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The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC)
Journal of Immunological Methods, 111 (1988) 89-94
89
Elsevier JIM04797
The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC) B. Micheel, P. Jantscheff, V. B~Sttger, G. Scharte, G. Kaiser, P. Stolley and L. Karawajew Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., DDR -1115 Berlin-Buch, G.D.R.
(Received 3 November 1987, accepted 3 February 1988)
Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmunobinding assays for the demonstration of antigens and antibodies. Key words: Monoclonal antibody; Fluorescein isothiocyanate; Hapten; Immunoassay
Introduction
Fluorescein isothiocyanate (FITC) is a commonly used marker for antibodies in immunofluorescence techniques (Riggs et al., 1958). The conjugation of FITC to proteins is relatively easy and does not, in general, destroy the biological activity of the labelled substances. Since FITC is strongly immunogenic specific antibodies can be readily produced to bind to the hapten and even in some cases to quench the fluorescence. In addition to polyclonal antibodies monoclonal anti-
bodies have also been described by several authors (Kranz and Voss, 1981; Haaijman et al., 1986). Because of the favourable properties of FITC we initiated a series of experiments using FITC as a general marker for different proteins in immunologic test systems and employing monoclonal antibodies to FITC as a universal indicator reagent. This paper describes the production of monoclonal anti-FITC antibodies and the use of iodinated anti-FITC antibodies for the demonstration of different antigens and antibodies.
Materials and methods Correspondence to: B. Micheel, Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., DDR-1115 Berlin-Buch, G.D.R. Abbreviations: AFP, a-fetoprotein; BSA, bovine serum albumin; CEA, carcinoembryonic antigen; FITC, fluorescein isothiocyanate; HAT, hypoxanthine, aminopterin, thymidine; HCG, human chorionic gonadotropin; HRP, horseradish peroxidase; I, iodine; Ig, immunoglobulin; MoAb(s), monoclonal antibody(ies); NCE, non-specific cross-reacting antigen; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PVC, polyvinyl chloride; RPMI 1640, Roswell Park Memorial Institute cell culture medium 1640; TRITC, tetramethyl rhodamine isothiocyanate.
Mice BALB/c Han mice of the colony of the Central Institute of Cancer Research, Berlin-Buch, were used throughout. Labelling of proteins with FITC and 1:Sl The labelling of proteins with FITC was performed using a modification of the method of Gani et al. (1980). In brief, FITC was dissolved in PBS (1 mg/ml) and then added to the solution
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
90 containing the protein using a ratio of 20 btg F I T C / m g protein. The p H was adjusted to 9.5 by 0.1 M Na3PO 4 and the mixture was agitated for 2 h at room temperature. The pH was controlled and readjusted at intervals of 5 min for the first 20 min and after that every 30 min. The labelling of proteins with 125I was performed by the Iodogen method (Fraker and Speck, 1978). Before labelling, monoclonal antibodies (MoAbs) were purified by ammonium sulphate precipitation or hydroxylapatite chromatography (Stanker et al., 1985).
Immunization Mice were immunized with FITC-labelled Helix pomatia haemocyanin by injecting intraperitoneally 100 ~tg of the material emulsified in complete Freund's adjuvant followed 3 months later with an intravenous injection of the same quantity without adjuvant. Fusion 4 days after the booster injection spleen cells from the immunized animals were fused with mouse myeloma cell line X63-Ag8.653 (Kearny et al., 1979). Different fusion protocols were used. The first fusion was achieved using a modification of the original P E G method (KiShler and Milstein, 1975). For selecting hybrid cells a selection medium was used, replacing the aminopterin in the original H A T medium by azaserine (Karsten and Rudolph, 1985). The second fusion was an antigen-directed electric field-mediated fusion. For this purpose the X63-Ag8.653 myeloma cells were labelled with FITC according to Karawajew et al. (1987). The fusion was performed using a slight modification of the method described by Karsten et al. (1985). Screening for anti-FITC antibodies Screening for monoclonal antibodies in hybridoma culture fluid was performed using a solid-phase radioimmunoassay. For this purpose FITC-labelled bovine serum albumin (FITC-BSA, 10 /~g/ml PBS) was adsorbed to polyvinyl chloride (PVC) plates (pill blisters). Remaining binding sites at the PVC surface were blocked by incubating with PBS containing 10% calf serum.
In the next step the plates were incubated with the culture fluids and then with 125I-labelled rabbit anti-mouse Ig antibodies (purified by affinity chromatography). Incubations were, in general, performed for 2 h at 37 ° C in a reaction volume of 50 /zl. Dilutions of the protein to be adsorbed to the solid phase were made up in PBS. All other dilutions were performed in PBS containing 10% calf serum. Between the incubations the plates were washed with tap water. Bound radioactivity was measured in an N Z - 3 1 0 / A gamma counter (Gamma, Budapest, Hungary).
Assays to demonstrate the specificity of the antiFITC MoAbs Two types of solid-phase assays were used to examine the specificity of the anti-FITC antibodies. In the first assay substances structurally related to FITC were adsorbed to the solid phase. The assay was then performed as in the screening tests to check for binding of anti-FITC MoAbs. The second assay was an inhibition test. Purified anti-FITC antibodies (100 # g / m l ) were adsorbed to the solid phase and the plates were then incubated with FITC-labelled BSA (2 /~g/ml). In the last step an incubation was performed using a mixture of 125I-labelled anti-FITC MoAbs (30 000 cpm) and the substances to be tested. Determination of subclasses of MoAbs The subclasses of anti-FITC MoAbs were determined by a haemagglutination-inhibition test (B~Sttger et al., to be published). Sheep erythrocytes were conjugated with normal mouse Ig by the chromium chloride method and MoAb-containing culture fluids were tested for their ability to inhibit the agglutination of these conjugated erythrocytes by mouse Ig subclass-specific antisera. Detection of soluble antigens using anti-FITC MoAbs Two-site binding solid-phase assays were used to detect soluble antigens. MoAbs reactive with the corresponding antigen were adsorbed to the PVC plates (as diluted ascitic fluids) and the plates were then incubated with the antigen solution. In
91
the next step an incubation was performed with FITC-labelled MoAbs detecting a different epitope on the antigen molecule and in the last step 125I-labelled anti-FITC MoAbs were added (40 000 cpm/50/~1).
MoAb from clone B13-AF9 was an IgG2b protein and MoAb from clone B13-DE1 was of the IgG1 subclass. Most of the experiments were performed with MoAbs from clone D13-DE1.
Detection of antibodies using anti-FITC MoAbs A solid-phase assay was used to detect murine MoAbs with the help of anti-FITC MoAbs. Affinity chromatography-purified goat anti-mouse Ig antibodies were adsorbed to the PVC plates (10 # g / m l in PBS). The coated plates were then incubated with the antibody solution and, in the next step, with FITC-labelled antigen. Finally, l=5I-labelled anti-FITC MoAbs were added (40 000 cpm/50 #1). Normal mouse serum was included in this solution to avoid any binding of the labelled anti-FITC antibodies to the solid phase-bound anti-mouse Ig antibodies.
Specificity of the monoclonal anti-FITC antibodies Both antibodies were checked for binding after the following substances were adsorbed to the solid phase: FITC, fluorescein, 6-carboxyfluorescein, rhodamine isothiocyanate B, tetramethyl rhodamine isothiocyanate (TRITC), TRITC-labelled BSA, bromcresol purple, m-cresol purple, phenol red, cresol red, phenolphthalein, o-cresolphthalein and bromphenol blue. In the case of FITC, fluorescein and 6-carboxyfluorescein adsorbed to the solid phase a strong binding of the anti-FITC antibodies was observed although the intensity was lower than that observed using FITC-labelled BSA adsorbed to the solid phase. Since antibody-containing ascitic fluids at a dilution of 10-1 and in some cases 10-2 also reacted with most of the other dyes adsorbed to the solid phase, inhibition experiments were performed using 100 and 200 #g dye/ml as inhibitor. Only m-cresol purple and phenol red showed definite inhibition. Since this was more pronounced with phenol red a comparison was made using phenol red and fluorescein in different dilutions as competitors. The results, shown in Fig. 1, demonstrate that a 1000-fold excess of phenol red
Radioimmunobinding assay using anti-FITC MoAbs with cells in suspension Samples of 5 X 105 formalin-fixed cells washed in PBS and suspended in 50 /~1 PBS containing 10% calf serum (cultured adherent cells prepared by trypsinization and fixation in PBS containing 0.2% formalin) were incubated with 50/~1 of the corresponding FITC-labelled antibody for 2 h at 4°C in microtitration plates. After washing the cells three times in PBS containing 10% calf serum by centrifugation they were incubated in 125I-labelled anti-FITC MoAbs (100000 cpm/50 /~1) for 2 h at 4°C. After another wash the cells were transferred into tubes and the radioactivity measured in a gamma counter.
Results
Production of monoclonal anti-FITC antibodies In both fusions about 80% of the wells showed colony growth but in both cases only about 5% of them produced antibodies which bound to FITClabelled BSA (and not to haemocyanin). Two clones producing strongly binding anti-FITC antibodies were used for further experiments (B13-AF9 from the normal fusion and B13-DE1 from the antigen-directed electrofusion).
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104 10-' 10-' 10 2 10-' 10° 10' 102 )Jg dye/m[ Fig. 1. Inhibition of binding of 125I-labelled anti-FITC MoAb to FITC-labelled BSA in a solid-phase two-site binding assay using different concentrations of fluorescein ( o o) and phenol red (O O).
92 was required to obtain the same inhibition as with fluorescein.
Two-site binding assay using monoclonal anti-FITC antibodies An assay was developed for the demonstration of human chorionic gonadotropin (HCG). Ascitic fluid (diluted 10 -3 ) containing the cross-reactive HCG-binding MoAb B9-AB9 (Micheel et al., to be published) was adsorbed to the solid phase and the HCG-specific FITC-labelled MoAb B9-BA8 (5 /~g/ml; Micheel et al., to be published) was used as the soluble phase antibody followed by 125I-labelled anti-FITC MoAb. First experiments using this assay for the demonstration of H C G in an HCG-containing standard sample suggested a detection limit of less than 5 m I U / m l (Fig. 2). Similar two site binding assays were developed for the demonstration of a-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As in the case of the H C G system the assays showed a level of sensitivity which was sufficient for clinical purposes (data not presented).
Solid-phase antibody binding assay using monoclonal anti-FITC antibodies Monoclonal anti-HCG antibodies in diluted ascitic fluid (containing cross-reactive antibody B9-AB9) were demonstrated using the incubation
cpm
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cpm 6000
4000
2000
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10~
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Antibody
10 '
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Fig. 3. Solid-phase radioimmunoassay for the demonstration of
HCG-binding MoAb in ascitic fluid. The presence of HCGbinding MoAb bound by a solid phase-attached anti-Ig is shown using FITC-labelled HCG and 125I-labelled anti-FITC MoAb (© ©) or 12SI-labelled HCG (O - - - e). sequence: goat anti-mouse Ig antibodies - antiH C G MoAbs - FITC-labelled H C G (0.3/~g/ml) - 125I-labelled anti-FITC MoAb. Fig. 3 shows the titration curve of a representative experiment compared with an assay using the conventional incubation sequence: anti-mouse Ig - anti-HCG MoAb - tzsI-labelled H C G (20 ng/ml). The assay using the anti-FITC MoAbs exhibited the same sensitivity as the conventional assay. The test was sensitive enough to demonstrate anti-HCG MoAbs in culture fluid and could therefore be used as a hybridoma screening assay. Similar types of assays were developed for the demonstration of MoAbs against A F P and BSA. In all cases the addition of 1% normal mouse serum was sufficient to avoid binding of the 125Ilabelled anti-FITC MoAbs to the anti-mouse Ig antibodies bound to the solid phase.
10000
Radioimmunobinding assays with cells using monoclonal anti-FITC antibodies
800E 6000
400C 200E
/
/o
20
40 fi0 80 C o n c e n t r a t i o n of HCG (m]U/ml)
100
Fig. 2. Two-site binding radioimmunoassay for the demonstration of HCG. The binding of FITC-labelled second MoAb to
antigen bound by a solid phase-attached first MoAb was detected using 125I-labelled anti-FITC MoAb. A standard curve is shown using a diluted HCG-containingstandard sample.
Formalin-fixed suspended cells of the colonic carcinoma line LS174T (Tom et al., 1976) were incubated with FITC-labelled MoAb D14-HD11 (B/Sttger et al., to be published) which reacts with CEA and non-specific cross-reacting antigen (NCA). In the next step 125I-labelled anti-FITC MoAb was added. Fig. 4 shows the titration curve of this experiment. A similar result was obtained using 125I-labelled rabbit anti-mouse Ig antibodies to demonstrate the binding of the FITC-labelled a n t i - C E A / N C A MoAb to cell surface antigens.
93 cDm 12000 1000C o 800£ 60O(: 400£ 200C
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~5
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0125 00625 ' ' ConcentrQtion of FITC [obeUed on'libody [~Jg/m[}
Fig. 4. Radioimmunobinding assay for the detection of cell surface antigens. The binding of different concentrations of FITC-labelled anti-CEA/rNCA MoAb to colonic carcinoma cells (LS 174) is demonstrated using 125I-labelled anti-FITC MoAb. The dot in the lower right corner indicates the background binding of ]25I-labelled anti-FITC MoAb to the cells.
From the absolute numbers of bound cpm it was deduced that the binding of anti-mouse Ig was considerably less than that of anti-FITC MoAb.
Discussion
The results obtained indicate that MoAbs against FITC are very valuable reagents for various types of immunoassay. The anti-FITC MoAbs showed no cross-reactivity with another fluorescence marker (TRITC), which confirms the results of Haaijman et al. (1986) who used their anti-FITC MoAbs for immunohistochemical purposes. A series of structurally related dyes were tested for cross-reactivity in our experiments and a weak but definite cross-reactivity was found with m-cresol purple and phenol red. However, compared to fluorescein, a 1000-fold excess of phenol red was required to obtain similar competition. Although the concentration of 10/xg phenol red/ml which is present in the RPMI 1640 used for our cultures may result in a significant inhibition, this did not, apparently, influence the binding of anti-FITC MoAbs to FITC-labelled BSA. All the anti-FITC MoAb-contaihing hybridoma culture fluids contained phenol red but the binding to FITC-labelled BSA was very efficient. The antibody binding experiments with cell suspensions were, moreover,
performed in phenol red-containing PBS. It is probable that the avidity of the anti-FITC MoAbs is higher when the FITC is bound to proteins compared to free fluorescein so that there may be an even greater difference in the inhibiting activity of phenol red and fluorescein. Anti-FITC MoAbs were found to be very valuable for different types of immunoassay, such as the demonstration of antigens and antibodies in solid-phase assays and for immunological tests with cells in suspension. For all these assays only one batch of ]25I-labelled anti-FITC MoAbs was required. The binding reagents for the different assays were readily labelled by FITC and could be stored without difficulty. There was no problem labelling different MoAbs with FITC when FITC was used only as a hapten. There were, however, sometimes problems maintaining the fluorescence of the bound molecule (data not shown). The disadvantage of an additional incubation step in two-site binding assays for the demonstration of antigens can easily be overcome by performing the last incubation with a mixture of the FITC-labelled MoAb and the 125I-labelled antiFITC MoAb. However, optimum conditions have yet to be devised for this type of assay. The same combined incubation should be possible in antibody screening tests using FITC-labelled antigen. There are potential applications for labelled anti-FITC MoAbs in immunocytology and immunohistology where undesirable cross-reactivities preclude the use of anti-Ig antibodies, e.g., mouse antibodies for mouse lymphoid tissues or human antibodies for human lymphoid tissues. However, whether the FITC-anti-FITC system can be used for the same purposes as the well established avidin-biotin system (Wilchek and Bayer, 1984) remains to be established. The anti-FITC MoAbs can also be used to quench the fluorescence of fluorescein (data not shown). Further tests will be needed to determine whether this can be exploited in sensitive immunoassays as was demonstrated by other authors using polyclonal anti-FITC antibodies (Nargessi et al., 1979; Zuk et al., 1979). In addition to the experiments presented here we have been able to show that our anti-FITC MoAbs can be labelled with horseradish peroxidase (HRP) and that such HRP-labelled anti-
94
FITC MoAbs can be used in enzyme immunoassays (data not shown). The development of enzyme immunoassays using bispecific antiF I T C / a n t i - H R P MoAbs is discussed in another paper (Karawajew et al., 1988).
Acknowledgements We thank S. Knop for ascites production and I. Walther for help in the electrofusion.
References Fraker, J.P. and Speck, J.C. (1977) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6tetrachloro-3,6-diphenylglycouril. Biochem. Biophys. Res. Commun. 80, 849. Gani, M.M., Hunt, T. and Summerell, J.M. (1980) A simple method of labelling mouse Thy-1 antibodies with FITC. J. Immunol. Methods 34, 133. Haaijman, l.J., Coolen, J., Kr~Sse, C.J.M., Pronk, G.J. and Ming, Z.F. (1986) Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry. Histochemistry 84, 363. Karawajew, L., Behrsing, O., Kaiser, G. and Micheel, B. (1988) Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP). J. Immunol. Methods 111, 95. Karsten, U. and Rudolph, M. (1985) Monoclonal antibodies against tumour-associated antigens: mycoplasma as a major technical obstacle and its possible circumvention by azaserine selection medium. Arch. Geschwulstforsch. 55, 305.
Karsten, U., Papsdorf, G., Roloff, G., Stolley, P., Abel, H., Walther, I. and Weiss, H. (1985) Monoclonal anti-cytokeratin antibody from a hybridoma clone generated by electrofusion. Eur. J. Cancer Clin. Oncol. 21,733. Kearny, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K. (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1547. Kt~hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Kranz, D.M. and Voss, Jr., E.W. (1981) Partial elucidation of an anti-hapten repertoire in BALB/c mice: comparative characterization of several monoclonal anti-fluorescyl antibodies. Mol. Immunol. 18, 889. Nargessi, R.D., Landon, J. and Smith, D.S. (1979) Use of antibodies against the label in non-separative nonisotopic immunoassay: 'indirect quenching' fluoroimmunoassay of proteins. J. Immunol. Methods 26, 307. Riggs, J.L., Seiwald, R.J., Burckhalter, J.H., Downs, C.M. and Metcalf, T.G. (1958) Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am. J. Pathol. 34, 1081. Stanker, L.H., Vanderlaan, M. and Juarez-Salinas, H. (1985) One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography. J. Immunol. Methods 76, 157. Tom, B.H., Rutzky, L.P., Jakstys, M.M., Oyasu, R., Kaye, C.I. and Kuhan, B.D. (1976) Human colonic adenocarcinoma cells. I. Establishment and description of a new line. In Vitro 12, 180. Wilchek, M. and Bayer, E.A. (1984) The avidin-biotin complex in immunology. Immunol. Today 5, 39. Zuck, R.F., Rowley, G.L. and Ullman, E.F. (1979) Fluorescence protection immunoassay: a new homogeneous assay technique. Clin. Chem. 25, 1554.
89
Elsevier JIM04797
The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC) B. Micheel, P. Jantscheff, V. B~Sttger, G. Scharte, G. Kaiser, P. Stolley and L. Karawajew Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., DDR -1115 Berlin-Buch, G.D.R.
(Received 3 November 1987, accepted 3 February 1988)
Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmunobinding assays for the demonstration of antigens and antibodies. Key words: Monoclonal antibody; Fluorescein isothiocyanate; Hapten; Immunoassay
Introduction
Fluorescein isothiocyanate (FITC) is a commonly used marker for antibodies in immunofluorescence techniques (Riggs et al., 1958). The conjugation of FITC to proteins is relatively easy and does not, in general, destroy the biological activity of the labelled substances. Since FITC is strongly immunogenic specific antibodies can be readily produced to bind to the hapten and even in some cases to quench the fluorescence. In addition to polyclonal antibodies monoclonal anti-
bodies have also been described by several authors (Kranz and Voss, 1981; Haaijman et al., 1986). Because of the favourable properties of FITC we initiated a series of experiments using FITC as a general marker for different proteins in immunologic test systems and employing monoclonal antibodies to FITC as a universal indicator reagent. This paper describes the production of monoclonal anti-FITC antibodies and the use of iodinated anti-FITC antibodies for the demonstration of different antigens and antibodies.
Materials and methods Correspondence to: B. Micheel, Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., DDR-1115 Berlin-Buch, G.D.R. Abbreviations: AFP, a-fetoprotein; BSA, bovine serum albumin; CEA, carcinoembryonic antigen; FITC, fluorescein isothiocyanate; HAT, hypoxanthine, aminopterin, thymidine; HCG, human chorionic gonadotropin; HRP, horseradish peroxidase; I, iodine; Ig, immunoglobulin; MoAb(s), monoclonal antibody(ies); NCE, non-specific cross-reacting antigen; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PVC, polyvinyl chloride; RPMI 1640, Roswell Park Memorial Institute cell culture medium 1640; TRITC, tetramethyl rhodamine isothiocyanate.
Mice BALB/c Han mice of the colony of the Central Institute of Cancer Research, Berlin-Buch, were used throughout. Labelling of proteins with FITC and 1:Sl The labelling of proteins with FITC was performed using a modification of the method of Gani et al. (1980). In brief, FITC was dissolved in PBS (1 mg/ml) and then added to the solution
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
90 containing the protein using a ratio of 20 btg F I T C / m g protein. The p H was adjusted to 9.5 by 0.1 M Na3PO 4 and the mixture was agitated for 2 h at room temperature. The pH was controlled and readjusted at intervals of 5 min for the first 20 min and after that every 30 min. The labelling of proteins with 125I was performed by the Iodogen method (Fraker and Speck, 1978). Before labelling, monoclonal antibodies (MoAbs) were purified by ammonium sulphate precipitation or hydroxylapatite chromatography (Stanker et al., 1985).
Immunization Mice were immunized with FITC-labelled Helix pomatia haemocyanin by injecting intraperitoneally 100 ~tg of the material emulsified in complete Freund's adjuvant followed 3 months later with an intravenous injection of the same quantity without adjuvant. Fusion 4 days after the booster injection spleen cells from the immunized animals were fused with mouse myeloma cell line X63-Ag8.653 (Kearny et al., 1979). Different fusion protocols were used. The first fusion was achieved using a modification of the original P E G method (KiShler and Milstein, 1975). For selecting hybrid cells a selection medium was used, replacing the aminopterin in the original H A T medium by azaserine (Karsten and Rudolph, 1985). The second fusion was an antigen-directed electric field-mediated fusion. For this purpose the X63-Ag8.653 myeloma cells were labelled with FITC according to Karawajew et al. (1987). The fusion was performed using a slight modification of the method described by Karsten et al. (1985). Screening for anti-FITC antibodies Screening for monoclonal antibodies in hybridoma culture fluid was performed using a solid-phase radioimmunoassay. For this purpose FITC-labelled bovine serum albumin (FITC-BSA, 10 /~g/ml PBS) was adsorbed to polyvinyl chloride (PVC) plates (pill blisters). Remaining binding sites at the PVC surface were blocked by incubating with PBS containing 10% calf serum.
In the next step the plates were incubated with the culture fluids and then with 125I-labelled rabbit anti-mouse Ig antibodies (purified by affinity chromatography). Incubations were, in general, performed for 2 h at 37 ° C in a reaction volume of 50 /zl. Dilutions of the protein to be adsorbed to the solid phase were made up in PBS. All other dilutions were performed in PBS containing 10% calf serum. Between the incubations the plates were washed with tap water. Bound radioactivity was measured in an N Z - 3 1 0 / A gamma counter (Gamma, Budapest, Hungary).
Assays to demonstrate the specificity of the antiFITC MoAbs Two types of solid-phase assays were used to examine the specificity of the anti-FITC antibodies. In the first assay substances structurally related to FITC were adsorbed to the solid phase. The assay was then performed as in the screening tests to check for binding of anti-FITC MoAbs. The second assay was an inhibition test. Purified anti-FITC antibodies (100 # g / m l ) were adsorbed to the solid phase and the plates were then incubated with FITC-labelled BSA (2 /~g/ml). In the last step an incubation was performed using a mixture of 125I-labelled anti-FITC MoAbs (30 000 cpm) and the substances to be tested. Determination of subclasses of MoAbs The subclasses of anti-FITC MoAbs were determined by a haemagglutination-inhibition test (B~Sttger et al., to be published). Sheep erythrocytes were conjugated with normal mouse Ig by the chromium chloride method and MoAb-containing culture fluids were tested for their ability to inhibit the agglutination of these conjugated erythrocytes by mouse Ig subclass-specific antisera. Detection of soluble antigens using anti-FITC MoAbs Two-site binding solid-phase assays were used to detect soluble antigens. MoAbs reactive with the corresponding antigen were adsorbed to the PVC plates (as diluted ascitic fluids) and the plates were then incubated with the antigen solution. In
91
the next step an incubation was performed with FITC-labelled MoAbs detecting a different epitope on the antigen molecule and in the last step 125I-labelled anti-FITC MoAbs were added (40 000 cpm/50/~1).
MoAb from clone B13-AF9 was an IgG2b protein and MoAb from clone B13-DE1 was of the IgG1 subclass. Most of the experiments were performed with MoAbs from clone D13-DE1.
Detection of antibodies using anti-FITC MoAbs A solid-phase assay was used to detect murine MoAbs with the help of anti-FITC MoAbs. Affinity chromatography-purified goat anti-mouse Ig antibodies were adsorbed to the PVC plates (10 # g / m l in PBS). The coated plates were then incubated with the antibody solution and, in the next step, with FITC-labelled antigen. Finally, l=5I-labelled anti-FITC MoAbs were added (40 000 cpm/50 #1). Normal mouse serum was included in this solution to avoid any binding of the labelled anti-FITC antibodies to the solid phase-bound anti-mouse Ig antibodies.
Specificity of the monoclonal anti-FITC antibodies Both antibodies were checked for binding after the following substances were adsorbed to the solid phase: FITC, fluorescein, 6-carboxyfluorescein, rhodamine isothiocyanate B, tetramethyl rhodamine isothiocyanate (TRITC), TRITC-labelled BSA, bromcresol purple, m-cresol purple, phenol red, cresol red, phenolphthalein, o-cresolphthalein and bromphenol blue. In the case of FITC, fluorescein and 6-carboxyfluorescein adsorbed to the solid phase a strong binding of the anti-FITC antibodies was observed although the intensity was lower than that observed using FITC-labelled BSA adsorbed to the solid phase. Since antibody-containing ascitic fluids at a dilution of 10-1 and in some cases 10-2 also reacted with most of the other dyes adsorbed to the solid phase, inhibition experiments were performed using 100 and 200 #g dye/ml as inhibitor. Only m-cresol purple and phenol red showed definite inhibition. Since this was more pronounced with phenol red a comparison was made using phenol red and fluorescein in different dilutions as competitors. The results, shown in Fig. 1, demonstrate that a 1000-fold excess of phenol red
Radioimmunobinding assay using anti-FITC MoAbs with cells in suspension Samples of 5 X 105 formalin-fixed cells washed in PBS and suspended in 50 /~1 PBS containing 10% calf serum (cultured adherent cells prepared by trypsinization and fixation in PBS containing 0.2% formalin) were incubated with 50/~1 of the corresponding FITC-labelled antibody for 2 h at 4°C in microtitration plates. After washing the cells three times in PBS containing 10% calf serum by centrifugation they were incubated in 125I-labelled anti-FITC MoAbs (100000 cpm/50 /~1) for 2 h at 4°C. After another wash the cells were transferred into tubes and the radioactivity measured in a gamma counter.
Results
Production of monoclonal anti-FITC antibodies In both fusions about 80% of the wells showed colony growth but in both cases only about 5% of them produced antibodies which bound to FITClabelled BSA (and not to haemocyanin). Two clones producing strongly binding anti-FITC antibodies were used for further experiments (B13-AF9 from the normal fusion and B13-DE1 from the antigen-directed electrofusion).
cpm I
6000I
ooot
\.
\
2000
0
o ~
i
i
i
h
i
i
L
104 10-' 10-' 10 2 10-' 10° 10' 102 )Jg dye/m[ Fig. 1. Inhibition of binding of 125I-labelled anti-FITC MoAb to FITC-labelled BSA in a solid-phase two-site binding assay using different concentrations of fluorescein ( o o) and phenol red (O O).
92 was required to obtain the same inhibition as with fluorescein.
Two-site binding assay using monoclonal anti-FITC antibodies An assay was developed for the demonstration of human chorionic gonadotropin (HCG). Ascitic fluid (diluted 10 -3 ) containing the cross-reactive HCG-binding MoAb B9-AB9 (Micheel et al., to be published) was adsorbed to the solid phase and the HCG-specific FITC-labelled MoAb B9-BA8 (5 /~g/ml; Micheel et al., to be published) was used as the soluble phase antibody followed by 125I-labelled anti-FITC MoAb. First experiments using this assay for the demonstration of H C G in an HCG-containing standard sample suggested a detection limit of less than 5 m I U / m l (Fig. 2). Similar two site binding assays were developed for the demonstration of a-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As in the case of the H C G system the assays showed a level of sensitivity which was sufficient for clinical purposes (data not presented).
Solid-phase antibody binding assay using monoclonal anti-FITC antibodies Monoclonal anti-HCG antibodies in diluted ascitic fluid (containing cross-reactive antibody B9-AB9) were demonstrated using the incubation
cpm
~ o ~ 12000
cpm 6000
4000
2000
10~
10~
10 ~
10 ~
Antibody
10 '
10 ~
10 9 10 '°
dilution
Fig. 3. Solid-phase radioimmunoassay for the demonstration of
HCG-binding MoAb in ascitic fluid. The presence of HCGbinding MoAb bound by a solid phase-attached anti-Ig is shown using FITC-labelled HCG and 125I-labelled anti-FITC MoAb (© ©) or 12SI-labelled HCG (O - - - e). sequence: goat anti-mouse Ig antibodies - antiH C G MoAbs - FITC-labelled H C G (0.3/~g/ml) - 125I-labelled anti-FITC MoAb. Fig. 3 shows the titration curve of a representative experiment compared with an assay using the conventional incubation sequence: anti-mouse Ig - anti-HCG MoAb - tzsI-labelled H C G (20 ng/ml). The assay using the anti-FITC MoAbs exhibited the same sensitivity as the conventional assay. The test was sensitive enough to demonstrate anti-HCG MoAbs in culture fluid and could therefore be used as a hybridoma screening assay. Similar types of assays were developed for the demonstration of MoAbs against A F P and BSA. In all cases the addition of 1% normal mouse serum was sufficient to avoid binding of the 125Ilabelled anti-FITC MoAbs to the anti-mouse Ig antibodies bound to the solid phase.
10000
Radioimmunobinding assays with cells using monoclonal anti-FITC antibodies
800E 6000
400C 200E
/
/o
20
40 fi0 80 C o n c e n t r a t i o n of HCG (m]U/ml)
100
Fig. 2. Two-site binding radioimmunoassay for the demonstration of HCG. The binding of FITC-labelled second MoAb to
antigen bound by a solid phase-attached first MoAb was detected using 125I-labelled anti-FITC MoAb. A standard curve is shown using a diluted HCG-containingstandard sample.
Formalin-fixed suspended cells of the colonic carcinoma line LS174T (Tom et al., 1976) were incubated with FITC-labelled MoAb D14-HD11 (B/Sttger et al., to be published) which reacts with CEA and non-specific cross-reacting antigen (NCA). In the next step 125I-labelled anti-FITC MoAb was added. Fig. 4 shows the titration curve of this experiment. A similar result was obtained using 125I-labelled rabbit anti-mouse Ig antibodies to demonstrate the binding of the FITC-labelled a n t i - C E A / N C A MoAb to cell surface antigens.
93 cDm 12000 1000C o 800£ 60O(: 400£ 200C
° o
~5
o'25
0125 00625 ' ' ConcentrQtion of FITC [obeUed on'libody [~Jg/m[}
Fig. 4. Radioimmunobinding assay for the detection of cell surface antigens. The binding of different concentrations of FITC-labelled anti-CEA/rNCA MoAb to colonic carcinoma cells (LS 174) is demonstrated using 125I-labelled anti-FITC MoAb. The dot in the lower right corner indicates the background binding of ]25I-labelled anti-FITC MoAb to the cells.
From the absolute numbers of bound cpm it was deduced that the binding of anti-mouse Ig was considerably less than that of anti-FITC MoAb.
Discussion
The results obtained indicate that MoAbs against FITC are very valuable reagents for various types of immunoassay. The anti-FITC MoAbs showed no cross-reactivity with another fluorescence marker (TRITC), which confirms the results of Haaijman et al. (1986) who used their anti-FITC MoAbs for immunohistochemical purposes. A series of structurally related dyes were tested for cross-reactivity in our experiments and a weak but definite cross-reactivity was found with m-cresol purple and phenol red. However, compared to fluorescein, a 1000-fold excess of phenol red was required to obtain similar competition. Although the concentration of 10/xg phenol red/ml which is present in the RPMI 1640 used for our cultures may result in a significant inhibition, this did not, apparently, influence the binding of anti-FITC MoAbs to FITC-labelled BSA. All the anti-FITC MoAb-contaihing hybridoma culture fluids contained phenol red but the binding to FITC-labelled BSA was very efficient. The antibody binding experiments with cell suspensions were, moreover,
performed in phenol red-containing PBS. It is probable that the avidity of the anti-FITC MoAbs is higher when the FITC is bound to proteins compared to free fluorescein so that there may be an even greater difference in the inhibiting activity of phenol red and fluorescein. Anti-FITC MoAbs were found to be very valuable for different types of immunoassay, such as the demonstration of antigens and antibodies in solid-phase assays and for immunological tests with cells in suspension. For all these assays only one batch of ]25I-labelled anti-FITC MoAbs was required. The binding reagents for the different assays were readily labelled by FITC and could be stored without difficulty. There was no problem labelling different MoAbs with FITC when FITC was used only as a hapten. There were, however, sometimes problems maintaining the fluorescence of the bound molecule (data not shown). The disadvantage of an additional incubation step in two-site binding assays for the demonstration of antigens can easily be overcome by performing the last incubation with a mixture of the FITC-labelled MoAb and the 125I-labelled antiFITC MoAb. However, optimum conditions have yet to be devised for this type of assay. The same combined incubation should be possible in antibody screening tests using FITC-labelled antigen. There are potential applications for labelled anti-FITC MoAbs in immunocytology and immunohistology where undesirable cross-reactivities preclude the use of anti-Ig antibodies, e.g., mouse antibodies for mouse lymphoid tissues or human antibodies for human lymphoid tissues. However, whether the FITC-anti-FITC system can be used for the same purposes as the well established avidin-biotin system (Wilchek and Bayer, 1984) remains to be established. The anti-FITC MoAbs can also be used to quench the fluorescence of fluorescein (data not shown). Further tests will be needed to determine whether this can be exploited in sensitive immunoassays as was demonstrated by other authors using polyclonal anti-FITC antibodies (Nargessi et al., 1979; Zuk et al., 1979). In addition to the experiments presented here we have been able to show that our anti-FITC MoAbs can be labelled with horseradish peroxidase (HRP) and that such HRP-labelled anti-
94
FITC MoAbs can be used in enzyme immunoassays (data not shown). The development of enzyme immunoassays using bispecific antiF I T C / a n t i - H R P MoAbs is discussed in another paper (Karawajew et al., 1988).
Acknowledgements We thank S. Knop for ascites production and I. Walther for help in the electrofusion.
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Karsten, U., Papsdorf, G., Roloff, G., Stolley, P., Abel, H., Walther, I. and Weiss, H. (1985) Monoclonal anti-cytokeratin antibody from a hybridoma clone generated by electrofusion. Eur. J. Cancer Clin. Oncol. 21,733. Kearny, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K. (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1547. Kt~hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Kranz, D.M. and Voss, Jr., E.W. (1981) Partial elucidation of an anti-hapten repertoire in BALB/c mice: comparative characterization of several monoclonal anti-fluorescyl antibodies. Mol. Immunol. 18, 889. Nargessi, R.D., Landon, J. and Smith, D.S. (1979) Use of antibodies against the label in non-separative nonisotopic immunoassay: 'indirect quenching' fluoroimmunoassay of proteins. J. Immunol. Methods 26, 307. Riggs, J.L., Seiwald, R.J., Burckhalter, J.H., Downs, C.M. and Metcalf, T.G. (1958) Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am. J. Pathol. 34, 1081. Stanker, L.H., Vanderlaan, M. and Juarez-Salinas, H. (1985) One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography. J. Immunol. Methods 76, 157. Tom, B.H., Rutzky, L.P., Jakstys, M.M., Oyasu, R., Kaye, C.I. and Kuhan, B.D. (1976) Human colonic adenocarcinoma cells. I. Establishment and description of a new line. In Vitro 12, 180. Wilchek, M. and Bayer, E.A. (1984) The avidin-biotin complex in immunology. Immunol. Today 5, 39. Zuck, R.F., Rowley, G.L. and Ullman, E.F. (1979) Fluorescence protection immunoassay: a new homogeneous assay technique. Clin. Chem. 25, 1554.