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SOLUBLE FIBROBLAST GROWTH REGULATOR
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[43] D e r i v a t i o n o f M o n o c l o n a l A n t i b o d y D i r e c t e d a g a i n s t Fibroblast Growth Regulator By JOHN L. WANG Introduction Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to a similar treatment with unconditioned medium. I This growth inhibitor activity was fractionated by ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography,Z'3 yielding one preparation designated FGR-s (13K), which stands for fibroblast growth regulator, soluble form. Analysis of the chemical and biological properties of this factor indicated that (1) it consists of a single polypeptide chain (Mr 13,000; pl -10) when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) under both reducing and nonreducing conditions; (2) it is an endogenous cell product, synthesized by the 3T3 cells and secreted or shed into the medium; (3) the dose-response curve of growth inhibition shows 50% inhibition at a concentration of approximately 3 ng/ml, corresponding to about 0.23 nM; and (4) it is not cytotoxic and its effects on target cells are reversible. Using a partially purified preparation of the growth inhibitor as immunogen, we have carried out in vitro immunization of rat splenocytes and have obtained a monoclonal antibody, designated 2A4, that specifically bound FGR-s (13K). 4 In this chapter, we describe in detail the derivation of the monoclonal antibody specifically directed against the FGR-s (13K) inhibitory protein. Isolation of Immunogen Fraction Cell Culture and Preparation of Conditioned Medium Swiss 3T3 fibroblasts (American Type Culture Collection, CCL92, Rockville, MD) are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 100/,~g/ml streptomycin, I p. A. Steck, P. G. Voss, and J, L. Wang, J. Cell Biol. 83, 562 (1979). z p. A. Steck, J. Blenis, P. G. Voss, and J. L. Wang, J. Cell Biol. 92, 523 (1982). 3 Y.-M. Hsu and J. L. Wang, J. Cell Biol. 102, 362 (1986). 4 Y.-M. Hsu, J. M. Barry, and J. L. Wang, Proc. Natl. Acad. Sci. U.S.A. 81, 2107 (1984).
METHODS IN ENZYMOLOGY.VOL. 198
Copyright ~ 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
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OTHER GROWTH FACTORS AND GROWTH INHIBITORS
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and 10% (v/v) calf serum (Microbiological Associates, Walkersville, MD). The cells are incubated in Corning plastic tissue culture flasks (Corning Glass Works, Nos. 25100, 25110, 25120, Corning, NY), maintained at 37° in a humidified atmosphere of 10% CO2. The 3T3 cells are passaged and used for a maximum of 3 months, after which they are discarded and a fresh sample grown from frozen cultures kept at - 8 0 °. Routinely, our stock culture of 3T3 cells is passaged every 2 days, before the cells reach a confluent state (5 x 10 4 cells/cm2). The growth medium is first removed, and the cells are washed and then incubated in phosphate-buffered saline (PBS) with 0.25% trypsin (Nutritional Biochemicals, Cleveland, OH) and 4 x 10 -4 M Versene at 37° for 10 min. The cells are dislodged from the growth surface, transferred, and centrifuged at 1320 g for 3 min. The trypsin solution is removed, and the cells are resuspended in DMEM-10% calf serum and seeded at the desired density. We have "conditioned" growth medium by exposing it to a confluent monolayer of cells and then tested its capacity to support growth and cell division in sparse cultures. Serum-free conditioned medium [CM(SF)] is prepared according to the protocol diagrammed in Fig. 1. Source cells, which are used to condition the medium, are seeded at an initial density of 2 x 104 cells/cm 2 and allowed to grow for at least 3 days. When cells reach a confluent monolayer, the medium is removed and "stimulation feeding" is performed by adding fresh DMEM-10% calf serum (0.1 ml/ c m 2 of growth surface). The purpose of this feeding is to induce one final round of cell division and assure a confluent monolayer. After 24 hr, the medium is again removed, and the cells are washed twice with serum-free DMEM. Then DMEM (I0 ml/150 c m 2 of growth area) is added to the cultures as medium to be conditioned. After 24 hr the medium is transferred and centrifuged at 1470 g for 10 min to remove cellular debris and particulate material. The supernatant is collected and used as CM(SF). In all experiments, serum-free unconditioned medium [UCM(SF)] is prepared in parallel from the same batch of DMEM and incubated under the same conditions, but in the absence of cells (Fig. 1). Radioactive growth inhibitor preparations are isolated from source cells cultured as described above. During the period of conditioning (Fig. 1), [35S]methionine is added (100 ~Ci/ml, 1000 Ci/mmol, Amersham Corp., Arlington Heights, IL) to DMEM containing unlabeled methionine at 3 /xg/ml (1/10 of the concentration of methionine normally found in DMEM).
Assays of Growth-Inhibitory Activity Target cells used to test growth-inhibitory activity are routinely seeded in DMEM containing 10% calf serum at an initial density of 5 x 103 cells/
[43]
SOLUBLE FIBROBLAST GROWTH REGULATOR
I Unconditioned Media ]
I Conditioned Media I
[Source Cells ] ~
+
453
(2 x 10 4cells/cm 2) 72 hr
GrowthMedia (0.1 ml/cm 2)
Confluent (4-5 x 104cells/cm2)
Stimulation Feeding ,~,~.~ ConditioningMedia
24hr DME (0-07ml/cm2)
"~
24 hr
i
No cells
Y media harvest
Centrifuge
CM (S
10 min
Unconditioned media harvest /
UCM (SF)
! ° ° ° ° c~)" "-
0000~ 0000C~
ooo.oq
24 hr
Assay
[ 3H]TdR incorporation protein content determination cell number determination
FIG. 1. Protocol used in the preparation of serum-flee conditioned medium [CM(SF)] and unconditioned medium [UCM(SF)]. Cell densities are expressed as the number of cells per square centimeter of growth surface in tissue culture dishes. The volumes of medium added at the various steps are expressed in this diagram as the milliliters of medium used per unit area of growth surface.
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OTHER GROWTH FACTORS AND GROWTH INHIBITORS
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cm z in a 96-well culture dish (Costar, No. 3596, Cambridge, MA). After overnight incubation, the cells are deprived of serum for 24 hr. Then the medium is removed, and the test fraction is added (75 tzl) along with 150 txl DMEM containing 5% (v/v) calf serum. DNA synthesis is assayed 24 hr later with a pulse of [3H]thymidine (l/zCi/culture, 1.9 Ci/mmol, SchwarzMann, Orangeburg, NY) for 3 hr at 37°. After the pulse the radioactive medium is removed, and the cells are washed 3 times with cold PBS and once with 10% trichloroacetic acid. The cells are then solubilized with 0.2 ml of 1% (w/v) SDS in 0.l N NaOH. After incubation at 37° for 10 min, cell lysates are added to 2 ml of scintillation cocktail [1 g dimethyl-l,4bis(2-5-phenyloxazolyl)benzene, 8 g 2,5-diphenyloxazole; 1333 ml Triton X-100, and 2666 ml of toluene] for scintillation counting. To assay increases in cell number after treatment with growth inhibitor fractions, target cells are prepared in the same fashion in 6- or 24-well culture dishes (Costar, No. 3406 or 3524). Cultures are then treated with the inhibitory fraction or a control fraction. At various times thereafter, the cells are washed 3 times with PBS and then removed from the growth surface by trypsin treatment. The cells are centrifuged, resuspended in PBS, diluted with trypan blue [0.08% (w/v) in PBS], and counted in a corpuscle counting chamber (Hausser Scientific, Blue Bell, PA). The viability of cells treated with inhibitor fractions is determined while the cells remain attached to the growth surface. After removal of growth medium, the cells are incubated with trypan blue for 10 min at room temperature. The staining solution is then removed, and the viable cells are counted using an Olympus inverted microscope.
Fractionation of Conditioned Medium Components Solid ammonium sulfate is added to serum-free conditioned medium to a saturation of 80% at room temperature. All subsequent operations are performed at 4°. The ammonium sulfate-precipitated mixture is centrifuged at 12,400 g for 15 min and the supernatant decanted. The precipitate collected from 800 ml of serum-free conditioned medium is resuspended in 2 ml of DMEM and fractionated on a column (1.4 × 110 cm) of Sephadex G-50 (Pharmacia, Piscataway, N J) equilibrated with DMEM. Fractions of 1.6 ml are collected. FGR-s represents the material eluting from the Sephadex G-50 column at a position corresponding to a molecular weight of 10,000-15,000. This region (Component C) contains material previously shown to be enriched in specific biological activity. 2 Polyacrylamide gel electrophoretic analysis in the presence of SDS is performed according to the procedure of LaemmliS; the acrylamide concentration of the running 5 U. K. Laemmli, Nature (London) 227, 680 (1970).
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gel is 16%. The gels, stained with the silver reagent, 6 show that the principal components of the FGR-s fraction are polypeptides of approximate Mr 10,000 and 13,000. This preparation is used for the immunization of rat spleen cultures for the generation of antibody-secreting cells, as well as for the screening of hybridoma clones. FGR-s can be further fractionated by ion-exchange chromatography on DEAE-cellulose. 3 To carry out ion-exchange chromatography, two minor modifications of the above procedure are made. First, gel filtration on Sephadex G-50 columns is performed in 5 mM Tris, pH 8.0, instead of DMEM. This allows the direct application of the effluent from the Sephadex column onto the ion-exchange column. Second, a wider range of fractions, centered approximately at FGR-s, is pooled and subjected to ion-exchange chromatography. The rationale for this is that we do not assay the individual fractions from the Sephadex G-50 column for activity or for polypeptide content on polyacrylamide gels. Therefore, the precise position corresponding to the FGR-s fractions is not determined and is compensated for by including material in fractions adjacent to FGR-s. This allows us to save material which would have been used in the assays and to save time, thereby minimizing losses of material owing to adsorption to test tubes. A column (0.8 x 2 cm) of DEAE-cellulose (Pharmacia) is equilibrated with 5 mM Tris, pH 8.0. The pooled material corresponding to the FGR-s fractions of the Sephadex G-50 column is applied to the ion-exchange column. After washing with starting buffer, a gradient (0 to 0.5 M NaC1 in 100 ml of 5 mM Tris, pH 8.0) is used to develop the column. Fractions of 1.7 ml are collected. When the fractions eluting from the ion-exchange column are assayed for growth-inhibitory activity, only the material flowing through the column (Component A) exhibits activity; the remainder of the components fail to show any appreciable activity. The sum of the growth-inhibitory activity in Component A accounts for 80% of the total activity applied to the column. There is a 6-fold enrichment in terms of specific activity in this fractionation step. Polyacrylamide gel electrophoresis analyses are carried out on the fractions derived from the DEAE-cellulose column. Component A yields a single polypeptide, migrating at a position corresponding to a molecular weight of 13,000. Identical results are obtained irrespective of whether the polyacrylamide gel electrophoresis is carried out under reducing [with 5% (v/v) 2-mercaptoethanol] or nonreducing conditions. Using known amounts of cytochrome c (Mr 12,500) as a standard for the silver staining
6 C. R. Merril, M. L. Dunau, and D. Goldman, Anal. Biochem. llO, 201 (1981).
456
O T H E R G R O W T H FACTORS A N D G R O W T H INHIB1TORS
180-200 gml female I
21-day-old female I I Thymocyte preparation 1 48-hr culture ~lr Conditioned] med um I
[43]
Confluent 3T3 cu ture FGR-s
Spleen cell preparation
• ~ 96-hr culture Harvest primed I spleen cell
INS-1 cells I
~ fusion
Selection of posit ve c ones FIG. 2. Procedure used for the generation of monoclonal antibodies directed against components of FGR-s usingthe in vitro immunizationmethod. technique, we estimate that I liter of conditioned medium derived from 3T3 cultures yields approximately 1/~g of the 13,000-dalton polypeptide. Generation of Hybridoma Cells In Vitro I m m u n i z a t i o n with F G R - s
The immunization procedure is adapted from Luben and Mohler 7 and is outlined in Fig. 2. Thymocytes from a 21-day-old female SpragueDawley rat (Charles River Co., Wilmington, MA) are isolated and used to condition the medium [5 x l06 cells/ml; 40 ml of DMEM containing 20% (v/v) fetal calf serum (Hazelton Dutchland, Denver, PA), 50/~M 2mercaptoethanol, 2 mM glutamine] for 48 hr at 37° in a humidified atmosphere of 10% COz. The cells are then removed by centrifugation (1320 g for 3 min). The supernatant medium is sterilized by filtration using a 0.2/~m filter. The conditioned medium, which is to be used for the culture of 7R. A. Luben and M. A. Mohler,Mol. lmmunol. 17, 635 (1980).
[43]
SOLUBLE FIBROBLAST GROWTH REGULATOR
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rat spleen cells in the presence of the immunogen, FGR-s, can be stored for up to 14 days at 4° . Spleen cells from a female Sprague-Dawley rat (-200 g) are suspended at a density of 1.0 × 10 7 cells/ml in DMEM containing 2 mM glutamine and 50/~M 2-mercaptoethanol. The spleen cell suspension (4 ml) is first mixed with 1 ml of FGR-s (-100 ng/ml) and incubated at 37°. After 45 min, 5 ml of thymocyte-conditioned medium (resupplemented with 2 mM glutamine and 50 txM 2-mercaptoethanol), 0.5 ml fetal calf serum, and 0.5 ml horse serum (GIBCO, Grand Island, NY) are added. The mixture is then cultured at 37° for 96 hr. The immune spleen cells are harvested and fused with the mouse myeloma cell line, P3/NS1/1-Ag4-1, as described below. Comment. The initial incubation of the immunogen fraction, FGR-s, with the splenocytes to be immunized in the absence of serum is an important modification of the general procedure as described by Luben and Mohler. 7 Because the amount of antigen available is low, we sought to maximize the probability of binding of the antigen by specific immunoglobulin receptors on the lymphocytes without interference by the large amounts of protein contained in serum. Thus, thymocyte-conditioned medium (which contains fetal calf serum) and freshly added fetal calf serum and horse serum are introduced only after the immunogen has had a chance to interact with the lymphocytes. Although the initial incubation in the absence of serum may result in a decrease of viability of the lymphocytes, it has been our experience that successful immunization with limiting amounts of antigen (e.g., -~ 100 ng) could best be achieved by this serumfree exposure to the lymphocytes.
In Vivo Immunization of Rats with 3T3 Cells We have also carried out immunization of rats with 3T3 cells to generate monoclonal antibodies that bind to whole 3T3 cells but are not specifically directed against the FGR-s fraction. Confluent monolayers of 3T3 cells (5 × 10 4 cells/cm 2) are scraped with a rubber policeman and washed with 10 ml of PBS. The cells are resuspended in PBS at a density of 1.0 x 10 7 cells/ml and emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI). Female Sprague-Dawley rats (-200 g) are immunized intraperitoneaUy with l-ml aliquots of the emulsion. The rats are given booster injections of similar doses in Freund's incomplete adjuvant at days 8 and 31. When the serum from an immunized rat is assayed as positive in the primary screening assay (see below), the rat is boosted again and sacrificed 7 days later. Immune spleen cells are prepared and fused with mouse myeloma cells.
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OTHER GROWTH FACTORS AND GROWTH INHIBITORS
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Fusion of Immune Spleen Cells with Myeloma Cells Procedures for the preparation of the fusion partners to generate hybridomas, for the fusion event, and for selection of hybrid cells in hypoxanthine-aminopterin-thymidine medium have been detailed in this series, 8 as well as in a volume on general immunological methods. 9 We follow these procedures closely. The mouse myeloma cell line we use is P3/ NSI/1-Ag4-1 (commonly known as NS-I) and is obtained from the Cell Distribution Center of The Salk Institute (La Jolla, CA). It is checked for continued resistance to 8-azaguanine prior to use. NS-1 cells in log-phase growth (-105 cells/ml) with a viability greater than 95% (trypan blue exclusion assay 9) are used. Immune spleen cells (from either in vitro immunization with FGR-s or in vivo immunization with 3T3 cells) and NS-1 myeloma cells are mixed at a ratio of 10:1 in a 50-ml centrifuge tube and centrifuged at 400 g for 10 min at room temperature to form a tight pellet. Polyethylene glycol 1500 (Gallard Schlessinger, Carle Place, NY) is added as a 50% (w/w) solution as detailed. 9 After fusion and washing, the cell suspension is distributed into individual wells of 96-well culture dishes (Costar, No. 3596) to yield master plates for selection. 9 After selection, individual cultures are screened for production of antibodies directed against 3T3 cells using the primary screening assay (see below). Positive cultures are expanded by transferring cells to l-ml cultures in 24-wel! dishes (Costar, No. 3524). Cells in positive cultures after two rounds of expansion are cloned in soft agar. 9 Screening for Relevant Antibody-Producing Hybridomas
Primary Screening Assay Antisera from the immunized rats or supernatants from selected hybridoma cultures are tested by the primary screening assay (Fig. 3). 3T3 cells (3 x 105 cells/well) are seeded in 96-well polyvinyl plates (Costar, No. 2595) and cultured at 37 ° for 24 hr. All further steps are done at room temperature. The attached 3T3 cells are fixed with 200 tzl of 0.25% (w/v) glutaraldehyde in PBS for 10 min. The cells are washed 3 times in PBS, and 200/zl of bovine serum albumin [3% (w/v), BSA] in PBS is added, incubated for 1 hr, and then washed as above. Antisera or supernatants from hybridoma cultures are added (50/xl), incubated for I hr, and washed. 8 G. Galfr~ and C. Milstein, this series, Vol. 73, p. 3. 9 B. B. Mishell and S. M. Shiigi, " S e l e c t e d M e t h o d s in Cellular I m m u n o l o g y . " Freeman, San Francisco, California, 1980.
[43]
SOLUBLE FIBROBLASTGROWTH REGULATOR
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3T3cells seed on ] 96-well plate
Fixed with 0.25% glutaraldehyde I
l Wash off glutaraldehyde, 1 Incubate with 3% BSA
Wash off BSA incubate with 50 p.I of 1° Antibody i
Wash off 1° Antibody, IgG incubate with rabbit anti-rat 1251-1abeled
,l Wash off unbound IgG
rabbit anti-rat 251-1abeled
by,O,oSOS
,he,,ex,b,ep,ate
I 12Slcounting Fie. 3. Outline of the primary screening assay. The primary ( I°) antibody is either serum or supernatant from hybridoma cultures. Finally, rabbit anti-rat 125I-labeled immunoglobulin [5 × 108 counts/min (cpm)/mg; 1 × 105 cpm; 50/zl] is added and incubated for 1 hr. After unbound radioactivity is removed by washing, the polyvinyl plates are cut into individual wells, and the amount of radioactivity in each well is measured using a LKB RIA y counter. This assay detects, in general, any rat antibody reactive against any component of 3T3 cells.
460
O T H E R G R O W T H FACTORS A N D G R O W T H I N H I B I T O R S
/q~
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30
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FIG. 4. Effect of serial dilution of immune serum in the primary screening assay. Immune and preimmune sera were serially diluted in PBS as indicated and then tested in the primary screening assay.
Figure 4 shows the results o f the p r i m a r y screening assay using the serum collected f r o m the rat immunized in uiuo with 3T3 cells whose spleen cells were subsequently used for fusion. Serial dilutions of the immune serum (1 : 128 to I : 2048) result in a linear decrese of the binding of z25I-labeled antibody. On the other hand, higher concentrations of immune serum (1 : 64 and I : 32 dilutions) also show lower binding. The reason for this decrease at high concentrations is not known. Nonetheless, it is obvious that the primary screening a s s a y can clearly distinguish normal serum (preimmune) from serum containing antibodies directed against 3T3 cells. H y b r i d o m a s which produce antibodies reacting with fixed 3T3 cells are detected by the p r i m a r y screening a s s a y and are cloned in soft agar.
[43]
SOLUBLE FIBROBLAST GROWTH REGULATOR
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The clones and their secreted products are numbered according to the sequence of establishment of the clones.4 Supernatants from the clones are tested using the primary screening assay; the clones exhibit 1.7- to 4.4fold greater binding activity compared to that seen using the supernatant from the parental myeloma NS-1 cell line.
Screening Assays for Hybridoma Cultures Producing Antibodies Directed against FGR-s A modification of the primary screening assay has been developed to detect hybridoma clones secreting antibodies directed specifically against a partially purified protein fraction (e.g., FRG-s in place of 3T3 cells in Fig. 3). This assay requires the efficient adsorption of the specific antigen fraction onto solid surfaces to minimize the amount of antigen required during the screening process. Moreover, because of the limited amount of material available, this screening assay is used only on hybridoma clones that show a positive reaction in the primary screening assay, thus indicating that these clones may be "putative positives" in terms of being clones which secrete monoclonal antibodies directed against our antigen of interest, FRG-s. Unlabeled FGR-s (200 ~1 of 100 ng/ml solution) is added to individual wells of Immulon-2 micro-ELISA plates (Dynatech, Alexandria, VA). Approximately 8 ng ( - 4 0 % of amount added) binds to each well. The wells are washed 3 times in PBS, and BSA (3% w/v) in PBS is then added and incubated for 1 hr. The wells are washed again as above. Supernatants (50 t~l) from hybridoma cultures or immunoglobulins purified from such supernatants are added and incubated for 1 hr. Finally, rabbit ~25I-labeled antibodies directed against rat immunoglobulin (5 x 108 cpm/mg; 1 x 105 cpm; 50 ~l) are added and incubated for 1 hr. After unbound radioactivity is removed by washing, the plates are cut into individual wells and the amount of radioactivity in each well is measured in a y counter. The supernatants from three hybridoma lines, designated DC4, 3C9, and 2A4, show positive reactions when assayed with FGR-s on Immulon2 plates. Compared to the supernatant of NS-1 myeloma cultures, each of the hybridoma supernatants exhibit 1.5- to 1.7-fold more binding. This binding is specific. When bovine serum albumin is substituted for FGR-s on the Immulon-2 plates, the binding of the supernatants from DC4, 3C9, 2A4, and NS-I cells are all the same. The supernatants of clones 2A4 and 3C9 are also positive when assayed with whole 3T3 cells fixed to the plates, exhibiting 1.4- to 1.6-fold more binding than the corresponding supernatant from NS-I cells. 4 Quite the opposite results are obtained with hybridoma clones derived
462
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
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from rats immunized in vivo with whole 3T3 cells and screened and selected on the basis of binding to whole 3T3 cells. A representative clone, designated 104, is used here for illustrative purposes. Clone 104 shows a strong reaction (2.4-fold) when assayed on whole 3T3 cells but a negligible reaction when assayed on FGR-s. These results suggest that the products of clones 2A4 and 3C9 are monoclonal antibodies directed against some component of FGR-s, which, in turn, is a constituent of whole 3T3 cells. 4
Properties of Monoclonal Antibody 2A4
Isolation and Character&ation of Antibody 2A4 Rabbit antibodies directed against rat immunoglobin are coupled to Affi-Gel 10 (Bio-Rad, Richmond, CA) using the procedure of Ikeda and Steiner.m Supernatant from the hybridoma clone 2A4 is passed through the column (l x 12 cm) 3 times. Material bound nonspecifically is removed by washing with 0.1 M phosphate buffer (pH 8.0). The specifically bound material is eluted with 0.1 M citrate buffer (pH 3.0), dialyzed overnight against PBS with 3 changes of buffer, and subjected to SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol (5%). Two polypeptides, corresponding to the heavy (Mr 55,000) and light (M r 23,000) chains of immunog|obulin G, are observed. All subsequent experiments are performed wtih the purified IgG molecule, designated Antibody 2A4. The isolated Antibody 2A4 is stored at - 2 0 °. Similar results are obtained with the antibody secreted by hybridoma clone 104. This rat lgG molecule is also purified, and Antibody 104 is used in all experiments parallel to Antibody 2A4 as a control antibody.
Identification of Target of Antibody 2A4 Antibody 2A4 is coupled to Affi-Gel 10 as described above, m The affinity column (0.6 x 4 cm) is used to fractionate FGR-s preparations to determine the molecular identity of the antigenic target of Antibody 2A4. When a [35S]methionine-labeled preparation of FGR-s is fractionated over this Antibody 2A4 column, the unbound flow-through fraction yields one predominant polypeptide (Mr 10,000). The material bound by Antibody 2A4 also shows one polypeptide band (Mr 13,000). These results indicate that Antibody 2A4 is specifically directed against the M r 13,000 polypeptide in the FGR-s fraction. 4 to y . lkeda and M. Steiner, J. Biol. Chem. 251, 6135 (1976).
[43]
SOLUBLE EIBROBLAST GROWTH REGULATOR
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Effect of Antibody 2A4 on Growth-Inhibitory Activity of FGR-s To test the possibility that Antibody 2A4 can neutralize the growthinhibitory activity of FGR-s, the effect of the inhibitor preparation on [3H]thymidine incorporation in target 3T3 cells is assayed in the presence and absence of the purified antibody. The growth-inhibitory effect of FGR-s on 3T3 cells is dependent on the concentration of the ligand added. 2 In the present assay, 60% inhibition is obtained with 20/zl of the FGR-s preparation. When the effect of FGR-s is assayed in the presence of Antibody 2A4 (25 ng/ml), however, the inhibition is completely abrogated. The level of DNA synthesis in this case is the same as that of control cultures, without any FGR-s or Antibody 2A4. Similarly, the addition of 25 ng/ml of Antibody 2A4 reduces the inhibitory effect of 50/xl of FGR-s from 80 to 35%. Thus, when the effect of FGR-s is assayed in the presence of Antibody 2A4, there is always a higher level of [SH]thymidine incorporation (i.e., a reduced level of growth inhibition). The effects of Antibody 2A4 on reversing the inhibition of FGR-s are specific. Antibody 104, which is not reactive with FGR-s polypeptides, fails to yield the same effect. These observations suggest that the results obtained with Antibody 2A4 are most probably not due to a growth factor contaminating the immunoglobulin fraction. The results on the neutralization of growth-inhibitory activity of FGR-s by Antibody 2A4, coupled with the fact that this antibody specifically recognizes the M r 13,000 polypeptide, strongly suggest that this polypeptide carries growth-inhibitory activity. 4 This conclusion is further supported by experiments that showed depletion of the growth-inhibitory activity from preparations of FGR-s (13K) on Antibody 2A4 affinity columns.
Depletion of Growth-Inhibitory Activity from FGR-s (13K) on Affinity Column Containing Antibody 2A4 Rabbit antibodies directed against rat immunoglobulin (40 rag) are coupled to Affi-Gel 10 (2 ml of beads). 1° The derivatized gel is used to prepare two different columns: (I) the supernatant (50 ml) of hybridoma clone 2A4 is passed over one column (0.4 x 2.5 cm) 3 times to bind Antibody 2A4 (Antibody 2A4 column); (2) the supernatant of the parent myeloma NS-I cell line is passed over the other column (NS-1 control column). Material bound nonspecificaily is removed by washing with 0. ! M phosphate buffer (pH 8.0). Finally, a preparation of FGR-s (13K) (8 ng/ ml, 2 ml) is percolated through either the Antibody 2A4 column or the NS1 control column. The original FGR-s (13K) preparation, as well as the pooled flow-through fractions, representing material not bound by the respective affinity columns, are assayed for growth-inhibitory activity.
464
O T H E R G R O W T H FACTORS A N D G R O W T H I N H I B I T O R S
[43]
The results show that the growth-inhibitory activity of FGR-s (13K) is depleted in the material passed through the Antibody 2A4 column. 3 In contrast, the NS-I control column has little effect on the growth-inhibitory activity. These results strongly suggest that FGR-s (13K) is directly responsible for the observed growth-inhibitory activity. The data also argue against any indirect mechanisms, such as stimulation of a growth factor receptor by Antibody 2A4, in the observed neutralization of FGR-s effects in the growth-inhibition assays. 4 Acknowledgments This work was supported by Grant GM 27203 from the National Institutes of Health and by Faculty Research Award FRA-221 from the American Cancer Society. 1 thank Dr. YenMing Hsu for all his help and Mrs. Linda Lang for the preparation of the manuscript.
SOLUBLE FIBROBLAST GROWTH REGULATOR
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[43] D e r i v a t i o n o f M o n o c l o n a l A n t i b o d y D i r e c t e d a g a i n s t Fibroblast Growth Regulator By JOHN L. WANG Introduction Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to a similar treatment with unconditioned medium. I This growth inhibitor activity was fractionated by ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography,Z'3 yielding one preparation designated FGR-s (13K), which stands for fibroblast growth regulator, soluble form. Analysis of the chemical and biological properties of this factor indicated that (1) it consists of a single polypeptide chain (Mr 13,000; pl -10) when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) under both reducing and nonreducing conditions; (2) it is an endogenous cell product, synthesized by the 3T3 cells and secreted or shed into the medium; (3) the dose-response curve of growth inhibition shows 50% inhibition at a concentration of approximately 3 ng/ml, corresponding to about 0.23 nM; and (4) it is not cytotoxic and its effects on target cells are reversible. Using a partially purified preparation of the growth inhibitor as immunogen, we have carried out in vitro immunization of rat splenocytes and have obtained a monoclonal antibody, designated 2A4, that specifically bound FGR-s (13K). 4 In this chapter, we describe in detail the derivation of the monoclonal antibody specifically directed against the FGR-s (13K) inhibitory protein. Isolation of Immunogen Fraction Cell Culture and Preparation of Conditioned Medium Swiss 3T3 fibroblasts (American Type Culture Collection, CCL92, Rockville, MD) are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 100/,~g/ml streptomycin, I p. A. Steck, P. G. Voss, and J, L. Wang, J. Cell Biol. 83, 562 (1979). z p. A. Steck, J. Blenis, P. G. Voss, and J. L. Wang, J. Cell Biol. 92, 523 (1982). 3 Y.-M. Hsu and J. L. Wang, J. Cell Biol. 102, 362 (1986). 4 Y.-M. Hsu, J. M. Barry, and J. L. Wang, Proc. Natl. Acad. Sci. U.S.A. 81, 2107 (1984).
METHODS IN ENZYMOLOGY.VOL. 198
Copyright ~ 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
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and 10% (v/v) calf serum (Microbiological Associates, Walkersville, MD). The cells are incubated in Corning plastic tissue culture flasks (Corning Glass Works, Nos. 25100, 25110, 25120, Corning, NY), maintained at 37° in a humidified atmosphere of 10% CO2. The 3T3 cells are passaged and used for a maximum of 3 months, after which they are discarded and a fresh sample grown from frozen cultures kept at - 8 0 °. Routinely, our stock culture of 3T3 cells is passaged every 2 days, before the cells reach a confluent state (5 x 10 4 cells/cm2). The growth medium is first removed, and the cells are washed and then incubated in phosphate-buffered saline (PBS) with 0.25% trypsin (Nutritional Biochemicals, Cleveland, OH) and 4 x 10 -4 M Versene at 37° for 10 min. The cells are dislodged from the growth surface, transferred, and centrifuged at 1320 g for 3 min. The trypsin solution is removed, and the cells are resuspended in DMEM-10% calf serum and seeded at the desired density. We have "conditioned" growth medium by exposing it to a confluent monolayer of cells and then tested its capacity to support growth and cell division in sparse cultures. Serum-free conditioned medium [CM(SF)] is prepared according to the protocol diagrammed in Fig. 1. Source cells, which are used to condition the medium, are seeded at an initial density of 2 x 104 cells/cm 2 and allowed to grow for at least 3 days. When cells reach a confluent monolayer, the medium is removed and "stimulation feeding" is performed by adding fresh DMEM-10% calf serum (0.1 ml/ c m 2 of growth surface). The purpose of this feeding is to induce one final round of cell division and assure a confluent monolayer. After 24 hr, the medium is again removed, and the cells are washed twice with serum-free DMEM. Then DMEM (I0 ml/150 c m 2 of growth area) is added to the cultures as medium to be conditioned. After 24 hr the medium is transferred and centrifuged at 1470 g for 10 min to remove cellular debris and particulate material. The supernatant is collected and used as CM(SF). In all experiments, serum-free unconditioned medium [UCM(SF)] is prepared in parallel from the same batch of DMEM and incubated under the same conditions, but in the absence of cells (Fig. 1). Radioactive growth inhibitor preparations are isolated from source cells cultured as described above. During the period of conditioning (Fig. 1), [35S]methionine is added (100 ~Ci/ml, 1000 Ci/mmol, Amersham Corp., Arlington Heights, IL) to DMEM containing unlabeled methionine at 3 /xg/ml (1/10 of the concentration of methionine normally found in DMEM).
Assays of Growth-Inhibitory Activity Target cells used to test growth-inhibitory activity are routinely seeded in DMEM containing 10% calf serum at an initial density of 5 x 103 cells/
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SOLUBLE FIBROBLAST GROWTH REGULATOR
I Unconditioned Media ]
I Conditioned Media I
[Source Cells ] ~
+
453
(2 x 10 4cells/cm 2) 72 hr
GrowthMedia (0.1 ml/cm 2)
Confluent (4-5 x 104cells/cm2)
Stimulation Feeding ,~,~.~ ConditioningMedia
24hr DME (0-07ml/cm2)
"~
24 hr
i
No cells
Y media harvest
Centrifuge
CM (S
10 min
Unconditioned media harvest /
UCM (SF)
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24 hr
Assay
[ 3H]TdR incorporation protein content determination cell number determination
FIG. 1. Protocol used in the preparation of serum-flee conditioned medium [CM(SF)] and unconditioned medium [UCM(SF)]. Cell densities are expressed as the number of cells per square centimeter of growth surface in tissue culture dishes. The volumes of medium added at the various steps are expressed in this diagram as the milliliters of medium used per unit area of growth surface.
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OTHER GROWTH FACTORS AND GROWTH INHIBITORS
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cm z in a 96-well culture dish (Costar, No. 3596, Cambridge, MA). After overnight incubation, the cells are deprived of serum for 24 hr. Then the medium is removed, and the test fraction is added (75 tzl) along with 150 txl DMEM containing 5% (v/v) calf serum. DNA synthesis is assayed 24 hr later with a pulse of [3H]thymidine (l/zCi/culture, 1.9 Ci/mmol, SchwarzMann, Orangeburg, NY) for 3 hr at 37°. After the pulse the radioactive medium is removed, and the cells are washed 3 times with cold PBS and once with 10% trichloroacetic acid. The cells are then solubilized with 0.2 ml of 1% (w/v) SDS in 0.l N NaOH. After incubation at 37° for 10 min, cell lysates are added to 2 ml of scintillation cocktail [1 g dimethyl-l,4bis(2-5-phenyloxazolyl)benzene, 8 g 2,5-diphenyloxazole; 1333 ml Triton X-100, and 2666 ml of toluene] for scintillation counting. To assay increases in cell number after treatment with growth inhibitor fractions, target cells are prepared in the same fashion in 6- or 24-well culture dishes (Costar, No. 3406 or 3524). Cultures are then treated with the inhibitory fraction or a control fraction. At various times thereafter, the cells are washed 3 times with PBS and then removed from the growth surface by trypsin treatment. The cells are centrifuged, resuspended in PBS, diluted with trypan blue [0.08% (w/v) in PBS], and counted in a corpuscle counting chamber (Hausser Scientific, Blue Bell, PA). The viability of cells treated with inhibitor fractions is determined while the cells remain attached to the growth surface. After removal of growth medium, the cells are incubated with trypan blue for 10 min at room temperature. The staining solution is then removed, and the viable cells are counted using an Olympus inverted microscope.
Fractionation of Conditioned Medium Components Solid ammonium sulfate is added to serum-free conditioned medium to a saturation of 80% at room temperature. All subsequent operations are performed at 4°. The ammonium sulfate-precipitated mixture is centrifuged at 12,400 g for 15 min and the supernatant decanted. The precipitate collected from 800 ml of serum-free conditioned medium is resuspended in 2 ml of DMEM and fractionated on a column (1.4 × 110 cm) of Sephadex G-50 (Pharmacia, Piscataway, N J) equilibrated with DMEM. Fractions of 1.6 ml are collected. FGR-s represents the material eluting from the Sephadex G-50 column at a position corresponding to a molecular weight of 10,000-15,000. This region (Component C) contains material previously shown to be enriched in specific biological activity. 2 Polyacrylamide gel electrophoretic analysis in the presence of SDS is performed according to the procedure of LaemmliS; the acrylamide concentration of the running 5 U. K. Laemmli, Nature (London) 227, 680 (1970).
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SOLUBLE FIBROBLAST GROWTH REGULATOR
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gel is 16%. The gels, stained with the silver reagent, 6 show that the principal components of the FGR-s fraction are polypeptides of approximate Mr 10,000 and 13,000. This preparation is used for the immunization of rat spleen cultures for the generation of antibody-secreting cells, as well as for the screening of hybridoma clones. FGR-s can be further fractionated by ion-exchange chromatography on DEAE-cellulose. 3 To carry out ion-exchange chromatography, two minor modifications of the above procedure are made. First, gel filtration on Sephadex G-50 columns is performed in 5 mM Tris, pH 8.0, instead of DMEM. This allows the direct application of the effluent from the Sephadex column onto the ion-exchange column. Second, a wider range of fractions, centered approximately at FGR-s, is pooled and subjected to ion-exchange chromatography. The rationale for this is that we do not assay the individual fractions from the Sephadex G-50 column for activity or for polypeptide content on polyacrylamide gels. Therefore, the precise position corresponding to the FGR-s fractions is not determined and is compensated for by including material in fractions adjacent to FGR-s. This allows us to save material which would have been used in the assays and to save time, thereby minimizing losses of material owing to adsorption to test tubes. A column (0.8 x 2 cm) of DEAE-cellulose (Pharmacia) is equilibrated with 5 mM Tris, pH 8.0. The pooled material corresponding to the FGR-s fractions of the Sephadex G-50 column is applied to the ion-exchange column. After washing with starting buffer, a gradient (0 to 0.5 M NaC1 in 100 ml of 5 mM Tris, pH 8.0) is used to develop the column. Fractions of 1.7 ml are collected. When the fractions eluting from the ion-exchange column are assayed for growth-inhibitory activity, only the material flowing through the column (Component A) exhibits activity; the remainder of the components fail to show any appreciable activity. The sum of the growth-inhibitory activity in Component A accounts for 80% of the total activity applied to the column. There is a 6-fold enrichment in terms of specific activity in this fractionation step. Polyacrylamide gel electrophoresis analyses are carried out on the fractions derived from the DEAE-cellulose column. Component A yields a single polypeptide, migrating at a position corresponding to a molecular weight of 13,000. Identical results are obtained irrespective of whether the polyacrylamide gel electrophoresis is carried out under reducing [with 5% (v/v) 2-mercaptoethanol] or nonreducing conditions. Using known amounts of cytochrome c (Mr 12,500) as a standard for the silver staining
6 C. R. Merril, M. L. Dunau, and D. Goldman, Anal. Biochem. llO, 201 (1981).
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O T H E R G R O W T H FACTORS A N D G R O W T H INHIB1TORS
180-200 gml female I
21-day-old female I I Thymocyte preparation 1 48-hr culture ~lr Conditioned] med um I
[43]
Confluent 3T3 cu ture FGR-s
Spleen cell preparation
• ~ 96-hr culture Harvest primed I spleen cell
INS-1 cells I
~ fusion
Selection of posit ve c ones FIG. 2. Procedure used for the generation of monoclonal antibodies directed against components of FGR-s usingthe in vitro immunizationmethod. technique, we estimate that I liter of conditioned medium derived from 3T3 cultures yields approximately 1/~g of the 13,000-dalton polypeptide. Generation of Hybridoma Cells In Vitro I m m u n i z a t i o n with F G R - s
The immunization procedure is adapted from Luben and Mohler 7 and is outlined in Fig. 2. Thymocytes from a 21-day-old female SpragueDawley rat (Charles River Co., Wilmington, MA) are isolated and used to condition the medium [5 x l06 cells/ml; 40 ml of DMEM containing 20% (v/v) fetal calf serum (Hazelton Dutchland, Denver, PA), 50/~M 2mercaptoethanol, 2 mM glutamine] for 48 hr at 37° in a humidified atmosphere of 10% COz. The cells are then removed by centrifugation (1320 g for 3 min). The supernatant medium is sterilized by filtration using a 0.2/~m filter. The conditioned medium, which is to be used for the culture of 7R. A. Luben and M. A. Mohler,Mol. lmmunol. 17, 635 (1980).
[43]
SOLUBLE FIBROBLAST GROWTH REGULATOR
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rat spleen cells in the presence of the immunogen, FGR-s, can be stored for up to 14 days at 4° . Spleen cells from a female Sprague-Dawley rat (-200 g) are suspended at a density of 1.0 × 10 7 cells/ml in DMEM containing 2 mM glutamine and 50/~M 2-mercaptoethanol. The spleen cell suspension (4 ml) is first mixed with 1 ml of FGR-s (-100 ng/ml) and incubated at 37°. After 45 min, 5 ml of thymocyte-conditioned medium (resupplemented with 2 mM glutamine and 50 txM 2-mercaptoethanol), 0.5 ml fetal calf serum, and 0.5 ml horse serum (GIBCO, Grand Island, NY) are added. The mixture is then cultured at 37° for 96 hr. The immune spleen cells are harvested and fused with the mouse myeloma cell line, P3/NS1/1-Ag4-1, as described below. Comment. The initial incubation of the immunogen fraction, FGR-s, with the splenocytes to be immunized in the absence of serum is an important modification of the general procedure as described by Luben and Mohler. 7 Because the amount of antigen available is low, we sought to maximize the probability of binding of the antigen by specific immunoglobulin receptors on the lymphocytes without interference by the large amounts of protein contained in serum. Thus, thymocyte-conditioned medium (which contains fetal calf serum) and freshly added fetal calf serum and horse serum are introduced only after the immunogen has had a chance to interact with the lymphocytes. Although the initial incubation in the absence of serum may result in a decrease of viability of the lymphocytes, it has been our experience that successful immunization with limiting amounts of antigen (e.g., -~ 100 ng) could best be achieved by this serumfree exposure to the lymphocytes.
In Vivo Immunization of Rats with 3T3 Cells We have also carried out immunization of rats with 3T3 cells to generate monoclonal antibodies that bind to whole 3T3 cells but are not specifically directed against the FGR-s fraction. Confluent monolayers of 3T3 cells (5 × 10 4 cells/cm 2) are scraped with a rubber policeman and washed with 10 ml of PBS. The cells are resuspended in PBS at a density of 1.0 x 10 7 cells/ml and emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI). Female Sprague-Dawley rats (-200 g) are immunized intraperitoneaUy with l-ml aliquots of the emulsion. The rats are given booster injections of similar doses in Freund's incomplete adjuvant at days 8 and 31. When the serum from an immunized rat is assayed as positive in the primary screening assay (see below), the rat is boosted again and sacrificed 7 days later. Immune spleen cells are prepared and fused with mouse myeloma cells.
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OTHER GROWTH FACTORS AND GROWTH INHIBITORS
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Fusion of Immune Spleen Cells with Myeloma Cells Procedures for the preparation of the fusion partners to generate hybridomas, for the fusion event, and for selection of hybrid cells in hypoxanthine-aminopterin-thymidine medium have been detailed in this series, 8 as well as in a volume on general immunological methods. 9 We follow these procedures closely. The mouse myeloma cell line we use is P3/ NSI/1-Ag4-1 (commonly known as NS-I) and is obtained from the Cell Distribution Center of The Salk Institute (La Jolla, CA). It is checked for continued resistance to 8-azaguanine prior to use. NS-1 cells in log-phase growth (-105 cells/ml) with a viability greater than 95% (trypan blue exclusion assay 9) are used. Immune spleen cells (from either in vitro immunization with FGR-s or in vivo immunization with 3T3 cells) and NS-1 myeloma cells are mixed at a ratio of 10:1 in a 50-ml centrifuge tube and centrifuged at 400 g for 10 min at room temperature to form a tight pellet. Polyethylene glycol 1500 (Gallard Schlessinger, Carle Place, NY) is added as a 50% (w/w) solution as detailed. 9 After fusion and washing, the cell suspension is distributed into individual wells of 96-well culture dishes (Costar, No. 3596) to yield master plates for selection. 9 After selection, individual cultures are screened for production of antibodies directed against 3T3 cells using the primary screening assay (see below). Positive cultures are expanded by transferring cells to l-ml cultures in 24-wel! dishes (Costar, No. 3524). Cells in positive cultures after two rounds of expansion are cloned in soft agar. 9 Screening for Relevant Antibody-Producing Hybridomas
Primary Screening Assay Antisera from the immunized rats or supernatants from selected hybridoma cultures are tested by the primary screening assay (Fig. 3). 3T3 cells (3 x 105 cells/well) are seeded in 96-well polyvinyl plates (Costar, No. 2595) and cultured at 37 ° for 24 hr. All further steps are done at room temperature. The attached 3T3 cells are fixed with 200 tzl of 0.25% (w/v) glutaraldehyde in PBS for 10 min. The cells are washed 3 times in PBS, and 200/zl of bovine serum albumin [3% (w/v), BSA] in PBS is added, incubated for 1 hr, and then washed as above. Antisera or supernatants from hybridoma cultures are added (50/xl), incubated for I hr, and washed. 8 G. Galfr~ and C. Milstein, this series, Vol. 73, p. 3. 9 B. B. Mishell and S. M. Shiigi, " S e l e c t e d M e t h o d s in Cellular I m m u n o l o g y . " Freeman, San Francisco, California, 1980.
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SOLUBLE FIBROBLASTGROWTH REGULATOR
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3T3cells seed on ] 96-well plate
Fixed with 0.25% glutaraldehyde I
l Wash off glutaraldehyde, 1 Incubate with 3% BSA
Wash off BSA incubate with 50 p.I of 1° Antibody i
Wash off 1° Antibody, IgG incubate with rabbit anti-rat 1251-1abeled
,l Wash off unbound IgG
rabbit anti-rat 251-1abeled
by,O,oSOS
,he,,ex,b,ep,ate
I 12Slcounting Fie. 3. Outline of the primary screening assay. The primary ( I°) antibody is either serum or supernatant from hybridoma cultures. Finally, rabbit anti-rat 125I-labeled immunoglobulin [5 × 108 counts/min (cpm)/mg; 1 × 105 cpm; 50/zl] is added and incubated for 1 hr. After unbound radioactivity is removed by washing, the polyvinyl plates are cut into individual wells, and the amount of radioactivity in each well is measured using a LKB RIA y counter. This assay detects, in general, any rat antibody reactive against any component of 3T3 cells.
460
O T H E R G R O W T H FACTORS A N D G R O W T H I N H I B I T O R S
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FIG. 4. Effect of serial dilution of immune serum in the primary screening assay. Immune and preimmune sera were serially diluted in PBS as indicated and then tested in the primary screening assay.
Figure 4 shows the results o f the p r i m a r y screening assay using the serum collected f r o m the rat immunized in uiuo with 3T3 cells whose spleen cells were subsequently used for fusion. Serial dilutions of the immune serum (1 : 128 to I : 2048) result in a linear decrese of the binding of z25I-labeled antibody. On the other hand, higher concentrations of immune serum (1 : 64 and I : 32 dilutions) also show lower binding. The reason for this decrease at high concentrations is not known. Nonetheless, it is obvious that the primary screening a s s a y can clearly distinguish normal serum (preimmune) from serum containing antibodies directed against 3T3 cells. H y b r i d o m a s which produce antibodies reacting with fixed 3T3 cells are detected by the p r i m a r y screening a s s a y and are cloned in soft agar.
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SOLUBLE FIBROBLAST GROWTH REGULATOR
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The clones and their secreted products are numbered according to the sequence of establishment of the clones.4 Supernatants from the clones are tested using the primary screening assay; the clones exhibit 1.7- to 4.4fold greater binding activity compared to that seen using the supernatant from the parental myeloma NS-1 cell line.
Screening Assays for Hybridoma Cultures Producing Antibodies Directed against FGR-s A modification of the primary screening assay has been developed to detect hybridoma clones secreting antibodies directed specifically against a partially purified protein fraction (e.g., FRG-s in place of 3T3 cells in Fig. 3). This assay requires the efficient adsorption of the specific antigen fraction onto solid surfaces to minimize the amount of antigen required during the screening process. Moreover, because of the limited amount of material available, this screening assay is used only on hybridoma clones that show a positive reaction in the primary screening assay, thus indicating that these clones may be "putative positives" in terms of being clones which secrete monoclonal antibodies directed against our antigen of interest, FRG-s. Unlabeled FGR-s (200 ~1 of 100 ng/ml solution) is added to individual wells of Immulon-2 micro-ELISA plates (Dynatech, Alexandria, VA). Approximately 8 ng ( - 4 0 % of amount added) binds to each well. The wells are washed 3 times in PBS, and BSA (3% w/v) in PBS is then added and incubated for 1 hr. The wells are washed again as above. Supernatants (50 t~l) from hybridoma cultures or immunoglobulins purified from such supernatants are added and incubated for 1 hr. Finally, rabbit ~25I-labeled antibodies directed against rat immunoglobulin (5 x 108 cpm/mg; 1 x 105 cpm; 50 ~l) are added and incubated for 1 hr. After unbound radioactivity is removed by washing, the plates are cut into individual wells and the amount of radioactivity in each well is measured in a y counter. The supernatants from three hybridoma lines, designated DC4, 3C9, and 2A4, show positive reactions when assayed with FGR-s on Immulon2 plates. Compared to the supernatant of NS-1 myeloma cultures, each of the hybridoma supernatants exhibit 1.5- to 1.7-fold more binding. This binding is specific. When bovine serum albumin is substituted for FGR-s on the Immulon-2 plates, the binding of the supernatants from DC4, 3C9, 2A4, and NS-I cells are all the same. The supernatants of clones 2A4 and 3C9 are also positive when assayed with whole 3T3 cells fixed to the plates, exhibiting 1.4- to 1.6-fold more binding than the corresponding supernatant from NS-I cells. 4 Quite the opposite results are obtained with hybridoma clones derived
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from rats immunized in vivo with whole 3T3 cells and screened and selected on the basis of binding to whole 3T3 cells. A representative clone, designated 104, is used here for illustrative purposes. Clone 104 shows a strong reaction (2.4-fold) when assayed on whole 3T3 cells but a negligible reaction when assayed on FGR-s. These results suggest that the products of clones 2A4 and 3C9 are monoclonal antibodies directed against some component of FGR-s, which, in turn, is a constituent of whole 3T3 cells. 4
Properties of Monoclonal Antibody 2A4
Isolation and Character&ation of Antibody 2A4 Rabbit antibodies directed against rat immunoglobin are coupled to Affi-Gel 10 (Bio-Rad, Richmond, CA) using the procedure of Ikeda and Steiner.m Supernatant from the hybridoma clone 2A4 is passed through the column (l x 12 cm) 3 times. Material bound nonspecifically is removed by washing with 0.1 M phosphate buffer (pH 8.0). The specifically bound material is eluted with 0.1 M citrate buffer (pH 3.0), dialyzed overnight against PBS with 3 changes of buffer, and subjected to SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol (5%). Two polypeptides, corresponding to the heavy (Mr 55,000) and light (M r 23,000) chains of immunog|obulin G, are observed. All subsequent experiments are performed wtih the purified IgG molecule, designated Antibody 2A4. The isolated Antibody 2A4 is stored at - 2 0 °. Similar results are obtained with the antibody secreted by hybridoma clone 104. This rat lgG molecule is also purified, and Antibody 104 is used in all experiments parallel to Antibody 2A4 as a control antibody.
Identification of Target of Antibody 2A4 Antibody 2A4 is coupled to Affi-Gel 10 as described above, m The affinity column (0.6 x 4 cm) is used to fractionate FGR-s preparations to determine the molecular identity of the antigenic target of Antibody 2A4. When a [35S]methionine-labeled preparation of FGR-s is fractionated over this Antibody 2A4 column, the unbound flow-through fraction yields one predominant polypeptide (Mr 10,000). The material bound by Antibody 2A4 also shows one polypeptide band (Mr 13,000). These results indicate that Antibody 2A4 is specifically directed against the M r 13,000 polypeptide in the FGR-s fraction. 4 to y . lkeda and M. Steiner, J. Biol. Chem. 251, 6135 (1976).
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SOLUBLE EIBROBLAST GROWTH REGULATOR
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Effect of Antibody 2A4 on Growth-Inhibitory Activity of FGR-s To test the possibility that Antibody 2A4 can neutralize the growthinhibitory activity of FGR-s, the effect of the inhibitor preparation on [3H]thymidine incorporation in target 3T3 cells is assayed in the presence and absence of the purified antibody. The growth-inhibitory effect of FGR-s on 3T3 cells is dependent on the concentration of the ligand added. 2 In the present assay, 60% inhibition is obtained with 20/zl of the FGR-s preparation. When the effect of FGR-s is assayed in the presence of Antibody 2A4 (25 ng/ml), however, the inhibition is completely abrogated. The level of DNA synthesis in this case is the same as that of control cultures, without any FGR-s or Antibody 2A4. Similarly, the addition of 25 ng/ml of Antibody 2A4 reduces the inhibitory effect of 50/xl of FGR-s from 80 to 35%. Thus, when the effect of FGR-s is assayed in the presence of Antibody 2A4, there is always a higher level of [SH]thymidine incorporation (i.e., a reduced level of growth inhibition). The effects of Antibody 2A4 on reversing the inhibition of FGR-s are specific. Antibody 104, which is not reactive with FGR-s polypeptides, fails to yield the same effect. These observations suggest that the results obtained with Antibody 2A4 are most probably not due to a growth factor contaminating the immunoglobulin fraction. The results on the neutralization of growth-inhibitory activity of FGR-s by Antibody 2A4, coupled with the fact that this antibody specifically recognizes the M r 13,000 polypeptide, strongly suggest that this polypeptide carries growth-inhibitory activity. 4 This conclusion is further supported by experiments that showed depletion of the growth-inhibitory activity from preparations of FGR-s (13K) on Antibody 2A4 affinity columns.
Depletion of Growth-Inhibitory Activity from FGR-s (13K) on Affinity Column Containing Antibody 2A4 Rabbit antibodies directed against rat immunoglobulin (40 rag) are coupled to Affi-Gel 10 (2 ml of beads). 1° The derivatized gel is used to prepare two different columns: (I) the supernatant (50 ml) of hybridoma clone 2A4 is passed over one column (0.4 x 2.5 cm) 3 times to bind Antibody 2A4 (Antibody 2A4 column); (2) the supernatant of the parent myeloma NS-I cell line is passed over the other column (NS-1 control column). Material bound nonspecificaily is removed by washing with 0. ! M phosphate buffer (pH 8.0). Finally, a preparation of FGR-s (13K) (8 ng/ ml, 2 ml) is percolated through either the Antibody 2A4 column or the NS1 control column. The original FGR-s (13K) preparation, as well as the pooled flow-through fractions, representing material not bound by the respective affinity columns, are assayed for growth-inhibitory activity.
464
O T H E R G R O W T H FACTORS A N D G R O W T H I N H I B I T O R S
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The results show that the growth-inhibitory activity of FGR-s (13K) is depleted in the material passed through the Antibody 2A4 column. 3 In contrast, the NS-I control column has little effect on the growth-inhibitory activity. These results strongly suggest that FGR-s (13K) is directly responsible for the observed growth-inhibitory activity. The data also argue against any indirect mechanisms, such as stimulation of a growth factor receptor by Antibody 2A4, in the observed neutralization of FGR-s effects in the growth-inhibition assays. 4 Acknowledgments This work was supported by Grant GM 27203 from the National Institutes of Health and by Faculty Research Award FRA-221 from the American Cancer Society. 1 thank Dr. YenMing Hsu for all his help and Mrs. Linda Lang for the preparation of the manuscript.
