[17]
ASSAYS FOR TRANSFORMING
G R O W T H F A C T O R ot
185
Summary This chapter outlines in detail the optimal conditions for the expression of human recombinant proteins in mouse cells using a bovine papillomavims-based mammalian expression vector. The procedures we have described were used to successfully express high levels of the human EGF precursor in our laboratory and the human insulin receptor in the laboratory of Whittaker. 4 Using this experimental approach we were able to demonstrate that expression of a cDNA for prepro-EGF produces a glycosylated membrane protein with biological activity. Acknowledgments I am grateful to Martha Reich for skilled technical assistance, Jonathan Whittaker for helpful discussions and valuable advice, Susan Heaver for secretarial assistance, and Ann M. Soderquist for critical reading of the manuscript.
[ 17] G e n e r a t i o n o f A n t i b o d i e s a n d A s s a y s for T r a n s f o r m i n g Growth Factor a By CATHERINE LUCAS, TIMOTHY S. BRINGMAN, a n d RIK DERYNCK
Introduction Two types of growth factors have been termed transforming growth factors (TGF) because they were discovered in an assay that evaluated their ability to elicit cellular transformation using an immortalized nonneoplastic fibroblast line. One of these, TGF-a, is a single-chain polypeptide structurally related to epidermal growth factor (EGF). Several TGF-a species are proteolytically derived from a transmembrane precursor form. The fully processed form is 50 amino acids long and lacks glycosylation, whereas the larger forms are glycosylated. All these forms and the transmembrane precursor are biologically active and interact with the same receptor as EGF. The best studied activity of TGF-a is its ability to stimulate DNA synthesis and mitosis of the many cell types that contain the corresponding cell surface receptors. 1-3TGF-fl is structurally unrelated t R. Derynck, Cell (Cambridge, Mass.) 54, 593 (1988). 2 R. Brachmann, P. B. Lindquist, M. Nagashima, W. Kohr, T. Lipari, M. Napier, and R. Derynck, Cell (Cambridge, Mass.) 56, 691 (1989). 3 S. T. Wong, L. F. Winchell, B. K. McCune, H. S. Earp, J. Teixido, J. Massagu6, B. Herman, and D. C. Lee, Cell (Cambridge, Mass.) 56, 495 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
186
EGF, TGF-a, AND RELATEDFACTORS
[17]
to TGF-a and binds to distinct receptors. 4'5 It is described in more detail in [29]-[32] in this volume. Much interest in both TGF-a and TGF-/3 has arisen because both types of factors have been shown to be functional in normal physiology and also have been implicated in malignant transformation and tumorigenesis. The availability of antibodies to transforming growth factor a is of utmost importance in studying its biology, not only by neutralizing its activities, but also in developing fast, reliable, and specific assays. This chapter describes the methodology we have used to develop antibodies and assays specific for TGF-a. Because both TGF-a and EGF bind to the same receptor, a receptorbinding assay would not be able to discriminate between the factors; however, it would have the advantage of measuring only active growth factors in a configuration able to bind the receptor. The lack of specificity for TGF-a in a receptor binding assay thus dictates the need to develop a TGF-a-specific antibody and immunoassay. Ideally such antibodies and assays should not cross-react with EGF. EGF and TGF-a share about a 30% sequence identity, and it is thus imperative that the lack of crossreactivity be documented. It would also be an advantage if the antibody only reacts with the biologically active molecules and interacts with an epitope formed by the secondary structure of the properly folded factor. Finally, the antibodies should be able to recognize the multiple forms of secreted TGF-a and the larger transmembrane form. A major problem in developing antibodies for TGF-a is the lack of naturally secreted TGF-a. The TGF-a secreted by human cell lines is present in such low concentrations in the medium that it becomes virtually impossible to purify sufficiently large quantities for immunization experiments. We have overcome this difficulty by expressing the 50 amino acid form of TGF-a as a fusion protein in E s c h e r i c h i a coli. The fusion protein is then purified, refolded into the proper disulfide configuration, and cleaved to release the 50 amino acid TGF-a. Preparation of Recombinant Transforming Growth Factor a The 50 amino acid form of human TGF-a can be obtained as follows. An expression vector is constructed in such a way that it directs the expression of a 69 amino acid TGF-a fusion protein in E. coli. The fusion 4 A. B. Roberts and M. B. Sporn, in "Peptide Growth Factors and Their Receptors" (M. B. Spore and A. B. Roberts, eds.), Handbookof ExperimentalPharmacology.Springer-Verlag, Heidelberg, in press. 5M. B. Sporn, A. B. Roberts, L. M. Wakefield,and B. de Crombrugghe,J. Cell Biol. 105, 1039 (1987).
[17]
ASSAYS FOR TRANSFORMING GROWTH FACTOR ot
187
protein consists of an 18 amino acid leader sequence followed by a unique methionine that precedes the 50 amino acid TGF-a. This fusion protein is isolated and purified from the recombinant E. coli and cleaved with cyanogen bromide. This treatment results in a cleavage following the methionine residue, thus releasing TGF-a. The TGF-a is then refolded in an oxidation-reduction buffer, and the biologically active TGF-a with the correct disulfide bridge configuration is purified to apparent homogeneity by HPLC. The bacterial expression of TGF-a and its purification have been detailed. 6,7
Generation of Antibodies
Polyclonal Antibodies Antisera against human TGF-a are prepared in rabbits using the purified and refolded recombinant human TGF-a as immunogen. A first injection on day 0 consists of 400/xg TGF-a emulsified in Freund's complete adjuvant and is given intradermally at multiple sites. Subsequent booster injections in Freund's incomplete adjuvant are given subcutaneously at multiple sites on day 60 with 175/xg TGF-a and on days 90, 150, 180, and 210 with 100 /zg TGF-a. The antibody titers are measured by indirect ELISA (for a general description of this procedure, see the production of monoclonal antibodies to TGF-/3 in [29], this volume) at 10 to 14 days after each injection. The antiserum is collected starting at day 190 and is designated 34D. This antiserum blocks the binding of TGF-a to the receptor and immunoprecipitates the 50 amino acid TGF-a. It also reacts with denatured and reduced 50 amino acid TGF-a in Western blots. The antibody is also able to immunoprecipitate the secreted glycosylated TGF-a species and to a lesser extent the nonglycosylated transmembrane precursor. We were not able, however, to immunoprecipitate the solubilized glycosylated transmembrane form using the antibody. 8
Antipeptide Antibodies As an alternative approach to produce an anti-TGF-a antiserum we injected rabbits with synthetic peptides that correspond to segments of the 50 amino acid TGF-a sequence. The peptides, synthesized using the 6 R. Derynck, A. B. bridge, Mass.) 38, 7 M. E. Winkler, T. 8 T. S. Bringman, P.
Roberts, M. E. Winkler, E. Y. Chen, and D. V. Goeddel, Cell (Cam287 (1984). S. Bringman, and B. J. Marks, J. Biol. Chem. 261, 13838 (1986). B. Lindquist, and R. Derynck, Cell (Cambridge, Mass.) 48, 429 (1987).
188
EGF, TGF-a, AND RELATEDFACTORS
[17]
solid-phase method, 9 are coupled via thioester linkages from the terminal cysteine to soybean trypsin inhibitor at a ratio of 5-10 mol of peptide per mole carrier protein. Rabbits are injected intradermally on day 0 with 1 mg of the conjugated peptide in Freund's complete adjuvant. Subsequent subcutaneous booster injections of 200/xg of the immunogen in Freund's incomplete adjuvant are given on day 30, and injections of 100 t~g immunogen are given on days 70, 100, 130, and 160. The antibody titer to each peptide is determined by indirect ELISA at 10 to 14 days after each injection. 8 The first antiserum, 34AP1, was against a peptide corresponding to the first 10 amino acids of the 50 amino acid TGF-a (CRFLVQEDKP), whereas antiserum 34AP2 was raised against the C-terminal octapeptide (CEHADLLA). Another TGF-ot peptide, which was used to raise the antiserum 34AP3, corresponds to a C-terminal loop of TGF-a (KVCHSGYVGARCEHADLLA). This 19 amino acid long peptide was disulfide bonded in order to mimick the TGF-o~ loop and linked via a N-terminal Lys residue to the carrier protein. I° Although we were able to raise peptide-specific antibodies, they are only moderately useful to immunoprecipitate TGF-a and are inferior to the above-described 34D antiserum. Finally an antiserum was raised against the 15-mer R H E K P S A L L K GRTAC, which corresponds to a sequence in the cytoplasmic segment of the TGF-a precursor. This peptide was also coupled via its Cys-residue to the soybean trypsin inhibitor carrier protein. We have used this antiserum 34E successfully to immunoprecipitate the cytoplasmic segment following the cleavage of the precursor as well as both the glycosylated and nonglycosylated transmembrane TGF-a precursors. 8 Monoclonal Antibodies One hundred micrograms of purified and refolded human TGF-ot in Freund's complete adjuvant is injected intraperitoneally into BALB/c mice. After 5 weeks each animal receives an additional subcutaneous injection with 20/zg TGF-o~ in Freund's incomplete adjuvant. Sixteen days later, the presence of TGF-ot antibodies is demonstrated by indirect ELISA in one of the mice. The mouse then receives an intravenous injection of 25 /zg TGF-ct, followed by three consecutive daily injections of 100/zg TGF-a. The next day the spleen is removed, and the splenocytes are fused 9 G. Barany and R. B. Merrifield, in "The Peptides" (E. Gross and J. Meienhofer, eds.), Vol. 2, p. 1. Academic Press, New York, 1980. l0 j. W. Littlefield, Science 145, 709 (1964).
[17]
ASSAYS FOR TRANSFORMING GROWTH FACTOR oz
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with P3X63Ag8.653 plasmacytoma cells H as described, n The fused cells are plated into the wells of 96-well polystyrene microtiter plates at a density of 2 × 105 cells/well followed by HAT (hypoxanthine, aminopterin, thymidine) selection ~° 1 day after the fusion. The resulting hybridomas are screened for the production of anti-TGF-a antibodies by indirect ELISA (see [29], this volume). This procedure gave rise to the hybridoma secreting the monoclonal antibody (MAb) TGF-oq. The hybridoma cells are grown as ascites in pristane-primed BALB/c mice. 13Characterization of the antibody showed that TGF-oq belongs to the IgGl type and has a K d of 10 -9 mol/liter. The antibody has very little cross-reactivity with EGF, since about 30,000-fold more EGF than TGF-~ is needed to displace 125I-labeled TGF-a binding in a radioimmunoassay (RIA). MAb TGF-aj is able to block effectively the binding of the 50 amino acid TGF-o~ to the receptor (whether this antibody neutralizes glycosylated TGF-o~ precursor forms is still unknown). We were not able to accurately determine the antibody epitope of TGF-o~; however, the antibody does not bind to the C-terminal loop of TGF-~ and does not recognize denatured and reduced TGF-o~.
Purification of Antibodies The immunoglobulin G fraction of the rabbit anti-TGF-a antisera or of the ascites fluid from anti-TGF-~ hybridomas is purified by adsorption to protein A-Sepharose. Whereas the rabbit antisera are loaded undiluted, we dilute the ascites fluids 5-fold with 3 M KCI, 0.1 M acetic acid, 0.1 M Tris-HCl (pH 8.5), 25 mM EDTA, 0.1% Tween 20 before loading onto the column. The protein A column is subsequently washed with phosphatebuffered saline (PBS) or with the dilution buffer in the case of the ascites fluid. The bound IgG is eluted with 0.15 M NaCI, 0.1 M acetic acid, neutralized to pH 8.0 with 1 M Tris, and dialyzed against PBS. The antibody preparation is concentrated to 10 mg/ml by ultrafiltration using an Amicon (Danvers, MA) PM10 membrane. We use peptide affinity column chromatography in order to purify the 34E antiserum. The affinity gel is made by coupling 20 mg of the oligopeptide used as immunogen to 10 ml oJ-aminobutyl-agarose (Sigma, St. Louis, MO). The matrix is activated with N-maleimidobenzoyl sulfosuccinimide ester (Pierce, Rockford, IL) and is then allowed to react with the synthetic peptide via its C-terminal cysteine using the manufacturer's suggested i1 j. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewski, J. Immunol. 123, 1548 (1979). lZ S. F. De St. Groth and D. Scheidegger, J. Imrnunol. Methods 35, 1 (1980). 13 M. Potter, J. G. Humphrey, and J. L. Walter, J. Natl. Cancer Inst. 49, 305 (1972).
190
EGF, TGF-a, AND RELATEDFACTORS
[17]
protocol. Five milliliters of serum is loaded on a 10-ml column, which is then washed with 50 ml PBS before elution of the bound antibodies with 0.15 M NaCI, 0.1 M acetic acid. The eluted antibody fractions are adjusted to pH 8.0 with 1 M Tris and dialyzed against PBS. Bovine serum albumin (BSA) is then added to 5 mg/ml. A typical purification of 5 ml antiserum yields about 0.6 mg affinity purified antibody. Assay of Transforming Growth Factor a
Enzyme-Linked lmmunosorbent Assay Quantitation of TGF-a is performed using a double-sandwich ELISA. We find that this assay, outlined below, is superior in both sensitivity and specificity to an RIA using our antibodies. Micro-ELISA plates (Nunc, Roskilde, Denmark) are coated with 200 ng per well of purified TGF-aj antibody in PBS for 16 hr at 4 °. After the plates are briefly rinsed with PBS-0.05% Tween 20, 100-/A aliquots of the samples in TBS-BSATween (0.15 M NaCI, 50 mM Tris-HC1, pH 7.4, 2 mM EDTA, 5 mg/ml BSA, 0.05% Tween 20) are incubated in the wells for 2 hr at 22°. A dilution series of pure refolded human TGF-a derived from E. coli (5 to 0.078 ng/ml) is included in duplicate on every plate. The plates are washed 5 times with PBS-Tween and then incubated for 2 hr at 22° with 100/zl/well of a 1 : 1000 dilution of the 34D IgG fraction in TBS-BSA-Tween. The plates are again washed 5 times in PBS-Tween and are incubated for 2 hr at 22° with 100/zl/well of a 10,000-fold dilution of horseradish peroxidaseconjugated goat anti-rabbit immunoglobulin in TBS-BSA-Tween. The plates are again washed 5 times and then allowed to react for 30 min with 100/A/well of 0.2 mg/ml o-phenylenediamine, 0.012% (v/v) H20: in 0.1 M phosphate-citrate (pH 5.0). The reaction is stopped by the addition of 50 t~l/well of 2.5 N HzSO 4. The absorbance is measured using a Titertek Multiscan autoreader interfaced with a Hewlett-Packard integral personal computer with Titercalc software. This allows the conversion of OD492 values to TGF-a concentrations. The ELISA does not detect TGF-a with incorrectly formed disulfide bridges, equivalent amounts of rat TGF-a, or 10 tzg/ml human EGF. The results obtained from this assay are in agreement with the values determined from an EGF receptor-binding assay. The ELISA allows accurate measurements of TGF-a between 0. I and 5 ng/ml.
Radioreceptor Assay The radioreceptor assay measures the binding of properly folded EGF or TGF-a to the common cell surface receptor and thus does not discrimi-
[18]
TGF-a STRUCTURE--FUNCTIONANALYSIS
191
hate between the two ligands. Mink lung epithelial-like cells (MvlLu, ATCC CCL64) are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and I% nonessential amino acids. Cells are seeded at 2 x 105/well in 96-well tissue culture plates with removable wells (Dynatech, Alexandria, VA), for 18 to 24 hr prior to assay. Immediately before the assay the cells are washed twice with DMEM containing 10 g/liter BES [N,N-bis(2-hydroxyethyl)-aminoethanesulfonic acid] buffer, pH 7.0 (Sigma), and 1 g/liter BSA (dilution buffer). The assay samples are serially diluted in the same buffer, and 100-~1 aliquots are added to each well. EGF is labeled with 125I by the chloramine-T method to a specific activity of 80-100/zCi//zg. One hundred microliters containing 10 6 counts/min (cpm) of 125I-labeled EGF diluted in the same dilution buffer is added to each well, and the plates are incubated at 22 ° for 70 min. The wells are washed 4 times with Hanks' balanced salt solution and are then counted with a y-counter. Under these conditions, 4-9% of the lZSI-labeled EGF binds to the cells, and 4 × 10-10 M EGF is needed to displace 50% of the specific binding, as analyzed by Scatchard analysis. Conclusion The development of antibodies and assays for transforming growth factor a has been difficult. In particular, the production of antibodies of sufficient titer and specificity, and especially of neutralizing antibodies, was only possible with the availability of larger quantities (recombinant) of the protein in the proper configuration. In spite of these difficulties, reagents of high quality were produced, and reliable procedures were developed for the measurement of TGF-a.
[18] M o l e c u l a r a n d B i o c h e m i c a l A p p r o a c h e s to Structure-Function Analysis of Transforming Growth Factor a
By
DEBORAH DEFEO-JONES,
JOSEPH Y. TAI, and ALLEN OL1FF
Introduction Transforming growth factor a (TGF-a) is a 50 amino acid peptide hormone. Medical interest in TGF-a stems from its biological activity as a stimulator of tumor cell growth. Transfection of nontransformed murine fibroblasts with a molecular clone of TGF-a cDNA converts these cells to METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by AcademicPress, Inc. All rights of reproductionin any form reserved.
ASSAYS FOR TRANSFORMING
G R O W T H F A C T O R ot
185
Summary This chapter outlines in detail the optimal conditions for the expression of human recombinant proteins in mouse cells using a bovine papillomavims-based mammalian expression vector. The procedures we have described were used to successfully express high levels of the human EGF precursor in our laboratory and the human insulin receptor in the laboratory of Whittaker. 4 Using this experimental approach we were able to demonstrate that expression of a cDNA for prepro-EGF produces a glycosylated membrane protein with biological activity. Acknowledgments I am grateful to Martha Reich for skilled technical assistance, Jonathan Whittaker for helpful discussions and valuable advice, Susan Heaver for secretarial assistance, and Ann M. Soderquist for critical reading of the manuscript.
[ 17] G e n e r a t i o n o f A n t i b o d i e s a n d A s s a y s for T r a n s f o r m i n g Growth Factor a By CATHERINE LUCAS, TIMOTHY S. BRINGMAN, a n d RIK DERYNCK
Introduction Two types of growth factors have been termed transforming growth factors (TGF) because they were discovered in an assay that evaluated their ability to elicit cellular transformation using an immortalized nonneoplastic fibroblast line. One of these, TGF-a, is a single-chain polypeptide structurally related to epidermal growth factor (EGF). Several TGF-a species are proteolytically derived from a transmembrane precursor form. The fully processed form is 50 amino acids long and lacks glycosylation, whereas the larger forms are glycosylated. All these forms and the transmembrane precursor are biologically active and interact with the same receptor as EGF. The best studied activity of TGF-a is its ability to stimulate DNA synthesis and mitosis of the many cell types that contain the corresponding cell surface receptors. 1-3TGF-fl is structurally unrelated t R. Derynck, Cell (Cambridge, Mass.) 54, 593 (1988). 2 R. Brachmann, P. B. Lindquist, M. Nagashima, W. Kohr, T. Lipari, M. Napier, and R. Derynck, Cell (Cambridge, Mass.) 56, 691 (1989). 3 S. T. Wong, L. F. Winchell, B. K. McCune, H. S. Earp, J. Teixido, J. Massagu6, B. Herman, and D. C. Lee, Cell (Cambridge, Mass.) 56, 495 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
186
EGF, TGF-a, AND RELATEDFACTORS
[17]
to TGF-a and binds to distinct receptors. 4'5 It is described in more detail in [29]-[32] in this volume. Much interest in both TGF-a and TGF-/3 has arisen because both types of factors have been shown to be functional in normal physiology and also have been implicated in malignant transformation and tumorigenesis. The availability of antibodies to transforming growth factor a is of utmost importance in studying its biology, not only by neutralizing its activities, but also in developing fast, reliable, and specific assays. This chapter describes the methodology we have used to develop antibodies and assays specific for TGF-a. Because both TGF-a and EGF bind to the same receptor, a receptorbinding assay would not be able to discriminate between the factors; however, it would have the advantage of measuring only active growth factors in a configuration able to bind the receptor. The lack of specificity for TGF-a in a receptor binding assay thus dictates the need to develop a TGF-a-specific antibody and immunoassay. Ideally such antibodies and assays should not cross-react with EGF. EGF and TGF-a share about a 30% sequence identity, and it is thus imperative that the lack of crossreactivity be documented. It would also be an advantage if the antibody only reacts with the biologically active molecules and interacts with an epitope formed by the secondary structure of the properly folded factor. Finally, the antibodies should be able to recognize the multiple forms of secreted TGF-a and the larger transmembrane form. A major problem in developing antibodies for TGF-a is the lack of naturally secreted TGF-a. The TGF-a secreted by human cell lines is present in such low concentrations in the medium that it becomes virtually impossible to purify sufficiently large quantities for immunization experiments. We have overcome this difficulty by expressing the 50 amino acid form of TGF-a as a fusion protein in E s c h e r i c h i a coli. The fusion protein is then purified, refolded into the proper disulfide configuration, and cleaved to release the 50 amino acid TGF-a. Preparation of Recombinant Transforming Growth Factor a The 50 amino acid form of human TGF-a can be obtained as follows. An expression vector is constructed in such a way that it directs the expression of a 69 amino acid TGF-a fusion protein in E. coli. The fusion 4 A. B. Roberts and M. B. Sporn, in "Peptide Growth Factors and Their Receptors" (M. B. Spore and A. B. Roberts, eds.), Handbookof ExperimentalPharmacology.Springer-Verlag, Heidelberg, in press. 5M. B. Sporn, A. B. Roberts, L. M. Wakefield,and B. de Crombrugghe,J. Cell Biol. 105, 1039 (1987).
[17]
ASSAYS FOR TRANSFORMING GROWTH FACTOR ot
187
protein consists of an 18 amino acid leader sequence followed by a unique methionine that precedes the 50 amino acid TGF-a. This fusion protein is isolated and purified from the recombinant E. coli and cleaved with cyanogen bromide. This treatment results in a cleavage following the methionine residue, thus releasing TGF-a. The TGF-a is then refolded in an oxidation-reduction buffer, and the biologically active TGF-a with the correct disulfide bridge configuration is purified to apparent homogeneity by HPLC. The bacterial expression of TGF-a and its purification have been detailed. 6,7
Generation of Antibodies
Polyclonal Antibodies Antisera against human TGF-a are prepared in rabbits using the purified and refolded recombinant human TGF-a as immunogen. A first injection on day 0 consists of 400/xg TGF-a emulsified in Freund's complete adjuvant and is given intradermally at multiple sites. Subsequent booster injections in Freund's incomplete adjuvant are given subcutaneously at multiple sites on day 60 with 175/xg TGF-a and on days 90, 150, 180, and 210 with 100 /zg TGF-a. The antibody titers are measured by indirect ELISA (for a general description of this procedure, see the production of monoclonal antibodies to TGF-/3 in [29], this volume) at 10 to 14 days after each injection. The antiserum is collected starting at day 190 and is designated 34D. This antiserum blocks the binding of TGF-a to the receptor and immunoprecipitates the 50 amino acid TGF-a. It also reacts with denatured and reduced 50 amino acid TGF-a in Western blots. The antibody is also able to immunoprecipitate the secreted glycosylated TGF-a species and to a lesser extent the nonglycosylated transmembrane precursor. We were not able, however, to immunoprecipitate the solubilized glycosylated transmembrane form using the antibody. 8
Antipeptide Antibodies As an alternative approach to produce an anti-TGF-a antiserum we injected rabbits with synthetic peptides that correspond to segments of the 50 amino acid TGF-a sequence. The peptides, synthesized using the 6 R. Derynck, A. B. bridge, Mass.) 38, 7 M. E. Winkler, T. 8 T. S. Bringman, P.
Roberts, M. E. Winkler, E. Y. Chen, and D. V. Goeddel, Cell (Cam287 (1984). S. Bringman, and B. J. Marks, J. Biol. Chem. 261, 13838 (1986). B. Lindquist, and R. Derynck, Cell (Cambridge, Mass.) 48, 429 (1987).
188
EGF, TGF-a, AND RELATEDFACTORS
[17]
solid-phase method, 9 are coupled via thioester linkages from the terminal cysteine to soybean trypsin inhibitor at a ratio of 5-10 mol of peptide per mole carrier protein. Rabbits are injected intradermally on day 0 with 1 mg of the conjugated peptide in Freund's complete adjuvant. Subsequent subcutaneous booster injections of 200/xg of the immunogen in Freund's incomplete adjuvant are given on day 30, and injections of 100 t~g immunogen are given on days 70, 100, 130, and 160. The antibody titer to each peptide is determined by indirect ELISA at 10 to 14 days after each injection. 8 The first antiserum, 34AP1, was against a peptide corresponding to the first 10 amino acids of the 50 amino acid TGF-a (CRFLVQEDKP), whereas antiserum 34AP2 was raised against the C-terminal octapeptide (CEHADLLA). Another TGF-ot peptide, which was used to raise the antiserum 34AP3, corresponds to a C-terminal loop of TGF-a (KVCHSGYVGARCEHADLLA). This 19 amino acid long peptide was disulfide bonded in order to mimick the TGF-o~ loop and linked via a N-terminal Lys residue to the carrier protein. I° Although we were able to raise peptide-specific antibodies, they are only moderately useful to immunoprecipitate TGF-a and are inferior to the above-described 34D antiserum. Finally an antiserum was raised against the 15-mer R H E K P S A L L K GRTAC, which corresponds to a sequence in the cytoplasmic segment of the TGF-a precursor. This peptide was also coupled via its Cys-residue to the soybean trypsin inhibitor carrier protein. We have used this antiserum 34E successfully to immunoprecipitate the cytoplasmic segment following the cleavage of the precursor as well as both the glycosylated and nonglycosylated transmembrane TGF-a precursors. 8 Monoclonal Antibodies One hundred micrograms of purified and refolded human TGF-ot in Freund's complete adjuvant is injected intraperitoneally into BALB/c mice. After 5 weeks each animal receives an additional subcutaneous injection with 20/zg TGF-o~ in Freund's incomplete adjuvant. Sixteen days later, the presence of TGF-ot antibodies is demonstrated by indirect ELISA in one of the mice. The mouse then receives an intravenous injection of 25 /zg TGF-ct, followed by three consecutive daily injections of 100/zg TGF-a. The next day the spleen is removed, and the splenocytes are fused 9 G. Barany and R. B. Merrifield, in "The Peptides" (E. Gross and J. Meienhofer, eds.), Vol. 2, p. 1. Academic Press, New York, 1980. l0 j. W. Littlefield, Science 145, 709 (1964).
[17]
ASSAYS FOR TRANSFORMING GROWTH FACTOR oz
189
with P3X63Ag8.653 plasmacytoma cells H as described, n The fused cells are plated into the wells of 96-well polystyrene microtiter plates at a density of 2 × 105 cells/well followed by HAT (hypoxanthine, aminopterin, thymidine) selection ~° 1 day after the fusion. The resulting hybridomas are screened for the production of anti-TGF-a antibodies by indirect ELISA (see [29], this volume). This procedure gave rise to the hybridoma secreting the monoclonal antibody (MAb) TGF-oq. The hybridoma cells are grown as ascites in pristane-primed BALB/c mice. 13Characterization of the antibody showed that TGF-oq belongs to the IgGl type and has a K d of 10 -9 mol/liter. The antibody has very little cross-reactivity with EGF, since about 30,000-fold more EGF than TGF-~ is needed to displace 125I-labeled TGF-a binding in a radioimmunoassay (RIA). MAb TGF-aj is able to block effectively the binding of the 50 amino acid TGF-o~ to the receptor (whether this antibody neutralizes glycosylated TGF-o~ precursor forms is still unknown). We were not able to accurately determine the antibody epitope of TGF-o~; however, the antibody does not bind to the C-terminal loop of TGF-~ and does not recognize denatured and reduced TGF-o~.
Purification of Antibodies The immunoglobulin G fraction of the rabbit anti-TGF-a antisera or of the ascites fluid from anti-TGF-~ hybridomas is purified by adsorption to protein A-Sepharose. Whereas the rabbit antisera are loaded undiluted, we dilute the ascites fluids 5-fold with 3 M KCI, 0.1 M acetic acid, 0.1 M Tris-HCl (pH 8.5), 25 mM EDTA, 0.1% Tween 20 before loading onto the column. The protein A column is subsequently washed with phosphatebuffered saline (PBS) or with the dilution buffer in the case of the ascites fluid. The bound IgG is eluted with 0.15 M NaCI, 0.1 M acetic acid, neutralized to pH 8.0 with 1 M Tris, and dialyzed against PBS. The antibody preparation is concentrated to 10 mg/ml by ultrafiltration using an Amicon (Danvers, MA) PM10 membrane. We use peptide affinity column chromatography in order to purify the 34E antiserum. The affinity gel is made by coupling 20 mg of the oligopeptide used as immunogen to 10 ml oJ-aminobutyl-agarose (Sigma, St. Louis, MO). The matrix is activated with N-maleimidobenzoyl sulfosuccinimide ester (Pierce, Rockford, IL) and is then allowed to react with the synthetic peptide via its C-terminal cysteine using the manufacturer's suggested i1 j. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewski, J. Immunol. 123, 1548 (1979). lZ S. F. De St. Groth and D. Scheidegger, J. Imrnunol. Methods 35, 1 (1980). 13 M. Potter, J. G. Humphrey, and J. L. Walter, J. Natl. Cancer Inst. 49, 305 (1972).
190
EGF, TGF-a, AND RELATEDFACTORS
[17]
protocol. Five milliliters of serum is loaded on a 10-ml column, which is then washed with 50 ml PBS before elution of the bound antibodies with 0.15 M NaCI, 0.1 M acetic acid. The eluted antibody fractions are adjusted to pH 8.0 with 1 M Tris and dialyzed against PBS. Bovine serum albumin (BSA) is then added to 5 mg/ml. A typical purification of 5 ml antiserum yields about 0.6 mg affinity purified antibody. Assay of Transforming Growth Factor a
Enzyme-Linked lmmunosorbent Assay Quantitation of TGF-a is performed using a double-sandwich ELISA. We find that this assay, outlined below, is superior in both sensitivity and specificity to an RIA using our antibodies. Micro-ELISA plates (Nunc, Roskilde, Denmark) are coated with 200 ng per well of purified TGF-aj antibody in PBS for 16 hr at 4 °. After the plates are briefly rinsed with PBS-0.05% Tween 20, 100-/A aliquots of the samples in TBS-BSATween (0.15 M NaCI, 50 mM Tris-HC1, pH 7.4, 2 mM EDTA, 5 mg/ml BSA, 0.05% Tween 20) are incubated in the wells for 2 hr at 22°. A dilution series of pure refolded human TGF-a derived from E. coli (5 to 0.078 ng/ml) is included in duplicate on every plate. The plates are washed 5 times with PBS-Tween and then incubated for 2 hr at 22° with 100/zl/well of a 1 : 1000 dilution of the 34D IgG fraction in TBS-BSA-Tween. The plates are again washed 5 times in PBS-Tween and are incubated for 2 hr at 22° with 100/zl/well of a 10,000-fold dilution of horseradish peroxidaseconjugated goat anti-rabbit immunoglobulin in TBS-BSA-Tween. The plates are again washed 5 times and then allowed to react for 30 min with 100/A/well of 0.2 mg/ml o-phenylenediamine, 0.012% (v/v) H20: in 0.1 M phosphate-citrate (pH 5.0). The reaction is stopped by the addition of 50 t~l/well of 2.5 N HzSO 4. The absorbance is measured using a Titertek Multiscan autoreader interfaced with a Hewlett-Packard integral personal computer with Titercalc software. This allows the conversion of OD492 values to TGF-a concentrations. The ELISA does not detect TGF-a with incorrectly formed disulfide bridges, equivalent amounts of rat TGF-a, or 10 tzg/ml human EGF. The results obtained from this assay are in agreement with the values determined from an EGF receptor-binding assay. The ELISA allows accurate measurements of TGF-a between 0. I and 5 ng/ml.
Radioreceptor Assay The radioreceptor assay measures the binding of properly folded EGF or TGF-a to the common cell surface receptor and thus does not discrimi-
[18]
TGF-a STRUCTURE--FUNCTIONANALYSIS
191
hate between the two ligands. Mink lung epithelial-like cells (MvlLu, ATCC CCL64) are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and I% nonessential amino acids. Cells are seeded at 2 x 105/well in 96-well tissue culture plates with removable wells (Dynatech, Alexandria, VA), for 18 to 24 hr prior to assay. Immediately before the assay the cells are washed twice with DMEM containing 10 g/liter BES [N,N-bis(2-hydroxyethyl)-aminoethanesulfonic acid] buffer, pH 7.0 (Sigma), and 1 g/liter BSA (dilution buffer). The assay samples are serially diluted in the same buffer, and 100-~1 aliquots are added to each well. EGF is labeled with 125I by the chloramine-T method to a specific activity of 80-100/zCi//zg. One hundred microliters containing 10 6 counts/min (cpm) of 125I-labeled EGF diluted in the same dilution buffer is added to each well, and the plates are incubated at 22 ° for 70 min. The wells are washed 4 times with Hanks' balanced salt solution and are then counted with a y-counter. Under these conditions, 4-9% of the lZSI-labeled EGF binds to the cells, and 4 × 10-10 M EGF is needed to displace 50% of the specific binding, as analyzed by Scatchard analysis. Conclusion The development of antibodies and assays for transforming growth factor a has been difficult. In particular, the production of antibodies of sufficient titer and specificity, and especially of neutralizing antibodies, was only possible with the availability of larger quantities (recombinant) of the protein in the proper configuration. In spite of these difficulties, reagents of high quality were produced, and reliable procedures were developed for the measurement of TGF-a.
[18] M o l e c u l a r a n d B i o c h e m i c a l A p p r o a c h e s to Structure-Function Analysis of Transforming Growth Factor a
By
DEBORAH DEFEO-JONES,
JOSEPH Y. TAI, and ALLEN OL1FF
Introduction Transforming growth factor a (TGF-a) is a 50 amino acid peptide hormone. Medical interest in TGF-a stems from its biological activity as a stimulator of tumor cell growth. Transfection of nontransformed murine fibroblasts with a molecular clone of TGF-a cDNA converts these cells to METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by AcademicPress, Inc. All rights of reproductionin any form reserved.
