WO1992007951A1

WO1992007951A1 - Quantitation of aldehyde-containing lesions in nucleic acids

Info

Publication number: WO1992007951A1
Application number: PCT/US1991/007813
Authority: WO
Inventors: Yoke Wah Kow
Original assignee: Yoke Wah Kow
Priority date: 1990-10-19
Filing date: 1991-10-17
Publication date: 1992-05-14

Abstract

Methods of quantifying aldehyde-containing lesions, such as abasic sites, in nucleic acid, and reagents specific for aldehyde groups of aldehyde-containing lesions are described. A nucleic acid is reacted with a reagent which specifically complexes with the aldehyde group of the lesion. The amount of complexed reagent is then detected as an indication of the quantity of aldehyde-containing lesions in the nucleic acid.

Description

OUANTITATION OF ALDEHYDE-CONTAINING LESIONS IN NUCLEIC ACIDS

Background of the Invention

Apyrimidinic/apurinic (AP) , or abasic, sites are the most common lesions produced by DNA. AP sites are formed as a result of cleavage of the N-glycosylic bond between the base and deoxyribose moiety. Abasic sites can be generated spontaneously under physiological conditions by hydrolysis of the N-glycosylic bond. In humans, it has been estimated that about 10,000 AP sites are generated spontaneously per cell per day. AP sites can also be produced following chemical modification of DNA bases by DNA damaging agents such as bleomycin, by alkylating agents, and by ionizing radiation.

AP sites are also intermediates in the base excision repair pathway initiated by the action of DNA N-glycosylases. Thus, the cellular steady state level of AP sites would be elevated as a consequence of base modifications and their subsequent repair. If left unrepaired, AP- sites can lead to cell death and/or mutation induction. AP sites are strong blocks to DNA synthesis in vitro, and are cytotoxic lesions when present in phage transfecting DNA. Although AP sites are bypassed inefficiently in bacterial cells, when bypass occurs, mutation can result since AP sites are non-instructive lesions. There are several methods currently available to quantitate AP sites in DNA. These procedures usually involve the estimation of single strand breaks formed as a result of alkali treatment of AP site-containing DNA, including alkali elution and DNA unwinding. Recently, Weinfeld e_fc .al. , (1990) Biochemistry 22.:1737-1743, using a 32[p]__postiabeling assay, was able to quantitate AP sites in DNA. AP sites can also be measured by the use of ¹⁴C-labeled methoxyamine. Talpaert-Borle, M. and Liuzzi, M.

(1983) Biochem. Bioohvs. Acta 7_1£:410-416; Liuzzi, M. and Talpaert-Borle, M. (1988) Int. J. Radiat. Biol. 54.:709-722. Because ¹⁴C-methoxyamine is only available at low specific activity, this method is relatively insensitive unless large quantities of DNA are available.

All these known procedures detect AP sites in DNA at the femtomole level. However, due to the chemical instability of AP sites, the lengthy procedures involved in the preparation of DNA samples can lead to strand breaks and possible oxidation of the aldehyde group, potentially reducing the number of measurable AP sites in the DNA.

Summary of the Invention This invention pertains to a method of quantifying aldehyde-containing lesions such as abasic sites in nucleic acids, such as DNA, and to reagents specific for aldehyde groups of an aldehyde-containing lesion for use in the method. The method comprises reacting the nucleic acid with a reagent that specifically reacts with the aldehyde group of the lesion (an "aldehyde reactive probe" or ARP) to form a detectable complex and then detecting the complexed reagent. The amount of complexed reagent is proportional to the quantity of aldehyde-containing lesions in the nucleic acid. The ARP is chemically specfic for aldehyde groups in nucleic acid. The ARP can be an O-alkyl hydroxylamine, preferably O-4-nitrobenzyl hydroxylamine and N-biotinyl N'-(aminooxy)acetyl hydrazide. The complexed ARP can be detected in a variety of different ways, including colorimetrically and immunologically. Colorimetric detection can be achieved by labelling the ARP with an enzyme such as a peroxidase that converts a nob-absorbing substrate to an absorbing product (color-forming enzyme) . For example, the ARP can be a biotinylated O-alkyl hydroxylamine such as N-biotinyl N'-(aminooxy)acetyl hydrazide. After aldehyde-specific complexation with the nucleic acid, the biotinylated ARP can be tagged with a biotinylated color-forming enzyme through avidin and the activity of the enzyme measured as an indication of the quantity of aldehyde-containing sites in the nucleic acid. Alternatively, the ARP can be detected immunologically by using an antibody specific to an epitope formed by the reaction of the ARP with the nucleic acid and measuring the complex-specific antibody bound as an indication of the quantity of aldehyde-containing lesions.

The invention also includes kits for performing the methods to quantify aldehyde-containing lesions in nucleic acids. For colorimetric assays, the kit can include a biotinylated ARP, a biotinylated enzyme, its chromogenic substrate and avidin. For an immunological assay, the kit can include an antibody specific for the epitope formed by complexation of the ARP and the nucleic acid and appropriate reagents for detection of the antibody.

The methods and reagents of this invention provide for simple and sensitive quantification of aldehyde-containing lesions in nucleic acids. The methods can be used to test for genotoxicity of various agents.

Brief Description of the Drawings Figure 1 illustrates the structure of N-biotinyl N' (aminooxy)acetyl hydrazide of the invention.

Figure 2 illustrates that the signal measured by colorimetric detection of a biotinylated ARP was proportional to the number of apyrimidinic/apurinic (AP) sites in the DNA sample.

Figure 3 illustrates that the reagent of the invention was able to detect AP sites in both single stranded DNA and double stranded DNA.

Figure 4A illustrates that the number of AP sites detected in uracil-containing fl single stranded DNA was proportional to the amount of uracil N-glycolsylase added.

Figure 4B illustrates that the number of AP sites detected after endonuclease III digestion of calf thymus DNA containing thymine glycols was linearly proportional to the original number of thymine glycols in the DNA sample.

Figure 5A illustrates that when fl single stranded DNA was exposed to ionizing radiation, the number of ARP sites detected was proportional to the x-ray dose. Figure 5B illustrates that the number of AP sites in φX RF DNA was proportional to increasing doses of x-rays.

Figure 5C illustrates that the number of AP sites in calf thymus DNA was proportional to increasing doses of x-rays.

Figure 6 illustrates the rate of production of ARP sites and thymine glycols in heat-inactivated E. coli cells. Figure 7 illustrates that the steady state production and subsequent repair of AP sites in E. coli cells was detectable by the assay of the invention.

Figure 8 illustrates hapten inhibition of anti-NBHA-dRp (5' phosphodeoxyibosyl O-4-nitrobenzyl hydroxylamine) antibody reactivity.

Figures 9A and 9B illustrate inhibition of anti-NBHA (O-4-nitrobenzyl hydroxylamine) antibody reactivity by calf thymus DNA containing NBHA-modified AP sites.

Figure 10 illustrates inhibition of anti-NBHA antibody reactivity by NBHA residues in DNA. The symbols are the same as for Figures 9A and 9B, and data were calculated from Figure 9B. Figure 11 illustrates specificity of anti-NBHA-dRp antibody.

Figure 12A illustrates the effect of DNA denaturation on antibody reactivity.

Figure 12B illustrates the reactivity of anti-NBHA-dRp antibody toward single stranded and duplex DNA. Figure 13 illustrates that the number of antibody reactive sites detected in fl single stranded DNA modified with NBHA and exposed to ionizing radiation, was proportional to the x-ray dose.

Detailed Description of the Invention

According to the methods of this invention, aldehyde-containing lesions in nucleic acid are detected by reacting the nucleic acid with an ARP and then detecting the amount of ARP complexed as an indication of the amount of aldehyde-containing sites in the nucleic acid. The ARP is chemically specific for aldehyde groups in the nucleic acid. The amount of ARP complexed to the nucleic acid is directly proportional to the quantity of aldehyde-containing lesions in the nucleic acid.

Aldehyde-containing lesions detectable by the method of this invention may be naturally occurring, or may be induced by such methods as x-irradiation, chemical treatment, or genetic alteration. Aldehyde-containing lesions are primarily apyrimidinic and apurinic (abasic) sites (AP) . These occur in nucleic acids as a result of cleavage of the N-glycosylic bond between the pyrimidine or purine base and the ribose. Other aldehyde-containing lesions such as formamidopyrimidine, which is an imidazole ring opened purine induced by ionizing radiation, can be detected by the method of this invention. Aldehyde-containing lesions detected in, for example, x-ray irradiated DNA, probably include AP sites, such as alkali labile sugar lesions, strand breaks containing AP sites at the terminus, as well as formamidopyrimidine. Certain classes of AP sites and strand breaks which do not contain an aldehyde group would not be detected by these methods. Since more than one type of x-ray damage is detectable by these methods, they provide a simple and sensitive method for detecting the presence of x-ray damage.

The nucleic acid can include DNA, RNA, rRNA, tRNA, and the like. Typically the nucleic acid is DNA. DNA is generally prepared for the assay as described by Ausubel e_£ al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. The ARP of this invention are reagents which specifically react with the aldehyde group of a lesion of a nucleic acid. In preferred embodiments, the ARP is an O-alkyl hydroxylamine. O-alkyl hydroxylamines react specifically with aldehyde groups in the nucleic acid. A particularly preferred ARP is O-4-nitrobenzyl hydroxylamine (NBHA) , which forms an iminic linkage with abasic sites. This reaction stabilizes the phosphodiester bond 3' to the original aldehyde-containing site, producing O-NBHA residues at the site.

The ARP and the nucleic acid are reacted by incubating them under conditions conducive for reaction of the ARP with the aldehyde groups of the nucleic acid. In general, the incubation is performed in a physiological buffer at room temperature. The ARP reacts with the aldehyde group to form a complex which can be detected in various ways. The ARP can be tagged with a detectable label such as an enzyme, radioisotope or fluorescent compound. In a preferred embodiment, the ARP is biotinylated. For example, a biotinylated O-alkyl hydroxylamine can be formed by reacting O-carboxymethyl hydroxylamine and biotin hydrazide in the presence of carbodiimide. The biotinylated ARP can be detected by means of a biotinylated enzyme bound to the complexed ARP through an avidin-mediated linkage. Preferred enzymes are peroxidases, such as horseradish peroxidase, but other types of enzyme, such as phosphatases, oxidases, galactosidases and ureases may be used.

The complexed ARP can also be detected immunologically. This can be done with an antibody that specifically binds to an epitope formed by the complexation of the ARP to the nucleic acid. For example, antibodies can be raised against an epitope formed by the complex of NBHA and an abasic site.

A monoclonal antibody is preferred. Monoclonal antibody specific for an ARP complex can be prepared by standard somatic cell hybridization technqiues _f(Milstein and Kohler (1975) Nature 256:495-497). Briefly, an appropriate animal, such as a mouse, is immunized with an analog of the ARP complex. For example, to produce an antibody against NBHA sites in DNA, 5'phosphodeoxyribosyl O-4-nitrobenzyl hydroxylamine can be conjugated to a protein such as BSA and used as the immunogen. After immunization, the animal is sacrificed, and cells are removed from its spleen and fused with myeloma cells or other immortalizing cells. The resulting antibody- producing cells (hybridomas) are screened to idenfity the antibodies of desired specificity. The antibodies can be screened for reactivity with the immunogen by an ELISA or similar assay. The antibody can then be evaluated for appropriate sensitivity and specificity in the detection of ARP sites in nucleic acids. For detection, the antibody can be labelled with an enzyme, a radioisotope, fluorescent compound or other detectable label. The enzyme can be peroxidase alkaline phosphatase, glucose oxidase, galactosidase, urease, and the like. In a preferred embodiment, the antibody is detected by means of a second antibody directed against it. For example, the second antibody can be directed against the Fc region of the first antibody (e.g., if the primary antibody is murine, a goat anti-mouse antibody can be used). In this format, the second antibody is labelled for detection as described above.

For convenience and standardization, the reagents for performing the assays of this invention may be assembled into assay kits. These kits contain, in individual containers, predetermined quantities of the reagents necessary to perform a quantitation of aldehyde-containing lesion sites in nucleic acid samples. The kits would also include appropriate standards and controls. The components of the kit depend on the format of the assay. For example, a kit for performing an enzymatic, colorimetric assay can comprise biotinylated ARP (e.g., biotinylated NBHA), a biotinylated enzyme (e.g., horseradish peroxidase), a chromogenic substrate for the enzyme (e.g.,

O-phenylenediamine) and avidin. For an immuπological assay, a kit can contain the ARP, an antibody specific for the ARP complex with the nucleic acid and a secondary antibody detectably labelled, for example, with an enzyme for colorimetric determina ion.

The methods of this invention offer several advantages depending on the embodiment of the assay. In embodiments which employ a biotinylated hydroxylamine as ARP, the chemical specificity for the aldehyde group in AP sites can be combined with the ease and sensitivity of the avidin/biotin system in an enzyme assay (e.g. , Elisa-like microtiter plate assay). For example, employing this method, 1 AP site per 10⁵ nucleotides can be detected where approximately 80 ng of DNA is bound per microtiter well. This detection limit translates to about 1.5 femtomoles of AP sites. Thus, the sensitivity of the assay is comparable to the current available methods for detection of AP sites. However, in contrast to other procedures, the assay is rapid and simple to perform, and offers the possibility of processing a large number of DNA samples through autqmation.

The antibody-based embodiment demonstrates about the same sensitivity as above and as other procedures for measuring AP sites including alkali elution (Brent e_£ al. (1978) DNA Repair Mechanisms, eds. Hanawalt, Friedberg, and Fox (Academic Press, New York), pp. 19-22), DNA unwinding (Kohn ≤£. al. (1981)

DNA Repair: A Laboratory Manual of Research

Procedures, eds. Friedberg and Hanawalt (Marcel Dekker, New York) , Vol. 1., Part B, pp. 379-401), 32p__postiabeling (Weinfeld e_£ al. (1990) Biochemistry 2£:1737-1743) . Some of these methods involve laborious procedures (Brent e_£ al. (1978) DNA Repair Mechanisms, eds. Hanawalt, Friedberg and Fox (Academic Press, New York), pp. 19-22 ; Kohn e_£ al. (1981) DNA Repair: A Laboratory Manual of Research Procedures, eds. Friedberg and Hanawalt (Marcel Dekker, New York), Vol. 1, Part B, pp. 379-401; Birnboim and Jevcak (1981) Cancer Res. 11:1889-1892 and Weinfeld ≤£ al- (1990) Biochemistry 21:1737-1743) and others require the use of radioactive materials (Weinfeld. e± al- (1990) Biochemistry 21:1737-1743; Talpaert-Borle and Liuzzi (1983) Biochem. Biophvs. Acta 740:410-416 and Liuzzi and Talpaert-Borle (1988) Int. J. Radiat. Biol. 54:709-722). In addition, the modification of AP sites by NBHA helps to stabilize the AP sites in the nucleic acid, and therefore, helps to reduce the loss of labile AP sites during purification of DNA from cells. Thus, this embodiment of the method should have broad applicability for detecting abasic sites in the DNA extract from damaged cells.

The methods of this invention have many different utilities. They allow researchers to detect and quantitate different types of damage to nucleic acids, thus facilitating the study of DNA repair and the role of DNA repair in the etiology of different diseases. In the clinic, the assays can be used to monitor the extent of genotoxicity resulting from radiotherapy or chemotherapy. The amount of lesions in DNA from individuals (in lymphocytes from blood samples) undergoing a specific therapy can be quantitated. The DNA can be treated with a battery of damage-specific enzymes, which will remove the lesions, leaving behind an AP site that is detectable. The assays will allow one to quantitatively assess the extent of DNA damage (and DNA repair) . Information derived from these measurements should help physicians establish an effective regime of chemotherapy for a cancer patient optimizing therapeutic gain while minimizing potential side effects of the chemotherapeutic agent. The methods of this invention can be used to monitor individuals for possible occupational exposure to genotoxic agents. People exposed to chemicals or radioactivity have a higher risk of genetic damage. DNA from individual workers

(lymphocytes from blood) can be monitored for DNA damage. This information can be used to set guidelines and to limit individual exposure.

The methods of this invention provide a useful supplement for the Ames' Test for mutagenicity. Every year, thousands of new chemicals are synthesized, and their genotoxicity is not known. The Ames' Test has been used to screen chemicals that might be mutagenic before they are released into the public sector. The assay of this invention can be used in conjunction with and, in some cases, in place of the Ames' Test. DNA in vitro or in vivo can be treated directly with the new chemical and damage to DNA can be quantitated. In addition, sensitivity can be increased by exposing sensitive cells (prokaryotes and eukaryotes) to these agents, and DNA can be used to assess the damage. Most importantly, human cell lines can be used as a model to assess a chemical's genotoxic effect on human DNA. The methods can also be used to test water supplies for possible contamination with genotoxic chemicals and to detect genotoxic chemicals in the environment, including possible contaminations in air or water resources.

The invention is illustrated further by the following examples.

EXEMPLIFICATION

EXAMPLE 1

Chemicals

O-4-nitrobenzyl, O-methyl, and O-carboxymethyl hydroxylamine were purchased from Aldrich Chemical Company Inc., 1001 West Saint Paul Avenue, Milwaukee, WI 53233. l-Ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDAC) , biotin hydrazide and methylmethanesulfonate were obtained from Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178.

Enzvmes.

Endonuclease III was prepared as described in Katcher and Wallace (1983) Biochemistry 22:4071-4081, and Kow and Wallace (1987) Biochemistry ϋ:8200-8206, from an E. coli strain harboring an overproducing endonuclease III plasmid pHITl obtained from R.P. Cunningham, SUNY, Albany. The procedure was changed by replacing the DNA agarose step with FPLC mono S chromatography, for which the reagents are available from Pharmacia Biochemicals, 800 Centennial Avenue, Piscataway, NJ 08854, following the methods of Asahara e± al. (1989) Biochemistr^y 2&: 444-4449. Uracil'N-glycosylase was prepared from an 1^ coli strain carrying an overproducing plasmid of uracil N-glycosylase according to the method of Lindahl e_fc. al. (1977) J. Biol. Chem. 251:3286-3294.

Nucleic acids.

Calf thymus DNA was obtained from Pharmacia Biochemicals, 800 Centennial Avenue, Piscataway, NJ 08854. PM2, φX-174 RF and fl DNA are prepared routinely in the laboratory. Calf thymus DNA containing AP sites was prepared by heat/acid depurination (Lindahl, T. and Nyberg, B. (1972) Biochemistry 11:3610-3618) and the number of AP sites was estimated by correlation with the rate of heat/acid depurination of PM2 DNA treated under the same conditions (Kow, Y.W. (1989) Biochemistry 23.:3280-3287) . The number of AP sites in PM2 DNA was assayed by alkali fluorometry (Kow, Y.W. (1989) Biochemistry 23.:3280-3287 and Futcher, S.D. and Morgan, A.R. (1979) Can. J. Biochem. 57:932-938). DNA containing other lesions including thymine glycols, pyrimidine dimers, uracil, urea residues, o-methyl hydroxylamine residues and reduced AP sites were prepared as previously described in Futcher and Morgan (1979) Can. J. Biochem. .£2:932-938, and Kow e al- (1990) Mutat. Res. 25.:147-156. E. coli DNA was isolated after lysis of the cells with sodium dodecyl sulfate (SDS) and lysozyme (Ausubel, F.M. e_t al.

(1989) Current Protocols in Molecular Biology. John Wiley & Sons, New York). After the RNAse and protease K treatment described in the Futcher and Kow references, the DNA was extracted with phenol twice and extensively dialyzed in 10 mM Tris-HCl (pH 7.5) plus 1 rriM EDTA. N-biotinyl N'-_aminooxv. acetyl hvdrazide. a biotinylated aldehyde reactive probe (ARP) .

Biotin hydrazide initially was used as a reagent for detecting AP sites in DNA. However, due to the reactivity of hydrazide, the signal to noise ratio was poor for DNA containing AP sites. In order to overcome this problem, biotin hydrazide was converted to an O-alkyl hydroxylamine derivative by the reaction of 0-carboxymethyl hydroxylamine with biotin hydrazide in the presence of carbodiimide (Rosenberg, M.B. e_t al- (1986) J. Neurochem. 46:641-648). O-Carboxymethyl hydroxylamine-HCl (1.365 g) was dissolved in 125 ml of distilled water. EDAC (2.395g) was added, and the pH of the solution was adjusted with pyridine to between 4.0 and 5.0. Then 320 mg of biotin hydrazide was added and the reactive solution was incubated overnight at room temperature. Following incubation, the pH was adjusted to 7.0 by NaOH and the solution was extracted three times with an equal volume of chloroform (CHCI3). The chloroform phase was discarded and the aqueous phase was evaporated to dryness under reduced pressure. The residue was extracted again with chloroform. The chloroform-extracted residue was then dissolved in 50 ml of distilled water, and the pH was adusted to neutrality.

50 g of AG1-X8 (Cl~ form) (Bio-Rad Laboratories, 1414 Harbour Way South, Richmond, CA 94804) was prepared by washing the sample resin twice with 200 ml of 2 M NaCI, and packing the washed resin into a column (2.5 x 50 cm). The resin was washed with four bed volumes of distilled water. Crude ARP solution was loaded onto the column to remove unreacted 0-carboxymethyl hydroxylamine. The first 50 ml of eluate was discarded and subsequent fractions of 50 ml were collected. Each of the fractions was tested for its ability to produce a colorimetric reading with calf thymus DNA containing AP sites previously bound to a microtiter plate (for colorimetric quantitation, see following section) .

Active fractions were pooled (totaling approximately 300 ml) and used as a reagent for AP sites without further purification. The chemical structure of the ARP reagent is tentatively assigned as shown in Figure 1.

Microtiter plate method for assaying AP sites. Immulon I microtiter plates, available from Dynatech Laboratories, 14340 Sully Field Circle, Chantilly VI 22021, were irradiated overnight with 40 watts from an unfiltered germicidal lamp (254 nm) at a distance of 30 cm from the source. The irradiated plates could be used for up to two months with no loss in DNA binding efficiency. 200 μl of calf thymus DNA containing AP sites (10 μg/ml) in phosphate buffered saline (137 mM NaCI, 2.7 mM KC1, 4.3 mM Na₂HP0 .7H2θ, and 1.4 mM KH2PO4) was added to each well, and incubated at 37°C for 2-3 hrs.

Alternatively, the microtiter plates were incubated at 4°C overnight to facilitate DNA binding. The plates were then washed four times with PBS-Tween buffer (PBS plus 0.5% Tween 20, Tween is available from Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178. Next, 100 μl of ARP reagent was added and the plates were further incubated at 37°C for an hour. The plate was washed four times with PBS-Tween to remove any unreacted ARP reagent, and 50 μl of horseradish peroxidase substrate (O-phenylenediamine) was added. Color development was stopped with 50 μl of 5N H2SO after an appropriate time interval and the absorbance at 490 nm was taken. Standard curves were determined with either fl or calf thymus DNA containing known amounts of AP sites. For determining the number of intermediary AP sites produced after the action of endonuclease III, 200 μl of calf thymus DNA containing different amounts of thymine glycol (10 μg/ml solution) was added to each of the wells of a microtiter plate as described above. After incubation at 37^βC for two hours, the plate was washed four times with PBS-Tween, after which 100 μl of diluted endonuclease III was added, and the sample incubated at 37°C for 10 minutes. In this example, endonuclease III was diluted into 10 mM Tris-HCl, 1 mM EDTA and 0.1 M KCl at 37^βC. The reaction was terminated by washing the microtiter plate four times with PBS-Tween after which 100 μl of ARP reagent was added and the number of AP sites determined as described above. For determining the number of AP sites produced after the action of uracil glycosylase, a procedure was used which is similar to that described for endonuclease III. The procedure was modified by using uracil-containing fl single stranded DNA, and uracil N-glycosylase was diluted appropriately into 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA. X-irradiation.

Calf thymus, φX RF, and fl DNA were x-irradiated at a concentration of 30 μg/ml in 10 mM potassium phosphate buffer (pH 7.5) using a Philips x-ray generator (Philips Electronic Instruments, Mount Vernon, NY) with a beryllium window Machlett tube (Machlett Laboratories, 1063 Hope Street, Stamford, CT 06907) operated at 50 kVp and 2 mA (9.6 Gy per minute) . The dose rate is routinely determined in the laboratory by a Fricke ferrous sulfate dosimetry (Fisher Scientific Company, Fair Lawn, NJ) and phage T4 survival.

For irradiation of E. coli cells, 20 ml of strain AB1157 cells (laboratory strain) in PBS (1 x lo⁹ per ml) were inactivated by heating at 90°C for 2 minutes and quick cooled to 4^βC. The dead cells were then diluted to a cell density of 2 x 10⁸ per milliliter and x-irradiated at 10^βC at the doses indicated in Figures 5A and 5B. Due to the length of the x-irradiation, heat inactivated !___ coli cells were employed so as to minimize any possible post-irradiation repair of the x-ray-induced lesions. Irradiated cells were then collected by centrifugation at 5000 rpm for 10 minutes and the DNA was isolated as described by Ausubel e_t al« (1989) Current Protocols in Molecular Biology. John Wiley & Sons, New York.

Methvlmethanesulfonate treatment.

10 ml (5 x 10⁸ cells) of actively growing E. coli (AB1157) cells in PBS buffer were treated with different concentration of methylmethanesulfonate (MMS) and incubated at 37°C for 30 minutes. The MMS was then removed by centrifugation (2000 rpm, SS34) at 4°C, and the resulting cell pellet was washed once with 10 ml of PBS. The cell pellet was collected at 2000 rpm (SS34) and resuspended in 10 ml PBS. For each treatment, 5 ml of the washed cells were further incubated at 37°C for 1 hour, and 5 ml was centrifuged at 2000 rpm, with the resulting cell pellet frozen at -20^βC. After one hour, the post-MMS treated cells were collected by configuration at 2000 rpm and the cell paste frozen at -20^βC. The frozen cell paste was later thawed and the DNA purified according to the published procedures of Ausubel e_£ al. noted above.

RESULTS

Specificity and sensitivity of the ARP assay. The ARP is a biotin-tagged derivative of O-carb^'oxymethyl hydroxylamine, thus the chemical specificity of ARP was expected to be similar to that of O-alkyl hydroxylamine. To determine whether this was the case, DNA containing different unique base modifications was prepared. These included thymine glycols, urea residues, uracil, pyrimidine dimers, O-methyl hydroxylamine-modified AP sites, NaBH4~reduced AP sites, as well as the simple AP sites produced by heat/acid depurination. Microtiter plates coated with calf thymus DNA containing each of these lesions were incubated with the ARP reagent. The amount of biotin bound in each of the wells was measured by an avidin/biotin complex (ABC) conjugated to horseradish peroxidase for colorimetric determination. The amount of signal measured was taken as a measure of the reactivity of the lesion with the ARP reagent. Table I shows that ARP reacted only with the simple AP site. No reactivity of the ARP reagent was observed with other lesions even after prolonged incubation with the reagent (data not shown) . Modification of the AP sites by sodium borohydride (NaBH4) reduction or methoxyamine completely eliminated the signal.

TABLE I Specificity of the ARP reagent

DNA substrates

Undamaged calf thymus DNA

Uracil containing fl DNA^b

UV (254 nm) irradiated calf thymus DNA^C Calf thymus DNA containing 1 AP site per molecule

Calf thymus DNA containing reduced AP sites^d

Calf thymus DNA containing methoxyamine modified AP sites^e

Calf thymus DNA containing

10 thymine glycol per molecule

^a control DNA ^b DNA contained approximately 200 uracil per molecule ^c DNA contained approximately 10 UV dimers per molecule ^d DNA contained approximately 10 AP sites per molecule was reduced with N BH4 ^e DNA contained approximately 10 AP sites per molecule was modified with methoxyamine As illustrated in Figure 2, the signal measured with the ABC-horseradish peroxidase assay was proportional to the number of AP sites in the DNA. In that case, fl DNA containing AP sites was prepared y heat/acid depurination. Reduced AP sites were prepared by sodium borohydride reduction. The fl DNA containing different numbers of AP sites (solid square) or reduced AP sites (solid triangle) was adsorbed to microtiter plates as described above. The DNA on the plate was then allowed to react with the ARP reagent and the amount of biotin on the plate was measured with avidin/biotin complexed to horseradish peroxidase as described above. Again, once the AP site was reduced with sodium borohydride, no reactivity with the ARP reagent was observed.

The ARP reagent was able to detect AP sites in both single and double stranded DNA, as illustrated in Figure 3. In that case, 200 μl of partially depurinated fl single stranded DNA (solid circle) or calf thymus duplex DNA (solid square) were used to coat the wells of a microtiter plate at 4°C overnight as described above. The ARP reagent (100 μl) was added and the amount of biotin on the plate was measured with avidin/biotin-horseradish peroxidase as described above. The reaction was much faster with single stranded DNA than with double stranded DNA (data not shown) .

Figure 3 further shows that the signal produced in depurinated fl DNA is approximately twice the amount produced in depurinated calf thymus DNA at all depurinating time intervals. This is in agreement with the fact that the rate of depurination in single stranded DNA is two-fold higher than double stranded DNA. AP sites formed in DNA after the action of DNA N-glycosylases are detectable by the ARP assay of the invention. Base excision repair enzymes, such as endonuclease III and uracil N-glycosylase, excise the damaged base from DNA, leaving behind an AP site containing aldehyde reactive sites. Figures 4A and 4B show that the ARP reagent exhibited no reaction with thymine glycol or uracil. In these cases, microtiter plates were precoated with fl DNA containing 2% uracil (Figure 4A) or calf thymus DNA containing different amounts of thymine glycols (Figure 4B) . For Figure 4A, uracil N-glycosylase was added and incubated at 37°C for 10 minutes. The enzyme was removed by washing the plates and the amount of ARP signal was then measured by the ARP assay. For Figure 4B, 100 μl of endonuclease III (solid square) was added in a buffer containing 0.1M KCl, 10 mM Tris-EDTA (pH 7.5) while to control plates^', 100 μl of the same buffer was added without endonuclease III (solid triangle). The plates were incubated at 37°C for 30 minutes and the wells were washed. The amount of ARP signal was measured by the ARP assay.

Upon digestion with endonuclease III and uracil N-glycosylase respectively, the intermediary AP sites formed were readily detectable with the ARP reagent. Using uracil-containing fl single stranded DNA, the number of AP sites detected was proportional to the amount of uracil N-glycosylase added (Figure 4A) . Similarly, with calf thymus DNA containing thymine glycols, the ARP signal observed after endonuclease III digestion was linearly proportional to the original number of thymine glycols in the DNA (Figure 4B) . AP sites detected in x-irradiated DNA.

Ionizing radiation produces a wide spectrum of DNA damages including DNA strand breaks, base damages as well as AP sites. The results illustrated in Figures 5A, 5B and 5C were obtained by irradiating fl single stranded (Figure 5A) , φX-RF (Figure 5B) and calf thymus duplex (Figure 5C) DNA in phosphate buffer at a concentration of 30 μg/ml at the indicated x-ray dose. The DNA was then adsorbed onto the microtiter wells and the number of AP sites produced was determined by the ARP assay (solid square) . For Figure 5A, the amount of thymine glycols (solid circle) was also measured by using anti-thymine glycol antibody following the procedures described by Hayes e_fc al- (1988) J. Mol. Biol. 2__01:239-246.

Figure 5A shows that when fl single stranded DNA was exposed to ionizing radiation, the number of ARP sites detected was proportional to the x-ray dose. Similarly, production of ARP sites in φX RF (Figure 5B) and calf thymus DNA (Figure 5C) was proportional to increasing doses of x-rays. The estimated detectable limit of ARP sites was obtained with an x-ray dose of approximately 50 rad. The rate of production of thymine glycols by x-rays, as detected by an anti-thymine glycol antibody, was significantly less than the rate of production of ARP sites. We estimated that at one Krad (10 Gray), seven ARP sites were produced as compared to one thymine glycol per fl DNA molecule. ARP sites in DNA extracted from X-irradiated E. coli cells.

When actively growing wild type E. coli cells (starved at 37°C for 30 minutes in PBS buffer) were x-irradiated, little or no detectable ARP signal was observed, suggesting that the lesions may have been repaired during processing of the irradiated cells to obtain the DNA sample. To circumvent this problem, Ej. coli cells (AB1157, 2 x 10⁹ per ml in PBS buffer) were preheated at 90°C for two minutes to inactivate the repair enzymes, then irradiated with the appropriate dose of x-rays. Figure 6 shows the rate of production of ARP sites and thymine glycols in heat-inactivated E. coli cells. ARP sites were detectable at about one krad, while thymine glycol was barely detectable even at one Krad of x-irradiation. The rate of production of ARP sites was proportional to the x-ray dose.

Production and Repair of AP sites in MMS-treated E. coli cells.

Methylmethanesulfonate (MMS) alkylates both adenine and guanine at multiple positions. Upon alkylation of the purines, the N-glycosylic bond becomes more labile, leading to an increased production of AP sites. Furthermore, repair of these alkylated DNA bases by N-glycosylases, such as 3-methyladenine DNA N-glycosylase, should lead to a further increase in the production of AP sites. To determine the number of AP sites formed in DNA after MMS treatment of ] _ coli cells, DNA was isolated from cells treated with different concentrations of MMS for 30 minutes at 37°C. MMS was then removed by centrifugation followed by washing with PBS buffer. Figure 7 illustrates the results, wherein DNA was extracted from half of the cell pellet (solid square), while the other half (solid triangle) was further incubated at 37°C for an additional hour to allow for repair. Cells were then collected by centrifugation and the DNA extracted and the number of AP sites formed determined by the ARP assay of this invention. Figure 7 shows that the steady state production and subsequent repair of AP sites in £__. coli cells was detectable by the ARP assay of this invention. At 25 μM MMS, where the survival of E coli cells was at about 80% (data not shown), a strong signal was observed. Since a five-fold lower signal can easily be measured, by extrapolation, ARP sites should be detectable in DNA from cells treated with 5 μM MMS. At this concentration, little or no killing of cells was observed (data not shown) .

EXAMPLE 2

Materials and Methods

Chemicals.

Deoxyribose 5-phosphate, deoxyribose, ribose, O-4-nitrobenzyl hydroxylamine, and O-benzyl hydroxlamine were purchased from Aldrich Chemical Company, 1001 West Saint Paul Avenue, Milwaukee, WI 53233. Preparation of haptens and antigen.

5'phosphodeoxyribosyl O-4-nitrobenzyl hydroxlyamine (NBHA-dRp) was prepared by incubating O-4-nitrobenzyl hydroxylamine (NBHA, 10 mM solution at pH 6-7) with 15 mM deoxyribose 5-phosphate overnight at room temperature. The solution was then used for hapten inhibition studies without further purification. Solutions of 5*phosphodeoxyribosyl O-benzyl hydroxylamine, deoxyriboxyl O-4-nitrobenzl hydroxylamine and deoxyribosyl O-benzyl hydroxylamine, each at 10 mM, were prepared in a similar manner without further purification. The reaction of O-alkyl hydroxylamine with aldehyde is known to be quantitative. NBHA-dRp was coupled to BSA through the 5'-phosphate by the carbodiimide procedure described by Rosenberg e_t al. (1986) J. Neurochem. !£.:641-648.

Preparation of the monoclonal antibody.

Immunization of BALB/c mice was performed according to Holmdahl £_£ al» (1985) J. Immunol.

Methods £1:379-384. The NBHA-dRp-BSA conjugate was dissolved in 0.05 mM sodium phosphate buffer (pH 7.5) to a concentration of 2 mg/ml. The solution was emulsified with an equal volume of complete Freund's adjuvant, available from GIBCO BRL, P.O. Box 68, Grand Island, NY 14072. A 50 μl aliquot of the emulsion was injected subcutaneously into each hind foot pad. Nine days after immunization, lymphocytes were isolated from the draining popliteal lymph nodes and fused with mouse myeloma P3 x 63-AG63-653. Screening of antibody production was done by ELISA assay using NBHA-dRp-RSA conjugate as the antigen. Ten out of 440 wells tested positive. The positive supernatants were subsequently assayed by ELISA for binding efficiency to calf thymus DNA containing NBHA-modified AP sites (200 sites per 100,000 bp) . Of the ten positive lines, one reacted equally well with undamaged calf thymus DNA as with NBHA-modified AP DNA, while two clones bound specifically to calf thymus DNA containing

NBHA-modified AP sites. The latter were subcloned twice by limited dilution. Four monoclonal antibody clones were obtained and one of them, 3-12G-12H-12H, was chosen for further study. The antibody was IgM, kappa as determined by Ouchterlony gel diffusion.

Preparation of NBHA-modified depurinated DNA.

Calf thymus DNA was alkylated with 0.3M MMS at 37°C for one hour. After exhaustive dialysis against Tris-HCl (pH 7.5), 1 mM EDTA, the DNA was partially depurinated by heating at 50^βC for an appropriate amount of time to produce DNA containing approximately 200 AP sites per 10,000 bp. DNA containing 2 to 16 AP sites per 10,000 bp was prepared according to Lindahl and Nyberg (1972) Biochemistry 11:3610-3618. Calf thymus DNA was dissolved in 0.1M NaCI, 0.01M sodium citrate (pH 5.0, 100 μg/ml) at 70^βC for 15, 30, 60 or 120 minutes to produce approximately 2, 4, 8 or 16 AP sites per 10,000 bp, respectively. The depurinated DNA preparations were modified by reaction with 5 mM NBHA in phosphate buffer (pH 7.2) at room temperature for two hours. The product was then ethanol precipitated in the presence of 2.5M sodium acetate. The precipitated DNA was washed once with 75% ethanol, then precipitated again to remove traces of NBHA. The precipitated DNA was re-dissolved in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Undamaged calf thymus DNA was treated similarly with NBHA to serve as the control.

Direct binding assay.

Direct binding assays were carried out as previously described by Haugen e_£ al. (1981) Proc.

Nat. Acad. Sci. U.S.A. 78:4124. Polystryene 96 wells microtiter plates, available from Corning 25855, Corning Incorporated Science Products Division, Corning NY 14831, or Immulon 1, available from Dynatech Laboratories, Inc., 14340 Sully Field Circle, Chantilly, VI 22021, were UV-irradiated overnight and coated with 200 μl of NBHA-modified calf thymus DNA in phosphate buffered saline (137 mM NaCI, 2.7 mM KCl, 4.3 mM Na2HPθ4, 7H 0, and 1.4 mM KH₂P0 ) for 2 hours at 37°C. After blocking the plate with 1% horse serum in PBS-Tween (PBS plus 0.5% Tween) for one hour at room temperature, the monoclonal antibody, diluted 250 fold in PBS-Tween, was added to the coated wells and incubated at 37°C for 2 hours. After washing with PBS-Tween, bound antibody was detected with horseradish peroxidase-conjugate goat-anti-mouse IgG + IgM (1:3000 dilution, TAGO, Inc., Burlingame, CA 94010) by the addition of hydrogen peroxide (H2O2) and O-phenylenediamine. Absorbance at 490 nm was then taken after stopping the reaction with 50 μl 8N H2SO at appropriate times. Inhibition assay.

Competitive enzyme immunoassay by haptens of calf thymus DNA containing NBHA-modified AP sites was performed with microtiter plates which were precoated with calf thymus DNA containing NBHA-modified AP sites (200 sites per 10,000 bp, 270 ng/ml) according to procedures previously described by Hubbard e_t al. (1989) Radiat. Res. 111:257-268.

Results

Selection of monoclonal antibody clones for study. Four monoclonal antibody clones specific for NBHA-modified AP sites were obtained and examined for their reactivities with NBHA-modified AP sites using a competitive enzyme immunoassay. Microtiter wells were preadsorbed with calf thymus DNA containing 40 NBHA-modified AP sites per 10,000 bp. In addition, the NBHA-modified DNA was diluted at various concentrations and 100 μl of aliquots were incubated at 37°C for one hour with 100 μl of each, of the antibody preparations. Aliquots of the incubated DNA samples were removed and added to the individual wells containing the adsorbed NBHA-modified AP DNA. ELISA assays were performed using horseradish peroxidase as the indicator enzyme. The results are shown in Table II.

TABLE II % Inhibition of Antibody Reactivity

Monoclonal Inhibitor Concentration Clone (μg / ml)

5 2.5 1.25 0.625 0.312 0.16 0.08

-12G-12H-12H — 64 -8H-2D-12G 52 51

4-8H-3H-6H 46 48

4-8H-12H-9A 29 23

As shown in Table II, clone 3-12G-12H-12H was consistently more reactive with competing NBHA-modified AP sites at all DNA concentrations tested1 At 0.08 μg/ml of competing NBHA-modified AP sites in DNA, 21% inhibition was observed for clone 3-12G-12H-12H as compared to 12%, 0% and 0% for clones 4-8H-2D-12G, 4-8H-3H-6H and 4-8H-12H-9A, respectively. Based on these data, clone 3-12G-12H-12H was selected for further characterization.

Antibodv specificity.

Various haptens were used as competitors of the binding of antibody to NBHA-modified AP sites in DNA. The results are shown in Figure 8 and Table III.

TABLE III

Hapten IC50

5'Phosphoribosyl-NBHA 0.3 μM

Deoxyribosyl-NBHA 5.0 μM Ribosyl-NBHA 5.0 μM

NBHA 7.0 μM

5'Phosphoribosyl-BHA No inhibit. at ImM

Deoxyribosyl-BHA No inhibit. at ImM

Ribosyl-BHA No inhibit. at ImM BHA No inhibit. at ImM

Deoxyribose 5-phosphate No inhibit. at lOmM

Ribose No inhibit. at lOmM

Deoxyribose No inhibit. at lOmM

IC50 is the concentration of inhibitor to effect a fifty percent inhibition of the antibody response. In this case, UV-irradiated microtiter plates were previously adsorbed with calf thymus DNA (200 ng/well) containing 200 NBHA-modified AP sites per 10,000 bp. Serial dilutions of haptens were incubated with 1:1000 dilution of antibody

((NH4)2Sθ4~preci itate supernatant), and 100 μl of the hapten-antibody reaction mix was added to each t>"lι _f__>r ua..h'nπ ui<-h PBS-Tween. the amount of bound antibody was determined with horseradish peroxidase-conjugated goat anti-mouse IgM (1:3000 dilution) in PBS-Tween. Haptens used in the experiments were 5*phosphoribosyl-NBHA (solid square), deoxyribosyl-NBHA (solid circle), ribosyl-NBHA (open circle), and NBHA (open square).

As shown in Table III and Figure 8, 5'deoxyribosyl-NBHA was approximately 20 times more effective as a competitor (IC50 = 0.3 μM) than NBHA, deoxyribosyl-NBHA and ribosyl-NBHA, which gave IC50 values of 5μm, 5μm and 7 μM, respectively. Haptens lacking a nitro group, such as benzyl hydroxylamine, ribosyl, deoxyribosyl, and 5'-phosphoribosylbenzyl hydroxylamine, showed no inhibition up to 1 mM. Deoxyribose, ribose, and deoxyribose 5-phosρhate showed no inhibition even up to 10 mM.

When DNA containing NBHA-modified AP sites was used to inhibit antibody reactivity, inhibition occurred with DNA containing as few as two NBHA-modified AP sites per 10,000 bp, at a concentration of 50 μg/ml of DNA. Inhibition of anti-NBHA antibody reactivity by calf thymus DNA containing NBHA-modifed AP sites was tested using UV-irradiated microtiter plates which were previously adsorbed with 200 μl of 160 ng/ml of calf thymus DNA containing NBHA-modified AP sites (200 sites per 10,000 bp) . Dilutions of calf thymus DNA containing various amounts of NBHA-modified AP sites were then incubated with a 1:1000 dilution of the antibody preparation.

Figure 9A illustrates the results of calf thymus DNA containing 0 (solid triangle), 2 (open circle), 4 (open square), 8 (solid circle), or 16 (solid square) NBHA-modified AP sites per 10,000 bp used to adsorb to the microtiter plate. Similarly, calf thymus DNA containing 25 (open circle), 50 (open square), 100 (closed circle), or 200 (solid square) NBHA-modified AP sites per 10,000 bp were used to obtain the results illustrated in Figure 9B. In both cases, 100 μl of the NBHA-treated DNA/antibody reactions were added to each well, and the bound antibody was detected with horseradish peroxidase-conjugated anti-mouse IgM diluted 1:3000 in PBS-Tween as described above.

The results reported in Figure 10 demonstrate that inhibition was proportional to the total number of AP sites regardless of the extent of depurination of DNA used. The symbols used in Figure 10 are the same as those used for Figures 9A and 9B, and the data were calculated from test results illustrated in Figure 9B.

The specificity of the anti-NBHA-dRp antibody of this invention was also tested. UV-irradiated microtiter plates were coated with 200 μl of DNA containing AP sites (solid circle), reduced AP sites (open square), NBHA-modified AP sites (solid square), and thymine glycols (open triangle) . After blocking the microtiter plate with 1% calf serum in PBS-Tween, the monoclonal antibody (1:250 dilution of the ascites) was added to the wells and the bound antibody was detected with horseradish peroxidase-conjugated goat anti-mouse IgG + IgM at a dilution of 1:3000 as described above. The results, reported in Figure 11 demonstrate, using a direct ELISA assay with native calf thymus DNA, that the antibody reacted only with NBHA-modified AP sites but not with DNA containing AP sites, reduced AP sites or thymine glycols.

The effect of DNA denaturation on antibody reactivity, and the reactivity of the antibody of this invention toward single stranded and duplex DNA were also tested. The results are illustrated in Figures 12A and 12B. To test the effect of DNA denaturation on antibody reactivity, native (solid square) or heat denatured (solid circle) calf thymus DNA containing NBHA-modified AP sites adsorbed to microtiter plates. Bound antibody was detected with the horseradish peroxidase-conjugated goat anti-mouse IgM secondary antibody as described above. As illustrated in Figure 12A, the immunoreactivity of the antibody NBHA-modified AP sites was about two fold higher with heat denatured calf thymus DNA as compared to native DNA.

This preference was substantiated by comparing single stranded fl (solid square) and double stranded calf thymus (solid circle) DNA, as illustrated in Figure 12B. In this case, DNA containing NBHA-modified AP sites (10 mg/ml) were adsorbed on the UV-irradiated microtiter plate. The amount of detectable NBHA residues were measured with anti-NBHA-dRp monoclonal antibody, and the signal obtained with NBHA-modified fl DNA was two-fold higher than NBHA-modified calf thymus DNA. This suggests that the antibody of this invention reacts more effectively with the lesion in single stranded DNA than in double stranded DNA. Antibodv sensitivity.

Calf thymus DNA containing two NBHA-modified AP sites per 10,000 bp gave an optical density reading of 0.65 after a two minute reaction time. Therefore, by extrapolation, the antibody should easily detect one AP site per 10,000 nucleotides. Since only approximately 70 to 100 ng of DNA was absorbed on each well, the antibody thus can detect approximately 10 femtomoles of AP sites.

Reaction of the antibody with damaged fl DNA.

To determine the number of AP sites detected by the antibody of this invention, fl single stranded DNA was x-irradiated in phosphate buffer at a concentration of 30 μg/ml at the indicated x-ray dosage. The x-irradiated DNA was treated with 10 mM NBHA for three hours at room temperature, and excess NBHA was removed by ethanol precipitation. The DNA was then adsorbed onto microtiter wells and the number of anti-NBHA reactive sites were determined. As illustrated in Figure 13, the number of antibody reactive sites detected was proportional to the x-ray dose. The estimated detectable limit of the antibody reactive sites was obtained with an x-ray dose of approximately 50 rad.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, by no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of quantifying aldehyde-containing lesions in nucleic acid, comprising the steps of: a. reacting the nucleic acid with a reagent that specifically reacts with the aldehyde group of the lesion in the nucleic acid to form a detectable complex of the reagent and the nucleic acid; and b. detecting the amount of reagent complexed to the nucleic acid as an indication of the quantity of aldehyde-containing lesions in the nucleic acid.

2. The method of claim 1, wherein the aldehyde- containing lesion is an abasic site.

3. The method of claim 1, wherein the nucleic acid is DNA.

4 . The method of claim 1, wherein the aldehyde- specific reagent is an O-alkyl hydroxylamine.

5. The method of claim 4, wherein the O-alkyl hydroxylamine is O-4-nitrobenzylhydroxylamine.

6. The method of claim 1, wherein the aldehyde- specific reagent is detectably labeled and the amount of reagent complexed with the nucleic acid is determined by detecting the amount of label associated with the nucleic acid.

7. A method of claim 1, wherein the aldehyde- specific reagent is biotinylated and the amount of biotinylated reagent complexed with the nucleic acid is determined by detecting the activity of a biotinylated enzyme bound to the biotinylated reagent through avidin mediated linkage.

8. A method of claim 1, wherein the amount of aldehyde-specific reagent complexed with nucleic acid is determined by i) reacting the nucleic acid with an antibody that specifically binds to the complex of the reagent and the nucleic acid; and ii) detecting the amount of antibody bound to the nucleic acid.

9. A method of quantifying abasic sites in DNA, comprising the steps of: a. reacting the DNA with a biotinylated, aldehyde-specific O-alkyl hydroxylamine to form a complex of the O-alkyl hydroxylamine and the abasic sites of the DNA; b. reacting the DNA with avidin to bind the biotinylated O-alkyl hydroxylamine complexed to the abasic sites; c. reacting the avidin-bound DNA with a biotinylated enzyme to enzymatically label the abasic sites; and d. measuring activity of the enzyme as indicative of the amount of abasic sites in the DNA.

10. The method of claim 9, wherein the biotinylated, aldehyde-specific O-alkyl hydroxylamine is N'-biotinyl N' (aminooxy)acetyl hydrazide.

11. The method of claim 9, wherein the biotinylated enzyme is horseradish peroxidase.

12. The method of claim 11, wherein enzymatic activity is measured colorimetrically.

13. A method of quantifying abasic sites in DNA, comprising the steps of: a. reacting the DNA with an aldehyde-specific O-alkyl hydroxylamine to form a complex of the O-alkyl hydroxylamine and the abasic sites of the DNA; b. reacting the DNA with an antibody that binds specifically to the complex of the O-alkyl hydroxylamine and the abasic sites; and c. detecting the amount of complex-specific antibody bound to the DNA as indicative of the amount of the abasic sites in the DNA.

14. The method of claim 13, wherein the aldehyde- specific O-alkyl hydroxylamine is O-4-nitrobenzyl hydroxylamine.

15. The method of claim 13, wherein the antibody is monoclonal.

16. The method of claim 13, wherein the amount of complex-specific antibody bound to the DNA is detected by i) reacting the DNA with a detectably labeled second antibody against the complex-specific antibody; and ii) measuring the activity of the label associated with the DNA as indicative of the amount of complex-specific antibody bound to the DNA.

17. The method of claim 16, wherein the antibody is labeled with an enzyme.

18. A detectably labeled reagent which specifically complexes to the aldehyde group of an abasic site of a nucleic acid.

19. A reagent of claim 18, comprising a biotinylated O-alkyl hydroxylamine, N-biotinyl

N'-(aminooxy)acetyl hydrazide.

20. A reagent of claim 19, comprising* 0-4-nitrobenzyl hydroxylamine.

21. A reagent which specifically complexes with the aldehyde group of an abasic site of DNA and forms an epitope for antibody binding to the complex.

22. A reagent of claim 21, comprising O-4-nitrobenzylhydroxylaπ.ine.

23. A monoclonal antibody specific for the complex of a reagent which specifically reacts with aldehyde groups in nucleic acid.

24. A monoclonal antibody of claim 23 wherein said reagent includes an O-alkyl hydroxylamine.

25. A monoclonal antibody of claim 24, wherein the hydroxylamine is O-4-nitrobenzylhydroxylamine.

26. A kit for performing a colorimetric assay to quantify aldehyde-containing lesions of DNA, comprising: a. a biotinylated reagent specific for the aldehyde group of the lesion of DNA; b. avidin; c. biotinylated enzyme, and d. chromogenic substrate for the enzyme.

27. The kit of claim 26, wherein the aldehyde- specific reagent is a biotinylated O-alkyl hydroxy1amine.

28. A kit of claim 26, wherein the enzyme is horseradish peroxidase.

29. A kit for performing an assay to quantify abasic sites in DNA, comprising: a. a reagent which specifically complexes to an aldehyde group of an abasic site of DNA and forms an epitope for antibody binding to the complex; and b. an antibody specific for the epitope.

30. The kit of claim 29, wherein the aldehyde- specific reagent is an O-alkyl hydroxylamine.

31. The kit of claim 39, wherein the O-alkyl hydroxylamine is O-4-nitrobenzylhydroxylamine.