Detection and quantitation of 8-OH-Adenine using monoclonal antibodies

The present invention relates generally to assays for 8-hydroxyadenine (8-OH-Ade) and, more particularly, to improved immunoassays for the detection and quantitation of 8-OH-Ade in biological specimens. The present invention further relates to new monoclonal antibodies directed against 8-OH-Ade for use in the immunoassays.

FIELD OF THE INVENTION
 The present invention relates generally to assays for 8-hydroxyadenine
 (8-OH-Ade) and, more particularly, to improved immunoassays for the
 detection and quantitation of 8-OH-Ade in biological specimens. The
 present invention further relates to new monoclonal antibodies directed
 against 8-OH-Ade for use in the immunoassays.
 BACKGROUND OF THE INVENTION
 8-OH-Ade is a modified nucleotide base resulting from single electron
 oxidation reactions. Steenken, "Purine bases, nucleosides and nucleotides:
 Aqueous solution redox chemistry and transformation of their radical
 cations e and OH Adducts," Chem. Rev., Vol. 89, pp. 503-520 (1989). The
 presence of this oxidized base has been detected in biological specimens
 in a manner consistent with toxicant exposure and carcinogenesis. Malins
 et al., "The Etiology of Cancer: hydroxyl radical-induced DNA lesions in
 histologically normal livers of fish from a population with liver tumors,"
 Aquat. Toxicol., Vol. 20, pp. 123-130 (1991) and Malins et al., "The
 Etiology of Breast Cancer," Cancer Vol. 71, No. 10, pp. 3036-3043 (1993).
 Previous methods of detecting and quantitating 8-OH-Ade have relied on
 analysis by high performance liquid chromatography-electrochemical
 detection (HPLC-ECD) (Shigenaga et al., "Urinary
 8-hydroxy-2'-Deoxyguanosine as a Biological Marker of In Vivo Oxidative
 DNA Damage," Proc. National. Acad. Sci. USA, Vol. 86, pp. 9697-9701
 (1989)) and gas chromatography-mass-spectrometry with selected ion
 monitoring (GC-MS/SIM) (Malins et al. (1993)) procedures. Recently, assays
 utilizing polyclonal antibodies have also been used to detect 8-OH-Ade.
 West et al., "Radioimmunoassay of 7, 8-dihydro-8-oxoadenine
 (8-hydroxyadenine)," Int. J. Radiat. Biol., Vol. 42, No. 5, pp. 481-490
 (1982). The present invention provides new and improved materials and
 methods for detecting and quantitating the 8-OH-Ade base structure in a
 biological specimen comprising a group of highly specific monoclonal
 antibodies against 8-OH-Ade which may be used in a variety of
 immunoassays.
 Oxygen-free radicals are the primary mediators of cellular free-radical
 reactions. They are produced in normal or pathological cell metabolism,
 and as a result of exposure to a variety of exogenous sources of oxidative
 stress such as tobacco smoke, fatty acids in foods, iron and copper ions,
 and ethanol. Furthermore, ultraviolet light and ionizing radiation can
 stimulate the generation of oxygen-free radicals.
 Oxygen-free radicals cause constant damage which the body's antioxidant
 defense systems usually repair so that a dynamic equilibrium is
 maintained. However, occasionally an overabundance of oxygen free radicals
 in the body occurs. Oxidative stress refers to the condition in which
 there is an overproduction of oxygen-free radicals or a deficiency in the
 antioxidant defense and repair mechanisms. Examples of short-term
 oxidative stress reactions include ischemia, reperfusion injury, acute
 inflammation and hyperoxia. Dreher et al., "Role of Oxygen Free Radicals
 in Cancer Development," European Journal of Cancer, Vol. 32A(1), pp. 30-38
 (1996).
 While short-term oxidative stress does not generally result in severe or
 debilitating illness, chronic oxidative stress can result in oxidative
 damage to an organisms DNA, which in turn has been associated with a
 variety of diseases. It has been established that reactive oxygen species
 play a significant role in mutagenesis, carcinogenesis and tumor
 promotion. Bhimani et al., "Inhibition of Oxidative Stress in HeLa Cells
 by Chemopreventive Agents," Cancer Research, Vol. 53, pp. 4528-4533
 (1993). The dismutation of superoxide yields hydrogen peroxide, which is
 highly reactive in vivo. Hydrogen peroxide reacts with partially reduced
 metal ions to form the hydroxyl radical which can directly inflict DNA
 damage. Dreher et al. (1996). If hydroxyl radicals are generated close to
 DNA, they can attack the purine and pyrimidine bases, causing mutations.
 Halliwell, "Free Radicals, Antioxidants, and Human Disease: Curiosity,
 Cause, or Consequence?," The Lancet, Vol. 344, pp. 721-724 (1994). A
 discussion of the mechanisms of oxygen-free radical related mutagenesis
 resulting from DNA damage can be found in Dreher et al., Role of
 Oxygen-Free Radicals in Cancer Development, European Journal of Cancer,
 Vol. 32A, No. 1, pp. 30-38 (1996).
 The involvement of reactive oxygen species in the development of cancer in
 humans is supported by the abundant presence of oxidative DNA
 modifications in cancer tissue. Loft et al., "Cancer Risk and Oxidative
 DNA Damage in Man," J. Mol. Med., Vol. 74, pp. 297-312 (1996). For
 example, in breast cancer, base lesion concentrations have been found to
 be substantial. Base lesions previously reported include 8-OH-Ade, among
 others. Malins, et al. (1990). It is believed that these base lesions play
 a pivotal roll in oncogenesis and may further serve as early predictors of
 breast cancer risk, in addition to a variety of other cancers, due to
 their inherently mutagenic and carcinogenic effects. See Halliwell (1994)
 and Bhimani et al. (1993) and Dreher et al. (1996).
 Reactive oxygen species have also been implicated in the etiology and
 pathophysiology of many other human diseases including cardiovascular
 disease, chronic inflammatory disease, neurodegenerative diseases,
 rheumatoid arthritis, systemic lupus, erythematosis and sickle cell
 anemia. Bhimani et al. (1993) and Halliwell (1994).
 The etiology of these diseases and their progression, as well as toxicant
 exposure, can be studied by measuring levels of oxidized DNA bases in
 biological specimens. Levels of oxidized DNA bases, such as 8-OH-Ade, may
 also be used as a predictor of risk of disease, thereby allowing
 preventive intervention before the clinical disease develops. Strickland
 et al., "Methodologies for Measuring Carcinogen Adducts in Humans," Cancer
 Epidemiology, Biomarkers and Prevention, Vol. 2, pp. 607-619 (1993).
 One of the most abundant and most studied oxidative modifications of
 DNA-bases is the C-8 hydroxylation of guanine. Loft et al., "Cancer Risk
 and Oxidative DNA Damage in Man", J. Mol. Med., Vol. 74, pp. 297-312
 (1996). Other abundant oxidatively modified purines and pyrimidines
 include 8-oxoadenine, 2-hydroxyadenine, Fapy-A, 5-hydroxy cytosine, and
 thymine glycol, among others. Loft et al. (1996). Of interest to the
 present invention in the modified purine 8-OH-Ade.
 The presence of oxidative damage in genomic and mitochondrial DNA obtained
 from biological specimens such as tissues and isolated cells has been
 studied by a variety of methods, including most commonly gas
 chromatography/mass spectroscopy with selective ion monitoring (GC/MS-SIM)
 and high-performance liquid chromatographic separation (HPLC) followed by
 detection by UV or electrochemistry. Four primary methods used for
 monitoring and quantifying oxidative DNA damage in biological specimens
 are discussed in detail in the review article by Strickland et al. (1993)
 and include immunoassay techniques, post-labeling, fluorescence
 spectroscopy and GC/MS.
 In view of the great interest in DNA adducts, including 8-OH-Ade, efforts
 have been made to make antibodies sensitive to these oxidized nucleosides.
 The use of antibodies to detect DNA adducts began in the mid-1970s.
 Strickland et al. (1993). A variety of assays utilizing the antibodies
 have been developed, including the competitive radioimmunoassay, solid
 phase enzyme immunoassay, competitive solid phase enzyme immunoassay,
 ELISA, and immunoaffinity chromatography. Strickland et al. (1993).
 Monoclonal antibodies have been used to detect, for example,
 1-methyladenosine and pseudouridine in urine (Matsuda et al., "An
 Immunohistochemical Analysis for Cancer of the Esophagus Using Monoclonal
 Antibodies specific for Modified Nucleosides," Cancer, Vol. 72, pp.
 3571-3578 (1993); various derivatives of guanosine and thymine
 (Adamkdewicz et al., "Monoclonal Antibody-Based Immunoanalytical Methods
 for Detection of Carcinogen-Modified DNA Components," Arc Sc. Publ., Vol.
 70, pp. 403-41 (1986); and 8-OH-guanine (Lee et al., "Identification of
 8-Hydroxyguanine Glycosylase Activity in Mammalian Tissues Using
 8-Hydroxyguanine Specific Monoclonal Antibody," Biochemical and
 Biophysical Research Communications, Vol. 196, No. 3, pp. 1545-1551 (1993)
 and Yin et al., "Determination of 8-Hydroxydeoxyguanosine by an
 Immunoaffinity Chromatography-Monoclonal Antibody-Base Elisa," Free Rad.
 Biol. & Med., Vol. 18(6), pp. 1023-1032 (1995)). In the article "Radial
 Immunoassay of 7, 8-Dihydro-8 Oxoadenine (8-Hydroxyadenine)," West et al.
 (1982), the authors discuss a specific radioimmunoassay for 8
 hydroxy-adenine, and its application in the study of irradiated adenine
 solutions as well as a preliminary measurements of the production of
 8-hydroxyadenine, using polyclonal antibodies.
 Although detection and quantitation of oxidized nucleoside basis by
 immunoassay is gaining popularity, the challenge is in producing a
 suitable antibody for a specific base. It is preferable to have a specific
 and highly sensitive antibody which exhibits little cross-reactivity with
 related molecules. Polyclonal antibodies lack specificity and assays using
 such antibodies lack sensitivity. Therefore, use of monoclonal antibodies
 are preferred. However, in practice, obtaining suitable monoclonal
 antibodies can be difficult. The technique of producing monoclonal
 antibodies by hybridoma technology is well known in the art. Nevertheless,
 the results obtained by this technique are unpredictable. Only by carrying
 out the process for making the monoclonal antibodies can the nature of the
 monoclonal antibodies be determined and ascertained. To the bast of the
 inventors knowledge, no assay has been developed for the detection and
 quantitation of 8-OH-Ade in a biological specimen using monoclonal
 antibodies.
 Therefore, what is needed in the art is a highly sensitive and specific
 assay for detecting the presence of and quantitating the amount of
 8-OH-Ade present in a biological specimen.
 SUMMARY OF THE INVENTION
 The present invention relates to methods and materials for the detection
 and quantitation 8-OH-Ade in biological specimens. Specifically, the
 present invention is directed to a group of highly specific monoclonal
 antibodies reactive with the modified nucleoside structure 8-OH-Ade, and
 to various immunoassays for 8-OH-Ade utilizing these monoclonal
 antibodies. The monoclonal antibodies of the present invention may be used
 in assays for diagnosing or monitoring the progression of certain types of
 cancer, in addition to a variety of other diseases associated with
 mutagenesis resulting from oxidative damage of DNA. Assays utilizing the
 monoclonal antibodies of the present invention may also be used to analyze
 or monitor toxicant exposure, such as from environmental sources. The
 monoclonal antibodies of the present invention may also be used to detect
 and quantitate epitopes of 8-OH-Ade.
 The monoclonal antibodies of the present invention were prepared with the
 immunogen 8-OH-adenosine coupled to keyhole limpet hemocyanin (KLH). It is
 believed that the monoclonal antibodies bind with the base portion of the
 structure (8-OH-Ade) and not the carbohydrate (ribose) or protein linkage
 region of the conjugate, because, as demonstrated, conjugates bound to
 nucleosides other than 8-OH-adenosine were unreactive with these
 antibodies. Therefore, the antibodies of the present invention can be used
 to detect and quantitate (by the use of a standard curve) the presence of
 8-OH-Ade in biological specimens of DNA. Procedures for such an assay
 include, for example, immobilizing the DNA, denaturing it to disrupt the
 base-pairing scheme exposing the free base structures, and quantitating
 the amount of 8-OH-Ade present per amount of DNA in a quantitative
 immunoassay.
 Thus, it is an object of the present invention to develop an immunoassay
 for 8-OH-Ade, and its epitopes, in a biological specimen.
 It is another object of the present invention to develop monoclonal
 antibodies specific against 8-OH-Ade.
 Yet another object of the present invention is to develop an immunoassay
 highly sensitive and specific for 8-OH-Ade, utilizing monoclonal
 antibodies against 8-OH-Ade.
 It is a further object of the present invention to prepare antibody
 producing hybridoma cells characterized by their production of monoclonal
 antibodies against 8-OH-Ade.
 Yet another object of the present is to provide an immunoassay for
 diagnosing diseases associated with oxidative DNA damage resulting in
 8-OH-Ade formation.
 It is a further object of the present invention to provide an immunoassay
 for diagnosing and monitoring the development of cancer in a patient.
 Another object of the present invention is to develop an immunoassay for
 monitoring a patient's response to treatment for diseases associated with
 oxidative DNA damage resulting in 8-OH-Ade formation.
 A further object of the present invention is to develop an immunoassay for
 monitoring the effects of toxicant exposure in a human or animal.
 It is also an object of the present invention to develop an immunoassay for
 detecting toxicant exposure in a human or animal.
 Other aspects and advantages of the present invention will be apparent upon
 consideration of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION
 As used herein, the term "8-OH-Ade" refers to both bound, e.g.,
 incorporated into DNA or RNA, and free forms of the nucleotide base.
 The present invention is directed to materials and methods for detecting
 8-OH-Ade in a sample of nucleic acids obtained from a biological specimen
 using monoclonal antibodies. In one aspect of the present invention,
 monoclonal antibodies are provided that are characterized by their
 specific reactivity with 8-OH-Ade. Representative embodiments of this
 aspect of the invention are the monoclonal antibodies identified below,
 produced by the hybridomas also identified below:
 TABLE I
 Monoclonal Antibodies of the Present Invention
 Antibody Hybridoma Accession No.
 8A1 8A1
 8A2 8A2
 8A3.E10 8AE.E10
 8A3.E11 8A3.E11
 8A4.B7 8A4.B7
 8A4.G10 8A4.G10
 8A5 8A5
 8A6 8A6 ATCCHB12189
 8A7 8A7
 8A8 8A8
 8A9 8A9 ATCCHB12188
 The hybridoma cells 8A6 and 8A9 were deposited with American Type Culture
 Collection located at 12301 Parklawn Drive, Rockville, Md. 20852 on Sep.
 13, 1996 and assigned the accession numbers indicated above.
 The preferred monoclonal antibodies for use in the assays of the present
 invention are antibodies 8A6 and 8A9, identified above. Antibody 8A5 is
 reactive primarily with 8-OH-Ade, but was also identified as reactive with
 8-OH-guanine. Thus, antibody 8A5 may be useful in detecting 8-OH-adducts
 of purines generally.
 Monoclonal antibodies of this invention can be prepared according to
 conventional methods by using 8-OH-adenosine conjugated to a carrier
 protein as an immunogen, as described in Current Protocols in Immunology,
 John Wiley & Sons, Inc. New York, N.Y. (1994), incorporated herein by
 reference. The synthesis of 8-OH-adenosine from 8-BR-adenosine is
 described in Cho et al., "Structure of Oxidatively Damaged Nucleic Acid
 Adducts. 3. Tautomerism, Ionization and Protonation of 8-Hydroxy-adenosine
 Studied by .sup.15 NMR Spectroscopy," Nucleic Acids Research, Vol. 19, pp.
 1041-1047 (1991), West et al. (1982) and in Example 1 herein. FIG. 1 shows
 results of GC-MS/SIM analysis of synthetic 8-OH-adenosine prepared in
 accordance with the method of the present invention after hydrolysis using
 60% formic acid and trimethylsilation. The product elutes as a major peak
 with a retention time of 9.7 min. There are only minor additional peaks in
 the total ion chromatogram, indicating high purity of this material. The
 retention time and mass spectrum of the synthetic 8-OH-adenosine are
 indistinguishable from a pure standard 8-OH-Ade, and monoclonal antibodies
 against 8-OH-adenosine react virtually equivalently against 8-OH-Ade.
 To prepare the immunogen the 8-OH-adenosine product can be readily coupled
 to carrier proteins through the available amino groups by methods known in
 the art, although other types of antigen carrying molecules may be used.
 In a preferred embodiment of the present invention, 8-OH-adenosine is
 coupled to keyhole limpet hemocyanin (KLH) by Schiff base formation with
 the lysine groups of KLH, followed by reduction with NaCNBH.sub.3. KLH is
 a preferred conjugation protein for such a coupling reaction since it aids
 in stimulating an immune response from attached ligands. Harlow et al.,
 Antibodies, Cold Spring Harbor Laboratory (1988). Because of this, such an
 antigen is not useful for hybridoma screening. Other antigen carrier
 molecules which may be suitable for practicing the present invention
 include, but are not limited to BSA, ovalbumin, nucleic acids, lipids,
 carbohydrates, and naturally occurring biological conjugates such as
 glucuronic acid conjugates.
 Immunization may be carried out according to conventional methods well
 known to those skilled in the art, such as by subcutaneously,
 intravenously or intraperitoneally injecting the 8-OH-adenosine conjugated
 to a carrier protein into an animal. More specifically, the immunogen may
 be diluted with PBS or physiological saline to a suitable concentration,
 and then injected into the animal, together with a suitable adjuvant if
 necessary. The immunogen should be injected several times (3 to 5 times)
 at an interval of 7 to 10 days with 50 to 100 .mu.g of immunogen in 0.1 ml
 total volume until the total volume injected reaches 100 .mu.l per animal.
 A conventional carrier may be used for the injection. Spleen cells
 isolated from the animal three days after the completion of the injection
 of the 8-OH-adenine conjugate are desirable for use as immune cells.
 In a preferred embodiment to the present invention balb/c mice were
 injected subcutaneously in multiple sites with 8-OH-adenosine-KLH
 conjugate, prepared by mixing the immunogen with PBS and Freund's
 incomplete adjuvant followed by emulsification. The mice were immunized
 four times at ten day intervals with a total volume of 100 .mu.l of
 immunogen per animal. Three days after fusion, spleen cells were then
 isolated for use as immune cells.
 The transformed mammalian cells immuned by an 8-OH-adenosine conjugate are
 then fused with mammal plasmacytoma to produce hybridomas. A clone
 recognizing 8-OH-adenosine is selected from the hybridomas and the target
 monoclonal antibody is then obtained from the clone. In the above process
 there are few limitations to the mammal cells to be transformed with the
 immune antigen. It is desired that the immune antigen be selected taking
 its compatibility with the mammal plasmacytoma be fused into
 consideration. Mice, rats, rabbits and the like are generally preferable
 for use.
 Various known myeloma cells can be used as mammal plasmacytoma to be fused
 with the above immune cells. Such myeloma cells include, for example, p3
 (pe/x63-Ag8) (Nature, 256:495-497 (1975)), P3-U1 (Current Topics of
 Microbiology and Immunology, 81:1-7 (1987)), NS-1 (Eur. J. Immunol.,
 6:511-519 (1976)), MPC-11 (Cell, 8405-415 (1976)), PS2/0 (Nature,
 276:269-270 (1978)), FO (J. Immunol. Meth., 35:1-21 (1980)), x63, 6, 5, 3
 (J. Immunol., 123:1548-1550 (1979)), S194 (J. Exp. Med., 148:313-323
 (1978)), and R210 (Nature, 277:131-133 (1979)) of rat, and the like. In a
 preferred embodiment of the present invention, mouse X63 myeloma cells are
 used.
 The fusion of the immune cell and the plasmacytoma can be carried out in
 accordance with known methods, (see Harlow et al. (1988)) in the presence
 of a fusion accelerator and in a conventional nutritious medium.
 Conventional fusion accelerators, such as polyethylene glycol (PEG) and
 sendai virus (HVJ) can be used. Optionally, adjuvants such as
 dimethylsulfoxide and the like may be used in order to promote the
 efficiency of the fusion. A conventional fusion ratio of about 1-10 immune
 cells per one plasmacytoma may be used. As a medium for the fusion, any
 medium used for the cultivation of the plasmacytoma, such as PRMI 1640
 medium and MEM medium, as well as other various media used for the
 cultivation of this type of cell, can be used. Serum obtained by removing
 serum complement from fetal calf serum (FCS) is a typical example of the
 type of medium that may be used.
 The fusion is carried out by thoroughly mixing a prescribed amount of the
 immune cells with the plasmacytoma and blending this mixture with a medium
 to which about 30-60% (w/v) of a PEG (e.g., PEG with an average molecular
 weight of 1,000-6,000) solution which has been heated to about 37.degree.
 C. in advance is added. The cultivation in the HAT medium is continued for
 a period sufficient for cells other than hybridoma (such as unfused cells)
 to die, usually for several days to several weeks. The hybridoma obtained
 is then subjected to a conventional limiting dilution method to detect the
 target cell lines producing the antibody of interest. In a preferred
 method of the present invention, the hybridoma is subjected to limited
 dilution cloning containing 8.times.10.sup.5 mouse thymocytes as feeder
 cells per well. RPMI medium with 10% FCS and 1 mM pyrovate and 2 mM
 glutamine thymocytes were used as feeder cells both for original fusion
 and in subcloning.
 The detection of the antibody-producing cell lines of the present invention
 may be carried out according to standard methods commonly used for the
 detection of antibodies, as described in the laboratory manual by Harlow
 et al. (1988) cited elsewhere herein, for example. Standard methods
 commonly used include the ELISA method, the plaque method, the spot
 method, the agglomeration reaction method, the Ouchterlony method, the
 radio immunoassay (RIA), and the like. Use of 8-OH-adenosine-conjugated
 BSA as an antigen for the detection is desirable.
 In accordance with a preferred embodiment of the present invention,
 supernatant was tested for binding to 8-OH-adenosine conjugated BSA in a
 solid phase binding assay. The initial differential screening of the
 fusion was conducted with an 8-OH-adenosine-BSA conjugate versus an
 adenosine-BSA conjugate, along with BSA alone. Reactivity of
 8-OH-adenosine selected clones from two independent fusions with alternate
 antigens was analyzed. The number of independent clones reactive with BSA
 coupled to one of three antigens is shown in Table II below:
 TABLE II
 Initial Differential Screening of Antibodies
 Fusion # 8-OH-adenosine/BSA Adenosine/BSA BSA Control
 1 23 0 0
 2 38 0 0
 As demonstrated, the 8-OH-adenosine hapten was highly immunogenic by virtue
 of the considerable number of positive specific wells obtained in both
 fusions.
 Hybridomas that showed reactive antibodies were further analyzed for
 binding specificity by comparing the reactivity of BSA conjugates linked
 to 8-OH-adenosine, native base structure, alternate oxidative products,
 irrelevant bases and oxidized products and a negative control. In
 accordance with the present invention and methods known in the art,
 antibody-producing cell lines were screened to obtain those cell lines
 that generate antibody having binding specificity for 8-OH-adenosine. See
 Example 4 and FIGS. 2 to 12. Hybridomas producing target monoclonal
 antibodies of the invention can be cultivated over generations in
 conventional media and can be stored in liquid nitrogen.
 Collection of monoclonal antibodies of the present invention from
 hybridomas of the invention can be performed by cultivating the hybridoma
 according to conventional methods and obtaining the monoclonal antibody as
 a supernatant, or by administering the hybridoma to a mammal with which
 the hybridoma is compatible, allowing the hybridoma to proliferate, and
 collecting the desired antibodies from the ascites fluid. The former
 method is adaptable to the production of high purity monoclonal antibody,
 and the latter to mass production of monoclonal antibody. Monoclonal
 antibodies thus obtained may be purified by means of salting, gel
 filtration, affinity chromatography, or in accordance with other methods.
 The monoclonal antibodies of the present invention are identified in Table
 I above. The isotype of each monoclonal antibody was determined by an
 Isostrip assay (Boehringer-Mannheim, Indianapolis, Ind.). The results were
 as shown in Table III below:
 TABLE III
 Antibody Isotype
 8Al IgM
 8A2 IgGl
 8A3.E10 IgGl
 8A3.E11 IgGl
 8A4.B7 IgGl
 8A4.G10 IgGl
 8A5 IgGl
 8A6 IgGl
 8A7 IgM
 8A8 IgGl
 8A9 IgGl
 The antibodies of the present invention can be used to detect and
 quantitate (by the use of a standard curve) the presence of 8-OH-adenine
 in biological specimens of DNA. Procedures for doing this would include
 immobilizing the DNA, denaturing it to disrupt the base-pairing scheme
 exposing the free base structures, and quantitating the amount of 8-OH-Ade
 present per amount of DNA in a quantitative immunoassay similar to those
 described below.
 The presence of 8-OH-Ade in a biological sample can be analyzed at a high
 sensitivity and precision and with a high specificity in a simple manner
 by the use of monoclonal antibodies of the invention in conventional
 immunoassay formats, such as enzymatic immunoassays (EIA), enzyme-linked
 immunosorbent assays (ELISA), radioimmunometric assays (RIA),
 immunoturbidimetric assays, or others known in the prior art. The lab
 manual by Harlow et al. (1988) discusses many of these methods. Because
 the monoclonal antibodies of the present invention react with 8-OH-Ade
 with specificity they are useful for the determination of 8-OH-Ade in
 clinical samples by immunoassay, thus enabling screening for various
 diseases and exposure to toxicants associated with elevated levels of
 8-OH-Ade and associated with mutagenesis resulting from oxidative DNA
 damage. Thus, the present invention further provides immunoassay methods
 for determining the presence or amount of 8-OH-Ade in a biological fluid
 sample using the monoclonal antibodies of the invention. The assay
 comprises immunochemical reagents for forming an immunoreaction product
 whose presence or amount relates, either directly or indirectly, to the
 presence or amount of 8-OH-Ade in the sample. Those skilled in the art
 will appreciate that there are numerous well known clinical diagnostic
 procedures in which the immunochemical reagents of this invention can be
 used to form an immunoreaction product whose presence and/or amount
 relates to the presence and/or amount of 8-OH-Ade present in a sample.
 While exemplary assay methods are described herein, the invention is not
 so limited. Various heterogeneous and homogenous protocols, either
 competitive or noncompetitive, can be employed in performing an assay of
 this invention.
 For example, the monoclonal antibodies of the present invention can be used
 in a direct solid phase immunoassay of antigen present in a biological
 specimen. DNA can be extracted from a tissue, cell or urine, for example,
 and subjected to a solid phase assay under conditions where the results
 with known amounts of DNA (e.g. by weight) are compared to a standard
 curve containing known amounts of antigen. This methodology could also be
 applied to impure DNA fractions or unfractionated biological specimens
 such as tissue, cells, or bodily fluid and the results normalized to
 another parameter such as protein concentration or nucleic acid using
 alternate means for determining the amount of nucleic acid present in the
 specimen (e.g. the amount of adenine present).
 Furthermore, the monoclonal antibodies of the present invention can be used
 in a quantitative immunohistochemical analysis of cells and tissues. For
 example, cells or tissue sections can be immobilized on glass slides under
 conditions which denature the cellular DNA, such as heating or drying the
 specimen. Antibody analysis can be conducted with, for example,
 fluorescently labeled 8-OH-Ade specific antibodies under conditions where
 the fluorescence intensity of the stained sections is proportional to the
 amount of 8-OH-Ade present in the specimen. A similar assay was previously
 described for monoclonal antibodies reactive with other types of modified
 nucleotide bases by Matsuda et al. (1993).
 Alternatively, the 8-OH-Ade specific antibodies can be immobilized and used
 to absorb or capture soluble antigen from known amounts of biological
 specimens such as cells, tissue, and fluids, including bodily fluids. This
 can be used as a concentration step prior to elution and a detection and
 quantitation step using other methodologies such as HPLC-ECM (Shigenaga et
 al. (1989)) or GC-MS/SIM (Malins et al. (1993)) procedures. In addition, a
 detection and quantitation step involving inhibition of antibody binding
 to antigen as discussed below could be applied.
 Furthermore, soluble antigen present in known amounts of biological
 specimens, including bodily fluids, can be detected and quantitated either
 directly or after an initial concentration step by determining the amount
 of this material required to provide inhibition of antibody binding to
 immobilized antigen. In these procedures, the specimen would be combined
 with monoclonal antibody of the present invention and incubated for a
 period of time sufficient to allow antibody complexes to form with the
 soluble antigen. The resulting mixture would be incubated with immobilized
 antigen and the amount of antibody binding to the immobilized antigen
 determined. The concentration of antigen present in the specimen would be
 determined by comparison to the effect with known amounts of 8-OH-Ade
 containing soluble fractions in either single determinations or in serial
 dilutions of the specimen. The dilution state required to relieve the
 inhibition of binding to the immobilized antigen to a proscribed level
 would be proportional to the concentration of 8-OH-Ade present in the
 specimen.
 In another illustrative embodiment, a double antibody or "sandwich"
 immunoassay format may be employed comprising the steps of (a) forming a
 first immunoreaction admixture by admixing a sample with a first antibody,
 e.g., a monoclonal antibody, wherein the antibody and 8-OH-Ade present in
 the sample are capable of forming a first immunoreaction product (the
 first antibody can be operatively linked to a solid matrix); (b)
 maintaining the first immunoreaction admixture so formed under biological
 assay conditions for a time period sufficient to form the first
 immunoreaction product (the first immunoreaction product can then be
 separated from the sample); (c) forming a second immunoreaction admixture
 by admixing the first immunoreaction product with a second antibody,
 monoclonal or polyclonal, which recognizes 8-OH-Ade; (d) maintaining the
 second immunoreaction admixture so formed under biological assay
 conditions for a period sufficient to form the second or "sandwich"
 immunoreaction product; and (e) determining the presence and, optionally,
 the amount of second immunoreaction product formed, and thereby the
 presence and, optionally, the amount of 8-OH-Ade in the sample.
 Preferably, the second antibody is labeled, preferably with an enzyme, and
 thus the second immunoreaction product formed will be a labeled product to
 facilitate determination of the second immunoreaction product.
 In preferred double antibody assay methods, the amount of immunoreaction
 product determined is related to the amount of immunoreaction product
 similarly formed and determined using a standard sample in place of the
 biological sample wherein the standard sample contains a known amount of
 8-OH-Ade in accordance with this invention. Alternatively, a synthetic
 secondary standard can be used.
 It is also preferred that the second antibody be directed to a site on the
 8-OH-Ade which is not the same as the site to which the first antibody is
 directed. For example, the first antibody can be directed to a site other
 than that which reacts with the monoclonal antibodies of the present
 invention.
 In any of the illustrative assays, the biological sample can be provided as
 a known or unknown quantity of urine, semen, seminal fluid, saliva,
 tissue, blood, or a blood derived product such as serum or plasma. Samples
 for study of oxidative DNA damage generally come from two main sources:
 urinary excretions of oxidized nucleosides and bases from DNA isolated
 target tissue or cells, such as lymphocytes. First, the DNA in the
 specimen must be immobilized, and then denatured to disrupt the base
 pairing scheme, exposing the tree base structures. The amount of antibody
 used can be known or unknown. The admixture is maintained under biological
 assay conditions for a predetermined period of from about 1 hour to about
 16 hours at a temperature of from about 4.degree. C. to about 37.degree.
 C., most preferably about 22.degree. C.
 Biological assay conditions are those that maintain the biological activity
 of the immunochemical reagents of this invention and the 8-OH-Ade. Those
 conditions can generally include a temperature range of from about
 4C..degree. to about 37.degree. C., a pH value range of from at least
 about 6.0 to about 8.0, with a preferred range of 7.0 to 7.4, and an ionic
 strength varying from about 50 mM to 500 mM. Upon routine experimentation,
 other biological assay conditions may be learned. Methods for optimizing
 such conditions are well known to those skilled in the art.
 Another assay format that is may be used in practicing the present
 invention is the precipitation assay. In this embodiment, the process
 comprises formation of an immunoreaction admixture by admixing a DNA
 sample obtained from a biological specimen with a monoclonal antibody of
 the invention to yield a precipitous immunoreaction product. The antibody
 can be operatively linked to a solid particulate such as a microparticle
 or bead, such that when antibody-antigen cross-linking occurs, the
 particulate matter aggregates, indicating the presence of the target
 material.
 Another method is immunoturbidimetry because of its adaptability to
 automatic analysis, enabling a large number of samples to be measured at
 one time. Specifically, an amount of 8-OH-Ade in a sample of DNA obtained
 from urine, blood, or the like can be determined by adding one or more of
 the monoclonal antibodies of the present invention to the sample for the
 reaction and by measuring changes in the absorbence before and after the
 reaction.
 Many other types of assays within the scope of this invention will be
 readily apparent to those skilled in the art.
 Furthermore, the monoclonal antibodies of the present invention may form
 part of a kit comprising the monoclonal antibody of the invention and a
 means for detecting an immunoreaction product comprising 8-OH-Ade and the
 monoclonal antibody. Instructions for use of a packaged immunochemical
 reagent are also typically included in such a kit.
 As used herein, the term "packaged" can refer to the use of a solid matrix
 or material such as glass, plastic, paper, fiber, foil and the like
 capable of holding within fixed limits an antibody of this invention.
 Thus, for example, a package can be a glass vial used to contain
 monoclonal milligram quantities of antibody of the present invention, or
 it can be a microliter plate well to which microgram quantities of a
 contemplated antibody has been operatively affixed. Alternatively, a
 package could include antibody-coated microparticles entrapped within a
 porous membrane or embedded in a test strip or dipstick, etc.
 Alternatively, the antibody can be directly coated onto a membrane, test
 strip or dipstick, etc. which contacts the sample fluid. Many other
 possibilities exist and will be readily recognized by those skilled in
 this art.
 Instructions for use typically include a tangible expression describing the
 reagent concentration or at least one assay method parameter such as the
 relative amounts of reagent and sample to be admixed, maintenance time
 periods for reagent/sample admixtures, temperature, buffer conditions and
 the like.
 In preferred embodiments, a diagnostic system of the present invention
 further includes a label or indicating means capable of signaling the
 formation of a complex containing an antibody of the present invention.
 The word "complex" as used herein refers to the product of a specific
 binding reaction such as an antibody-antigen or receptor-ligand reaction.
 Exemplary complexes are immunoreaction products.
 As used herein, the terms "label" and "indicating means" in their various
 grammatical forms refer to single atoms and molecules that are either
 directly or indirectly involved in the production of a detectable signal
 to indicate the presence of a complex. Any label or indicating means can
 be linked to or incorporated in an expressed protein, peptide, or antibody
 molecule that is part of the present invention, or used separately, and
 those atoms or molecules can be used alone or in conjunction with
 additional reagents. Such labels are themselves well known in the
 diagnostic art.
 The labeling means can be a fluorescent labeling agent that chemically
 binds to antibodies or antigens without denaturing them to form a
 fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable
 fluorescent labeling agents are fluorochromes such as fluorescein
 isocyanate (FIC), fluorescein isothiocyanate (FITC),
 5-dimethylamine-1-natpthalenesulfonyl chloride (DANSC),
 tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200
 sulphonyl chloride (RB 200 SC) and the like. A description of
 immunofluorescence analysis techniques is found in DeLuca,
 "Immunofluorescence Analysis," Antibody As a Tool, Marchalonis et al.,
 Eds., John Wiley & Sons, Ltd., pp. 189-231 (1982).
 The indicating group may also be an enzyme such as horseradish peroxidase
 (HRP), glucose oxidase, or the like. In such cases where the principle
 indicating group is an enzyme such as HRP or glucose oxidase, additional
 reagents are required to indicate that a receptor-ligand complex
 (immunoreacant) has formed. Such additional reagents for HRP include
 hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
 An additional reagent useful with glucose oxidase is
 2,2,-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).
 Radioactive elements are also useful labeling agents and may be used in
 practicing the present invention. An exemplary radiolabeling agent is a
 radioactive element that produces gamma ray emissions. Elements which
 themselves emit gamma rays, such as 1.sup.24 I, 1.sup.25 I, 1.sup.28 I,
 1.sup.32 I and 5.sup.1 Cr represent one class of gamma ray
 emission-producing radioactive element indicating groups. Particularly
 preferred is 1.sup.25 I. Another group of useful labeling means are those
 elements such as 1.sup.1 C, 1.sup.8 F, 1.sup.5 O and 1.sup.3 N which
 themselves emit positrons. Also useful is a beta emitter, such as 1.sup.11
 indium or 3H.
 The linking of labels, i.e., labeling of peptides and proteins, is well
 known in the art. For instance, monoclonal antibodies produced by a
 hybridoma can be labeled by metabolic incorporation of
 radioisotope-containing amino acids provided as a component in the culture
 medium. See, for example, Galfre et al., Meth. Enzyol., 73:3-46 (1981).
 The techniques of protein conjugation or coupling through activated
 functional groups are also applicable. See, for example, Aurameas et al.,
 Scand. J. Immunol., 8(7):7-23 (1978); Rodwell et al., Biotech., 3:889-894
 (1984); and U.S. Pat. No. 4,493,795.
 The diagnostic test kit can also include, preferably as a separate package,
 a "specific binding agent," which is a molecular entity capable of
 selectively binding an antibody of this invention or a complex containing
 such a species, but is not itself an antibody of this invention. Exemplary
 specific binding agents are second antibody molecules, complement proteins
 or fragments thereof. Preferably the specific binding agent binds the
 antibody when it is present as part of a complex.
 In preferred embodiments, the specific binding agent is labeled. However,
 when the diagnostic system includes a specific binding agent that is not
 labeled, the agent is typically used as an amplifying means or reagent. In
 these embodiments, the labeled specific binding agent is capable of
 specifically binding the amplifying means when the amplifying means is
 bound to a complex.
 The diagnostic kits of the present invention can be used in an "ELISA"
 format to detect the quantity of 8-OH-Ade in biological samples of DNA
 obtained from biological specimens such as cells, plasma, saliva, serum,
 semen, seminal fluid tissue, urine or blood. "ELISA" refers to an enzyme
 linked immunosorbent assay such as those discussed above, which employ an
 antibody or antigen bound to a solid phase and an enzyme-antigen or
 enzyme-antibody conjugate to detect and quantify the amount of an antigen
 present in a sample. A description of the ELISA technique is found in
 Harlow et al. (1988).
 Thus, in preferred embodiments a monoclonal antibody with inherent
 specificity for 8-OH-Ade can be affixed to a solid matrix to form a solid
 support. A reagent is typically affixed to a solid matrix by adsorption
 from an aqueous medium, although other modes of affixation applicable to
 proteins and peptides well known to those skilled in the art can be used.
 Useful solid matrices are also well known in the art. Such materials are
 water insoluble and include the cross-linked dextran available under the
 trademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, N.J.); agarose;
 polystyrene beads about 1 micron to about 5 millimeters in diameter;
 polyvinyl chloride, polystyrene, cross-linked polyacrylamide,
 nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or
 tubes, plates or the wells of a microtiter plate such as those made from
 polystyrene or polyvinylchloride.
 The immunoreagents of any diagnostic system described herein can be
 provided in solution, as a liquid dispersion or as a substantially dry
 powder, e.g., in lyophilized form. Where the indicating means is an
 enzyme, the enzyme's substrate can also be provided in a separate package.
 A solid support such as the above-described microtiter plate and one or
 more buffers can also be included as separately packaged elements in the
 diagnostic assay systems of this invention.
 The packaging materials discussed herein in relation to diagnostic systems
 are those customarily utilized in diagnostic systems. Such materials
 include glass and plastic (e.g., polyethylene, polypropylene and
 polycarbonate) bottles, vials, plastic and plastic-foil laminated
 envelopes and the like.
 Thus, the monoclonal antibodies of the present invention may be used in a
 variety of immunoassays to detect and quantitate 8-OH-Ade in biological
 specimens. These assays may be useful for research, diagnosis of disease,
 prognosis and tracking of response to treatment. The following examples
 illustrate a preferred method for making the monoclonal antibodies of the
 present invention.
 EXAMPLE 1
 Preparation of 8-OH-adenosine
 8-OH-adenosine was prepared according to the method previously described in
 Cho et al.(1991). 8-Br-Adenosine (14 mmol) (Sigma, St. Louis, Mo.) was
 dissolved in 40 ml of dry DMSO and added to a mixture containing sodium
 benzyloxide. This reagent was made by reacting 1 g of sodium metal with 35
 ml of benzyl alcohol in 100 ml of DMSO. The reaction mixture was heated at
 65.degree. C. for 24 hours, cooled to room temperature, acidified with
 glacial acetic acid, and poured into 1 L of anhydrous ether. Preparation
 of 8-OH-adenosine is a single step reaction due to the acid labile nature
 of the initial C8-benzyloxylation product which yields 8-OH-adenosine
 directly. The precipitate was collected, dissolved in methanol, and
 absorbed onto 5 g of silica gel (Merck, Darmstadt, Germany). The solvent
 was removed on a rotary evaporator, and the residue applied to a 500 ml
 bed volume silica gel column in a solvent composed of CHCl.sub.3 :CH.sub.3
 OH (4:1). The column was eluted with this solvent and fractions were
 collected. The elution of products was monitored by thin layer
 chromatography and appropriate fractions pooled. The fractions which
 contained 8-OH-adenosine were identified after GC-MS/SIM analysis of the
 purified components using standard methodology as described in Malins et
 al. (1993). The product was recrystallized from water and yielded about
 0.5 g of pure 8-OH-adenosine as a white powder.
 EXAMPLE 2
 Coupling of 8-OH-adenosine to Keyhole Limpet Hemocyanin (KLH) and Bovine
 Serum Albumin (BSA)
 The 8-OH-adenosine product can be readily coupled to carrier proteins
 through available amino groups. Coupling of this hapten to carrier protein
 was conducted through the ribose moiety of the nucleoside after mild
 oxidization by NaIO.sub.4 at pH 4.5 using a sodium phosphate buffer. The
 progress of the oxidation reaction was followed by the change in mobility
 of the UV-absorbing spots after thin layer chromatography on silica gel
 plates using a solvent system composed of CHCl.sub.3 :CH.sub.3 OH (2:1).
 The oxidized product migrates as a faster moving spot on the chromatogram.
 This introduces vicinal aldehyde groups capable of forming Schiff bases
 with primary amines.
 The antigen for immunization was prepared by Schiff base formation with
 lysine groups of keyhole limpet hemocyanin (KLH) followed by reduction
 with NaCNBH.sub.3. the antigen for hybridoma screening was prepared by
 coupling the IO.sub.4.sup.- oxidized derivative to BSA. This placed the
 specific ligand of interest on a molecule that was ideal for solid-phase
 immunoassays to be used in the hybridoma screening process.
 Hapten conjugates of BSA using a variety of negative control nucleotide
 base structures were prepared using these same procedures with
 commercially available periodate oxidized nucleosides (Sigma, St. Louis,
 Mo.). Commercially obtained periodate oxidized nucleosides used were
 adenosine, cytosine, uracil, and guanosine. In addition, 8-OH-guanosine,
 which was prepared according to the method described by Cho et al., Chem.
 Res. Toxicol., Vol. 3, pp. 445-452, (1990), was also periodate oxidized
 and coupled to BSA. All BSA conjugates containing approximately 15 hapten
 molecules per protein subunit.
 EXAMPLE 3
 Preparation of Anti-8-OH-Ade Antibodies
 The methods employed were standard procedures as described in the
 laboratory manual Harlow et al. (1988). Balb/c mice were injected
 subcutaneously in multiple sites with 8-OH-adenosine conjugated keyhole
 limpet hemocyanin. The immunogen was prepared by mixing 2 mg of
 8-OH-adenosine-KLH conjugate in 900 .mu.l of phosphate buffered saline
 (PBS), 100 .mu.l of 2 mg/ml MDP (N-acetylmuramy 1-L-alanyl-D-isoglutamine
 (Pierce, Rockford, Ill.), and 1 ml of Freund's incomplete adjuvant (Sigma,
 St. Louis, Mo.) followed by emulsification. The mice were immunized 4
 times at 10 day intervals with 100 .mu.l of immunogen, the last 3 days
 before fusion. Spleen cells were isolated and fused with mouse X63 myeloma
 cells.
 EXAMPLE 4
 Solid Phase Immunoassay
 Once clones of hybridoma cells appeared after HAT selection, supernatant
 was tested for binding to 8-OH-adenosine conjugated BSA in a solid phase
 binding assay on 96-well Probind plates. Wells containing reactive
 antibodies were moved to 24-well plates and expanded for more detailed
 analysis of binding specificity. Each reactive antibody was tested for
 specificity in side-by-side comparison of reactivity with BSA conjugates
 linked to the specific antigen, native base structure, alternate oxidative
 products, irrelevant bases and oxidized products, and a negative control.
 Wells showing antibody of proper specificity were selected and cloned to
 yield monoclonal antibody producing cell lines.
 The 8-OH-adenosine-conjugated BSA was deposited on 96-well Probind plates
 by coating each well with 50 .mu.l of a solution containing 50 .mu.g
 protein conjugate per ml of 50 mM sodium phosphate buffer, pH 7.5, 5 mm
 MgCl.sub.2, 15 mM NaN.sub.3 and incubated overnight. The plates were
 blocked with PBS containing 5% BSA for 2 hours, followed by incubation
 with antibody containing culture supernatant for 18 hours. The plates were
 then washed extensively with PBS, followed by incubation with 1:500
 diluted rabbit anti-mouse whole Ig (ICN Immunobiologicals, Costa Mesa,
 Calif.) for 1 hour. The plates were again extensively washed with PBS and
 incubated with 1.sup.25 I-protein A (90,000 cpm/well) for 1 hour. The
 plates were washed again with PBS and the amount of 1.sup.25 I in each
 well was determined in a gamma counter.
 FIGS. 2 through 12 show results of solid phase immunoassays using senior
 dilutions of nucleoside-BSA conjugates and cloned hybridoma culture
 supernatants as the antibody source. The results indicate that antibodies
 8A1, 8A2, 8A3.E10, 8A3.E11, 8A4.B7, 8A4.G10, 8A6, and 8A9 of the present
 invention were absolutely specific for 8-OH-adenosine haptens coupled to
 BSA. No cross reactivity was observed with BSA conjugated to either
 adenosine, cytosine, uracil, guanosine, or 8-OH-guanosine. A broader
 specificity was observed with 8A5 (FIG. 8) in which substantial reactivity
 with 8-OH-guanosine was also observed, and with 8A7 (FIG. 10) and 8A8
 (FIG. 11) which also reacted with guanosine and adenosine, and
 8-OH-guanosine, respectively.
 In experiments to titrate antigen reactivity, the conjugate protein was
 serially diluted in the above buffer alone. As shown in FIGS. 2 to 12, the
 antibodies were capable of detecting very low amounts of 8-OH-Ade. In
 particular, antibodies 8A1, 8A2, 8A6, 8A7, 8A8 and 8A9 were reactive with
 8-OH-Ade levels present in quantities of less than 5 fmol per assay well.
 EXAMPLE 5
 Inhibition of Antibody Binding by Soluble Ligands
 Culture supernatants containing hybridomas of known titer were mixed with
 soluble ligands which were serially diluted 1:2 in a 96well plate starting
 with a concentration of 1 mM down to 5 .mu.M and incubated overnight at
 4.degree. C. followed by incubation for 2 hours on an 8-OH-adenosine-BSA
 antigen coated plate (50 .mu.l of 50 .mu.g/ml protein conjugate).
 Conditions of the assay were otherwise the same as described in Example 4
 above.
 FIGS. 13 to 23 show results of inhibition of antibody binding to
 8-OH-adenosine conjugated BSA (50 .mu.g of protein/ml as coating mixture)
 in a solid phase assay using commercial 8-OH-AMP (Sigma, St. Louis, Mo.)
 (Prepared from 3',5'cyclic 8-OH-AMP after hydrolysis by nucleotidase),
 AMP, or 4,6-diamino-5-formamidopyridine (Fapy-A). Fapy-A is a ring-opening
 oxidation product from single electron oxidations and is an alternate
 reaction product of the same reaction which leads to 8-OH-adenosine. In
 these experiments the titer of antibody present in the culture supernatant
 used was adjusted to yield near maximal, but not saturating levels of
 antibody binding so that the antibody titer was not in large excess over
 the antigen coated onto the plate. This is demonstrated in FIG. 24. The
 results indicate that in the cases of antibodies 8A1, 8A2, 8A3.E10,
 8A3E11, 8A4.B7, 8A4.G10, 8A6, 8A7, 8A8, and 8A9 specific inhibition of
 antibody binding was observed only for 8-OH-AMP as its concentration was
 varied from 1 mM to 5 .mu.M. No apparent inhibition with any antigen under
 the concentration range used was observed for 8A5. The concentration of
 soluble 8-OH-AMP capable of inhibiting the solid phase antibody binding
 was variable among the antibodies, with 8A6 and 8A9 showing the lowest
 concentrations of the soluble ligand capable of binding inhibition.
 To determine if changes in experimental conditions can modulate the
 concentration of soluble ligand capable of causing significant binding
 inhibition, the ratio of soluble 8-OH-AMP ligand to immobilized
 8-OH-adenosine present in BSA conjugates was varied and the effect on the
 concentration of soluble ligand capable of inhibiting antibody binding
 determined. The results for antibody 8A6 is shown in FIGS. 25 and 26. FIG.
 25 shows results under standard conditions with plates coated with the BSA
 conjugate at a concentration of 50 .mu.g/ml compared to an amount 32-fold
 lower shown in FIG. 26. These results indicate that this change in the
 ratio of soluble to bound ligand reduced the concentration of soluble
 ligand needed to provide a similar amount of binding inhibition by a
 factor of about 16. A similar study was also conducted with antibody 8A9
 as shown in FIGS. 27 and 28 with more striking results. The concentration
 of soluble ligand capable of providing the same amount of inhibition in
 comparisons of the standard condition (50 .mu.l of 50 .mu.g/ml protein
 coated per well) versus a 64-fold dilution (i.e. 50 .mu.l of 0.78 .mu.g/ml
 protein) was itself 64-fold lower. Thus, the ratio of bound vs. soluble
 antigen in the inhibition assay can be adjusted to provide a more
 sensitive assay and the assay can be adjusted to detect binding inhibition
 caused by very low concentrations of soluble antigen. This is done by
 reducing the amount of bound antigen used in the assay and amplifying the
 signal due to primary antibody binding to it so that low concentrations of
 soluble antigen can effectively competitively bind to the antigen binding
 site on the antibody and result in detectable binding inhibition. Such an
 assay can be used to determine the presence and concentration of soluble
 antigen present in biological specimens and bodily fluids including, but
 not limited to, blood or urine.
 EXAMPLE 6
 Binding of Antibodies 8A6 and 8A9 to Physiological DNA Specimens
 The ability of this panel of monoclonal antibodies to detect 8-OH-Ade
 lesions in physiological DNA was tested. For convenience, antibodies 8A6
 and 8A9 were selected for this analysis. DNA specimens were analyzed for
 the levels of 8-OH-Ade present using GC-MS/SIM methodology (Malins et al.
 (1993)). Control DNA contained 1.2 8-OH-Ade lesions per 10.sup.5 normal
 bases. Test DNA contained 4.7 8-OH-Ade lesions per 10.sup.5 normal bases.
 DNA specimens (50 ng) were dissolved in 100 .mu.l of 1 M ammonium acetate
 and slot blotted onto nitrocellulose membrane (0.45 micron) using a
 Minifold II slot-blot system (Schleicher & Schuell, Keene, N.H.) according
 to manufacturer's instructions. The blots were removed and heated in an
 oven at 80.degree. C. for 1 hour to denature the DNA. The blots were
 blocked using 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 5%
 non-fat dry milk overnight at room temperature, followed by treatment with
 culture supernatants containing monoclonal antibody for 3 hours at room
 temperature. The blots were washed extensively with blocking buffer and
 followed by incubation with 1:1000 diluted rabbit anti-mouse whole Ig
 secondary antibody (ICN, Costa Mesa, Calif.) for 1 hour at room
 temperature. This antibody was diluted in blocking buffer without milk.
 After extensive washing with blocking buffer, the blots were incubated
 with 1.sup.25 I-Protein (20,000 cpm per 50 .mu.l) for 1 hour at room
 temperature. 1.sup.25 I-Protein A was also diluted in blocking buffer
 without milk. After extensive washing with blocking buffer, the blots were
 dried and exposed to x-ray film overnight to visualize spots. The labeled
 spots were cut out and counted in a gamma counter. Net cpm 1.sup.25
 I-Protein A bound was calculated after subtraction of background.
 Background counts were determined by counting an equal area of the blot
 which contained no DNA.
 TABLE IV
 Analysis of Binding of Antibodies 8A6 and 8A9 to Denatured
 DNA Specimens of Known 8-OH-Ade Concentration
 Net cpm .sup.125 I-Protein A
 Bound
 DNA #8-OH-Ade fmol 8-OH-Ade/ Antibody Antibody
 Specimen 105 bases assay 8A6 8A9
 Control 1.2 0.5 882 910
 DNA
 Test DNA 4.7 1.8 3671 4662
 The data obtained is shown in Table IV below. The amount of 8-OH-Ade
 present at each blot is shown, as is the net cpm of 1.sup.25 I-Protein A
 bound. This value is proportional to the amount of 8A6 or 8A9 antibody
 bound to the DNA on the blot. This data shows increased antibody binding
 to test DNA compared to the control. In addition, each antibody is capable
 of producing a clear signal above background when 8-OH-Ade is present in
 the range of 2 fmol/assay. Thus, these data confirm that the antibodies
 bind directly to physiological DNA and are capable of detecting low fmol
 quantities of 8-OH-Ade per assay.
 In summary, the present invention comprises a panel of highly specific
 monoclonal antibodies reactive with the modified nucleoside structure
 8-OH-adenosine. The results of the inventors' experiments further define
 that it is the base portion of the structure (8-OH-Ade) and not the
 carbohydrate (ribose) or protein linkage region of the conjugate which is
 involved in antibody binding. This is known because conjugates with
 alternate nucleosides other than 8-OH-adenosine were unreactive with these
 antibodies, as demonstrated in Example 4 and FIGS. 2 to 12. In addition,
 direct binding of antibodies to 8-OH-Ade present in physiological DNA was
 demonstrated (see Example 6). In some instances minor specificities of
 certain antibodies were found (all of which correlate with antibody
 binding to the base structure). Of particular interest in this regard is
 antibody 8A5 which is reactive primarily with 8-OH-Ade but also detects
 8-OH-guanosine. Also of interest are antibodies 8A7 and 8A8 which show
 some reactivity to guanosine. Thus, monoclonal antibody 8A5 in particular
 (or one similar to it), and antibodies 8A7 and 8A8 may be useful in
 detecting 8-OH-adducts of purines generally.
 The results obtained by the inventors are typical for binding specificities
 of monoclonal antibodies. That is, specificity is defined by certain
 epitope portions of a larger molecule. Beyond the specific epitope, there
 is no recognition and no bias as to the chemical form containing the
 epitope for antigen presentation to the antibody. Similar results were
 demonstrated by the inventors for anti-carbohydrate antibodies (Holmes and
 Greene, Arch. Biochem. Biophys., 288, 87-96, 1991). In those instances,
 the aglycone portion carrying the antigen (e.g. from lipid to protein) was
 demonstrated to be irrelevant and antibody binding independent from it.
 The base ligand structure as the specific epitope for the antibodies of the
 present invention is further supported by antibody binding inhibition
 studies with soluble ligands. Soluble 8-OH-adenosine containing ligands
 specifically inhibited solid phase antibody binding, as demonstrated by
 the results shown in FIGS. 13 to 23. Soluble 8-OH-AMP (a single nucleotide
 moiety component of nucleic acids) inhibited antibody binding but adenine
 (from AMP) or Fapy-A structures did not. Thus, the antibodies of the
 present invention recognized antigen presented by both phosphorylated
 native nucleotides and periodate oxidized nucleosides. This last condition
 is suitable to cause ribose ring opening and, after reduction of a Schiff
 base with a protein amino group, yields a very different and
 non-physiological structure. Given these results, the only possible
 conclusion is that the monoclonal antibodies of the present invention are
 specific for the base structure (8-OH-Ade) alone and are independent of
 the nature of the group or structure the base is coupled to. Therefore,
 these antibodies are useful in detecting and quantitating the amount of
 8-OH-Ade present in biological specimens by virtue of their ability to
 specifically bind to this ligand.
 The foregoing illustrative examples relate to the measurement of 8-OH-Ade
 in a biological sample using monoclonal antibodies. While the preferred
 embodiment of the present invention has been described in terms of
 specific conditions and format, it is understood that numerous variations
 and modifications will occur to those skilled in the art upon
 consideration of the present invention without departing from the spirit
 and scope of the invention.