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[14] 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 to Basic F i b r o b l a s t G r o w t h F a c t o r a n d Its A p p l i c a t i o n B y YOSH1NO YOSHITAKE, KOUICH1 MATSUZAKI, KATSUZO N I S H I K A W A
and
Introduction
Basic fibroblast growth factor (bFGF), an endothelial cell mitogen and angiogenesis factor, has been isolated from a wide variety of tissues with the aid of heparin-Sepharose (HS) chromatography and has been used in studies of its biological role in the proliferation and differentiation of various types of cells.l,2 bFGF was first reported to be a single-chain polypeptide composed of 146 amino acids, 3 but later forms that were truncated or extended at the amino terminus were also isolated. The primary structure is highly conserved in human, bovine, ovine, and rat bFGF. Monoclonal antibodies (MAb) are useful in studies of growth factors because they are specific, homogeneous, and easy to produce in large amounts. Neutralizing MAb are powerful tools in studies of the physiological roles of these factors. This chapter describes techniques for preparation of MAb to bFGF, for studies on the properties of the MAb, and for their application in in viuo studies concerning the physiological role of bFGF.
Preparation of Bovine Basic Fibroblast Growth Factor Basic FGF is purified from bovine brain by the method of Gospodarowicz 4 involving ammonium sulfate precipitation, CM-Sephadex chromatography, and a modification of HS chromatography.5 The protein concentration in the eluate with 1.4 M NaCI/10 mM Tris-HCl (pH 7.2) from the HS column is determined with bovine serum albumin (BSA) (Sigma, St. Louis, MO) as a standard with the Pierce (Rockford, IL) BCA protein I D. Gospodarowicz, G. Neufeld, and L. Schweigerer, J. Cell. Physiol. 5 (Suppl.), 15 (1987). 2 j. Folkman and M. Klagsbrun, Science 235, 442 (1987). 3 F. Esch, A. Baird, N. Ling, N. Ueno, F. Hill, L. Denoroy, R. Klepper, D. Gospodarowicz, P. B6hlen, and R. Guillemin, Proc. Natl. Acad. Sci. U.S.A. 82, 6507 (1985). 4 D. Gospodarowicz, this series, Vol. 147, p. 106. s K. Nishikawa, Y. Yoshitake, and S. Ikuta, this series, Vol. 146, p. 11.
METHODS IN ENZYMOLOGY, VOL. 198
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assay reagent, which gives an accurate determination in the presence of high concentrations of NaCI in accordance with the Pierce Chemical Company research report. This fraction is diluted with 0.1% (w/v) bovine serum albumin (BSA) (Sigma A7511, fatty acid-free) in phosphate-buffered saline (PBS) (BSA/PBS), frozen once for sterilization at - 4 0 ° for at least 16 hr, and used for assays to stimulate both DNA synthesis in BALB/c 3T3 cells and growth of bovine brain capillary endothelial (BCE) cells and for radioimmunoassay (see below). This fraction is also dialyzed against water, lyophilized, dissolved in PBS, and then used for immunization, ELISA, and radioiodination (see below).
Radioiodination of Basic Fibroblast Growth Factor Purified bovine bFGF or recombinant bovine bFGF (The Radiochemical Centre, Amersham, UK) is labeled with ~25I by the chloramine-T method of Kan e t al. 6 with some modifications. Bovine bFGF (2/zg) in 40 /zl of PBS, 10 p.l of a 50 mCi/ml solution of Na125I, 2 /xl of 1% 3-[(3cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) solution in water, 37/zl of 0.4 M sodium phosphate buffer (pH 7.4), and 10 tzl of 1 mg/ml of chloramine-T solution in water are mixed and incubated for 1 min at room temperature. A volume of 10 txl of the same chloramine-T solution is again added, and the reaction mixture is incubated for another 1 min. The reaction is stopped by adding 100/xl of 20 mM dithiothreitol in 50 mM sodium phosphate buffer (pH 7.4), followed by successive addition of 100/zl of 10 mM NaI in water. The ~25I-labeled bFGF is then separated from free 1251by HS affinity chromatography by the method of Neufeld and Gospodarowicz. 7 The reaction mixture is applied to a column (0.25 ml) equilibrated with 0.3 M NaC1/10 mM Tris-HCl (pH 7.2)/0.1% CHAPS, and the column is washed with the same buffer until the radioactivity of the eluate becomes negligible ( - 7 ml). 125I-Labeled bFGF is eluted with 2 M NaC1/10 mM Tris-HCl (pH 7.2)/0.1% CHAPS, and the eluate can be stored at - 2 0 °. The recovery of ~25I in labeled bFGF is about 5-60%. The recovery with recombinant bovine bFGF is much better than that with purified bovine brain bFGF; however, the reason for this discrepancy is unclear. 125I-Labeled bFGF (2 × 105 cpm/ng) is used for assay after appropriate dilution with BSA/PBS within 4 weeks. After longer storage, 125I-labeled bFGF must be separated on a HS column from free 125Iwhich is liberated 6 M. Kan, D. DiSorbo, J. Hou, H. Hoshi, P. E. Mansson, and W. L. McKeehan, J. Biol. Chem. 263, 11306 (1988). 7 G. Neufeld and D. Gospodarowicz, J. Biol. Chem. 200, 13860 (1985).
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during storage. 125I-Labeled bFGF and unlabeled bFGF have identical biological activities as judged by stimulation of DNA synthesis in BALB/c 3T3 cells. CHAPS (0.02%) does not interfere with the iodination reaction. The detergent improves recovery during chromatography and storage of 125I-labeled bFGF. 8
Production of Monoclonal Antibodies 9 Media and Cell Lines 5
RPMI 1640 + 10% fetal calf serum (FCS) for maintenance ofmyeloma and hybridoma cell lines: 90% RPMI 1640 (Flow Lab, McLean, VA) supplemented with 2 g/liter of sodium bicarbonate, 100 U/ml of penicillin, 100/xg/ml of streptomycin, 15 mM N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (HEPES) (pH 7.3), 2 mM glutamine, 1 mM sodium pyruvate, 2.5 mg/ml of glucose, and 50 /xM 2-mercaptoethanol; 10% heat-inactivated fetal calf serum (FCS) (Flow Lab). Before use, sterilized and concentrated stock solutions ofglutamine (100 × ), sodium pyruvate (100 x ), glucose (200 × ), and 2-mercaptoethanol (2000 x ) in water are added to RPMI 1640. RPMI 1640 + 10% FCS + HAT (hypoxanthine, aminopterin, and thymidine) for selection of hybridoma cells 5 RPMI 1640 + 10% FCS + HT (hypoxanthine and thymidine) for growth of hybridoma cells 5 Mouse myeloma P3X63Ag8U.1 (P3U1) cells, kept in RPMI 1640 + 10% FCS and subcultured every 3-4 days Immunization o f Mice
Female BALB/c mice, 6 weeks old, are immunized 5 times at 2- to 4week intervals by subcutaneous injections with 10/zg of bovine bFGF in Freund's complete or incomplete adjuvant. After the third injection, a drop of blood is taken from the retroorbital venous plexus (see below, Application of Monoclonal Antibodies), and the plasma is separated and tested by ELISA for its ability to bind to bFGF as described below. If a further increase of the antibody titer is not observed after further immunization, the mouse is intraperitoneally injected with 10/zg of bFGF in PBS. Four days later, it is sacrificed, and the spleen is excised. 8 y. Matuo, N. Nishi, K. Matsumoto, K. Miyazaki, K. Matsumoto, F. Suzuki, and K. Nishikawa, this volume [48]. 9 K. Matsuzaki, Y. Yoshitake, Y. Matuo, H. Sasaki, and K. Nishikawa, Proc. Natl. Acad. Sci. U.S.A. 86, 9911 (1989).
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Generation of Hybridomas and Cloning Because detailed procedures for cell fusion to generate MAb have been described previously in this series, ~° and brief procedures have been also described for anti-human epidermal growth factor (hEGF) MAb, TM only points of modification are mentioned here. Mixtures of 108 spleen cells from an immunized mouse and 5 x 107 P3U1 cells are fused in 0.5 ml of 50% (w/v) polyethylene glycol 4000 (Merck, Darmstadt, Germany) in Dulbecco's modified Eagle's medium (DME) for 90 sec at 37°. The fused cells are washed with DME and then RPMI 1640 + 10% FCS by centrifugation and' resuspended in RPMI 1640 + 10% FCS + HAT medium. A suspension of the cells equivalent to 5 x 105 original spleen cells in 0.1 ml of medium is distributed into the wells of two 96-well multiwell plates (Falcon, 0.32 cm 2) in which thymocytes from normal mice (about 3 x 105 cells/well) have been cultured in 0.1 ml of the same medium for 1-2 days as the feeder layer. One-half of the culture medium of each well is replaced by fresh medium every other day. Hybridoma cell growth should be observed about 10 days after cell fusion. A sample of 50/zl of the conditioned medium is taken for assay of antibody activity by ELISA. ELISA for Basic Fibroblast Growth Factor Antibody n'j3 1. Wells of a Micro Test III flexible assay plate (Falcon 3912, Becton Dickinson & Co., Lincoln Park, N J) are coated overnight at 4° with 50/zl of a 2/zg/ml solution of purified bovine bFGF in PBS. 2. The liquid is withdrawn, and the wells are filled with 1% BSA (Sigma fraction V) solution in PBS and left to stand for 1 hr at room temperature. 3. The liquid is again withdrawn, and the wells are washed 3 times with 0.3 ml of 0.1% (w/v) Tween 20/PBS. 4. Samples of 50/xl of conditioned media from the hybridoma cultures or diluted plasma are added to the wells, and the plates are incubated overnight at 4° or for 4 hr at room temperature. 5. The liquid is withdrawn, and the wells are washed 4 times with 0.3 ml of 0.1% Tween 20/PBS. 6. Then 100 /zl of horseradish peroxidase-labeled rabbit anti-mouse IgG (Dako, Glostrup, Denmark) diluted 1 : 2000 in PBS is added, and the plates are incubated for 2 hr at room temperature. 7. The liquid is withdrawn, and the wells are washed 4 times with 0.3 ml of 0.1% Tween 20/PBS. 10G. Galfr6 and C. Milstein, this series, Vol. 73, p. 3. II y. Yoshitake and K. Nishikawa,Arch. Biochem. Biophys. 263, 437 (1988). 12S. L. Massoglia,J. S. Kenney,and D. Gospodarowicz,J. Cell. Physiol. 132, 531 (1987). 13j. y. Douillardand T. Hoffman,this series, Vol. 92, p. 168.
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8. A volume of 50 ~1 of peroxidase-substrate solution [4.9 ml of 0. I M citric acid, 5.1 ml of 0.2 M Na2HPO4, 10 ml water, 8 mg o-phenylenediamine dihydrochloride, and 4 ~1 of H202 (30%), pH 5.0] is added to the wells, and the plates are incubated for 15 min at room temperature in the dark. The reaction is stopped by adding 50 ~1 of 2 M H 2 S O 4. 9. The peroxidase reaction is measured by the increase in absorbance at 492 nm in an ELISA reader.
Determination of lmmunoglobulin Classes Immunoglobulin classes and subclasses can be determined by the double-immunodiffusion technique (micro-Ouchterlony method) as described in this series, 5 but it is more convenient to use a mouse monoclonal antibody isotyping kit (Amersham). A typing stick carries goat antibodies specific for the different types of mouse immunoglobulin classes and subclasses. Merely 0.5 ml of conditioned medium of hybridoma culture is sufficient to detect antibodies by peroxidase-labeled anti-mouse antibody.
Purification of Monoclonal Antibodies Since detailed procedures for purification of mouse MAb have been described elsewhere in this series, TM here the description is restricted to that for MAb of the IgG~ class from ascites fluid by protein G-Sepharose. Ascites fluid (5 ml) is diluted with a equal volume of 20 mM sodium phosphate buffer (pH 7.0) and applied to a column of protein G-Sepharose (gel bed, 3 ml) (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with 20 mM sodium phosphate buffer (pH 7.0). The column is washed with 70 ml of the same buffer, and IgG1 can be eluted with 10 ml of 10 mM glycine-HCl buffer (pH 2.7). The fractions of 1 ml are collected in tubes which contain 70/xl of 1 M Tris-HC1 buffer (pH 9.0) for neutralization. Recovery of IgG~ in fractions 4 and 5 is about 80%. The antibody concentration is conventionally determined by measuring the absorbance at 280 nm, assuming an A ~ value of 14. The column can be regenerated by washing with 5 ml of 10 mM glycine-HCl buffer (pH 2.7) and 30 ml of 20 mM sodium phosphate buffer (pH 7.0). Properties of Monoclonal Antibodies
Determination of Specificity and Dissociation Constant (Radioimmunoassay) 1. The reaction mixture, assembled in a 12 x 75 mm disposable polystyrene tube, contains 0.35 ml of 0.1 M sodium phosphate buffer (pH 7.4), J4 p. Parham, this series, Vol. 92, p. 110.
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0.05 ml of 1-2 ng/ml of 125I-labeled bFGF [8000-15,000 counts/min (cpm)] in BSA/PBS, 0.05 ml of unlabeled bFGF or acidic FGF in BSA/PBS to give a final concentration of 1-200 ng/ml, and 0.05 ml of 0.12-1.5/zg/ml of purified MAb in BSA/PBS. The mixture is incubated for 16 hr at 4 °. 2. Then 0.1 ml of 1% normal mouse serum in PBS and 0.1 ml of 0.77 mg/ml goat anti-mouse immunoglobulins (Dako) in PBS are added to the reaction mixture. 3. The mixture is incubated for 4 hr at 4 °. 4. Then 1 ml of 2% (w/v) polyethylene glycol 6000 in water is added to the reaction mixture. 5. The mixture is centrifuged at 1200 g for 30 min at 4 °, and the supernatant is discarded. 6. The radioactivity in the precipitate is counted in a y-counter. The value for nonspecific binding of radioactivity ( - 9 % of the total tracer added) in the absence of MAb is subtracted. Values of B (specific binding of ~25I-labeled bFGF in the presence of various concentrations of unlabeled bFGF) divided by B0 (specific binding in the absence of unlabeled bFGF), that is, B/Bo values, are plotted against the logarithm of the concentration of unlabeled bFGF. The concentration of MAb in the reaction mixture is adjusted to give a B0 value of 30-50% of the total tracer added. The apparent K d value can be calculated by Scatchard plot analysis of the displacement curves of bFGF.
Effects o f Monoclonal Antibodies on Biological Activities of Basic Fibroblast Growth Factor Addition of bFGF to cultured quiescent BALB/c 3T3 cells or cultured endothelial cells stimulates DNA synthesis or proliferation, respectively. Therefore, it is possible to determine whether the MAb inhibit these biological activities of bFGF. Detailed procedures for assay of DNA synthesis in BALB/c 3T3 cells 5 and for growth experiments with endothelial cells 4:5 have been described in this series. Here, a growth experiment using capillary endothelial cells isolated from bovine brain cortex (BCE cells) is described. BCE cells are isolated and maintained as described by Goetz et al.16 with minor modifications. The procedure involves dispersion of brain tissue by collagenase-dispase, pelleting clusters of BCE ceils by centrifugation in medium containing 25% BSA, and retaining the clusters on a column of glass beads. The modifications are as follows: The cells are 15 M. Klagsbrun, R. Sullivan, S. Smith, R. Rybka, and Y. Shing, this series, Vol. 147, p. 95. 16 I. E. Goetz, J. Warren, C. Estrada, E. Roberts, and D. Krause, In Vitro Cell. Dev. Biol. 21, 172 (1985).
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cultured in RPMI 1640 medium containing heat-inactivated fetal calf serum (FCS) and 1 ng/ml of bovine bFGF in plates that have been coated with type IV collagen (see below). Because a pure cell population of BCE cells can be obtained by isolating a primary colony formed from a single cluster containing 2-4 cells, this detailed method is also described.
Media and Solutions RPMI 1640 + 20% FCS + bFGF for primary culture of BCE cells (primary culture medium): 80% RPMI 1640 supplemented with 2 g/liter of sodium bicarbonate, 100 U/ml of penicillin, 100/zg/ml of streptomycin, 2.5/zg/ml of amphotericin B, and 15 mM HEPES (pH 7.3); 20% heat-inactivated FCS; 1 ng/ml of bovine bFGF. An appropriate volume of bovine bFGF solution in BSA/PBS should be added to the medium before use. RPMI 1640 + 10% FCS + bFGF for maintenance of BCE cells (maintenance medium): 90% of RPMI 1640, but without amphotericin B; 10% heat-inactivated FCS; 1 ng/ml of bovine bFGF Type IV collagen solution (10/zg/ml) for coating plates: 5 mg of type IV collagen (Sigma) is dissolved in 500 ml of PBS containing 100 U/ml of penicillin and 100/xg/ml of streptomycin by stirring overnight. The solution is sterilized by filtration through a 0.22-/xm filter. Trypsin-EDTA solution: 0.05% trypsin in PBS containing 0.02% EDTA
Isolating Progeny of Single Cluster of BCE Cells 1. Falcon plates (100 mm) are coated with 4 ml of a 10/xg/ml type IV collagen solution for 2 hr at 37°, and the solution is withdrawn. 2. The clusters of BCE cells, derived from about 10 g of brain cortex which are separated from glass beads, are suspended in 10 ml of primary culture medium. A volume of 0.2-0.5 ml of the suspension is added to coated plates containing I0 ml of the same medium. The cells are cultured at 37° in a humidified atmosphere of 5% CO2 in air. 3. On days 5 to 7, the sites of isolated colonies formed from single clusters are marked on the bottom of the plate; 5 to 10 colonies should be selected. 4. The medium is withdrawn, and the plate is washed with 5 ml of PBS. 5. A stainless steel cloning ring of 5-7 mm inside diameter and 10 mm in height, which has been dipped in silicone grease in a glass dish and autoclaved, is placed over the marked colony with forceps. 6. A drop of trypsin-EDTA solution is added to the ring, and the plate is incubated for 2 min at room temperature. 7. A small volume of primary culture medium is added to the ring, and
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the cells are gently suspended and transferred to a well of a 24-well multiwell tray (Falcon, 2 cm 2) coated with type IV collagen and containing 1 ml of the same medium. After 4-5 days, the cells grow to subconfluency. 8. The cells are washed with PBS, trypsinized at room temperature for 2-3 min, suspended in maintenance medium, and subcultured in the same medium. 9. Within 5 passages the cells are harvested, centrifuged, and suspended in a maintenance medium containing 8% dimethyl sulfoxide at a concentration of 1-2 × l 0 6 cells/ml. The cells are frozen and stored at - 8 0 °" Frozen cells can be thawed, subcultured every 3-4 days, and used for growth experiments within l0 passages. The cells should be checked for the presence ofvon Willebrand factor (Factor VIII) by immunohistochemical techniques.
Growth Experiment 1. BCE cells in subconfluent culture in maintenance medium are trypsinized. 2. The cells are suspended in maintenance medium which contains 1 ng/ml of bFGF, and inocula of 2 × 10 4 cells in 5 ml of the medium are introduced into 60-mm Falcon plates that have been coated with type IV collagen as described above. 3. Purified MAb (1-5 mg/ml) at a final concentration of 0.1-25/zg/ml is added. 4. After 5 days, the medium is withdrawn, and the cells are washed with PBS and trypsinized in 0.5 ml of trypsin-EDTA solution at 37° for 15 min. 5. The cells are suspended in 9.5 ml of PBS and counted in a Coulter counter. All measurements should be done at least in duplicate. The cell number after 5 days in the presence of 1 ng/ml of bFGF is about 4-6 × 105/plate. Addition of appropriate concentrations of neutralizing MAb to bFGF should inhibit the growth of these cells. When the cells are inoculated at higher density (105 cells), the cells can grow even in the absence of bFGF, although the growth rate is somewhat lower. If the neutralizing MAb to bFGF inhibits this growth, an autocrine action of bFGF which is produced by these cells may be indicated. Application of Monoclonal Antibodies When a specific MAb to bFGF has been obtained it can be used for various general techniques: identification (Western blot, immunoprecipitation), quantitative determination (radioimmunoassay, ELISA), purifica-
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tion (immunoaffinity chromatography), and determination of localization in cells or tissues (immunohistochemical staining). Furthermore, when a hybridoma cell line which produces a MAb to bFGF that can block its biological activity (neutralizing MAb) has been obtained, it can be used for study of the physiological role of bFGF not only in vitro but also in vivo. After a large quantity of pure MAb is available, it can be injected into animals; however, high levels of antibody activity in the blood of the animal can also be continuously maintained by injection of the hybridoma cells. Here in vivo methods using a hybridoma cell line are described. Generation o f Athymic Mice Having High Level of Anti-Basic Fibroblast Growth Factor Activity in Blood 9 I. Inocula of 5 x 105 hybridoma cells are introduced into 150-mm Falcon plates with 20 ml of RPMI 1640 + 10% FCS (see above). After 3 days, hybridoma cells grow to subconfluency ( - 1 . 6 x 10 7 cells/plate). 2. The cell pellet collected by centrifugation (500 g, 10 min) is suspended in 10 ml of serum-free RPMI 1640, and the cell suspension is again centrifuged. 3. The cell pellet is suspended in an appropriate volume of serum-free RPMI 1640 (5 x 10 7 cells/ml). 4. A volume of 0.2 ml of the cell suspension (10 7 cells) is injected subcutaneously into the back of an athymic mouse (BALB/c AJcL-nu; 6-10 weeks old) by a syringe with 25-gauge needle. 5. The hybridoma cells grow on the back of the mouse, and a solid tumor appears within 1 week. The tumor-bearing mouse can survive for 3-4 weeks, and the tumor grows to about 15% of the body weight of the host animal at the final stage. 6. At 4- to 6-day intervals, a drop of blood is taken from the retroorbital venous plexus of anesthetized mouse by a heparinized glass capillary (1.45-1.65 x 75 mm). The capillary is sealed with clay at one end and centrifuged at 1200 g for 10 min. The plasma is taken from the capillary by a microsyringe and diluted with BSA/PBS containing 0.02% NAN3. Although the solid tumor of the hybridoma cells can be also produced in BALB/c mice, the athymic mouse is more favorable for studies of the in vivo growth of tumors from various species including humans. Assay o f Anti-Basic Fibroblast Growth Factor Antibody Activity in Plasma The reaction mixture contains 0.35 ml of 0.1 M sodium phosphate buffer (pH 7.4), 0.05 ml of 1-2 ng/ml ~25I-labeled bFGF (8000-15,000 cpm) in BSA/PBS, 0.1 ml of purified MAb in BSA/PBS to give a final
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concentration of 0.2-1000 ng/ml or plasma at an appropriate dilution in BSA/PBS containing 0.02% NaN 3. The procedure for the binding experiment is the same as that for radioimmunoassay (see above). Values of radioactivity in the precipitate divided by the total tracer added are plotted against the logarithm of the concentration of purified MAb or plasma. The concentration of MAb in plasma can be calculated from the titration curves with purified MAb as a standard. Example. 9 We produced two neutralizing MAb to bovine bFGF. The immunoglobulin class and subclass of the MAb are IgG~/K. The MAb are named bFM-1 and bFM-2. In a competitive binding assay of the MAb with labeled bovine bFGF, half-maximal displacement is observed with unlabeled bovine bFGF at 6.4 and 64 ng/ml for bFM-I and bFM-2, respectively. The apparent dissociation constants (Kd) for bovine bFGF are 8.7 × 10 lJ (bFM-1) and 1.6 x 10 -9 M (bFM-2). The antibodies are highly specific for bFGF from bovine, human, rat, and mouse sources and do not cross-react with bovine acidic FGF. bFM-2 cross-reacts with heatinactivated bFGF, whereas bFM-1 does not, suggesting that bFM-1 recognizes the conformation of the bFGF molecule necessary for its biological activity and that bFM-2 recognizes a linear epitope of the bFGF molecule. Therefore, bFM-2 can be used for detection of inactivated bFGF by immunoblotting. The MAb inhibit growth of cultured BCE cells in both the presence and absence of exogenously added bFGF, indicating not only the neutralizing property of the MAb but also the autocrine action of bFGF in in vitro growth of these cells, bFM-1 also inhibits angiogenesis in chorioallantoic membranes of chick embryos induced by bFGF. Injections of the hybridoma cell lines producing bFM-I or bFM-2 into the backs of athymic mice result in development of solid tumors and a sustained high level of anti-bFGF activity in the blood of the tumor-bearing mice. The activity in blood increases with an increase in tumor mass. The concentrations of bFM-1 and bFM-2 in the plasma of these mice at the final stage are 2.9-4.3 and 35-50 mg/ml, respectively. These MAb should be useful for studies on bFGF, including the physiological role and the conformation-function relationships of this factor. Acknowledgments This work was supported in part by a grant-in-aid for cancer research by the Ministry of Education, Science and Culture of Japan.
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[14] 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 to Basic F i b r o b l a s t G r o w t h F a c t o r a n d Its A p p l i c a t i o n B y YOSH1NO YOSHITAKE, KOUICH1 MATSUZAKI, KATSUZO N I S H I K A W A
and
Introduction
Basic fibroblast growth factor (bFGF), an endothelial cell mitogen and angiogenesis factor, has been isolated from a wide variety of tissues with the aid of heparin-Sepharose (HS) chromatography and has been used in studies of its biological role in the proliferation and differentiation of various types of cells.l,2 bFGF was first reported to be a single-chain polypeptide composed of 146 amino acids, 3 but later forms that were truncated or extended at the amino terminus were also isolated. The primary structure is highly conserved in human, bovine, ovine, and rat bFGF. Monoclonal antibodies (MAb) are useful in studies of growth factors because they are specific, homogeneous, and easy to produce in large amounts. Neutralizing MAb are powerful tools in studies of the physiological roles of these factors. This chapter describes techniques for preparation of MAb to bFGF, for studies on the properties of the MAb, and for their application in in viuo studies concerning the physiological role of bFGF.
Preparation of Bovine Basic Fibroblast Growth Factor Basic FGF is purified from bovine brain by the method of Gospodarowicz 4 involving ammonium sulfate precipitation, CM-Sephadex chromatography, and a modification of HS chromatography.5 The protein concentration in the eluate with 1.4 M NaCI/10 mM Tris-HCl (pH 7.2) from the HS column is determined with bovine serum albumin (BSA) (Sigma, St. Louis, MO) as a standard with the Pierce (Rockford, IL) BCA protein I D. Gospodarowicz, G. Neufeld, and L. Schweigerer, J. Cell. Physiol. 5 (Suppl.), 15 (1987). 2 j. Folkman and M. Klagsbrun, Science 235, 442 (1987). 3 F. Esch, A. Baird, N. Ling, N. Ueno, F. Hill, L. Denoroy, R. Klepper, D. Gospodarowicz, P. B6hlen, and R. Guillemin, Proc. Natl. Acad. Sci. U.S.A. 82, 6507 (1985). 4 D. Gospodarowicz, this series, Vol. 147, p. 106. s K. Nishikawa, Y. Yoshitake, and S. Ikuta, this series, Vol. 146, p. 11.
METHODS IN ENZYMOLOGY, VOL. 198
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assay reagent, which gives an accurate determination in the presence of high concentrations of NaCI in accordance with the Pierce Chemical Company research report. This fraction is diluted with 0.1% (w/v) bovine serum albumin (BSA) (Sigma A7511, fatty acid-free) in phosphate-buffered saline (PBS) (BSA/PBS), frozen once for sterilization at - 4 0 ° for at least 16 hr, and used for assays to stimulate both DNA synthesis in BALB/c 3T3 cells and growth of bovine brain capillary endothelial (BCE) cells and for radioimmunoassay (see below). This fraction is also dialyzed against water, lyophilized, dissolved in PBS, and then used for immunization, ELISA, and radioiodination (see below).
Radioiodination of Basic Fibroblast Growth Factor Purified bovine bFGF or recombinant bovine bFGF (The Radiochemical Centre, Amersham, UK) is labeled with ~25I by the chloramine-T method of Kan e t al. 6 with some modifications. Bovine bFGF (2/zg) in 40 /zl of PBS, 10 p.l of a 50 mCi/ml solution of Na125I, 2 /xl of 1% 3-[(3cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) solution in water, 37/zl of 0.4 M sodium phosphate buffer (pH 7.4), and 10 tzl of 1 mg/ml of chloramine-T solution in water are mixed and incubated for 1 min at room temperature. A volume of 10 txl of the same chloramine-T solution is again added, and the reaction mixture is incubated for another 1 min. The reaction is stopped by adding 100/xl of 20 mM dithiothreitol in 50 mM sodium phosphate buffer (pH 7.4), followed by successive addition of 100/zl of 10 mM NaI in water. The ~25I-labeled bFGF is then separated from free 1251by HS affinity chromatography by the method of Neufeld and Gospodarowicz. 7 The reaction mixture is applied to a column (0.25 ml) equilibrated with 0.3 M NaC1/10 mM Tris-HCl (pH 7.2)/0.1% CHAPS, and the column is washed with the same buffer until the radioactivity of the eluate becomes negligible ( - 7 ml). 125I-Labeled bFGF is eluted with 2 M NaC1/10 mM Tris-HCl (pH 7.2)/0.1% CHAPS, and the eluate can be stored at - 2 0 °. The recovery of ~25I in labeled bFGF is about 5-60%. The recovery with recombinant bovine bFGF is much better than that with purified bovine brain bFGF; however, the reason for this discrepancy is unclear. 125I-Labeled bFGF (2 × 105 cpm/ng) is used for assay after appropriate dilution with BSA/PBS within 4 weeks. After longer storage, 125I-labeled bFGF must be separated on a HS column from free 125Iwhich is liberated 6 M. Kan, D. DiSorbo, J. Hou, H. Hoshi, P. E. Mansson, and W. L. McKeehan, J. Biol. Chem. 263, 11306 (1988). 7 G. Neufeld and D. Gospodarowicz, J. Biol. Chem. 200, 13860 (1985).
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during storage. 125I-Labeled bFGF and unlabeled bFGF have identical biological activities as judged by stimulation of DNA synthesis in BALB/c 3T3 cells. CHAPS (0.02%) does not interfere with the iodination reaction. The detergent improves recovery during chromatography and storage of 125I-labeled bFGF. 8
Production of Monoclonal Antibodies 9 Media and Cell Lines 5
RPMI 1640 + 10% fetal calf serum (FCS) for maintenance ofmyeloma and hybridoma cell lines: 90% RPMI 1640 (Flow Lab, McLean, VA) supplemented with 2 g/liter of sodium bicarbonate, 100 U/ml of penicillin, 100/xg/ml of streptomycin, 15 mM N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (HEPES) (pH 7.3), 2 mM glutamine, 1 mM sodium pyruvate, 2.5 mg/ml of glucose, and 50 /xM 2-mercaptoethanol; 10% heat-inactivated fetal calf serum (FCS) (Flow Lab). Before use, sterilized and concentrated stock solutions ofglutamine (100 × ), sodium pyruvate (100 x ), glucose (200 × ), and 2-mercaptoethanol (2000 x ) in water are added to RPMI 1640. RPMI 1640 + 10% FCS + HAT (hypoxanthine, aminopterin, and thymidine) for selection of hybridoma cells 5 RPMI 1640 + 10% FCS + HT (hypoxanthine and thymidine) for growth of hybridoma cells 5 Mouse myeloma P3X63Ag8U.1 (P3U1) cells, kept in RPMI 1640 + 10% FCS and subcultured every 3-4 days Immunization o f Mice
Female BALB/c mice, 6 weeks old, are immunized 5 times at 2- to 4week intervals by subcutaneous injections with 10/zg of bovine bFGF in Freund's complete or incomplete adjuvant. After the third injection, a drop of blood is taken from the retroorbital venous plexus (see below, Application of Monoclonal Antibodies), and the plasma is separated and tested by ELISA for its ability to bind to bFGF as described below. If a further increase of the antibody titer is not observed after further immunization, the mouse is intraperitoneally injected with 10/zg of bFGF in PBS. Four days later, it is sacrificed, and the spleen is excised. 8 y. Matuo, N. Nishi, K. Matsumoto, K. Miyazaki, K. Matsumoto, F. Suzuki, and K. Nishikawa, this volume [48]. 9 K. Matsuzaki, Y. Yoshitake, Y. Matuo, H. Sasaki, and K. Nishikawa, Proc. Natl. Acad. Sci. U.S.A. 86, 9911 (1989).
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Generation of Hybridomas and Cloning Because detailed procedures for cell fusion to generate MAb have been described previously in this series, ~° and brief procedures have been also described for anti-human epidermal growth factor (hEGF) MAb, TM only points of modification are mentioned here. Mixtures of 108 spleen cells from an immunized mouse and 5 x 107 P3U1 cells are fused in 0.5 ml of 50% (w/v) polyethylene glycol 4000 (Merck, Darmstadt, Germany) in Dulbecco's modified Eagle's medium (DME) for 90 sec at 37°. The fused cells are washed with DME and then RPMI 1640 + 10% FCS by centrifugation and' resuspended in RPMI 1640 + 10% FCS + HAT medium. A suspension of the cells equivalent to 5 x 105 original spleen cells in 0.1 ml of medium is distributed into the wells of two 96-well multiwell plates (Falcon, 0.32 cm 2) in which thymocytes from normal mice (about 3 x 105 cells/well) have been cultured in 0.1 ml of the same medium for 1-2 days as the feeder layer. One-half of the culture medium of each well is replaced by fresh medium every other day. Hybridoma cell growth should be observed about 10 days after cell fusion. A sample of 50/zl of the conditioned medium is taken for assay of antibody activity by ELISA. ELISA for Basic Fibroblast Growth Factor Antibody n'j3 1. Wells of a Micro Test III flexible assay plate (Falcon 3912, Becton Dickinson & Co., Lincoln Park, N J) are coated overnight at 4° with 50/zl of a 2/zg/ml solution of purified bovine bFGF in PBS. 2. The liquid is withdrawn, and the wells are filled with 1% BSA (Sigma fraction V) solution in PBS and left to stand for 1 hr at room temperature. 3. The liquid is again withdrawn, and the wells are washed 3 times with 0.3 ml of 0.1% (w/v) Tween 20/PBS. 4. Samples of 50/xl of conditioned media from the hybridoma cultures or diluted plasma are added to the wells, and the plates are incubated overnight at 4° or for 4 hr at room temperature. 5. The liquid is withdrawn, and the wells are washed 4 times with 0.3 ml of 0.1% Tween 20/PBS. 6. Then 100 /zl of horseradish peroxidase-labeled rabbit anti-mouse IgG (Dako, Glostrup, Denmark) diluted 1 : 2000 in PBS is added, and the plates are incubated for 2 hr at room temperature. 7. The liquid is withdrawn, and the wells are washed 4 times with 0.3 ml of 0.1% Tween 20/PBS. 10G. Galfr6 and C. Milstein, this series, Vol. 73, p. 3. II y. Yoshitake and K. Nishikawa,Arch. Biochem. Biophys. 263, 437 (1988). 12S. L. Massoglia,J. S. Kenney,and D. Gospodarowicz,J. Cell. Physiol. 132, 531 (1987). 13j. y. Douillardand T. Hoffman,this series, Vol. 92, p. 168.
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8. A volume of 50 ~1 of peroxidase-substrate solution [4.9 ml of 0. I M citric acid, 5.1 ml of 0.2 M Na2HPO4, 10 ml water, 8 mg o-phenylenediamine dihydrochloride, and 4 ~1 of H202 (30%), pH 5.0] is added to the wells, and the plates are incubated for 15 min at room temperature in the dark. The reaction is stopped by adding 50 ~1 of 2 M H 2 S O 4. 9. The peroxidase reaction is measured by the increase in absorbance at 492 nm in an ELISA reader.
Determination of lmmunoglobulin Classes Immunoglobulin classes and subclasses can be determined by the double-immunodiffusion technique (micro-Ouchterlony method) as described in this series, 5 but it is more convenient to use a mouse monoclonal antibody isotyping kit (Amersham). A typing stick carries goat antibodies specific for the different types of mouse immunoglobulin classes and subclasses. Merely 0.5 ml of conditioned medium of hybridoma culture is sufficient to detect antibodies by peroxidase-labeled anti-mouse antibody.
Purification of Monoclonal Antibodies Since detailed procedures for purification of mouse MAb have been described elsewhere in this series, TM here the description is restricted to that for MAb of the IgG~ class from ascites fluid by protein G-Sepharose. Ascites fluid (5 ml) is diluted with a equal volume of 20 mM sodium phosphate buffer (pH 7.0) and applied to a column of protein G-Sepharose (gel bed, 3 ml) (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with 20 mM sodium phosphate buffer (pH 7.0). The column is washed with 70 ml of the same buffer, and IgG1 can be eluted with 10 ml of 10 mM glycine-HCl buffer (pH 2.7). The fractions of 1 ml are collected in tubes which contain 70/xl of 1 M Tris-HC1 buffer (pH 9.0) for neutralization. Recovery of IgG~ in fractions 4 and 5 is about 80%. The antibody concentration is conventionally determined by measuring the absorbance at 280 nm, assuming an A ~ value of 14. The column can be regenerated by washing with 5 ml of 10 mM glycine-HCl buffer (pH 2.7) and 30 ml of 20 mM sodium phosphate buffer (pH 7.0). Properties of Monoclonal Antibodies
Determination of Specificity and Dissociation Constant (Radioimmunoassay) 1. The reaction mixture, assembled in a 12 x 75 mm disposable polystyrene tube, contains 0.35 ml of 0.1 M sodium phosphate buffer (pH 7.4), J4 p. Parham, this series, Vol. 92, p. 110.
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0.05 ml of 1-2 ng/ml of 125I-labeled bFGF [8000-15,000 counts/min (cpm)] in BSA/PBS, 0.05 ml of unlabeled bFGF or acidic FGF in BSA/PBS to give a final concentration of 1-200 ng/ml, and 0.05 ml of 0.12-1.5/zg/ml of purified MAb in BSA/PBS. The mixture is incubated for 16 hr at 4 °. 2. Then 0.1 ml of 1% normal mouse serum in PBS and 0.1 ml of 0.77 mg/ml goat anti-mouse immunoglobulins (Dako) in PBS are added to the reaction mixture. 3. The mixture is incubated for 4 hr at 4 °. 4. Then 1 ml of 2% (w/v) polyethylene glycol 6000 in water is added to the reaction mixture. 5. The mixture is centrifuged at 1200 g for 30 min at 4 °, and the supernatant is discarded. 6. The radioactivity in the precipitate is counted in a y-counter. The value for nonspecific binding of radioactivity ( - 9 % of the total tracer added) in the absence of MAb is subtracted. Values of B (specific binding of ~25I-labeled bFGF in the presence of various concentrations of unlabeled bFGF) divided by B0 (specific binding in the absence of unlabeled bFGF), that is, B/Bo values, are plotted against the logarithm of the concentration of unlabeled bFGF. The concentration of MAb in the reaction mixture is adjusted to give a B0 value of 30-50% of the total tracer added. The apparent K d value can be calculated by Scatchard plot analysis of the displacement curves of bFGF.
Effects o f Monoclonal Antibodies on Biological Activities of Basic Fibroblast Growth Factor Addition of bFGF to cultured quiescent BALB/c 3T3 cells or cultured endothelial cells stimulates DNA synthesis or proliferation, respectively. Therefore, it is possible to determine whether the MAb inhibit these biological activities of bFGF. Detailed procedures for assay of DNA synthesis in BALB/c 3T3 cells 5 and for growth experiments with endothelial cells 4:5 have been described in this series. Here, a growth experiment using capillary endothelial cells isolated from bovine brain cortex (BCE cells) is described. BCE cells are isolated and maintained as described by Goetz et al.16 with minor modifications. The procedure involves dispersion of brain tissue by collagenase-dispase, pelleting clusters of BCE ceils by centrifugation in medium containing 25% BSA, and retaining the clusters on a column of glass beads. The modifications are as follows: The cells are 15 M. Klagsbrun, R. Sullivan, S. Smith, R. Rybka, and Y. Shing, this series, Vol. 147, p. 95. 16 I. E. Goetz, J. Warren, C. Estrada, E. Roberts, and D. Krause, In Vitro Cell. Dev. Biol. 21, 172 (1985).
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cultured in RPMI 1640 medium containing heat-inactivated fetal calf serum (FCS) and 1 ng/ml of bovine bFGF in plates that have been coated with type IV collagen (see below). Because a pure cell population of BCE cells can be obtained by isolating a primary colony formed from a single cluster containing 2-4 cells, this detailed method is also described.
Media and Solutions RPMI 1640 + 20% FCS + bFGF for primary culture of BCE cells (primary culture medium): 80% RPMI 1640 supplemented with 2 g/liter of sodium bicarbonate, 100 U/ml of penicillin, 100/zg/ml of streptomycin, 2.5/zg/ml of amphotericin B, and 15 mM HEPES (pH 7.3); 20% heat-inactivated FCS; 1 ng/ml of bovine bFGF. An appropriate volume of bovine bFGF solution in BSA/PBS should be added to the medium before use. RPMI 1640 + 10% FCS + bFGF for maintenance of BCE cells (maintenance medium): 90% of RPMI 1640, but without amphotericin B; 10% heat-inactivated FCS; 1 ng/ml of bovine bFGF Type IV collagen solution (10/zg/ml) for coating plates: 5 mg of type IV collagen (Sigma) is dissolved in 500 ml of PBS containing 100 U/ml of penicillin and 100/xg/ml of streptomycin by stirring overnight. The solution is sterilized by filtration through a 0.22-/xm filter. Trypsin-EDTA solution: 0.05% trypsin in PBS containing 0.02% EDTA
Isolating Progeny of Single Cluster of BCE Cells 1. Falcon plates (100 mm) are coated with 4 ml of a 10/xg/ml type IV collagen solution for 2 hr at 37°, and the solution is withdrawn. 2. The clusters of BCE cells, derived from about 10 g of brain cortex which are separated from glass beads, are suspended in 10 ml of primary culture medium. A volume of 0.2-0.5 ml of the suspension is added to coated plates containing I0 ml of the same medium. The cells are cultured at 37° in a humidified atmosphere of 5% CO2 in air. 3. On days 5 to 7, the sites of isolated colonies formed from single clusters are marked on the bottom of the plate; 5 to 10 colonies should be selected. 4. The medium is withdrawn, and the plate is washed with 5 ml of PBS. 5. A stainless steel cloning ring of 5-7 mm inside diameter and 10 mm in height, which has been dipped in silicone grease in a glass dish and autoclaved, is placed over the marked colony with forceps. 6. A drop of trypsin-EDTA solution is added to the ring, and the plate is incubated for 2 min at room temperature. 7. A small volume of primary culture medium is added to the ring, and
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the cells are gently suspended and transferred to a well of a 24-well multiwell tray (Falcon, 2 cm 2) coated with type IV collagen and containing 1 ml of the same medium. After 4-5 days, the cells grow to subconfluency. 8. The cells are washed with PBS, trypsinized at room temperature for 2-3 min, suspended in maintenance medium, and subcultured in the same medium. 9. Within 5 passages the cells are harvested, centrifuged, and suspended in a maintenance medium containing 8% dimethyl sulfoxide at a concentration of 1-2 × l 0 6 cells/ml. The cells are frozen and stored at - 8 0 °" Frozen cells can be thawed, subcultured every 3-4 days, and used for growth experiments within l0 passages. The cells should be checked for the presence ofvon Willebrand factor (Factor VIII) by immunohistochemical techniques.
Growth Experiment 1. BCE cells in subconfluent culture in maintenance medium are trypsinized. 2. The cells are suspended in maintenance medium which contains 1 ng/ml of bFGF, and inocula of 2 × 10 4 cells in 5 ml of the medium are introduced into 60-mm Falcon plates that have been coated with type IV collagen as described above. 3. Purified MAb (1-5 mg/ml) at a final concentration of 0.1-25/zg/ml is added. 4. After 5 days, the medium is withdrawn, and the cells are washed with PBS and trypsinized in 0.5 ml of trypsin-EDTA solution at 37° for 15 min. 5. The cells are suspended in 9.5 ml of PBS and counted in a Coulter counter. All measurements should be done at least in duplicate. The cell number after 5 days in the presence of 1 ng/ml of bFGF is about 4-6 × 105/plate. Addition of appropriate concentrations of neutralizing MAb to bFGF should inhibit the growth of these cells. When the cells are inoculated at higher density (105 cells), the cells can grow even in the absence of bFGF, although the growth rate is somewhat lower. If the neutralizing MAb to bFGF inhibits this growth, an autocrine action of bFGF which is produced by these cells may be indicated. Application of Monoclonal Antibodies When a specific MAb to bFGF has been obtained it can be used for various general techniques: identification (Western blot, immunoprecipitation), quantitative determination (radioimmunoassay, ELISA), purifica-
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tion (immunoaffinity chromatography), and determination of localization in cells or tissues (immunohistochemical staining). Furthermore, when a hybridoma cell line which produces a MAb to bFGF that can block its biological activity (neutralizing MAb) has been obtained, it can be used for study of the physiological role of bFGF not only in vitro but also in vivo. After a large quantity of pure MAb is available, it can be injected into animals; however, high levels of antibody activity in the blood of the animal can also be continuously maintained by injection of the hybridoma cells. Here in vivo methods using a hybridoma cell line are described. Generation o f Athymic Mice Having High Level of Anti-Basic Fibroblast Growth Factor Activity in Blood 9 I. Inocula of 5 x 105 hybridoma cells are introduced into 150-mm Falcon plates with 20 ml of RPMI 1640 + 10% FCS (see above). After 3 days, hybridoma cells grow to subconfluency ( - 1 . 6 x 10 7 cells/plate). 2. The cell pellet collected by centrifugation (500 g, 10 min) is suspended in 10 ml of serum-free RPMI 1640, and the cell suspension is again centrifuged. 3. The cell pellet is suspended in an appropriate volume of serum-free RPMI 1640 (5 x 10 7 cells/ml). 4. A volume of 0.2 ml of the cell suspension (10 7 cells) is injected subcutaneously into the back of an athymic mouse (BALB/c AJcL-nu; 6-10 weeks old) by a syringe with 25-gauge needle. 5. The hybridoma cells grow on the back of the mouse, and a solid tumor appears within 1 week. The tumor-bearing mouse can survive for 3-4 weeks, and the tumor grows to about 15% of the body weight of the host animal at the final stage. 6. At 4- to 6-day intervals, a drop of blood is taken from the retroorbital venous plexus of anesthetized mouse by a heparinized glass capillary (1.45-1.65 x 75 mm). The capillary is sealed with clay at one end and centrifuged at 1200 g for 10 min. The plasma is taken from the capillary by a microsyringe and diluted with BSA/PBS containing 0.02% NAN3. Although the solid tumor of the hybridoma cells can be also produced in BALB/c mice, the athymic mouse is more favorable for studies of the in vivo growth of tumors from various species including humans. Assay o f Anti-Basic Fibroblast Growth Factor Antibody Activity in Plasma The reaction mixture contains 0.35 ml of 0.1 M sodium phosphate buffer (pH 7.4), 0.05 ml of 1-2 ng/ml ~25I-labeled bFGF (8000-15,000 cpm) in BSA/PBS, 0.1 ml of purified MAb in BSA/PBS to give a final
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concentration of 0.2-1000 ng/ml or plasma at an appropriate dilution in BSA/PBS containing 0.02% NaN 3. The procedure for the binding experiment is the same as that for radioimmunoassay (see above). Values of radioactivity in the precipitate divided by the total tracer added are plotted against the logarithm of the concentration of purified MAb or plasma. The concentration of MAb in plasma can be calculated from the titration curves with purified MAb as a standard. Example. 9 We produced two neutralizing MAb to bovine bFGF. The immunoglobulin class and subclass of the MAb are IgG~/K. The MAb are named bFM-1 and bFM-2. In a competitive binding assay of the MAb with labeled bovine bFGF, half-maximal displacement is observed with unlabeled bovine bFGF at 6.4 and 64 ng/ml for bFM-I and bFM-2, respectively. The apparent dissociation constants (Kd) for bovine bFGF are 8.7 × 10 lJ (bFM-1) and 1.6 x 10 -9 M (bFM-2). The antibodies are highly specific for bFGF from bovine, human, rat, and mouse sources and do not cross-react with bovine acidic FGF. bFM-2 cross-reacts with heatinactivated bFGF, whereas bFM-1 does not, suggesting that bFM-1 recognizes the conformation of the bFGF molecule necessary for its biological activity and that bFM-2 recognizes a linear epitope of the bFGF molecule. Therefore, bFM-2 can be used for detection of inactivated bFGF by immunoblotting. The MAb inhibit growth of cultured BCE cells in both the presence and absence of exogenously added bFGF, indicating not only the neutralizing property of the MAb but also the autocrine action of bFGF in in vitro growth of these cells, bFM-1 also inhibits angiogenesis in chorioallantoic membranes of chick embryos induced by bFGF. Injections of the hybridoma cell lines producing bFM-I or bFM-2 into the backs of athymic mice result in development of solid tumors and a sustained high level of anti-bFGF activity in the blood of the tumor-bearing mice. The activity in blood increases with an increase in tumor mass. The concentrations of bFM-1 and bFM-2 in the plasma of these mice at the final stage are 2.9-4.3 and 35-50 mg/ml, respectively. These MAb should be useful for studies on bFGF, including the physiological role and the conformation-function relationships of this factor. Acknowledgments This work was supported in part by a grant-in-aid for cancer research by the Ministry of Education, Science and Culture of Japan.
