Cytometry 11:498-505 (1990)

0 1990 Wiley-Liss, Inc.

Selective Cloning of Hybridoma Cells for Enhanced Immunoglobulin Production Using Flow Cytometric Cell Sorting and Automated Laser Nephelometry Philip Marder,' Ronald S. Maciak, Rebecca L. Fouts, Robert S. Baker, and James J. Starling Lilly Research Laboratories, Indianapolis, Indiana 46285 Received for publication September 5, 1989; accepted January 19, 1990.

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescenceactivated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouseIgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG)intensity sorting regions yield-

Monoclonal antibodies (MAb) have become major constituents of the burgeoning field of biotechnologyrelated products. It has become apparent that for the development and general distribution of MAb diagnostic and therapeutic products, large-scale production techniques will be necessary. Although MAbs can be produced in vivo by collection of ascitic fluid, mass cell culture is in many ways preferable (15).Due to the significant production expenses incurred in these large-scale cultures, obtaining maximal yields of reactive immunoglobulin (Ig) is indeed important (14,221. To date, most reports in this area describe methods for improving Ig yields by optimizing media formulations and by controlling environmental culture conditions (15). Lacking, however, are discussions of the selection and maintenance of initial variant hybridoma starter cultures capable of secreting increased MAb concentrations. Inasmuch a s genetic and

ed cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell depositioin provides positive attributes of severall traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures. Key terms: Flow cytometry, monoclonal antibody, cell separation

phenotypic variations do exist in monoclonally derived hybridoma cultures (2,5,24),continued observation and reselection of opt,in?al clones from these strains has a potentially large economic impact. In this manuscript we present a n alternative method for the isolation, of MAb-producing hybridoma cell lines. The method involves selective cloning of hybridoma cells that express enhanced cell surface immunoglobulin (SIg) levels by flow cytometric cell sorting (FCCS) and monitoring the derived clones' Ig secretion levels by automaled laser nephelometry. By using

'Address reprint requests to Philip Marder, Lilly Research Laboratories, Department MC620, Lilly Corporate Center, Indianapolis IN 46285.

SELECTIVE (’1,ONING OF HYIZRIDOMAS

these techniques we have detected heterogeneity in l g secretion from previously (manually) cloned cultures and, in addition, have successfully isolated hybridoma cell lines that secrete increased quantities of Ig. In the following study we provide a n example of how selective hybridoma cloning can provide improved starter cultures that could greatly improve l g production, and thus significantly decrease costs associated with mass scale monoclonal antibody production.

MATERIALS AND METHODS Hybridomas The establishment of the L-KS series (Ll-KS, L2-KS, and L4-KS) of monoclonal antibodies has been described in detail elsewhere (17). Briefly, BALBic (Harlan Sprague-Dawley, Indianapolis IN) mice were immunized three times at 1 week intervals with 3-20 x lo6 cell culture-derived P3IUCLA human lung adenocarcinoma cells. Three days after a final booster injection, the mouse spleens were removed, and the resulting single cell suspension (3 x lo7) was fused t o HL-1 Friendly .653 (1.5 x lo7) non-Ig-secreting myeloma cells (Ventrex Laboratories, Portland ME) using a 50% polyethylene glycol solution. After 7-10 d growth in hypoxanthine-, aminopterin-, and thymidine-containing medium, the resultant spent culture media (from growing clones) were screened by solid-phase immunoradiometric assay (RIA) for reactivity, described below. After further confirmatory assays, MAbcontaining media from clones of interest were subisotyped using a n antibody capture assay kit (Hyclone, Ogden UT). Cultures producing IgG isotypes were then cloned either by limiting dilution (3,121, soft agar (4,19), or the FCCS techniques described below. Three hybridoma lines emerged from these initial screens: L1-KS, which secretes IgG,,; L2-KS, secreting IgG,; and L4-KS, secreting IgG,,. All three MAbs were shown to react with the 40,000 dalton glycoprotein of P3IUCLA cells, which is defined by KSlI4 MAb reactivity (21). Flow Cytometric Cell Sorting All flow cytometric cell sorting was done with an Epics C (Coulter Epics Division, Hialeah, FL) flow cytometer-cell sorter (FCCS). Hybridoma cells were excited with 600 mW of 488 nm light emitted from a n argon ion laser. The resulting emitted cell-associated fluorescence was captured through a 488 nm laser blocking interference filter and a 515 nm long pass absorbance filter, both obtained from Coulter Epics Division. The optical alignment of the FCCS instrument was optimized, and the fluorescence readings obtained were standardized for each experiment using uniform, fluorescently stable fluorospheres (Coulter Epics Division). The flow cytometer was outfitted with an automated cloning accessory called a n AUTO-CLONE (Coulter Epics Division) that can be programmed to dispense a single particle or cell into selected compartments of a standard 96 well microtiter culture plate.

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The sort delay timing was adjusted per manufacturer’s instructions. In a test evaluation of the AUTOCLONE, a single fluorosphere was programmed t o be deposited into wells of three separate microtiter plates. Upon fluorescence microscopic inspection (data not shown), each of the 288 wells had exactly 1 fluorosphere, with no doublets and no misses. The cell-contact components of the FCCS were sterilized prior to cloning by flushing with 95% ethanol for 30 min followed by exhaustive rinsing in sterile phosphate buffer saline sheath fluid. Cells were prepared for sorting based on surface Ig reactivity by harvest of cultured hybridoma cells and incubation with F(ab’), fragments of goat-anti-mouseIgG-FITC (AMIGG-FITC; Cappel, cat. #1311-0081, West Chester, PA) for 45 min on ice followed by two washes in culture medium. In some experiments, hybridoma cells were harvested from culture, washed in medium, and sorted based on forward angle light scatter (FALS) alone (without AMIGG-FITC incubation). Propidium iodide (PI; Sigma Chemical Co., St. Louis, MO; 1 pgiml final concentration) was added to some cell preparations just prior to sorting to help define and select viable cells (7,8,10). Mean log fluorescence intensities were computed for fluorescence histograms using the EASY-88 (Coulter Epics Division) computer system. Subpopulations of cells to be sorted were FCCS-defined by either rectilinear gates or by polygonal bitmaps of collected two-parameter (FALS vs. log green fluorescence) histograms. A three-droplet sorting procedure was employed to help assure maximum purity (per manufacturer’s instructions). In all AUTOCLONE sorting experiments, single hybridoma cells were deposited into microtiter wells that were seeded 24 h previously with 5 x lo6 C3HIHeJ thymocyte feeder cells in 200 pl of complete cloning media (HL-1 media, Ventrex Laboratories), with added 10% fetal bovine serum, 50 mM L-glutamine, and 5.0 mcgiml gentamycin, all from GIBCO (Grand Island, NY).

RIA Screening of Hybridoma Supernatants After 7-10 d of growth, media from wells displaying positive hybridoma growth were screened for antibody reactive to fixed P3iUCLA cells by solid phase RIA. Twenty-five microliters of culture supernatant was added to gluteraldehyde-fixed P3iUCLA cells in a flexible microtiter plate and incubated for 1 h a t room temperature, as previously described (18).After extensive washing, 25 ~1 (100,000 cpm) of affinity-purified 1261labeled (6) F(ab’), fragments of rabbit anti-mouse IgG (Miles Laboratories, Elkhart IN) was added t o each well for 1 h a t room temperature. The plates were washed again, and the wells were cut out and counted on a gamma counter. Automated Laser Nephelometry In most cases, spent culture media from microwells showing positive growth were also checked for their

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Frc. 1. Forward angle light scatter (FALS) histogram of the L-KS mixture of hybridoma cells. Equal concentrations of L1-KS, 122-KS, and L4-KS hybridoma cells were mixed, flow-analyzed, and AUTOCLONE-sorted from the region indicated by the hatched area.

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1 magnitude of Ig secretion by automated laser nephelometry (ALN). Several reports have described the use of nephelometry for measuring Ig production by lymphoid cell lines and hybridomas (13,23). In this study we used a n automated Behring Laser Nephelometer (Behring Diagnostics, La Jolla, CA). Briefly, the instrument operates by automatically adding to disposable cuvettes programmed amounts of sample (hybridoma supernatant) or standard (subclass MAbs or myeloma proteins purchased from Sigma), specific antiserum (goat anti-mouse IgG, heavy and light chain specific; ICN Immunobiologicals, Lisle, IL), and diluent composed of 4% polyethelene glycol (cat. no. P-2139, Sigma) in 0.01 M PBS containing 0.05% Tween 20 and 0.1% thimersol. The cuvettes are automatically transported during a 30 min incubation period to the nephelometer. The magnitude of scattered incidence light (which is proportional to the quantity of antibody-antigen complexes formed) from each cuvette is measured and recorded in voltage units. The built-in computer collects data from the entire assay and computes sample potency by interpolation from an included standard curve. With sensitivity down to 1 pgiml and automated operation, this type of analysis provides a feasible approach to screening large numbers of hybridoma supernatants for magnitude of Ig secretion.

RESULTS Automated Flow Sorting of L-KS Hybridoma Mixtures Due to the paucity of published reports of using FCCS technology for hybridoma cell cloning, the first experiments were designed to determine the suitability of using the AUTO-CLONE for isolating single hybridoma cells while maintaining viability and antibody secretion levels in expanded cultures. Toward this goal, a mixture of equal concentrations of L1-KS, L2-KS, and L4-KS hybridoma cells was clone-sorted based on FALS alone (Fig. 1).This mixture contained cells from cultures that had been previously cloned (three times) manually using the limiting dilution approach (17) and

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FIG.2. Flow chart for the L-KS mixture experiment. Equal numbers of L1-KS, LZ-KS, and L3-KS hybridoma cells were FCCS-cloned, assayed, and recloned as diagrammed. Note that the low frequency of Ig-secreting clones observed in the initial FCCS sort was not repeated in the second round of' sorting.

had been selected f;w single-subisotype-specific Ig secretion. If the AUTO-CLONE were to perform as expected, each of the expanded cloned cultures should have produced a single subisotype Ig with no dual secretors present. Figure 2 depicts the experimental flow. Although it had been anticipated that antibody secretion would be maintained in these thrice-cloned hybridomas, in fact, only 32% of the newly generated clones produced any Ig a t all. When several of the + Igsecreting clones were recloned on the sorter, the resultant cultures subsequently maintained their Ig production, implying that the automated sorting process itself was not responsib'le for eradicating Ig synthesis in our earlier attempt. Thus, by using these techniques the frequency of Ig-producing cells in these cultures could be estimated, and through recloning, more homogeneous Ig secretion activity could be maintained.

Surface Immunoglobulin Expression on IL-KS Hybridomas Since FCCS technology is capable of analyzing and selecting cells based on several simultaneously measured parameters, we investigated the concept of using the AUTO-CLONE for selective cloning of hybridoma cells, that is, instead of employing the traditional method of randomly diluting cells to monoclonality (limiting dilution), we wanted to determine if we could

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SLLECTIVE ('LONING OF HYKRIUOMAS

L1-KS II

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L2-KS IV

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Autofluores Control AMIGG F ( a b')2- F I T C 10 mcglml AMIGG F(abD)2-FITC 100 mcg/ml AMIGG F(abD)2-FITC 1000 mcglml AMIGGMA -FITC 40 mcg/ml AMIGGMA -FITC 400 mcg/ml

FIG.3. Surface membrane reactivity of L-KS hybridoma cultures with anti-mouse immunoglobulins Clonal isolates of L-KS hybridomas were incubated with varying dilutions of F(ab'), fragments of antimouse IgG (AMIGG-FITC) or a whole antibody fraction of anti-mouse IgG, IgA, IgM (AMIGGMA-FITC) for 45 min on ice, washed, and analyzed on the Epics C. The mean log fluorescence intensity for each sample is displayed on each histogram of log green fluorescence vs. relative number of cells.

specifically select individual cells for expansion with a characteristic detectable by FCCS. Potentially, this technique could be used to limit the number of cultures to be screened while increasing the odds of generating clones with a desired (high Ig secretion) characteristic. In Fig. 3, flow cytometric data are displayed from a n experiment in which four subclones of the L-KS series were incubated with varying dilutions of commercial FITC-conjugated goat-anti-mouse IgG (AMIGG-FITC). This data indicates that 1) relatively high concentrations of AMIGG-FITC are required (as compared with standard FCCS indirect immunofluorescence assays) in order to observe significant membrane staining of these hybridomas; 2) the L1, L2, and L4 hybridomas react with AMIGG-FITC reagents to differing degrees; 3) L1-KS IV culture (selected in a previous FCCS cloning) displays significantly greater surface Ig staining than the L1-KS 11; 4) L2-KS IV cells displays the greatest reaction to the AMIGG-FITC reagents and does so in a n apparent bimodal manner. From this data, we decided to concentrate on the L2-KS hybridoma series

to study this bimodality of surface Ig expression and examine its relationship to Ig secretion levels in progeny cultures.

Selective Cloning of Hybridoma Cells Based on Surface Ig Expression In Fig. 4 the AMIGG-FITC FCCS staining pattern of the L2-KS-parent is shown. This population was derived from a frozen stock of the original fusion culture, and thus had not been cloned. Heterogeneity of surface Ig staining was observed in this primary culture a s had been observed in its L2-KS-IV progeny (Fig. 3 ) .We had decided to go back to the parent culture to select variants from a population with the greatest potential heterogeneity, a s had been shown by others (2,5,24).We clone-sorted (one plate each) hybridoma cells from three sorting regions: "dim," "medium," and bright" subpopulations of these AMIGG-FITC reactors. Figure 5 summarizes the Ig secretion data (as measured by ALN) from the expanded clones derived from this sort. Most importantly, none of clones derived from the

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35-LZ-KS 32.3 I

FIG.4. Fluorescence sorting regions used in FCCS cloning of the L2-KS-parental culture The LP-KS population was incubated with F(ab’),-AMIGG-FITC (100 Kgiml) and washed a s previously described. A single cell from either “Dim,” “Medium,” or “Bright” regions was deposited into selected microwells and expanded in culture.

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FIG. 5. Immunoglobulin levels secreted by FCCS-derived LZ-KS clones. Monoclonal cultures derived in experiments described in Fig. 4 were assayed for magnitude of secreted Ig by laser nephelometry after macroscopic growth appeared. The clones arising from the “Dim” region yielded 50 total cultures (overlapping on the figure), “Medium” 29, and “Bright” 2. The lower limit of assay sensitivity was 3.1 mcgiml of secreted MAb.

“dim” sort (50 total) produced measurable Ig ( > 3.125 pg/ml), whereas 72% of the clones derived from the “medium” intensity region were Ig secretors. While both of the clones that grew out of the “bright” sorting region were Ig secretors, a low cloning efficiency was observed and may have resulted from a decreased level of viability in this subpopulation. Representative clones from this sort were expanded and analyzed for surface Ig expression a s illustrated in Fig. 6. Clones derived from both the “bright” and “medium” AMIGG-FITC reacting subpopulations maintained their brighter AMIGG-FITC reaction levels as compared with the “dim” subpopulation, indicating that their surface Ig phenotype was maintained through one round of sorting. In addition, Ig secretion levels from subsequent sorts of these subclones indicated that the “dim” clones never produced measurable Ig (data not shown).

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FIG.6 . Surface membrane fluorescence of L2-KS hybridoma subclones. Clonal isolates of L2-KS hybridomas were incubated with F(ab’),-AMIGG-FITC (100 Fgiml), washed, and analyzed on the Epics C. 35.L2-KS was isolated from the “Dim” sorting region (Fig. 41, 75LP-KS from the “Medium,” and 82-L2-KS from the “Bright.” The mean log fluorescence intensity for each subclone is displayed on each histogram of log green fluorescence vs. relative number of cells.

Enhanced Cloning Efficiency by Using Propidium Iodide Exclusion All of the above-discussed FCCS cloning experiments utilized rectilinear sorting regions to define and select cells that displayed increased AMIGG-FITC reactivity and FALS characteristics of “viable” cells (10). In an effort to refine further this clone-sort selection process, propidium iodide (PI) was added to the AMIGG-FITCreacted hybridomas; just before sorting. Nonviable cells cannot metabolically exclude PI (7,8,10),and therefore (using the optical filter arrangement described above)

SELECTIVE CLONING O F HYBRIDOMAS

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FIG.7. Two-parameter histogram of forward angle light scatter (abscissa)vs. log green fluorescence (ordinate)for an L-KS hybridoma cell population stained with F(ab’),-AMIGG-FITC (100 pgiml) and propidium iodide (PI; 1 Wgiml). The bitmapped sorting region (4% of the total) is defined by the polygon labeled “PI neg,” which represents brightly fluorescing FITC-stained cells that are negative for PI incorporation.

have fluorescence intensities considerably higher than AMIGG-FITC-alone reacted samples. Two-parameter histograms were acquired on these samples, and bitmapped sorting regions were established that included the brightest AMIGG-FITC reacted cells but excluded the PI fluorescing (nonviable) component. As illustrated in Fig, 7, inclusion of PI into the sorting mixture indeed allowed us to exclude nonviable cells from the so-called FALS viable region. In this representative experiment, 59% percent of the sorted wells developed positive hybridoma cell growth compared with the 24% composite cloning efficiency observed in previous experiments that did not utilize the PI exclusion technique.

DISCUSSION In this manuscript we have presented a n alternative method for the clonal isolation of antibody-secreting hybridoma cells. We have described how use of a n automated single cell deposition device (AUTO-CLONE) in conjunction with a commercial flow cytometer-cell sorter can be used for selective isolation of antibodyproducing hybridoma cells. In addition, we have also described how the use of automated laser nephelometry can assist in the quantitative selection of high immunoglobulin secreting clones. The derivation of single cell cultures is in itself primary to all assumptions made about monoclonal anti-

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bodies in general. That is, to obtain MAb of a single molecular species, the investigator assumes that his cell isolation techniques will yield cultures that are derived from individual cells. The isolation techniques can be divided into two general categories: ones based on dilutionistatistical methods and those based on direct cell micromanipulation. The dilutionistatistical approaches (limiting dilution, soft agar) provide a relatively simple approach for producing multiple numbers of cloned cultures but make assumptions concerning the probability (Poisson) of single cell distribution without accounting for the biological advantage of multiple cell growth (11).The other hybridoma cloning approach, direct cell micromanipulation (16,201 provides more certainty in the deposition of single cells, yet with the expense of much increased labor intensity and thus is capable of producing far fewer monoclonal cultures. The FCCS cloning techniques described in our study provide advantages of both the dilutionistatistical and direct micromanipulative approaches. As in the direct micromanipulative techniques, each cell is individually located (visually in the manual approach and electronically by FCCS) and separated (aspiration in the manual approach and electrostatically in FCCS) into a single cell culture. Cloning by FCCS however, allows for multiple single cell seedings in a short time (less than 3 min for 96 cultures), which is similar in culture production volume to the dilution/statistical approaches. Thus, one obtains the certainty of single cell manipulation with the advantage of seeding multiple cultures. In contrast to both classical techniques, FCCS cloning allows for potential selective separation of cells to clone. That is, FCCS is capable of delineating phenotypic characteristics (fluorescence, light scatter) of individual cells and making microsecond decisions to deposit or dispose cells of interest. Add to this the optional microtiter plate positioning tool (AUTOCLONE), and a semiautomatic selective tool for hybridoma isolation is available. Subsequent to obtaining multiple (microtiter) cultures through FCCS, or any other type of hybridoma cloning, the resultant supernatants must be analyzed for class and subclass and quantity of MAb secreted. This has been accomplished in many labs by RIA or enzyme-linked immunosorbant assays (ELISA). Alternatively, in our study we chose ALN as the tool for quantifying Ig secretion levels from established clones. Our experience has suggested that ALN is particularly suited for cloning applications because of speed, simplicity, range of expected results, and low labor intensity. In contrast to ELISA and RIA, ALN does not involve preincubations, blocking reactions, enzyme or radionuclide conjugations, substrate additions, or other reagents and incubations capable of adding uncertainty to a n assay. The assay simply depends on the flocculation of antigen-antibody reactions and light scatter measurements. In contrast to ELISA and RIA assays, ALN assays are completed in less than 2 h time. While ALN is less sensitive in terms of Ig detec-

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tion limits (1-100 pglml) than RIA or ELISA, its de- Mr. John Mastro fix technical help with the competitection range conveniently falls within the expected tive RIA tests. concentrations of hybridoma culture Ig secretion. FiLITERATURE CITED nally, by using a commercial, automated device, dilu1. Alarcon-Segovia D, Fishbein E - Determination by laser nephetions, standard curve interpolations, and final results lometry of immunoglobulins by mononuclear cells in culture. Rev are obtainable with little operator intervention. Invest Clin 32:295--2917,1980. It has been commonly assumed that once a hybri2. Andreeff M, Bartal A, Feit C, Hirshaut Y: Clonal stability and doma cell has been twice cloned, an immortalized sinheterogeneity of hybridomas: analysis by multiparameter flow gle parent cell will give rise to a n endless continuum of cytometry. Hybridoma 4:277-287, 1985. 3. Coller HA, Coller BS: Statistical analysis of repetitive subcloning genetically and phenotypically identical daughter by the limiting dilutim technique with a view toward ensuring cells, yielding a continuous and homogeneous supply of hybridoma monoclonality. Hybridoma 2:91-96, 1983. MAb. Cloned hybridoma cultures are often observed, 4. Cotton RG, Secher DS, Milstein C: Somatic mutation and the however, to lose Ig secretion activity gradually. Our origin of antibody diversity. Clonal variability of the immunogiobulin produced by MOPCZl cells in culture. E u r J Immunol 3: study clearly shows that multicloned hybridomas are 135-140, 1973. not always homogeneous cultures and may in fact be 5. Hadas E, Theilen 2 : Production of monoclonal antibodies. The composed of cells that are phenotypically diverse and effect of hybridoma concentration on the yield of antibody-prodisplay a wide range of Ig secretion capacity. The oriducing clones. J Immlunol Methods 96:3-6, 1987. gin of this diversity is not clear, but may involve a slow 6. Hunter R: Standardization of the chloramine-T method of protein iodination. Proc SOCE:xp Biol Med 133:989-992, 1970. loss of chromosomal material from hybrid cells ( 2 ) .We 7. Jacobs DB, Pipho C: Use of propidium iodide staining and flow have provided in this study a technique for selecting cytometry to measure antibody-mediated cytotoxicity: resolution specific component cells from such mixtures that will of complement-sensitive and resistant target cells. J Immunol more likely produce higher concentrations of secreted Methods 62101-108, 1983. 8. Krishan A: Rapid flow cytofluorometric analysis of mammalian Ig. Our results indicate that selection of hybridoma cell cycle by propidium iodide staining. J Cell Biol 66:188-193 cells exhibiting high Ig membrane expression can pro1975. duce cultures with enhanced Ig secretory capacity, sim9 Leibson PJ, Loken M R , Panem S, Schreiber H: Clonal evolution of ilar in manner to that observed for some myeloma cell myeloma cells leads t,o quantitative changes in immunoglobulin lines (9). It should be noted that although varying secretion and surfa.ce antigen expression. Proc Natl Acad Sci 76: 2937-2941, 1979. amounts of Ig secretion may occur from such cultures, no change in the relative affinity of these antibodies 10 Loken MR, Stall AM: Flow cytometry as a n analytical and preparative tool in immunology. J Immunol Methods 50:R85-R112, has ever been observed (by competitive RIA; data not 1982. shown). Although we have never observed a culture 11 McCullough KC, But’cherRN, Parkinson D: Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease that was negative for surface Ig that secreted MAb, we virus (FMDV). 11. Cloning conditions. J Biol Stand 11:183-194, unfortunately have observed some of our “good” secret1983. ing cultures drift toward decreased Ig production (and 12 McKearn TJ: Cloning of hybridoma cells by limiting dilution in lower fluorescence levels) even from thrice-FCCSfluid phase. In: Monoclonal Antibodies, Kennett RH, McKearn cloned cultures (data not shown). This does not preTJ, Bechtol K B (edsl. Plenum Press, New York, 1980, pp 374380. clude using FCCS isolation techniques but more likely reinforces the premise that routine FCCS analysis and 13 Munoz J , Virella C:, Fundenberg HH: I381 Immunonephelometric assay for immunoglobulins released by cultured lymphocytes. In: reisolation may be even more important for certain culMethods in Enzymollogy, Vol 73, Langone JJ, Van Vunakis H tures with a proclivity for instability. In addition, we (eds.).Academic Press, New York, 1981, pp 578-580. have observed a less strict relationship between bright 14 Reuveny S, Velez D, Macmillan JD, Miller L: Comparison of cell propagation methods for their effect on monoclonal antibody yield surface Ig fluorescence and MAb secretion for some fermentors. J Irnniunol Methods 86:61-69, 1986 cultures (data not shown), that is, sorting bright sur- 15 in Samoilovich SR, Dugan CB, Macario AJ: Hybridoma technology: face fluorescing cells has not always produced high Ignew developments of practical interest. J Immunol Methods 101: secreting clones. Our feeling, however, is that in no 153-170, 1987. case would we likely obtain an inferior secretor using 16 Sijens R J , Thoma:; AA, Jackers A, Boeye A: Clonal isolation of hybridomas by manual single-cell isolation. Hybridoma 2231the FCCS techniques and thus have now incorporated 234, 1983. the techniques described in this study for the clonal 17 Starling JJ, Maciak. RS, Hinson NA, Nichols CL, Briggs SL, isolation of all new hybridomas of interest in our labBaker, AL, Laguzza BC: In vivo efficacy of monoclonal antibodydrug conjugates of three different subisotypes which bind the huoratory.

ACKNOWLEDGMENTS The authors would like to thank Mr. John Daley for lending encouragement toward the initial acquisition of a n AUTO-CLONE, Ms. Kathy Knebel for useful discussions on AMIGG-FITC staining of hybridoma cells, Dr. Stephen Hatfield for assistance in the verification of single particle deposition by the AUTO-CLONE, and

man tumor-associated antigen defined by the KSlI4 antibody. Cancer Immunol 1.mrnunother 28:171-178, 1989. 18 Starling JJ, Sieg SM, Beckett ML, Schellhammer PF, Ladaga LE, Wright GL: Monoclonal antibodies to human prostate and bladder tumor-associated :antigens. Cancer Res 42:3084-3089, 1982. 19 Starling JJ, Simrell CR, Klein PA, Noonan KD: Production of monoclonal antibodies against a cell surface concanavalin A binding glycoprotein. ,I Supramol Struct 11:563-577, 1979 20 Underwood PA, Bean PA: Hazards of the limiting-dilution method of cloning hybridomas. J Immunol Methods 107:119-128, 1988.

SELECTIVE CLONING OF HYBRIDOMAS 21. Varki NM, Reisfeld RA, Walker LE: Antigens associated with a human lung adenocarcinoma defined by monoclonal antibodies. Cancer Res 44:681-687, 1984. 22. Velez D, Reuveny S, Miller L, Macmillan JD: Kinetics of monoclonal antibody production in low serum growth medium. J Immunol Methods 86:45-52, 1986. 23. Virella G, Munoz JK, Robinson J E J r , Goust JM: Assay of immu-

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noglobulins in supernatants of lymphoid cell lines by conventional laser nephelometry. Z Immunitaetsforsch Immunobiol 155: 279-286, 1979. 24. Westerwoudt R J , Naipal AM, Harrison CM: Improved fusion technique. 11. Stability and purity of hybrid clones. J Immunol Methods 68:89-101, 1984.