Journal of Immunological Methods, 128 (1990) 211-217
211
Elsevier JIM05520
Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays M e e n u W a d h w a , R o b i n T h o r p e , C h r i s t o p h e r R. Bird a n d A n d r e w J.H. G e a r i n g National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, U.K.
(Received26 June 1989, revised received20 October 1989, accepted 5 December 1989)
Murine monoclonal antibodies and a sheep polyclonal antiserum against recombinant human granulocyte colony-stimulating factor (GCSF) have been produced. These have been used to develop an immunoassay which can detect 250 p g / m l (25 U) of both natural and recombinant human GCSF. The assay involves forming a complex between GCSF and a monoclonal anti-GCSF, binding of the complex to microtitre wells coated with sheep anti-GCSF and detection of the bound complex with 125I-labelled sheep anti-mouse IgG. Unlike the classical bone marrow assay and other cell line based bioassays for GCSF, the immunoassay was specific for the cytokine, showing no cross-reactivity with GM-CSF, IL-6, IL-3 or I L - l a and -ft. The assay does not exhibit interfering matrix effects when used for the estimation of human GCSF in serum. Key words: Granulocyte colony-stimulatingfactor; Immunoradiometricassay; Monoclonal antibody
Introduction
Colony-stimulating factors (CSFs) are glycoproteins which regulate the survival, proliferation and differentiation of haematopoietic cells. Four human CSFs have now been identified and their genomic c D N A clones isolated - granulocyte macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), macrophage CSF (M-CSF or CSF-1) and multi-CSF or IL-3 (Kawasaki et al., 1985; Wong et al., 1985; Nagata et al., 1986; Yang et al., 1986). The CSFs differ with respect to the cell hneage primarily affected and their biological responses.
Correspondence to: M. Wadhwa, Division of Immunobiology, NIBSC, Blanche Lane, South Mimms, Herts EN6 3QG, U.K.
Classically, CSFs are assayed in vitro by their abihty to stimulate formation of colonies of differentiated cells from progenitor cells of the bone-marrow in soft agar (Metcalf, 1984). The sensitivity of these assays is variable depending on the source and condition of the bone marrow. Moreover, such assays respond to all CSFs and are subject to positive and negative interference by other cytokines such as IL-1 and IL-6. As colony assays are time-consuming and very prone to error, alternative assays have now been developed relying on the ability of CSFs to stimulate prohferation of cell hnes derived from human myeloid leukemias, e.g., AML-193 or TALL-101 (Santoh et al., 1987) and murine lines such as NFS60 or W E H I 3BD + which respond to human GCSF (Nicola et al., 1983). Although more reproducible than bone marrow assays, some of these cell lines can be unstable and also non-specific. To over-
0022-1759/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
212 come the problems encountered in bioassays, we have developed a sensitive and specific immunoradiometric assay for GCSF using monoclonal and polyclonal antibodies.
Materials and methods
Mouse bone marrow proliferation assay Low density non-adherent mouse bone marrow cells were obtained by fractionation on a FicollHypaque gradient. After adjusting the cell density to 4 x 105 cells/ml, a cell suspension (100 #1) was added to each well containing different dilutions of GCSF and to the control wells. The cultures were incubated for 44 h, pulsed for 4 h with 0.5 #Ci/well [3H]thymidine and harvested. The radioactivity incorporated into DNA was measured in a liquid scintillation counter. For neutralization assays, differing dilutions of antiserum were incubated with GCSF for approximately 1 h at 37 °C before addition of the cell suspension. AML-193 assay for GCSF As this cell line is GMCSF dependent, it must be cultured in medium containing GMCSF (2 ng/ml). Details of this cell line have been published previously (Santoli et al., 1987). For assay, cells were starved overnight in medium devoid of GMCSF. After washing, the cells were seeded at 104/well in 96-well plates in the presence or absence of recombinant GCSF. After an incubation period of 44 h, the cells were pulsed for 4 h with 0.5 ttCi/well [3H]thymidine and harvested. The radioactivity incorporated into DNA was subsequently measured by liquid scintillation counting. For neutralization assays, differing dilutions of antiserum were incubated with GCSF for approximately 1 h at 37 °C before addition of the cell suspension. Production of antiserum Polyclonal antiserum to GCSF was raised in sheep by intramuscular injection of 500 #g of the rDNA-derived GCSF emulsified in Freund's complete adjuvant. 1 month after the primary injection, boosts of 200 #g of GCSF in Freund's in-
complete adjuvant were given at regular intervals of 1, 2 and 6 months. The sheep were bled 8 and 10 days after the final boost.
Production of monoclonal antibodies A mouse was injected subcutaneously with 25 #g GCSF emulsified in Freund's complete adjuvant. On days 30-60 after the primary immunization 10 #g GCSF emulsified in Freund's incomplete adjuvant were injected intraperitoneally. On day 150, the mouse was injected intraveneously with 10 #g GCSF in saline and on day 155 the spleen was removed for fusion. Cell fusion was performed essentially as described in Johnstone and Thorpe (1987). Spleen cells were fused with the HAT sensitive myeloma line NSO to produce hybridomas which secreted monoclonal antibodies specific for GCSF. Supernatants from wells containing almost confluent hybridomas were screened for antibodies against GCSF by a solid-phase radiobinding assay (Johnstone and Thorpe, 1987). The positive wells demonstrating greater than ten times background counts were cloned in soft agar and screened again. Specific clones were grown in culture, cryopreserved or injected into the peritoneal cavity of pristane primed BALB/c mice to produce ascitic fluid. Sofid-phase radiobinding assay Test antiserum or supernatants from wells containing almost confluent hybridomas from the fusion were incubated for at least 2 h in flexible 96-well microtitre plates which had been coated with GCSF by overnight incubation (5 #g/ml). Positive binding was assessed using ~25I-labelled donkey anti-sheep or sheep anti-mouse IgG. Immunoblotting The specificity of the antibodies to GCSF was assessed following separation by polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. After transfer, the protein binding sites on the nitrocellulose sheets were blocked by incubation for 30 rain with 3% bovine haemoglobin dissolved in phosphate-buffered saline (HbPBS). The blot was then incubated overnight with the antiserum (10 #1) diluted in 25 ml Hb-PBS, washed with Hb-PBS and then incubated for a
213 further 3 h with a25I-labelled donkey anti-sheep or sheep anti-mouse IgG (106 cpm/track). After extensive washing with PBS, the blots were dried and exposed to Kodak X-omat film for 2 days at - 7 0 °C in a cassette equipped with a single tungstate intensifying screen.
Purification of monoclonal antibodies IgG was purified from ascitic fluid by precipitation using 45% saturation with ammonium sulphate followed by FPLC using a Mono-Q ion exchange column (Pharmacia) (Clezardin et al., 1985). Purification of antibodies from polyclonal antiserum GCSF (2 mg) was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) (0.5 g) according to the manufacturer's instructions. This column was used to affinity purify anti-GCSF antibodies from the polyclonal antiserum. Bound antibodies were eluted with 0.1 M glycine pH 2.5 (Johnstone and Thorpe, 1987). Radioiodination of antibodies Antibodies were radiolabelled using chloramine-T as catalyst and Dowex 1X-8 ion exchanger to remove non-protein-bound 1251 (Johnstone and Thorpe, 1987). For each iodination 40 /~g of antibody and 200/xCi Na125I were used. Immunoradiometric assays (IRMA)for GCSF Flexible 96-well microtitre plates were used in immunoradiometric assays for GCSF. Wells were coated with 'capture' antibody by incubation with the antibody (5 # g / m l in phosphate-buffered saline (PBS)) overnight at 4°C. The plates were washed three times with PBS containing 3% bovine haemoglobin (Hb-PBS) and the residual proteinbinding sites blocked by incubating the plates with Hb-PBS for 30 min at room temperature. After a further wash with Hb-PBS, the wells were incubated at room temperature for 3 h with sampies or dilutions of GCSF in Hb-PBS. After washing, bound GCSF was detected by incubation with a a25I-labelled developing antibody (300,000 cpm/well) diluted in Hb-PBS for at least 1 h. After a subsequent wash, wells were excised with a hot-wire plate cutter and the radioactivity estimated in a gamma-counter.
The two-site IRMA described above was modified slightly to develop a more sensitive assay for GCSF (Overall et al., 1989). Instead of adding antigen directly to the wells, the antigen was complexed with the antibody in separate 96-well plates for 1.5 h at 37 o C. This complex was subsequently transferred to the coated plate and incubated for 2 h at 37 o C. Finally, the plate was washed and the bound GCSF complex detected with 125I-sheep anti-mouse IgG.
Results
Monoclonal antibodies The GCSF fusion yielded three hybridoma fines which secreted antibody able to bind to the immunogen. Following cloning in soft agar a single clone which secreted monoclonal antibodies capable of binding strongly to antigen was selected from each master line and coded 3B6, 3D1 and 3C6. The latter was found to be slightly cross-reactive with ovalbumin and other antigens and was therefore not used for further studies. The two other GCSF-specific clones were injected into pristane-primed mice to produce ascitic fluid containing a high concentration of antibody. These fluids were purified by ammonium sulphate precipitation and ion exchange FPLC to yield homogeneous IgG as shown by SDS-PAGE. Both antibodies were of the IgG2a subclass. Immunoblotting studies demonstrated that both the antibodies were capable of binding specifically to GCSF (Fig. 1). Neither antibodies displayed any cross-reactivity with GM-CSF, IL-3, IL-1 or IL-6. Further characterization of the antibodies indicated that although both 3B6 and 3D1 bound to GCSF equally well in binding assays, only the 3D1 antibody was capable of neutralising the proliferative activity of GCSF in the mouse bonemarrow assay (Fig. 2). Polyclonal antiserum The antiserum raised in sheep against GCSF did not exhibit any cross-reactivity with GMCSF, IL-3 or other cytokines tested (results not shown). In neutralisation experiments, the antiserum completely abolished the proliferative response to GCSF in both the AML-193 assay (Fig. 3) and the
214
2~00, []
Gt:~F
8
GCSF+~6
100
1000
_
15000. O
5OOO 10
Fig. 1. Immunoblotting of recombinant cytokines using GCSF-specific monoclonal antibody, 3D1. A = IL-1; B = IL-3; C = IL-6; D = GCSF; E = GMCSF and F = GCSF. An identical result was obtained using the monoclonal antibody 3B6. The GCSF preparations run in tracks D and F were from different sources.
mouse bone marrow assay (not shown) although the pre-bleed serum had no effect.
Immunoassay The GCSF-specific antibodies and polyclonal antisera were used to develop a sensitive and
3000
O"-----o " ~ %.
• •
G G+3B~12 G+3DII16
1
10
2000-
1000'
~
.0001 .001 .01
~
.1
100 1000
NG~L
Fig. 2. Neutralising effect of the monoclonal antibodies 3B6 and 3D1 on GCSF-induced proliferation of mouse bone marrow cells. The antibodies were used at 1/500 dilution.
10000 100000
Fig. 3. Neutralisation of GCSF-induced proliferation of the AML-193 cell line by the G-CSF-specific polyclonal antiserum. The antiserum was used at a 1/500 dilution.
specific IRMA for GCSF. To establish an IRMA, a combination of antibodies which can recognise different epitopes on the GCSF molecule is required. Surprisingly, all the combinations of monoclonal antibodies failed to detect GCSF. Since radioiodination can denature antibodies and impair their ability to bind the antigen, the antibodies were tested directly in a solid-phase binding assay. However, labelling of the antibodies did not influence the efficiency of binding. A two-site assay employing a combination of sheep polyclonal antiserum and the monoclonal antibodies was also tested. Using the sheep antibody as the capture reagent and either of the two labelled monoclonal antibodies as the detector antibody or vice-versa proved to be too insensitive. An assay using the sheep antibodies for both capture and detection produced a dose-response curve with a detection limit of 2.5 n g / m l . It is possible that binding the antibodies to the plates induced a conformational change in the antibody and interfered with the recognition of the G-CSF molecule. In an attempt to increase the sensitivity of the assay, the assay was modified to include a step involving the formation of a liquid-phase complex between G C S F and monoclonal anti-GCSF. This
215 TABLE I
100000
MEASUREMENT OF GCSF LEVELS IN SUPERNATANTS FROM DIFFERENT CELL LINES USING THE GCSF IMMUNOASSAY Cell line
GCSF concentration in supernatants (pg/ml)
10000
Human foreskin fibroblasts Human monocytes Rabbit monocytes Murine P388 D1 cells Human T cells Human B cells Jurkat cells
E 0
1000
Non-stimulated
Stimulated
Stimulus a
250
10000
IL-la
< 250 < 250 < 250 < 250 < 250 < 250
8 000 < 250 < 250 < 250 < 250 < 250
LPS LPS LPS PHA EBV PHA
a IL-la = interleukin-la, LPS = lipopolysaccharide, PHA = phytohaemagglutinin, EBV = Epstein-Barr Virus. 100 0
100
1000
10000
100000
pg/ml
al., 1988; N i o c h e et al., 1988) c o n d i t i o n e d m e d i a f r o m several h u m a n cell lines were m o n i t o r e d for G C S F activity ( T a b l e I).
Fig. 4. Dose-response curve of rDNA-derived human GCSF using the sheep antibody to capture GCSF-3D1 monoclonal antibody complexes and 12SI-labelledsheep anti-mouse IgG as detector antibody.
complex was then b o u n d to microtitre wells coated with sheep a n t i - G C S F a n d s u b s e q u e n t l y detected with 125I-labelled sheep a n t i - m o u s e IgG. This detection m e t h o d gave a fairly steep dose-response curve (Fig. 4). Both of the m o n o c l o n a l a n t i b o d i e s were able to detect G C S F i n this assay. Of the two antibodies, 3D1 gave a steeper dose-response curve w h e n complexed with G C S F (results n o t shown) a n d was therefore used i n the s u b s e q u e n t immunoassays. O p t i m u m results were o b t a i n e d i n i m m u n o a s says using 20 # g / m l of the 3D1 a n t i b o d y ; a lower c o n c e n t r a t i o n of the a n t i b o d y gave a less sensitive assay. Optimally, i n c u b a t i o n periods of 1.5 h with the a n t i g e n a n d 2 h with the p o l y c l o n a l a n t i s e r u m were required; shorter periods c o n s i d e r a b l y red u c e d the sensitivity of the assays. This assay was f o u n d to b e highly reproducible a n d c a p a b l e of detecting G C S F activity i n different r D N A derived preparations. Since a variety of cell types c a n p r o d u c e G C S F (Broudy et al., 1987; F i b b e et
100000
10000 E
@. u
1000
100 10
100
1000
10000
100000
I~/m! Fig. 5. The effect of 'spiking' rDNA-derived human GCSF with 25% human serum using the GCSF immunoassay employing the 3D1 antibody antigen complex and the sheep antibody to capture antigen. Open squares - no serum; dosed squares with serum.
216
Other cytokines interfere in the classical bone marrow assay and therefore it was necessary to test the immunoassay for specificity. Addition of relatively high concentrations of IL-la, IL-lfl, MCSF, GM-CSF, IL-3, IL-4 and IL-6 produced only a background response. The assay was apparently unaffected by components in human serum since spiking of the standard curve with 25% serum did not interfere with the dose response (Fig. 5). This assay has been used successfully to evaluate GCSF levels in human sera.
Discussion We have developed a sensitive and specific irnmunoassay for the detection of GCSF using a combination of monoclonal and polyclonal antibodies. This assay involves formation of antigenantibody complexes, binding of the complex to sheep anti-GCSF and subsequent detection of the bound complex with 125I-labelled anti-mouse IgG. The assay is quantitative between 250 pg/ml and 50 ng/ml and is specific for GCSF. Because of the speed, sensitivity, specificity and ease of performance, this assay can be routinely used for monitoting GCSF levels and obviates many of the problems associated with bioassays. As a careful evaluation of the antibodies for use in immunoassays is crucial, the antibodies were characterized by binding and neutralization assays. Both the monoclonal and polyclonal antibodies were specific for GCSF, there was no cross-reaction with other cytokines including IL-6 with which it shows some sequence homology (Van Snick et al., 1988). Although the monoclonal antibodies appeared to be similar using solid-phase radiobinding assays and immunoblotting, the antibodies performed differently in neutralization experiments. The 3D1 antibody completely inhibited the proliferation of the bone marrow cells induced by GCSF and 3B6 had no effect. The sheep antiserum was also effective in neutralizing the GCSF stimulated proliferation of bone marrow cells and the AML-193 cell line. The present immunoassay has been used to assay rDNA-detived preparations of GCSF from different manufacturers and natural GCSF in conditioned media from human cell lines. Super-
natants from lipopolysaccharide (LPS)-stimulated human monocytes which were shown to contain IL-1 by bioassay using the NOB-1 cell line (Gearing et al., 1987) contained about 8 n g / m l of GCSF. Unstimulated monocyte supernatants did not contain detectable amounts of GCSF. The release of GCSF by monocytes in response to a variety of stimuli has also been observed by other workers (Nioche et al., 1988). We have demonstrated that human fibroblasts produced small amounts of GCSF (250 pg/ml) and these levels increased to 10 n g / m l following stimulation with IL-la. Using the colony assay Kaushansky and coworkers (1988) also detected similar increases in GCSF following IL-1 stimulation of fibroblasts. None of the activated human T cell or B cell lines produced detectable GCSF. Supernatants from the Jurkat cell line following PHA stimulation were devoid of GCSF, although they contained relatively high amounts of IL-2. The assay appeared to be specific for the human cytokine since conditioned media from LPS activated rabbit monocytes and the murine cell line P388D1 gave only background counts despite containing high levels of IL-1. The present assay should prove useful in monitoring levels of GCSF in culture and tissue fluids.
Acknowledgements We would like to thank Immunex Corporation, Amgen, Schering Research (Schering-Plough Corporation), Cetus Corporation and Dr. T. Kishimoto for supplying cytokines, Dr. P. Herzog (Monash University, Australia) for helpful suggestions and Lisa Hudson for typing the manuscript.
References Broudy, V.C., Kaushansky, K., Harlan, J.M. and Adamson, J.W. (1987) Interleukin-1 stimulates human endothelial cells to produce granulocyte-macrophage colony stimulating factor and granulocyte-colony stimulating factor J. Immtmol. 139, 464-468. Clezardin, P., McGregor, J.L., Manach, M., Bounkerche, H. and Dechavanne, M. (1985) One-step procedure for the rapid isolation of mouse monoclonal antibodies and their
217 antigen binding fragments by fast protein LC on a Mono Q anion-exchange column. J. Chromatogr. 319, 67-77. Fibbe, W.E., Van Damme, J., Billiau, A., Goselink, H.M., Voogt, P.J., Van Eeden, G., Ralph, P., Altrock, B.W. and Falkenberg, J.H.F. (1988) Interleukin-1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony-stimulating factor. Blood 7, 430-435. Gearing, A.J.H., Bird, C.R., Bristow, A., Poole, S. and Thorpe, R. (1987) A simple sensitive bioassay for interleukin-1 which is unresponsive to 103 U / m l of interleukin-2. J. Immunol. Methods 99, 7-11. Johnstone, A. and Thorpe, R. (1987) Immunochemistry in Practice, 2nd edn. Blackwell Scientific Publications, London. Kaushansky, K., Lin, N. and Adamson, J.W. (1988) Interleukin 1 stimulates fibroblasts to synthesize granulocytemacrophage and granulocyte colony-stimulating factors. J. Clin. Invest. 81, 92-97. Kawasaki, E.S., Lander, M.B., Wang, A.M., Van Arsdell, J., Warren, M.K., Coyne, M.Y., Schweickart, V.L., Lee, M.T., Wilson, K.J., Boosman, A., Stanley, A.R., Ralph, P. and Mark, D.F. (1985) Molecular cloning of a complementary DNA encoding human macrophage specific colony stimulating factor (CSF-1). Science 230, 291-296. Metcalf, D. (1984) The Hemopoietic Colony Stimulating Factors. Elsevier, Amsterdam. Morstyn, G., and Burgess, A.W. (1988) Hemopoietic growth factors: A review. Cancer Res. 48, 5624-5637. Nagata, S., Tsuchiya, M., Asano, S., Kaziro, Y., Yamazaki, T., Yamamoto, O., Hirata, Y,, Kubota, N., Oheda, M., Nomura, H. and Ono, M. (1986) Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature 319, 415-418. Nicola, N.A., Metcalf, D., Matsumoto, M. and Johnson, G.R.
(1983) Purification of a factor inducing differentiation in murine myelo-monocytic leukemia cells. J. Biol. Chem. 258, 9017-9023. Nioche, S., Tazi, A., Lecossier, D. and Hance, A.J. (1988) Production of granulocyte colony-stimulating factor by human cells: T lymphocyte-dependent and T lymphocyte-independent release of G-CSF by blood monocytes. J. Immunol. 18, 1021-1026. Overall, M.L., Marzuki, S. and Hertzog, P.J. (1989) Comparison of different ELISAs for the detection of monoclonal antibodies to human interferon-a. Implications for antibody screening. J. Immunol. Methods 119, 27-33. Santoli, D., Yang, Y.C., Clark, S.C., Kreider, B.L., Caracciolo, D. and Rovera, G. (1987) Synergistic and antagonistic effects of recombinant human interlenkin 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors on the growth of GM-CSF-dependent leukemic cell lines. J. Immunol. 139, 3348-3354. Van Snick, J., Cayphas, S., Szikora, J.P., Renauld, J.C., Van Roost, E., Boon, T. and Simpson, R.J. (1988) cDNA cloning of murine interleukin HPI: homology with human interleukin-6. Eur. J. Immunol. 18, 193-197. Wong, G.G., Witek, J.S., Temple, P.A., Wilkens, K.M., Leary, A.C., Lowenberg, D.P., Jones, S.S., Brown, E.L., Kay R.M., Orr, E.C., Shoemaker, C., Golde, D.W., Kaufman, R.J., Hewick, R.M., Wang, E.A. and Clark, S.C. (1985) Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 228, 810-815. Yang, Y.C., Cialetta, A.B., Temple, P.A., Chung, M.P., Kovacic, S., Witek-Giannotti, J.S., Leafy, A.C. and Kriz, R. (1986) Human IL-3 (MUlti-CSF): Identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 47, 3-10.
211
Elsevier JIM05520
Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays M e e n u W a d h w a , R o b i n T h o r p e , C h r i s t o p h e r R. Bird a n d A n d r e w J.H. G e a r i n g National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, U.K.
(Received26 June 1989, revised received20 October 1989, accepted 5 December 1989)
Murine monoclonal antibodies and a sheep polyclonal antiserum against recombinant human granulocyte colony-stimulating factor (GCSF) have been produced. These have been used to develop an immunoassay which can detect 250 p g / m l (25 U) of both natural and recombinant human GCSF. The assay involves forming a complex between GCSF and a monoclonal anti-GCSF, binding of the complex to microtitre wells coated with sheep anti-GCSF and detection of the bound complex with 125I-labelled sheep anti-mouse IgG. Unlike the classical bone marrow assay and other cell line based bioassays for GCSF, the immunoassay was specific for the cytokine, showing no cross-reactivity with GM-CSF, IL-6, IL-3 or I L - l a and -ft. The assay does not exhibit interfering matrix effects when used for the estimation of human GCSF in serum. Key words: Granulocyte colony-stimulatingfactor; Immunoradiometricassay; Monoclonal antibody
Introduction
Colony-stimulating factors (CSFs) are glycoproteins which regulate the survival, proliferation and differentiation of haematopoietic cells. Four human CSFs have now been identified and their genomic c D N A clones isolated - granulocyte macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), macrophage CSF (M-CSF or CSF-1) and multi-CSF or IL-3 (Kawasaki et al., 1985; Wong et al., 1985; Nagata et al., 1986; Yang et al., 1986). The CSFs differ with respect to the cell hneage primarily affected and their biological responses.
Correspondence to: M. Wadhwa, Division of Immunobiology, NIBSC, Blanche Lane, South Mimms, Herts EN6 3QG, U.K.
Classically, CSFs are assayed in vitro by their abihty to stimulate formation of colonies of differentiated cells from progenitor cells of the bone-marrow in soft agar (Metcalf, 1984). The sensitivity of these assays is variable depending on the source and condition of the bone marrow. Moreover, such assays respond to all CSFs and are subject to positive and negative interference by other cytokines such as IL-1 and IL-6. As colony assays are time-consuming and very prone to error, alternative assays have now been developed relying on the ability of CSFs to stimulate prohferation of cell hnes derived from human myeloid leukemias, e.g., AML-193 or TALL-101 (Santoh et al., 1987) and murine lines such as NFS60 or W E H I 3BD + which respond to human GCSF (Nicola et al., 1983). Although more reproducible than bone marrow assays, some of these cell lines can be unstable and also non-specific. To over-
0022-1759/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
212 come the problems encountered in bioassays, we have developed a sensitive and specific immunoradiometric assay for GCSF using monoclonal and polyclonal antibodies.
Materials and methods
Mouse bone marrow proliferation assay Low density non-adherent mouse bone marrow cells were obtained by fractionation on a FicollHypaque gradient. After adjusting the cell density to 4 x 105 cells/ml, a cell suspension (100 #1) was added to each well containing different dilutions of GCSF and to the control wells. The cultures were incubated for 44 h, pulsed for 4 h with 0.5 #Ci/well [3H]thymidine and harvested. The radioactivity incorporated into DNA was measured in a liquid scintillation counter. For neutralization assays, differing dilutions of antiserum were incubated with GCSF for approximately 1 h at 37 °C before addition of the cell suspension. AML-193 assay for GCSF As this cell line is GMCSF dependent, it must be cultured in medium containing GMCSF (2 ng/ml). Details of this cell line have been published previously (Santoli et al., 1987). For assay, cells were starved overnight in medium devoid of GMCSF. After washing, the cells were seeded at 104/well in 96-well plates in the presence or absence of recombinant GCSF. After an incubation period of 44 h, the cells were pulsed for 4 h with 0.5 ttCi/well [3H]thymidine and harvested. The radioactivity incorporated into DNA was subsequently measured by liquid scintillation counting. For neutralization assays, differing dilutions of antiserum were incubated with GCSF for approximately 1 h at 37 °C before addition of the cell suspension. Production of antiserum Polyclonal antiserum to GCSF was raised in sheep by intramuscular injection of 500 #g of the rDNA-derived GCSF emulsified in Freund's complete adjuvant. 1 month after the primary injection, boosts of 200 #g of GCSF in Freund's in-
complete adjuvant were given at regular intervals of 1, 2 and 6 months. The sheep were bled 8 and 10 days after the final boost.
Production of monoclonal antibodies A mouse was injected subcutaneously with 25 #g GCSF emulsified in Freund's complete adjuvant. On days 30-60 after the primary immunization 10 #g GCSF emulsified in Freund's incomplete adjuvant were injected intraperitoneally. On day 150, the mouse was injected intraveneously with 10 #g GCSF in saline and on day 155 the spleen was removed for fusion. Cell fusion was performed essentially as described in Johnstone and Thorpe (1987). Spleen cells were fused with the HAT sensitive myeloma line NSO to produce hybridomas which secreted monoclonal antibodies specific for GCSF. Supernatants from wells containing almost confluent hybridomas were screened for antibodies against GCSF by a solid-phase radiobinding assay (Johnstone and Thorpe, 1987). The positive wells demonstrating greater than ten times background counts were cloned in soft agar and screened again. Specific clones were grown in culture, cryopreserved or injected into the peritoneal cavity of pristane primed BALB/c mice to produce ascitic fluid. Sofid-phase radiobinding assay Test antiserum or supernatants from wells containing almost confluent hybridomas from the fusion were incubated for at least 2 h in flexible 96-well microtitre plates which had been coated with GCSF by overnight incubation (5 #g/ml). Positive binding was assessed using ~25I-labelled donkey anti-sheep or sheep anti-mouse IgG. Immunoblotting The specificity of the antibodies to GCSF was assessed following separation by polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. After transfer, the protein binding sites on the nitrocellulose sheets were blocked by incubation for 30 rain with 3% bovine haemoglobin dissolved in phosphate-buffered saline (HbPBS). The blot was then incubated overnight with the antiserum (10 #1) diluted in 25 ml Hb-PBS, washed with Hb-PBS and then incubated for a
213 further 3 h with a25I-labelled donkey anti-sheep or sheep anti-mouse IgG (106 cpm/track). After extensive washing with PBS, the blots were dried and exposed to Kodak X-omat film for 2 days at - 7 0 °C in a cassette equipped with a single tungstate intensifying screen.
Purification of monoclonal antibodies IgG was purified from ascitic fluid by precipitation using 45% saturation with ammonium sulphate followed by FPLC using a Mono-Q ion exchange column (Pharmacia) (Clezardin et al., 1985). Purification of antibodies from polyclonal antiserum GCSF (2 mg) was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) (0.5 g) according to the manufacturer's instructions. This column was used to affinity purify anti-GCSF antibodies from the polyclonal antiserum. Bound antibodies were eluted with 0.1 M glycine pH 2.5 (Johnstone and Thorpe, 1987). Radioiodination of antibodies Antibodies were radiolabelled using chloramine-T as catalyst and Dowex 1X-8 ion exchanger to remove non-protein-bound 1251 (Johnstone and Thorpe, 1987). For each iodination 40 /~g of antibody and 200/xCi Na125I were used. Immunoradiometric assays (IRMA)for GCSF Flexible 96-well microtitre plates were used in immunoradiometric assays for GCSF. Wells were coated with 'capture' antibody by incubation with the antibody (5 # g / m l in phosphate-buffered saline (PBS)) overnight at 4°C. The plates were washed three times with PBS containing 3% bovine haemoglobin (Hb-PBS) and the residual proteinbinding sites blocked by incubating the plates with Hb-PBS for 30 min at room temperature. After a further wash with Hb-PBS, the wells were incubated at room temperature for 3 h with sampies or dilutions of GCSF in Hb-PBS. After washing, bound GCSF was detected by incubation with a a25I-labelled developing antibody (300,000 cpm/well) diluted in Hb-PBS for at least 1 h. After a subsequent wash, wells were excised with a hot-wire plate cutter and the radioactivity estimated in a gamma-counter.
The two-site IRMA described above was modified slightly to develop a more sensitive assay for GCSF (Overall et al., 1989). Instead of adding antigen directly to the wells, the antigen was complexed with the antibody in separate 96-well plates for 1.5 h at 37 o C. This complex was subsequently transferred to the coated plate and incubated for 2 h at 37 o C. Finally, the plate was washed and the bound GCSF complex detected with 125I-sheep anti-mouse IgG.
Results
Monoclonal antibodies The GCSF fusion yielded three hybridoma fines which secreted antibody able to bind to the immunogen. Following cloning in soft agar a single clone which secreted monoclonal antibodies capable of binding strongly to antigen was selected from each master line and coded 3B6, 3D1 and 3C6. The latter was found to be slightly cross-reactive with ovalbumin and other antigens and was therefore not used for further studies. The two other GCSF-specific clones were injected into pristane-primed mice to produce ascitic fluid containing a high concentration of antibody. These fluids were purified by ammonium sulphate precipitation and ion exchange FPLC to yield homogeneous IgG as shown by SDS-PAGE. Both antibodies were of the IgG2a subclass. Immunoblotting studies demonstrated that both the antibodies were capable of binding specifically to GCSF (Fig. 1). Neither antibodies displayed any cross-reactivity with GM-CSF, IL-3, IL-1 or IL-6. Further characterization of the antibodies indicated that although both 3B6 and 3D1 bound to GCSF equally well in binding assays, only the 3D1 antibody was capable of neutralising the proliferative activity of GCSF in the mouse bonemarrow assay (Fig. 2). Polyclonal antiserum The antiserum raised in sheep against GCSF did not exhibit any cross-reactivity with GMCSF, IL-3 or other cytokines tested (results not shown). In neutralisation experiments, the antiserum completely abolished the proliferative response to GCSF in both the AML-193 assay (Fig. 3) and the
214
2~00, []
Gt:~F
8
GCSF+~6
100
1000
_
15000. O
5OOO 10
Fig. 1. Immunoblotting of recombinant cytokines using GCSF-specific monoclonal antibody, 3D1. A = IL-1; B = IL-3; C = IL-6; D = GCSF; E = GMCSF and F = GCSF. An identical result was obtained using the monoclonal antibody 3B6. The GCSF preparations run in tracks D and F were from different sources.
mouse bone marrow assay (not shown) although the pre-bleed serum had no effect.
Immunoassay The GCSF-specific antibodies and polyclonal antisera were used to develop a sensitive and
3000
O"-----o " ~ %.
• •
G G+3B~12 G+3DII16
1
10
2000-
1000'
~
.0001 .001 .01
~
.1
100 1000
NG~L
Fig. 2. Neutralising effect of the monoclonal antibodies 3B6 and 3D1 on GCSF-induced proliferation of mouse bone marrow cells. The antibodies were used at 1/500 dilution.
10000 100000
Fig. 3. Neutralisation of GCSF-induced proliferation of the AML-193 cell line by the G-CSF-specific polyclonal antiserum. The antiserum was used at a 1/500 dilution.
specific IRMA for GCSF. To establish an IRMA, a combination of antibodies which can recognise different epitopes on the GCSF molecule is required. Surprisingly, all the combinations of monoclonal antibodies failed to detect GCSF. Since radioiodination can denature antibodies and impair their ability to bind the antigen, the antibodies were tested directly in a solid-phase binding assay. However, labelling of the antibodies did not influence the efficiency of binding. A two-site assay employing a combination of sheep polyclonal antiserum and the monoclonal antibodies was also tested. Using the sheep antibody as the capture reagent and either of the two labelled monoclonal antibodies as the detector antibody or vice-versa proved to be too insensitive. An assay using the sheep antibodies for both capture and detection produced a dose-response curve with a detection limit of 2.5 n g / m l . It is possible that binding the antibodies to the plates induced a conformational change in the antibody and interfered with the recognition of the G-CSF molecule. In an attempt to increase the sensitivity of the assay, the assay was modified to include a step involving the formation of a liquid-phase complex between G C S F and monoclonal anti-GCSF. This
215 TABLE I
100000
MEASUREMENT OF GCSF LEVELS IN SUPERNATANTS FROM DIFFERENT CELL LINES USING THE GCSF IMMUNOASSAY Cell line
GCSF concentration in supernatants (pg/ml)
10000
Human foreskin fibroblasts Human monocytes Rabbit monocytes Murine P388 D1 cells Human T cells Human B cells Jurkat cells
E 0
1000
Non-stimulated
Stimulated
Stimulus a
250
10000
IL-la
< 250 < 250 < 250 < 250 < 250 < 250
8 000 < 250 < 250 < 250 < 250 < 250
LPS LPS LPS PHA EBV PHA
a IL-la = interleukin-la, LPS = lipopolysaccharide, PHA = phytohaemagglutinin, EBV = Epstein-Barr Virus. 100 0
100
1000
10000
100000
pg/ml
al., 1988; N i o c h e et al., 1988) c o n d i t i o n e d m e d i a f r o m several h u m a n cell lines were m o n i t o r e d for G C S F activity ( T a b l e I).
Fig. 4. Dose-response curve of rDNA-derived human GCSF using the sheep antibody to capture GCSF-3D1 monoclonal antibody complexes and 12SI-labelledsheep anti-mouse IgG as detector antibody.
complex was then b o u n d to microtitre wells coated with sheep a n t i - G C S F a n d s u b s e q u e n t l y detected with 125I-labelled sheep a n t i - m o u s e IgG. This detection m e t h o d gave a fairly steep dose-response curve (Fig. 4). Both of the m o n o c l o n a l a n t i b o d i e s were able to detect G C S F i n this assay. Of the two antibodies, 3D1 gave a steeper dose-response curve w h e n complexed with G C S F (results n o t shown) a n d was therefore used i n the s u b s e q u e n t immunoassays. O p t i m u m results were o b t a i n e d i n i m m u n o a s says using 20 # g / m l of the 3D1 a n t i b o d y ; a lower c o n c e n t r a t i o n of the a n t i b o d y gave a less sensitive assay. Optimally, i n c u b a t i o n periods of 1.5 h with the a n t i g e n a n d 2 h with the p o l y c l o n a l a n t i s e r u m were required; shorter periods c o n s i d e r a b l y red u c e d the sensitivity of the assays. This assay was f o u n d to b e highly reproducible a n d c a p a b l e of detecting G C S F activity i n different r D N A derived preparations. Since a variety of cell types c a n p r o d u c e G C S F (Broudy et al., 1987; F i b b e et
100000
10000 E
@. u
1000
100 10
100
1000
10000
100000
I~/m! Fig. 5. The effect of 'spiking' rDNA-derived human GCSF with 25% human serum using the GCSF immunoassay employing the 3D1 antibody antigen complex and the sheep antibody to capture antigen. Open squares - no serum; dosed squares with serum.
216
Other cytokines interfere in the classical bone marrow assay and therefore it was necessary to test the immunoassay for specificity. Addition of relatively high concentrations of IL-la, IL-lfl, MCSF, GM-CSF, IL-3, IL-4 and IL-6 produced only a background response. The assay was apparently unaffected by components in human serum since spiking of the standard curve with 25% serum did not interfere with the dose response (Fig. 5). This assay has been used successfully to evaluate GCSF levels in human sera.
Discussion We have developed a sensitive and specific irnmunoassay for the detection of GCSF using a combination of monoclonal and polyclonal antibodies. This assay involves formation of antigenantibody complexes, binding of the complex to sheep anti-GCSF and subsequent detection of the bound complex with 125I-labelled anti-mouse IgG. The assay is quantitative between 250 pg/ml and 50 ng/ml and is specific for GCSF. Because of the speed, sensitivity, specificity and ease of performance, this assay can be routinely used for monitoting GCSF levels and obviates many of the problems associated with bioassays. As a careful evaluation of the antibodies for use in immunoassays is crucial, the antibodies were characterized by binding and neutralization assays. Both the monoclonal and polyclonal antibodies were specific for GCSF, there was no cross-reaction with other cytokines including IL-6 with which it shows some sequence homology (Van Snick et al., 1988). Although the monoclonal antibodies appeared to be similar using solid-phase radiobinding assays and immunoblotting, the antibodies performed differently in neutralization experiments. The 3D1 antibody completely inhibited the proliferation of the bone marrow cells induced by GCSF and 3B6 had no effect. The sheep antiserum was also effective in neutralizing the GCSF stimulated proliferation of bone marrow cells and the AML-193 cell line. The present immunoassay has been used to assay rDNA-detived preparations of GCSF from different manufacturers and natural GCSF in conditioned media from human cell lines. Super-
natants from lipopolysaccharide (LPS)-stimulated human monocytes which were shown to contain IL-1 by bioassay using the NOB-1 cell line (Gearing et al., 1987) contained about 8 n g / m l of GCSF. Unstimulated monocyte supernatants did not contain detectable amounts of GCSF. The release of GCSF by monocytes in response to a variety of stimuli has also been observed by other workers (Nioche et al., 1988). We have demonstrated that human fibroblasts produced small amounts of GCSF (250 pg/ml) and these levels increased to 10 n g / m l following stimulation with IL-la. Using the colony assay Kaushansky and coworkers (1988) also detected similar increases in GCSF following IL-1 stimulation of fibroblasts. None of the activated human T cell or B cell lines produced detectable GCSF. Supernatants from the Jurkat cell line following PHA stimulation were devoid of GCSF, although they contained relatively high amounts of IL-2. The assay appeared to be specific for the human cytokine since conditioned media from LPS activated rabbit monocytes and the murine cell line P388D1 gave only background counts despite containing high levels of IL-1. The present assay should prove useful in monitoring levels of GCSF in culture and tissue fluids.
Acknowledgements We would like to thank Immunex Corporation, Amgen, Schering Research (Schering-Plough Corporation), Cetus Corporation and Dr. T. Kishimoto for supplying cytokines, Dr. P. Herzog (Monash University, Australia) for helpful suggestions and Lisa Hudson for typing the manuscript.
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