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Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay
Immunology Letters, 28 (1991) 79-84 Elsevier
IMLET 01559
Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay N. D. D a n i l o v a a n d R. G. Vasilov Department of Medical Diagnostics, Institute of Biotechnology, Moscow, US.S.R. (Received 5 December 1990; accepted 21 December 1990)
1. Summary
Spleen cells from BALB/c mice immunised with KLH-theophylline conjugate were fused with a mouse myeloma cell line P3-X63-Ag8.653, and antibody-producing hybrids were identified by enzyme immunoassay. Three cell clones were obtained, each capable o f producing a unique monoclonal antibody to theophylline. Using these monoclonal antibodies, an immunoassay system for theophylline was developed.
methylxanthine, 1,3-dimethyluric acid, 1-methyluric acid and caffeine, which is contained in food. There are several publications describing the production of poly- and monoclonal antibodies to theophylline [1-3], but those antibodies, however, cross-react with some of the above mentioned compounds. In this study, we developed and characterised specific high-affinity murine monoclonal antibodies to theophylline. 3. Materials and Methods
2. Introduction
3.1. Preparation o f hapten-protein conjugates
Theophylline is a drug commonly used in the prevention and treatment of asthma, apnea and obstructive lung diseases. To be effective theophylline must be in serum within the narrow therapeutic concentration range o f 1 0 - 20 #g/ml, so the use oftheophylline requires careful dosage based on measurements of drug concentrations. Current methods for measuring theophylline in serum are based on immunological techniques. They require highly specific antibodies, because body fluids contain some other natural xanthines, such as xanthine, uric acid and hypoxanthine, which are structurally similar to theophylline, and which, if recognised by the antitheophylline antibody, would produce errors in assays. Also, good antibodies should not cross-react with theophylline metabolite compounds: 3-
Theophylline was coupled to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA). KLH-theophylline (KLH-theo) was used as immunogen: BSA-theophylline (BSA-theo) as antigen in the enzyme immunoassay. Theophylline was turned to 8-aminotheophylline [4] which was converted using nitrous acid to the corresponding diazonium compound [4]. Forty mg of diazonium salt was added to an aqueous suspension of 100 mg KLH. The mixture was stirred at 0°C for 2 h at a mildly alkaline pH, then dialysed against water. Coupling yield was determined by addition of [3H]theophylline in tracer amounts into the reaction mixture. A conjugate consisting o f 200 theophylline molecules per molecule o f K L H was prepared. The coupling reaction with BSA was performed in 1 ml of water (pH adjusted to 8 with NaOH) by slowly adding 120/z125°70 glutaraldehyde to the stirring solution of 50 mg 8-aminotheophylline and 100 mg BSA. The reaction mixture was set aside for 2 h at room temperature and 10 mg NaBH4 was added. The con-
Key words: Theophylline;Immunoassay; Monoclonal antibody Correspondence to: Dr. N. D. Danilova,Department of Medical Diagnostics, Institute of Biotechnology, Nauchny proezd 8, Moscow, 117246, U.S.S.R.
0165-2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V.
79
jugate was dialysed against water. The average number of theophylline molecules per molecule of BSA in BSA-theo was 20. 3.2. Enzyme conjugate The enzyme conjugate was prepared by chemically coupling 8-aminotheophylline to horseradish peroxidase (PO). One mg of peroxidase (Sigma) was dissolved in 0.1 ml of water and 1 mg sodium periodate in 25/A of water was added. The mixture was incubated for 2 h at room temperature in the dark, filtered through 0.5 cm Sephadex G-10 layer and mixed with 8-aminotheophylline solution (1 mg in 0.1 ml 0.01 M NaOH). Three hours later 0.1 mg NaBH 4 was added. After I h incubation the conjugate was dialysed against water. 3.3. Immunisation and cell fusion BALB/c mice were immunized intraperitoneally with 50/xg KLH-theo in complete Freund's adjuvant. Six weeks later the mice were boosted with similar doses of antigen with incomplete Freund's adjuvant. Seven days after the boost, mice were bled from the retroorbital sinus in order to detect antitheophylline antibodies in the serum. A mouse with highly positive serum was injected 3 days prior to fusion intraperitoneally with 100/zg antigen in saline. The fusion was carried out with a non-secreting myeloma cell line P3-X63-Ag8.653. Myeloma cells were grown in RPMI-1640 medium (Flow Lab) supplemented with 10% foetal calf serum (FCS). Immune spleen cells were fused with myeloma cells at a ratio of 1:1 using 50% polyethyleneglyco14000 (Merck). Following fusion, cells were suspended in HAT media and distributed into flat-bottomed, 96-well microtitre plates (Nunc), contained feeder layeres of mouse peritoneal macrophages, about 50/A per well at a density of 2 × 105 cells/well. Fresh HAT medium was added to the cells every 3 days. The wells were scanned for the presence of hybrid cell colonies, the supernatants from those wells were harvested for screening by immunoassay. Selected antibody-producing clones were subcloned by limiting dilution, and positive wells were selected for expansion and further cloning.
80
3.4. Detection of antibodies Hybridomas were screened for the production of monoclonal antitheophylline antibodies by enzymelinked immunosorbent assay (ELISA). 96-well microtitre plates (Nunc) were incubated with BSAtheo diluted in coating buffer (0.05 M Nacarbonate-bicarbonate, pH 9.6), I00/zl per well overnight at 4 °C. The plates were washed 3 times with water and 50 #1 of culture supernatants were added to the wells containing 50/zl of assay buffer (phosphate-buffered saline, 0.2% BSA, 0.05% Tween-20). The plates were incubated for 1 h at room temperature, washed, incubated 45 min with solution of rabbit anti-mouse IgG coupled to horseradish peroxidase (Daco Laboratories) in assay buffer, washed and the bound peroxidase was visualized by reaction with phenylenediamine-H20 2 (4 mg OPD and 4 #1 H20 2 per 10 ml of 0.1 M sodium citrate buffer) (substrate buffer). The reaction was stopped with an addition of 10% H2SO 4. The results were read at 492 nm on a multiscan spectrophotometer. To determine heavy chain isotype of the antitheophylline antibodies, IgG from goat anti-mouse isotypic sera was purified and coupled to peroxidase. The antibodies coated with BSA-theo were incubated 45 min with these reagents and the following assay was identical to the ELISA described above. 3.5. Purification of hybridoma antibody from
ascites Selected hybridomas were intraperitoneally injected into BALB/c mice previously primed with pristane. Ascites collected from individual mice were pooled, centrifuged to remove cells and stored at - 2 0 ° C until used. Antitheophylline antibodies were purified by ion exchange chromatography on DEAE-cellulose. Purity of the resulting antibody fraction was assessed by SDS-gel electrophoresis. 3.6. Antibody specificity determination The specificity of each anti-theophylline antibody was examined by the inhibition of binding of antibody to BSA-theo in ELISA. Different amounts of one of various theophylline analogues: caffeine, 1,3dimethyluric acid, hypoxanthine, 1-methyluric acid, 3-methylxanthine, uric acid, xanthine were added to
the adsorbed antigen and mixed with an antibody aliquot. After a 30 min incubation the plates were washed and the second antibody was added. The assay was continued as described above.
washed. 250/~1 of substrate buffer was added and after 10 min incubation the reaction was stopped with 1 ml of 5070 H2SO4 . Colour development was read at 492 nm. Thirty samples were assayed by this method and by high-performance liquid chromatography [6].
3.7. Affinity determination For the measurement of gaff of monoclonal antibodies the approach described by Beatty [5] was used. This method compares the As0 o f two sigmoid curves of antibody serial dilution on plates coated with two different antigen concentrations.
4. Results Spleen cells from BALB/c mice immunised with KLH-theo were fused with a myeloma cell line P3X63-Ag8.653. Two fusions were performed and the fusion frequencies were about 100°70. Hybridoma cell supernatants were screened for their reactivity to theo-BSA in ELISA. Cells from 41 positive wells were subcloned by limiting dilution and after screening 18 positive clones were obtained. Subcloned hybridoma cell lines were expanded in culture and the supernatants were examined for their ability to bind free theophylline. The ability to bind theophylline was evidenced by a decrease in antibody binding to the immobilised BSA-theo antigen in the presence of free theophylline. Three clones actively secreting anti-theophylline antibodies were selected. Specificities and affinities of these antibodies for theophyl-
3.8. Enzyme immunoassay for theophylline Polystyrene beads (Precision Plastic Ball Co.) were coated with monoclonal antibody 2G3 (1/~g/ml in coating buffer overnight at 4°C) and washed with water. The theophylline standards were prepared in human serum in the following concentrations of the drug: 5, 10, 20, 30/zg/ml. Five microliters o f standard or sample was added into a test tube and mixed with 250/zl of enzyme conjugate in assay buffer (1:200). One bead was added to each tube, incubated 30 min at room temperature and
OD
2G3
1.6
~( ~
1.4
\
1.2
\
1.0
2C10
1G9
"
~
0.8 0.6
0.4 0.2
\ ~
0
,
' 0.05' 0.5 '5
50
0.0~0.5 ~i 50 Concentration of inhibitors
0.05 0.5 5
~50 p.g/ml
Fig. 1. Inhibition of binding of monoclonal antibodies to BSA-theo by: e, theophylline; o, caffeine; z~, l-methyluricacid; A, 3methylxanthine; n, 1,3-dimethyluricacid. Cross-reactivitiesfor these antibodies are expressedas the molar ratio of theophyllineto analogue levelsgiving 50% inhibition of antibody binding and are shown in Table 1. 81
line were determined. Antibody specificity was defined by ELISA from the inhibition curves for theophylline and structurally related analogues. Theophylline inhibited the binding all of three antibodies very efficiently. Xanthine, hypoxanthine and uric acid were not recognised by the antibodies. Very different inhibition profiles were demonstrated for theophylline metabolites and caffeine. Examples of inhibition profiles for these monoclonal antibodies are shown in Fig. 1. The 2G3 and 1G9 hybridomas were produced in ascites, and the antibodies were purified by ion exchange chromatography. The 2G3 antibody was found to be of the IgG2a subclass, and 1G9 belonged to the IgGl subclass. Affinity constants were measured in ELISA for binding to theo-BSA using different concentrations of BSA-theo in the coating solution. The Karl value was 2 x 101° for the 2G3 antibody, 5 x 109 for the 1G9 and 109 l/mol for the 2C10. The 2G3 antibody-coated polystyrene beads in combination with theophylline-conjugated peroxidase were used to determine theophylline concentration in human serum. Theophylline concentration in the sample was determined from a standard curve (Fig. 2).
CO 1.6 1.4
1.2 1.0 0.8 0.6
0.4 0.2 5
10 20 30 theophylline concentration
40 p,g/ml
Fig. 2. Standard curve for competitive binding enzyme immunoassay for theophylline. This method is suitable to accurately quantitate theophylline concentrations from 1-40/zg/ml. The correlation between the data obtained by this method and highpressure chromatography is excellent (r=0.97).
immunogen. For hybridoma screening by ELISA, theophylline coupled to BSA through position 8 by a -NH-(CH2) 5 -NHbond was used. Use of these conjugates with different carrier proteins and different types of bonds enabled us to exclude anticarrier antibody interference. Three hybridoma clones secreting monoclonal antibodies for theophylline have been produced. The affinity constants for the monoclonal antibodies range from 109 to 2 × 101° 1/mol. The fine specificities of these antibodies for the theophylline metabolites and for the natural and dietary xanthines were determined in an inhibition enzyme immunoassay. The 2G3 antibody
5. Discussion
In order to produce monoclonal anti-theophylline antibodies, two theophylline-protein conjugates were prepared. A theophylline-KLH conjugate, containing a - N = N - bond between the protein and position 8 of the theophylline moiety, was used as
TABLE 1 Specificities of monoclonal a~atibodies. Compound
Theophylline (1,3-dimethylxanthine) Uric acid Xanthine Hypoxanthine 1-Methyluric acid 3-Methylxanthine 1,3-Dimethyluric acid Caffeine (1,3,7-trimethylxanthine)
82
Antibody 2G3
2C10
IG9
100 0.0001 0.0001 0.0001 10 0.002 7 0.001
100 0.0001 0.0001 0.0001 0.0001 0.0001 500 100
100 0.0001 0.0001 0.0001 80 8 4000 2
was highly specific for theophylline. No significant inhibition of binding by related compounds was observed, and there was little binding to 1-methyluric acid and 1,3-dimethyluric acid. The methyl group in position 1 in the xanthine molecule, therefore, seems to be critical for recognition by antibodies. The 2C10 antibody bound caffeine as well as the theophylline and 1,3-dimethyluric acid stronger than theophylline, but 1-methyluric acid and 3 methylxanthine were not recognized by the antibody. Therefore, the simultaneous presence of 1- and 3-methyl groups appears to be necessary for the binding. The specificity of the 1G9 antibody depends on the C8 carboxy group, because this antibody bound 1,3-dimethyluric acid 40 times more efficientlythan theophylline. Detailed structure-function analysis of the binding of antibodies to theophylline and its derivatives will be possible when the sequence data is correlated with X-ray crystallographic data from one or more antitheophylline antibodies. The results suggest that the 2G3 antibody has the
appropriate affinity and specificity to be useful for quantitating theophylline in serum, and in fact the quantitative polystyrene bead ELISA developed in this study could be employed as a diagnostic assay to monitor the levels of theophyUine in human serum. The antibodies described herein can be a useful system for the study of antigen-antibody interactions. References [1] Cook, C. E., Twine, M. E., Myers, M. andAmerson, E. (1976) Res. Commun. Chem. Pathol. Pharm. 13, 497. [2] Singh, P. and Hu, M. W. (1980) Theophylline antigens and antibodies. U.S. Patent 4.230.805. [3] Geltosky, J. E. (1985) Hybridoma cell lines and monoclonal antibodies to theophylline. U.S. Patent 4.521.510. [4] Jones, J. W. and Robins, R. K. (1960) J. Am. Chem. Soc. 82, 3773. [5] Beatty, J. D., Beatty, B. G. and Vlahos, W. G. (1987) J. Immunol. Methods 100, 173. [6] Kester, M. B., Saccar, C. L., Rocci, M. and Mansmann, H. C. (1986) J. Chromatogr. 380, 99.
83
IMLET 01559
Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay N. D. D a n i l o v a a n d R. G. Vasilov Department of Medical Diagnostics, Institute of Biotechnology, Moscow, US.S.R. (Received 5 December 1990; accepted 21 December 1990)
1. Summary
Spleen cells from BALB/c mice immunised with KLH-theophylline conjugate were fused with a mouse myeloma cell line P3-X63-Ag8.653, and antibody-producing hybrids were identified by enzyme immunoassay. Three cell clones were obtained, each capable o f producing a unique monoclonal antibody to theophylline. Using these monoclonal antibodies, an immunoassay system for theophylline was developed.
methylxanthine, 1,3-dimethyluric acid, 1-methyluric acid and caffeine, which is contained in food. There are several publications describing the production of poly- and monoclonal antibodies to theophylline [1-3], but those antibodies, however, cross-react with some of the above mentioned compounds. In this study, we developed and characterised specific high-affinity murine monoclonal antibodies to theophylline. 3. Materials and Methods
2. Introduction
3.1. Preparation o f hapten-protein conjugates
Theophylline is a drug commonly used in the prevention and treatment of asthma, apnea and obstructive lung diseases. To be effective theophylline must be in serum within the narrow therapeutic concentration range o f 1 0 - 20 #g/ml, so the use oftheophylline requires careful dosage based on measurements of drug concentrations. Current methods for measuring theophylline in serum are based on immunological techniques. They require highly specific antibodies, because body fluids contain some other natural xanthines, such as xanthine, uric acid and hypoxanthine, which are structurally similar to theophylline, and which, if recognised by the antitheophylline antibody, would produce errors in assays. Also, good antibodies should not cross-react with theophylline metabolite compounds: 3-
Theophylline was coupled to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA). KLH-theophylline (KLH-theo) was used as immunogen: BSA-theophylline (BSA-theo) as antigen in the enzyme immunoassay. Theophylline was turned to 8-aminotheophylline [4] which was converted using nitrous acid to the corresponding diazonium compound [4]. Forty mg of diazonium salt was added to an aqueous suspension of 100 mg KLH. The mixture was stirred at 0°C for 2 h at a mildly alkaline pH, then dialysed against water. Coupling yield was determined by addition of [3H]theophylline in tracer amounts into the reaction mixture. A conjugate consisting o f 200 theophylline molecules per molecule o f K L H was prepared. The coupling reaction with BSA was performed in 1 ml of water (pH adjusted to 8 with NaOH) by slowly adding 120/z125°70 glutaraldehyde to the stirring solution of 50 mg 8-aminotheophylline and 100 mg BSA. The reaction mixture was set aside for 2 h at room temperature and 10 mg NaBH4 was added. The con-
Key words: Theophylline;Immunoassay; Monoclonal antibody Correspondence to: Dr. N. D. Danilova,Department of Medical Diagnostics, Institute of Biotechnology, Nauchny proezd 8, Moscow, 117246, U.S.S.R.
0165-2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V.
79
jugate was dialysed against water. The average number of theophylline molecules per molecule of BSA in BSA-theo was 20. 3.2. Enzyme conjugate The enzyme conjugate was prepared by chemically coupling 8-aminotheophylline to horseradish peroxidase (PO). One mg of peroxidase (Sigma) was dissolved in 0.1 ml of water and 1 mg sodium periodate in 25/A of water was added. The mixture was incubated for 2 h at room temperature in the dark, filtered through 0.5 cm Sephadex G-10 layer and mixed with 8-aminotheophylline solution (1 mg in 0.1 ml 0.01 M NaOH). Three hours later 0.1 mg NaBH 4 was added. After I h incubation the conjugate was dialysed against water. 3.3. Immunisation and cell fusion BALB/c mice were immunized intraperitoneally with 50/xg KLH-theo in complete Freund's adjuvant. Six weeks later the mice were boosted with similar doses of antigen with incomplete Freund's adjuvant. Seven days after the boost, mice were bled from the retroorbital sinus in order to detect antitheophylline antibodies in the serum. A mouse with highly positive serum was injected 3 days prior to fusion intraperitoneally with 100/zg antigen in saline. The fusion was carried out with a non-secreting myeloma cell line P3-X63-Ag8.653. Myeloma cells were grown in RPMI-1640 medium (Flow Lab) supplemented with 10% foetal calf serum (FCS). Immune spleen cells were fused with myeloma cells at a ratio of 1:1 using 50% polyethyleneglyco14000 (Merck). Following fusion, cells were suspended in HAT media and distributed into flat-bottomed, 96-well microtitre plates (Nunc), contained feeder layeres of mouse peritoneal macrophages, about 50/A per well at a density of 2 × 105 cells/well. Fresh HAT medium was added to the cells every 3 days. The wells were scanned for the presence of hybrid cell colonies, the supernatants from those wells were harvested for screening by immunoassay. Selected antibody-producing clones were subcloned by limiting dilution, and positive wells were selected for expansion and further cloning.
80
3.4. Detection of antibodies Hybridomas were screened for the production of monoclonal antitheophylline antibodies by enzymelinked immunosorbent assay (ELISA). 96-well microtitre plates (Nunc) were incubated with BSAtheo diluted in coating buffer (0.05 M Nacarbonate-bicarbonate, pH 9.6), I00/zl per well overnight at 4 °C. The plates were washed 3 times with water and 50 #1 of culture supernatants were added to the wells containing 50/zl of assay buffer (phosphate-buffered saline, 0.2% BSA, 0.05% Tween-20). The plates were incubated for 1 h at room temperature, washed, incubated 45 min with solution of rabbit anti-mouse IgG coupled to horseradish peroxidase (Daco Laboratories) in assay buffer, washed and the bound peroxidase was visualized by reaction with phenylenediamine-H20 2 (4 mg OPD and 4 #1 H20 2 per 10 ml of 0.1 M sodium citrate buffer) (substrate buffer). The reaction was stopped with an addition of 10% H2SO 4. The results were read at 492 nm on a multiscan spectrophotometer. To determine heavy chain isotype of the antitheophylline antibodies, IgG from goat anti-mouse isotypic sera was purified and coupled to peroxidase. The antibodies coated with BSA-theo were incubated 45 min with these reagents and the following assay was identical to the ELISA described above. 3.5. Purification of hybridoma antibody from
ascites Selected hybridomas were intraperitoneally injected into BALB/c mice previously primed with pristane. Ascites collected from individual mice were pooled, centrifuged to remove cells and stored at - 2 0 ° C until used. Antitheophylline antibodies were purified by ion exchange chromatography on DEAE-cellulose. Purity of the resulting antibody fraction was assessed by SDS-gel electrophoresis. 3.6. Antibody specificity determination The specificity of each anti-theophylline antibody was examined by the inhibition of binding of antibody to BSA-theo in ELISA. Different amounts of one of various theophylline analogues: caffeine, 1,3dimethyluric acid, hypoxanthine, 1-methyluric acid, 3-methylxanthine, uric acid, xanthine were added to
the adsorbed antigen and mixed with an antibody aliquot. After a 30 min incubation the plates were washed and the second antibody was added. The assay was continued as described above.
washed. 250/~1 of substrate buffer was added and after 10 min incubation the reaction was stopped with 1 ml of 5070 H2SO4 . Colour development was read at 492 nm. Thirty samples were assayed by this method and by high-performance liquid chromatography [6].
3.7. Affinity determination For the measurement of gaff of monoclonal antibodies the approach described by Beatty [5] was used. This method compares the As0 o f two sigmoid curves of antibody serial dilution on plates coated with two different antigen concentrations.
4. Results Spleen cells from BALB/c mice immunised with KLH-theo were fused with a myeloma cell line P3X63-Ag8.653. Two fusions were performed and the fusion frequencies were about 100°70. Hybridoma cell supernatants were screened for their reactivity to theo-BSA in ELISA. Cells from 41 positive wells were subcloned by limiting dilution and after screening 18 positive clones were obtained. Subcloned hybridoma cell lines were expanded in culture and the supernatants were examined for their ability to bind free theophylline. The ability to bind theophylline was evidenced by a decrease in antibody binding to the immobilised BSA-theo antigen in the presence of free theophylline. Three clones actively secreting anti-theophylline antibodies were selected. Specificities and affinities of these antibodies for theophyl-
3.8. Enzyme immunoassay for theophylline Polystyrene beads (Precision Plastic Ball Co.) were coated with monoclonal antibody 2G3 (1/~g/ml in coating buffer overnight at 4°C) and washed with water. The theophylline standards were prepared in human serum in the following concentrations of the drug: 5, 10, 20, 30/zg/ml. Five microliters o f standard or sample was added into a test tube and mixed with 250/zl of enzyme conjugate in assay buffer (1:200). One bead was added to each tube, incubated 30 min at room temperature and
OD
2G3
1.6
~( ~
1.4
\
1.2
\
1.0
2C10
1G9
"
~
0.8 0.6
0.4 0.2
\ ~
0
,
' 0.05' 0.5 '5
50
0.0~0.5 ~i 50 Concentration of inhibitors
0.05 0.5 5
~50 p.g/ml
Fig. 1. Inhibition of binding of monoclonal antibodies to BSA-theo by: e, theophylline; o, caffeine; z~, l-methyluricacid; A, 3methylxanthine; n, 1,3-dimethyluricacid. Cross-reactivitiesfor these antibodies are expressedas the molar ratio of theophyllineto analogue levelsgiving 50% inhibition of antibody binding and are shown in Table 1. 81
line were determined. Antibody specificity was defined by ELISA from the inhibition curves for theophylline and structurally related analogues. Theophylline inhibited the binding all of three antibodies very efficiently. Xanthine, hypoxanthine and uric acid were not recognised by the antibodies. Very different inhibition profiles were demonstrated for theophylline metabolites and caffeine. Examples of inhibition profiles for these monoclonal antibodies are shown in Fig. 1. The 2G3 and 1G9 hybridomas were produced in ascites, and the antibodies were purified by ion exchange chromatography. The 2G3 antibody was found to be of the IgG2a subclass, and 1G9 belonged to the IgGl subclass. Affinity constants were measured in ELISA for binding to theo-BSA using different concentrations of BSA-theo in the coating solution. The Karl value was 2 x 101° for the 2G3 antibody, 5 x 109 for the 1G9 and 109 l/mol for the 2C10. The 2G3 antibody-coated polystyrene beads in combination with theophylline-conjugated peroxidase were used to determine theophylline concentration in human serum. Theophylline concentration in the sample was determined from a standard curve (Fig. 2).
CO 1.6 1.4
1.2 1.0 0.8 0.6
0.4 0.2 5
10 20 30 theophylline concentration
40 p,g/ml
Fig. 2. Standard curve for competitive binding enzyme immunoassay for theophylline. This method is suitable to accurately quantitate theophylline concentrations from 1-40/zg/ml. The correlation between the data obtained by this method and highpressure chromatography is excellent (r=0.97).
immunogen. For hybridoma screening by ELISA, theophylline coupled to BSA through position 8 by a -NH-(CH2) 5 -NHbond was used. Use of these conjugates with different carrier proteins and different types of bonds enabled us to exclude anticarrier antibody interference. Three hybridoma clones secreting monoclonal antibodies for theophylline have been produced. The affinity constants for the monoclonal antibodies range from 109 to 2 × 101° 1/mol. The fine specificities of these antibodies for the theophylline metabolites and for the natural and dietary xanthines were determined in an inhibition enzyme immunoassay. The 2G3 antibody
5. Discussion
In order to produce monoclonal anti-theophylline antibodies, two theophylline-protein conjugates were prepared. A theophylline-KLH conjugate, containing a - N = N - bond between the protein and position 8 of the theophylline moiety, was used as
TABLE 1 Specificities of monoclonal a~atibodies. Compound
Theophylline (1,3-dimethylxanthine) Uric acid Xanthine Hypoxanthine 1-Methyluric acid 3-Methylxanthine 1,3-Dimethyluric acid Caffeine (1,3,7-trimethylxanthine)
82
Antibody 2G3
2C10
IG9
100 0.0001 0.0001 0.0001 10 0.002 7 0.001
100 0.0001 0.0001 0.0001 0.0001 0.0001 500 100
100 0.0001 0.0001 0.0001 80 8 4000 2
was highly specific for theophylline. No significant inhibition of binding by related compounds was observed, and there was little binding to 1-methyluric acid and 1,3-dimethyluric acid. The methyl group in position 1 in the xanthine molecule, therefore, seems to be critical for recognition by antibodies. The 2C10 antibody bound caffeine as well as the theophylline and 1,3-dimethyluric acid stronger than theophylline, but 1-methyluric acid and 3 methylxanthine were not recognized by the antibody. Therefore, the simultaneous presence of 1- and 3-methyl groups appears to be necessary for the binding. The specificity of the 1G9 antibody depends on the C8 carboxy group, because this antibody bound 1,3-dimethyluric acid 40 times more efficientlythan theophylline. Detailed structure-function analysis of the binding of antibodies to theophylline and its derivatives will be possible when the sequence data is correlated with X-ray crystallographic data from one or more antitheophylline antibodies. The results suggest that the 2G3 antibody has the
appropriate affinity and specificity to be useful for quantitating theophylline in serum, and in fact the quantitative polystyrene bead ELISA developed in this study could be employed as a diagnostic assay to monitor the levels of theophyUine in human serum. The antibodies described herein can be a useful system for the study of antigen-antibody interactions. References [1] Cook, C. E., Twine, M. E., Myers, M. andAmerson, E. (1976) Res. Commun. Chem. Pathol. Pharm. 13, 497. [2] Singh, P. and Hu, M. W. (1980) Theophylline antigens and antibodies. U.S. Patent 4.230.805. [3] Geltosky, J. E. (1985) Hybridoma cell lines and monoclonal antibodies to theophylline. U.S. Patent 4.521.510. [4] Jones, J. W. and Robins, R. K. (1960) J. Am. Chem. Soc. 82, 3773. [5] Beatty, J. D., Beatty, B. G. and Vlahos, W. G. (1987) J. Immunol. Methods 100, 173. [6] Kester, M. B., Saccar, C. L., Rocci, M. and Mansmann, H. C. (1986) J. Chromatogr. 380, 99.
83