which is used because it is biotin free. Deoxyribonuclease I (DNase) is added to the DME (final concentration 10-50/~g/ml) to reduce cell aggregation caused by the release of intracellular components. In later stages the enzyme is removed by washing, as DNase would prove toxic if introduced into cells during fusion. To test the degree of biotinylation, cells are stained with fluoresceinconjugated avidin. Two million P3 x 63/Ag8.653 myeloma labeled cells are analyzed with a fluorescence-activated cell sorter. Greater than 98% of the cells are biotinylated. Two fractions containing either heavily or lightly biotinylated cells are cloned in soft agar. There are no differences in viability between the two fractions. In four different cell lines tested (P3 x63/Ag8.653, SP2/0, S194, and FOX-NY), the fluorescence intensity remains maximal up to 3 hr after biotinylation. P3 ×63/Ag8.653 cells retain approximately 10-30% of the original intensity after overnight incubation following biotinylation, while the other three lines lose virtually all of the label. Model fusion experiments (using an appropriate cell surface marker) and growth experiments (assessed by trypan blue exclusion) have demonstrated that both the capacity to undergo membrane fusion and the viability of myeloma cells are not significantly altered by biotinylation using BNHS. Avidinylation. Alternatively, myeloma ceils can be avidinylated. The heterobifunctional cross-linker N-succinimidyl 3-(2-pyridylthio)propiohate (SPDP) is used to covalently attach avidin to myeloma cell surfaces. Avidin is reacted with a 5-fold molar excess of SPDP for 1 hr at 23 °, and free reagent is removed by gel filtration on a Sephadex G-25 column with PBS. The degree of conjugation is determined from the absorbance of the avidin-SPDP solution at 253 and 282 nm. A 4 : 1 ratio of avidin to SPDP is typical. J2 A b o u t 10 7 log-phase myeloma cells grown in medium containing 0.1 m M 2-mercaptoethanol are reacted with 1 /~M avidin-SPDP for I hr at 23 ° without agitation. Cells are collected by centrifugation through a cushion of solution made from equal volumes of isosmotic sucrose and DME. The degree of labeling by this method is measured by the binding of biotinylated fluorescein-conjugated lysozyme. The fraction of cells labeled is usually greater than 80%, but the intensity of staining is about 10-

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fold lower compared with biotinylated myeloma ceils stained with fluorescein-conjugated avidin. The density of avidin on myeloma cell surfaces can be greatly increased by prior reaction of myeloma cells with reduced SPDP. This is prepared by dissolving 1 mg of SPDP and 0.5 mg of DTT in 30 /zl of dimethylformamide and then diluting into 30 ml of PBS. Myeloma cells are then incubated in this solution for 15 min at 23°, centrifuged through sucrose-DME, and resuspended in PBS. The cells are then incubated with 1 /xM of avidin-SPDP as described above. This method results in staining intensities equivalent to biotinylated cells stained with fluorescein-conjugated avidin. Comments. Although more complex, avidinylating of myeloma cells permits the use of biotinylated antigen to cross-link spleen and myeloma cells for fusion. Often large or complex proteins are more easily biotinylated than avidinylated. We try to avoid bridging biotinylated antigen and biotinylated myeloma cells with avidin. This process is usually uncontrolled and is not reproducible. Large aggregates of cells are often formed. Therefore, when biotinylated antigen is used, it is preferable to use avidinylated myeloma cells.

Spleen Cells For purposes of immunization, antigenic proteins are adsorbed to either bentonite or alumina. They may also be emulsified in Freund's adjuvant; however, we have found that this gives rise to additional problems with intraperitoneal adhesions. Small molecules, such as haptens or peptides, are first conjugated to keyhole limpet hemocyanin (KLH) before adsorption to the carrier substance. In some cases, animals, primed with injections of KLH alone before immunization with a KLH-antigen conjugate, develop serum titers to the antigen more rapidly. 25 The animals commonly used for monoclonal antibody production are either C57BL/6 or BALB/c mice. The route of administration is usually by intraperitoneal injection. However, when the antigen used is very precious, the final immunization can be given by intravenous or intrasplenic injection of a very small amount of antigen 3 days prior to fusion. The immunization schedule can be varied a great deal, depending on the availability of the antigen and considerations of time and experimental goals. It has been shown that a greater variety of higher-affinity antibodies are produced from hyperimmunized animals, as compared to those produced following acute exposure to the antigen, z6 A typical schedule may include 3 or 4 injections given 1-2 weeks apart. However, in our initial z~ C. Berek, G. M. Griffiths, and C. Milstein, Nature (London) 316, 412 (1985).

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experiments, we were interested in raising antibodies to very rare entities that are extremely hard to isolate. We therefore immunize animals only twice, 1 week apart, with microgram quantities of antigen each time. Both injections are intraperitoneal, and the final one is given 3 days prior to fusion. Three days after the final immunization, spleens are removed. Cells are dissociated on a fine wire mesh using a rubber policeman. Red blood cells may be lysed with freshly prepared ammonium chloride solution (0.84%); however, we find that the presence of red blood cells does not noticeably affect fusion in a high-voltage field. The lymphoid cells are collected in DME containing 50/xg/ml DNase. Incubation with the antigen-avidin (or antigen-biotin) conjugate is carried out for a minimum of 30 rain up to 2 hr, at 4 ° (to prevent capping and internalization of the conjugate).

Combining Cells Treated spleen cells are mixed with biotinylated (or avidinylated) myeloma cells at a ratio of 4:127 and centrifuged. The mixed cell pellet is loosened and spun for 10 sec at 50 g. The pellet is incubated for 30 min at either 23 or 37 ° and then diluted in DME. An aliquot containing up to 2 x 10 7 cells is underlaid with sucrose and centrifuged at 200 g for 6 min at 23 °. Cells are then resuspended in 0.5 ml isosmotic sucrose (300-320 mOsm) and placed into the cell fusion chamber (Fig. 4). It should be noted that harsh resuspension techniques (such as pipetting) should be avoided, since many steps in the preselection process involve mechanical agitation and disruption. Instead, gentle disruption by tapping or flicking the tube should be used. Since divalent cations promote nonspecific cell aggregation, they should be excluded. Cell Fusion After completion of the preparation steps described above, one is left with a suspension of treated myeloma and spleen cells in isosmotic sucrose. In order to avoid heating of the cells during high-voltage fusion, sucrose is used because of its low electrical conductance. The suspension contains the relatively few B cell-antigen-(avidin-biotin)-myeloma cell complexes. Typically, a 0.5-ml suspension, containing about 50 million cells, is introduced into the fusion chamber (Fig. 4). The cells are exposed to 2-4 pulses of 5 ~sec duration at a field intensity of 3 kV/cm. The 27 We find, however, that the ratio o f cell types is not as critical in high-voltage fusion as it is in fusions using PEG (see Ref. 3).

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FIG. 4. Electrofusion chamber. Cells are fused in 0.5 ml of a suspension held in a chamber created by sandwiching two stainless steel disks around a well drilled into a 3 mm thickness of polycarbonate, which are then clamped between two brass electrodes. The suspension is introduced into the chamber through a 22-gauge port. The chamber is also vented with another small port to prevent the formation of air bubbles. (Top) Unassembled apparatus; (bottom) apparatus assembled and ready for use.

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electrical pulse consists of a square wave generated by a commercial highvoltage pulse generator. The field is monitored with a storage oscilliscope connected to the electrodes through a 100x reduction probe. The field polarity is alternated between the pulses to reduce bulk cell transport toward one electrode or the other. Following fusion, cells are maintained at 37° for 30 min in DME. Membrane pores generated during electrofusion undergo spontaneous repair, and this process is temperature dependent. Cells are then plated into 96-well tissue culture plates with DME containing aminopterin, which kills all unfused myeloma cells over the course of the next few days. Unfused spleen cells die off in a matter of days in culture. Growth of the hybridomas is usually detected from 1 to 6 weeks following electrofusion. Comment. We were surprised to obtain predominantly IgG antibodies following the above acute immunization regimen, rather than those of the IgM class, which is what we had predicted. We hypothesize that the bioselective method, utilizing binding of antigen to surface antibody, facilitates fusion of those B cells stimulated to produce high-affinity antibodies (i.e., those of the IgG class), while the more weakly binding antibodyproducing spleen cells are left behind.

[77] C o m p l e x N e o g l y c o p r o t e i n s

By MING-CHUAN SHAO, LING-MEI CHEN, and FINN WOLD Introduction Biotinylated glycans bound to avidin or streptavidin represent useful glycoprotein models for the study of both glycan processing by Golgi enzymes and glycoprotein interactions with lectins/receptors. Although we have used primarily asparagine-linked glycans derived from pronase digestion of ovalbumin, in principle, any glycan isolated from a protease digest of a glycoprotein (with the amino acid or a short peptide still attached) should be amenable to the reaction of the free a-amino group with activated biotinyl derivatives and the subsequent tight binding to avidin or streptavidin. Once a particular avidin preparation has been characterized, the stoichiometry of binding and the stability of the neoglycoprotein complex are reproducible, constant features of that preparation, and after any METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.