Immunology Letters, 30 (1991) 23-26 Elsevier IMLET 01653

A monoclonal antibody to DNA modified with osmium tetroxide/2,2' -bipyridine Alexander E. Kabakov, Vladimir K. Podgorodnichenko and Alexander M. Poverenny Institute of Medical Radiology, Academy of Medical Sciences, Obninsk, Kaluga Region, U.S.S.R. (Received 14 April 1991; accepted 13 May 1991)

1. Summary

A murine monoclonal antibody (IgG) has been generated that binds to DNA modified with osmium tetroxide in the presence of 2,2' -bipyridine and does not interact with unmodified DNA. Reactivity of the antibody was tested by gel retardation assay, ELISA, dot-binding assay and immunoblotting. The results obtained suggest that the antibody does not crossreact with modified or unmodified RNA or proteins. The high specificity of the binding reaction is due to the specific recognition of modified deoxythymidine residue by the monoclonal antibody. A possible way of using the antibody produced is discussed. 2. Introduction

The chemical modification of DNA with osmium tetroxide (OsO4) has long been under investigation [1- 5]. In the presence of pyridine or 2,2' -bipyridine, the OsO 4 forms stable adducts with pyrimidine bases (Fig. 1) [2,6]. Thymine and cytosine in the double helix of B-DNA are protected from the reaction and this reagent cannot modify the usual native DNA. Therefore, OSO4/2,2'-bipyridine (or pyridine) can be used as a probe for some non-B-DNA structures. It has been reported that the singlestranded and double-stranded distorted regions [3, 4], cruciform structures [7, 8], B-Z junctions Key words: DNA modification; Osmium tetroxide; Structural probe; Monoclonal antibody

Correspondence to: Dr. A. E. Kabakov, Dept. of Radiation Biochemistry, Institute of Medical Radiology, Korolev's Str., 4, Obninsk 249020, Kaluga Region, U.S.S.R.

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Fig. 1. Adduct between thymine and OsO4/2,2'-bipyridine complex. Two molecules of pyridine can replace 2,2'-hipyridine in the complex.

[9-12] and some unusual homopurine/homopyrimidine sequences [13-16], are targets for modification. Apparently, specific antibodies to adducts could be applied to detect modified DNA to immunolocalize certain DNA structures. Rabbit polyclonal antibodies to DNA, modified with OsO 4 in the presence of pyridine and 2,2'-bipyridine, have been described previously [6]. In this communication we describe the generation and characterization of a monoclonal antibody that specifically binds to DNA modified with OsO4/2,2'-bipyridine (bipy,Os-DNA) in vitro. 3. Materials and Methods

3.1. Chemical modification Heat-denatured calf thymus DNA, poly[d(A-T)], poly[d(I-C)] (all from Sigma); high-molecularweight RNA from yeast (Serva); bovine serum albumin (BSA, Calbiochem) were modified with 2.2 mM OsO4 and 2.2 mM 2,2'-bipyridine (both from Merck) according to Palecek et al. [6]. The modifica-

0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.

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tion was performed in phosphate-buffered saline (PBS, pH 7.5) and the reaction mixture was incubated for 24 h at 26 °C. Surplus reagents were removed by dialysis against PBS.

3.2. Preparation of monoclonal antibody Bipy,Os-DNA complexed with methylated BSA was used for immunization. BALB/c mice were immunized intraperitoneally (60 #g DNA per mouse) at 3-week intervals. Three days after the 5th injection, the spleen cells were isolated and fused with X63.Ag 8.653 myeloma cells using 50070polyethylene glycol 1500 (Serva). The composition of the selective growth medium has been described [17]. Supernatants from primary hybridomas were tested by enzyme-linked immunosorbent assay (ELISA). The selected hybridoma was cloned twice and ascites were obtained in mice. Isotypes of immunoglobulins (Ig) were determined using a typing kit (Miles). IgG fraction was isolated from ascitic fluids by chromatography on DEAE-cellulose.

3.3. Studies of specificity Antibody reactivity was analyzed by direct and competitive binding in ELISA. 96-well plates (Nunc), pretreated with DEAE-dextran (0.1/~g per well), were coated with nucleic acids or polynucleotides (0.075 - 0.6/~g per well) dissolved in 0.05 M sodium carbonate buffer (pH 9.6) containing 0.004 M EDTA. Some wells untreated with DEAE-dextran were coated with bipy,Os-BSA. Antigen-coated plates were blocked with PBS containing 0.004 M EDTA, 0.05°7o Tween-20 and 0.4°70 gelatin. Antimouse IgG peroxidase-conjugates (Amersham) were diluted 1:1500. All other steps of the ELISA technique have been described [6]. Antibody-mediated retardation of DNA electrophoretic mobility was performed in 0.8070 agarose gel using purified IgG and staining with ethidium bromide [6]. The range of sensitivity of binding reaction was determined by dot-binding assay [18] using a nitrocellulose membrane (Schleicher and Schuell). Various quantities of antigens were immobilized on the nitrocellulose strips (2 #1 per dot) and the filters were baked in a vacuum oven for 2 h at 80°C. Then filters were blocked with 0.05 M Tris-HC1 buffer (TBS) containing 0.15 M NaCI, 0.004 M EDTA, 0.05°70 Tween-20 24

and 0.4% gelatin (pH 7.5) and incubated for 1 h at 37 °C with a monoclonal antibody. After washing with TBS/Tween the filters were treated with antimouse Ig biotinylated antibody (dilution 1:400) and then with streptavidin-biotin-peroxidase complex (Amersham B-S system) diluted 1:300. 0.05% 4chloro-l-naphthol and 0.01% H 2 0 2 were used for color development. RNase-free water for all the RNA solutions used was prepared by incubation with 0.001% diethylpyrocarbonate followed by autoclave treatment. Reactivity of an antibody with unmodified proteins was examined by immunoblotting with total HeLa cell extract [19]. 4. Results

A total of 408 primary hybridomas were screened. A stable hybridoma line, designated 4D10, was produced, that secretes a monoclonal antibody belonging to the IgG1 subclass and interacting with bipy,Os-DNA. The retardation of the electrophoretic mobility of bipy,Os-DNA by the antibody 4D10 is shown in Fig. 2. This effect correlates with IgG concentration, while the mobility of unmodified DNA turned out to be unresponsive to the presence of the antibody, even at maximum concentration (Fig. 2). The ELISA data are established in Fig. 3. The results of direct and competitive binding show that antibody 4D10 reacts with bipy,Os-DNA and bipy,Os-

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Fig. 2. Electrophoretic retardation of modified calf thymus DNA by antibody 4DI0. Lane 1, denatured unmodified DNA; lane 2, denatured bipy,Os-DNA; lanes 3 - 5, 0.05 #g of bipy,OsDNA after incubation with 0.05 (lane 3), 0.25 (line 4) and 1.25/~g (line 5) of the IgG in a volume of 25/~1; line 6, denatured unmodified DNA (0.05/zg) after incubation with 1.25 tzg of the IgG.

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native DNA or unmodified RNA. The results of immunoblotting revealed that antibody 4DI0 does not cross-react with HeLa cell proteins (data not shown).

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Fig. 3. Reactivity of antibody 4D10 (ELISA). (A) Direct binding of the IgG (10/~g/ml) to various antigens adsorbed on plates. (B) Competitive binding of the IgG to bipy,Os-DNA adsorbed on plates (0.3/~g per well) in the presence of various inhibitors, o, bipy,Os-DNA; e, bipy,Os-poly[d(A-t)]; z~, bipy,Os-RNA; A, denatured unmodified DNA; o, bipy,Os-poly[d(l-C)]; D, bipy,Os-BSA. 1

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Fig. 4. Dot-binding assay with antibody 4D10. Antigens were adsorbed on the membrane with 5-fold dilution (from 3125 pg in the initial point to 0.04 pg in the final point). Line a, denaturated bipy,Os-DNA; line b, bipy,Os-poly[d(A-T)]; line c, bipy,Os-RNA; line d, denaturated unmodified DNA. Ascitic fluid diluted 1:600 was used as antibody source.

poly[d(A-T)], but does not interact with unmodified denatured DNA or modified RNA, poly[d(I-C)] or BSA (Fig. 3). The strong reaction of the IgG with bipy,Os-poly[d(A-T)] and the lack of reaction with modified RNA and poly[d(I-C)], led us to suggest that the adduct between deoxythymidine and the OsO4/2,2'-bipyridine complex plays a significant role in specific recognition of modified DNA by antibody 4D10. Fig. 4 demonstrates the possibility of detecting very low quantities of modified DNA (0.04 pg per spot) by means of antibody 4D10. In the dot-binding assay, as well as in ELISA, the antibody reacted with bipy,Os-poly[d(A-T)] and did not interact with bipy,Os-RNA and unmodified DNA (Fig. 4). Antibody 4D10 also does not bind to DNA treated with OsO4 without 2,2' -bipyridine (thymine glycols are the major product of this reaction [1]). No cross-reaction was observed with methylated BSA,

The results show that a highly specific monoclonal antibody to bipy,Os-DNA has been produced. Since the antibody does not interact with cell proteins or with modified or unmodified RNA, it can be used to detect DNA modified in situ. The OsOa/2,2'-bipyridine complex was known to be a specific reagent for selective labeling of DNA directly in living cells [11, 16, 20]. The application of the modification and antibody described to the immunolocalization of certain thymine-containing DNA structures in plasmids and chromosomes seems a possibility. Furthermore, the highly sensitive and specific recognition of modified DNA by antibody 4D10 may be employed for non-radioactive immunodetection of DNA in DNA/DNA and DNA/RNA hybrids. The absence of cross-reactivity with unmodified DNA, RNA and cell proteins may provide an opportunity to use the antibody for visualization of in situ hybridization. Development of the immunodetection of cDNA probes using chemical modification with OsO4/2,2'-bipyridine and monoclonal antibody 4D10 is the next objective of our studies.

References [1] Gates, E T. and Linn, S. (1977) J. Biol. Chem. 252, 2802. [2] Chang, C. H., Beer, M. and Marzilli, L. G. (1977) Biochemistry 16, 33. [3] Lukasova, E., Jelen, E and Palecek, E. (1982) Gen. Physiol. Biophys. 1, 53. [4] Lukasova, E., Vojtiskova, M., Jelen, E, Sticzay, T. and Palecek, E. (1984) Gen. Physiol. Biophys. 3, 175. [5] Glikin, G. C., Vojtiskova, M., Rena-Descalzy, L. and Palecek, E. (1984) Nucleic Acids Res. 12, 1725. [6] Palecek, E., Krejcova, A., Vojtiskova, M., Podgorodnichenko, V., Ilyina, T. and Poverenny, A. (1989) Gen. Physiol. Biophys. 8, 491. [7] Lilley, D. M. J. and Palecek, E. (1984) EMBO J. 3, 1187. [8] McClellan, J. A. and Lilley, D. M. J. (1987) J. Mol. Biol. 197, 707. [9] Galazka, G., Palecek, E., Wells, R. D. and Klysik, J. (1986) J. Biol. Chem. 261, 7092. [10] Palecek, E., Boublikova, P., Galazka, G. and Klysik, J. (1987)

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Gen. Physiol. Biophys. 6, 327. [11] Palecek, E., Rasovska, E. and Boublikova, P. (1988) Biochem. Biophys. Res. Commun. 150, 731. [12] Boublikova, P. and Palecek, E. (1989) Gen. Physiol. Biophys. 8, 475. [13] Collier, D. A., Griffin, J. A. and Wells, R. D. (1988) J. Biol. Chem. 263, 7397. [14] Hanvey, J. C., Klysik, J. and Wells, R. D. (1988) J. Biol. Chem. 263, 7386. [15] Weinreb, A., Collier, D. A., Birshtein, B. K. and Wells, R. D. (1990) J. Biol. Chem. 256, 1352.

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[16] Karlovsky, P., Pecinka, P., Vojtiskova, M., Makaturova, E. and Palecek, E. (1990) FEBS Lett. 274, 39. [17] Lovborg, U. (1982) Monoclonal Antibodies: Production and Maintenance. Heinemann, London. [18] Hawkes, R., Niday, E. and Gordon, J. (1982)Anal. Biochem. 119, 142. [19] Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350. [20] McClellan, J. D., Boublikova, E, Palecek, E. and Lilley, D. M. J. (1990) Proc. Natl. Acad. Sci. USA 87, 8373.

2,2'-bipyridine.

A murine monoclonal antibody (IgG) has been generated that binds to DNA modified with osmium tetroxide in the presence of 2,2'-bipyridine and does not...
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