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WO1992015707A1 - Titrage immunologique pour anticorps contre l'adn oxyde et utilisations - Google Patents

Titrage immunologique pour anticorps contre l'adn oxyde et utilisations Download PDF

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Publication number
WO1992015707A1
WO1992015707A1 PCT/US1992/001419 US9201419W WO9215707A1 WO 1992015707 A1 WO1992015707 A1 WO 1992015707A1 US 9201419 W US9201419 W US 9201419W WO 9215707 A1 WO9215707 A1 WO 9215707A1
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Prior art keywords
antibodies
oxidized
antibody
base
bsa
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PCT/US1992/001419
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English (en)
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Krystyna Frenkel
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New York University
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Publication of WO1992015707A1 publication Critical patent/WO1992015707A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the invention relates immunoassay methods for detecting antibodies specific for oxidized DNA bases. Such methods are useful in the diagnosis and monitoring of inflammatory and autoimmune diseases.
  • Chronic inflammation is known to be involved in a large number of diseases, including autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) as well as in cancer (J. C. Fantone et al.. Am. J. Pathol. 107. 397 (1982) ; B. A. Freeman et al.. Lab Invest. 47. 412 (1982) ; Y. Niwa et al.. Inflammation £, 163 (1985) ; C. E. Cross et al.. Ann. Int. Med. 107. 526 (1987); S. Blount et al.. Clin. exp. Immunol. 81. 384 (1990)).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • Oxidative stress contributes to the pathogenesis of a broad array of diseases, including autoimmune diseases, as described above, cardiac disease such as ischemia/reperfusion injury (Jolly, S.R. et al.. Circ. Res. 54;277-285 (1984); McCord, J.M. New Eng. J. Med. 312:159-163 (1985), and neoplastic abnormalities (Vuillau e, M., Mutat. Res.186;43-72 (1987); Malins, P.C. et al.. Cane. Res. 51:5430-5432 (1991)). Inflammatory conditions are associated with increased infiltration of phagocytic cells into reactive tissue sites.
  • Phagocytic cells in particular polymor- phonuclear leukocytes (PMNs) or neutrophils, generate large amounts of active oxygen species when activated by a variety of stimuli including immune complexes or complement-derived fragments (Fantone et al. , supra: Freeman et al.. supra: Niwa et al.. supra: Cross et al.. supra; Badewy et al.. supra: Klebanoff, supra: Babior, supra: Weitzman et al.. supra; K. Frenkel, Environ. Health Persp. 81. 45 (1989); K. T. Oldha et al.. Free Rad. Biol. Med. 4. 387 (1988)).
  • PMNs polymor- phonuclear leukocytes
  • the reactive oxygen species include superoxide anion ra ⁇ dicals that dismutate either spontaneously or enzymatically to hydrogen peroxide (Hj0 2 ) (K. Frenkel, supra) .
  • Peroxide has been shown to activate the complement pathway in the presence of Fe or Cu ions (M. Shingu et al.. Permatologica 179 (Suppl. 1) . 107. (1989)).
  • H 2 0 2 that traverses cellular and nu ⁇ clear membranes almost like water, and reaches the nuclear PNA (K. Frenkel, supra: R. Meneghini, Mutation Res. 195. 215 (1988) ; B. Halliwell et al. , Arch. Biochem. Biophys.
  • H 2 0 2 reacts at sites that contain bound Fe or Cu, leading to the formation of hydroxyl radical (*OH) -like species that cause site- specific damage to PNA. Indeed, H 2 0 2 generated by a number of cel- lular processes is known to cause PNA strand breaks and to oxidize PNA bases. Products of this oxidation include 5-hydroxymethyl uracil (HMtJ) , thymine glycol (TG) and 8-hydroxyguanine (8-OHG) (Frenkel, supra: P. R. Putton et al.. Carcinogenesis 6:1279 (1985); K.
  • HMtJ 5-hydroxymethyl uracil
  • TG thymine glycol
  • 8-OHG 8-hydroxyguanine
  • Sera of SLE patients may contain increased amounts of catalase (M. Shingu et al.. supra) ; this enhancement is characteristic of oxidative stress (G. Storz et al.. TIG 6(11) . 363
  • PNA or polynucleotides as the analyte are PNA or polynucleotides as the analyte.
  • this document does not suggest the use of nucleotides containing oxidized bases. Importantly, the methods disclosed would not be useful with oxidized bases because reactive groups (e.g., alcohols) on the oxidized base's ring structure would interfere in the covalent attachment via the sugar moiety to the inorganic polymer. Furthermore, this reference does not suggest the use of nucleosides "artificially polymerized" by binding to a carrier such as a protein, wherein the carrier is then non-covalently attached to a solid support. Furthermore, this document only envisions inorganic supports.
  • the present inventors conceived of the notion that chronic enhanced production of oxidants in SLE or other disease states, particularly chronic inflammation, causes formation of strand breaks in PNA and oxidation of PNA bases at levels that ex ⁇ ceed the antioxidant and PNA repair capacities of the cells.
  • the released fragments of damaged PNA and, in particular, oxidized bases present in those fragments can serve as antigenic determi- nants, and induce the production of autoantibodies which react with the PNA that contains such bases.
  • the present inventors showed that sera of patients suspected of having SLE or other inflammatory diseases contained antibodies that recognized oxidized PNA bases. This serves as the basis of a novel composition and immunoassay for detecting these antibodies.
  • Such assays are useful in diagnosis and prognosis of inflammatory conditions, as well as any other conditions including neoplasia, as well as potentially damaging exposure to ionizing or UV irradiation, or cancer chemotherapy drugs, which result in oxidative PNA damage.
  • the present invention is thus directed to a composition useful for detecting or measuring antibodies specific for an oxidized PNAbase, comprising a nucleoside or nucleotide containing an oxidized base immobilized on a solid phase support, wherein the base is not part of an oligonucleotide or polynucleotide molecule.
  • the nucleoside or nucleotide containing the oxidized base is covalently linked to a protein which is immobilized on the support.
  • a protein which is immobilized on the support.
  • Non-human proteins such as bovine serum albumin, human serum albumin, ovalbumin or milk protein, are preferred when the composition is to be used for assaying human antibodies.
  • Oxidized PNA bases useful for the compositions and methods of the present invention include 5-hydroxymethyl uracil, thymine glycol and 8-hydroxyguanine.
  • any of a number of support materials are useful for immobilizing the oxidized PNA base according to the present invention, including glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellu ⁇ loses, polyacrylamide, agarose, and magnetite.
  • a preferred support is polystyrene, preferably in the form of a microtiter plate.
  • the present invention is also directed to an immunoassay method for detecting the presence or measuring the concentration of an antibody specific for an oxidized PNA base, comprising contacting a sample suspected of containing such an antibody with a nucleotide or nucleoside containing the oxidized base, wherein the base is not part of an oligonucleotide or polynucleotide molecule, and measuring the amount of antibody in the sample which is specific for the oxidized base, thereby detecting or measuring the antibody.
  • the immunoassay method comprises: (a) contacting a sample suspected of containing the antibody with a composition as described above; (b) allowing antibodies in the sample to bind to the oxidized base; (c) adding a detectably labeled binding partner for the antibody to the bound antibodies and allowing the binding partner to bind to the antibodies; and (d) measuring the amount of bound or unbound labeled binding partner, thereby detecting or measuring the antibody.
  • the nucleoside or nucleotide containing the oxidized base is covalently linked to a protein which is immobilized on the support.
  • Preferred binding partners in the above immunoassay method include an antibody specific for a human immunoglobulin and a bacterial protein capable of binding to a human immunoglobulin molecule.
  • a preferred detectable label for the above immunoassay method is an enzyme.
  • the present invention is further directed to a method of diagnosing an inflammatory or neoplastic disease or condition in a subject comprising obtaining from the subject a sample of a biological fluid and detecting the presence of antibodies specific for an oxidized PNA base using a method as described above, wherein the presence of the antibodies indicates that the inflammatory disease or condition exists.
  • the present invention also includes a method of monitoring the progression or the regression of an inflammatory disease or condition in a subject comprising obtaining from the subject on at least two occasions separated in time by at least seven days a first and a second sample of a biological fluid and measuring in each sample the concentration of antibodies specific for an oxidized PNA base using a method as described above, wherein an increase in the concentration of the antibodies from the first sample to the second sample is associated with progression of the disease or condition and wherein a decrease in the concentration of the antibodies from the first sample to the second sample is associated with regression of the disease or condition.
  • kits for detecting the presence or measuring the concentration of antibodies specific for an oxidized PNA base the kit being compartmentalized to receive in close confinement one or more containers, the kit comprising: (a) a first container containing a nucleoside or nucleotide containing the oxidized base, immobilized on, or capable of being immobilized on, a solid phase support; and (b) instructions for performing an immunoassay as described above.
  • the above kit may additionally comprise: (c) a second container containing a detectably labeled binding partner for the antibodies.
  • the above kit may additionally comprise: (d) a third container containing an agent capable of reacting with the detectably labeled binding partner to yield a detectable reaction product.
  • a preferably binding partner for the above kit is (a) an antibody specific for a human immunoglobulin or (b) a bacterial protein capable of binding to a human immunoglobulin molecule.
  • the detectable label is selected from the group consisting of an enzyme, a radionuclide, a fluorescent label, a chemiluminescent label and a bioluminescent label.
  • Preferred enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Figure 1 is a graph showing specific binding to 5-hydroxymethyl-2' -deoxyuridine coupled to bovine serum albumin (HMdU-BSA) and non-specific binding to mock-coupled bovine serum albumin (M-BSA) of antibodies present in ANA + serum of a lupus patient, and recognition of these antibodies by goat anti-human IgM, but not by anti-IgG, as determined by ELISA.
  • HdU-BSA bovine serum albumin
  • M-BSA mock-coupled bovine serum albumin
  • Serum #5 (2.5K dilution; 1 ml) was incubated in the absence (NT) or presence of 800 ⁇ g HMdU-BSA at 37°C for 1 h, then an aliquot was applied to plates coated with M-BSA (Panel A) or HMdU-BSA (Panels B and C) . After incubation at 37°C for 2 h, anti-human IgM (Panels A and B) or anti-IgG (Panel C) was added, followed by incubation with the substrate.
  • Figure 2 is a graph showing the effect of HMdU-BSA pre- treatment on binding of antibodies present in ANA + sera of patient with lupus (#12) and collagen vascular disease (#27)3 to wells coated with HMdU-BSA, as determined by ELISA.
  • Sera 2.5K dilution;
  • BSA(D; ⁇ ) -coated plates followed by goat anti-human IgM and the substrate. Results are expressed as mean values of percent of remaining antibody activity ⁇ SEM, measured as A ⁇ .
  • Figure 3 is a graph showing the effect of pretreating ANA "1" serum with HMdU-BSA and dT-BSA on the binding of antibodies to HMdU or dT.
  • ANA + serum (#27; diluted 1:2,500; 1 ml) was incubated overnight at 4°C in the absence (NT) or presence of 200 ⁇ g or 400 ⁇ g of either HMdU-BSA, dT-BSA or a mixture of both. This preincubated serum was then tested for binding to microplates coated with either HMdU-BSA or dT-BSA.
  • Figure 4 is a graph showing the effects of concentration of ANA + sera on specific and non-specific binding to HMdU-BSA- and M-BSA-coated plates, respectively.
  • Figure 5 is a graph showing specific and non-specific binding of three different types of human sera (ANA + , ANA * and NS) to plates coated with HMdU-BSA and M-BSA, respectively, as de ⁇ termined by ELISA.
  • Panel A shows the results with NS;
  • Panel B shows results with ANA + sera;
  • Panel C shows results with ANA " sera. Filled symbols represent specific binding. Open symbols represent non-specific binding.
  • Figure 6 is a graph showing the specific binding of pa- tients 1 sera to HMdU-BSA-coatedwells; Panel A represents confirmed SLE (ANA + /SLE + ) . Panel B represents other ANA + patients in whom SLE was not confirmed (ANA + /SLE ”) . Panel C represents miscellaneous inflammatory autoimmune diseases. Panel D represents patients with bullous pemphigoid (BP) . The dotted area shows the range of the mean ⁇ SEM of NS binding to HMdU-BSA (taken from Table 1) .
  • IIF indirect immunofluorescence
  • IC intracellular
  • BMZ basement membrane zone
  • Results are expressed as mean A t go values (ELISA) vs. combined serum titer against monkey and guinea pig esophagi (IIF) .
  • Figure 8 shows the chemical structures of thymidine (dT) (left) and 5-hydroxymethyl-2' -deoxyuridine (HMdU) (right). In free (2' -deoxribonucleoside) form, the substituent labeled as R is hydrogen.
  • BSA bovine serum albumin
  • Figure 9 is a scattergram of the mean values of anti-HMdU antibodies (A ⁇ / ⁇ l serum) present in human sera categorized according to disease type.
  • Figure 10 is a scattergram of the mean values of anti-dT antibodies ( ⁇ / ⁇ l serum) present in human sera categorized according to disease type, analyzed at the same time as anti-HMdU antibodies, as shown in Figure 9.
  • An immunoassay for antibodies specific for oxidized PNA bases typically comprises incubating a biological sample from a subject suspected of having a condition associated with such antibodies in the presence of an "antigen containing reagent" which includes an oxidized base, and detecting the binding to the oxidized base of the antibody which is in the sample.
  • PNA bases include the pyrimidine bases thymine (also known as 5-methyl uracil) , cytosine, and 5-methyl cytosine, and the purine bases adenine and guanine.
  • the oxidized form of these basis is used according to the present invention in the form of a deoxyribonucleoside, containing the sugar deoxyribose, or a deoxyribonucleotide, containing deoxyribose and phosphate.
  • Preferred oxidized PNA bases useful for the compositions and methods of the present invention include 5-hydroxymethyl uracil (which is oxidized thymine) , thymine glycol and 8-hydroxyguanine.
  • 5-hydroxymethyl uracil which is oxidized thymine
  • thymine glycol thymine glycol
  • 8-hydroxyguanine dozens of other oxidized PNA bases formed by oxidation of PNA are known in the art (R. Teoule, Int. J. Radiation Biol. .51:573-589 (1987); G.W. Teebor et al.. Int. J. Radiation Biol. 5_4:131-150 (1988) , which references are hereby entirely incorporated by reference) .
  • the oxidation relative to the native PNA base is generally associated with a substituent of the purine or pyrimidine ring structure, for example oxidation of the methyl group to hydroxymethyl in the conversion of thymine to HMU.
  • the oxidation is in the base, and not in the sugar or phosphate portion of the nucleoside or nucleotide.
  • the oxidized bases may either be obtained commercially or synthesized chemically using methods well-known in the art and are preferably used in the form of a nucleoside or nucleotide to allow conjugation to a carrier molecule, preferably a protein. While use of 5-hydroxymethyl uracil is exemplified in particular detail herein, any of these oxidized bases may be used according to the teachings of the present invention.
  • cis-thymidine glycol also known as 5,6-dihydroxy-5,6- dihydrothymidine monophosphate, pdTG
  • 8-hydroxyguanosine 8-OHrG
  • 8-hydroxy-2'-deoxyguanosine monophosphate 8-OHdGMP
  • cis-Thymine glycol, thymidine glycol and its monophosphate are commonly synthesized through formation of bromohydrins or by 0s0 oxidation (Leadon et al.. 1983 supra: Rajagopalan et al. , supra) .
  • the synthesis involves KMh0 4 as an oxidizing agent following a procedure of Iida et al. (Biochim. Biophvs. Acta 211:1-13 (1970)) as modified by
  • a neutral pH method For coupling of pdTG to BSA, a neutral pH method is preferred.
  • the periodate coupling method (Erlanger, B. et al.. Proc. Natl. Acad. Sci. 52:68 (1964)) requires alkaline conditions, under which the thymine glycol ring is unstable.
  • the neutral pH method the isomers of cis-pdTG are separately coupled to BSA according to Hamagishi et al. (J. Biochem. 88:1785-1792)) using 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide (EPCI) as a coupling agent.
  • pdTG-1/BSA (I) and pdTG-2/BSA (II) are separated from free pdTG by passing the preparations through desalting gel (BioRad P6-PG) columns.
  • the same method is used for preparation and purification of p[ 3 H]dTG/BSA I and II.
  • Pialysis of p[ 3 H]dTG/BSA I and II showed that only a very small fraction of tritium was released, thus proving that virtually all radioactivity is bound to BSA.
  • Samples of the 3 H-containing conjugates were treated with 0.1N HC1 at room temperature overnight and analyzed by HPLC. No free dTG was found under these conditions.
  • conjugates may be established by UV spectrophotometry and specific activity. For example, conjugates I and II have been found to contain 27 nmol pdTG-1/nmol BSA and 24 nmol pdTG-2/nmol BSA, respectively. Conjugates of ovalbumin have been found to contain lower levels of substitution than BSA. Conjugates may be lyophilized and stored at -20°C. Coupling of guanosine (rG) to BSA was described by Erlanger et al.
  • 8-OHrG may be synthesized by treating 8-bromoguanosine dissolved in acetic acid with a 10- fold excess of freshly fused sodium acetate at 118°C for 3 hrs (Ikehara, M. et al.. Che .
  • That marker was synthesized using light-sensitization of methylene blue as a source of singlet oxygen. (although the latter method is good, the yield of 8-OHrG is very low) .
  • the early-eluting peak co-chromatographed with the 8-OHrG marker. Products are separated into two fractions, early- and late-eluting from HPLC, and both are separately coupled to BSA according to Erlanger et al. (supra) .
  • This method is based on periodate oxidation of guanosine, followed by addition of BSA and reduction of the BSA complex with borohydride at alkaline pH. Coupling of 8-OHrG to protein using this method has been reported (Pegan, P.
  • the coupling may be performed using EPCI, as described above for pdTG.
  • 8-OHdGMP is synthesized from dGMP by oxidation with either (1) the Udenfriend reagent consisting of FeS0 4 /EPTA and ascorbic acid with oxygen passing through the reaction mixture, or (2) H 2 0 2 in the presence of ascorbate; both at pH 6.8, as described by Kasai et al. (Nucleic Acids Res. 12:2137- 2145 (1984)) and Frenkel et al. (Anal. Biochem. 196:126-136 (1991)) .
  • the oxidized PNA base-containing composition is an oxidized base-containing nucleoside or nucleotide conjugated to a protein.
  • the protein be of a species other than that of the species being tested.
  • a non- uman protein such as bovine serum albumin is preferred.
  • bovine serum albumin is preferred.
  • human serum albumin would be used.
  • One of ordinary skill in the art will be able to determine which protein is appropriate or desirable for the particular assay application without undue experimentation, such as horse serum, ovalbumin or large proteins in non-fat dry milk.
  • the oxidized PNA base bound to a carrier molecule such as a protein
  • a carrier molecule such as a protein
  • a solid support such as nitrocellulose or polystyrene.
  • This immobilized form of the oxidized base antigen is then allowed to interact with a biological sample, such that any specific antibodies in the sample will bind to the immobilized antigen.
  • the support may then be washed with suitable buffers followed by treatment with a detectably labeled binding partner for the antibody.
  • a preferred binding partner is an anti-immunoglobulin antibody produced in a different species.
  • a human antibody for example, "second antibody, " for example, a goat anti- human immunoglobulin antibody which is detectably labeled may be used.
  • the solid phase support may then be washed with the buffer to remove unbound antibody.
  • the amount of bound label on said solid support may then be detected by conventional means appropriate to the type of label used (see below) .
  • Such a “second antibody” may be specific for a particular human immunoglobulin isotype, for example IgM, IgGl, IgG2, and the like, thus permitting identification of the isotype or isotypes of antibodies in the sample which are specific for the oxidized base.
  • the second antibody may be specific for an idiotype of the sample antibody.
  • antibodies as binding partners for the sample antibody being detected, other known binding partners for human immunoglobulins, such as the staphylococcal protein A or streptococcal protein G, well-known in the art, may be used.
  • a biological sample suspected of containing an antibody specific for an oxidized base may be treated with a solid support or carrier which is capable of immobilizing soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the oxidized base reagent, which may be detectably labeled. If the oxidized base reagent is not detectably labeled, a second reagent containing a detectably labeled binding partner for the oxidized base reagent is provided to the complex.
  • the solid phase support may then be washed with the buffer to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means.
  • Possible binding partners for the oxidized base reagent include antibodies, for example monoclonal antibodies (mAb) specific for the oxidized base or the complex of the oxidized base with any structure to which it is bound such as a protein substituted with oxidized bases.
  • Another type of binding partner may be a mAb specific for an epitope of a non-human protein portion of a protein-oxidized base complex.
  • solid phase support any support capable of binding antigen or antibodies or other binding partners according to the present invention.
  • supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads, 96-well polystyrene microplates and test strips, all well-known in the art. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • a preferred type of assay to detect an antibody specific for an oxidized base is an enzyme immunoassay (EIA) , or enzyme-linked immunosorbent assay (ELISA) .
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the reagents useful in the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detectable label may be a radiolabel, and the assay thus used termed a radioimmunoassay (RIA) , which is well known in the art. See, for example, Yalow, R. et al.. Nature 184:1648 (1959); Work, T.S., et al.. Laboratory Techniques in Biochemistry and Molecular Biology. North Holland Publishing Company, NY, 1978, entirely incorporated by reference herein.
  • RIA radioimmunoassay
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: 3 H, 125 I, 131 I, 35 S, 14 C, and preferably 125 I.
  • the antigen or antibody reagents useful in the present invention with a fluorescent compound.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ⁇ -phthaldehyde and fluorescamine.
  • the reagents can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lan- thanide series. These metals can be attached to an antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (PTPA) or ethylenediaminetetraacetic acid (EPTA) .
  • PTPA diethylenetriaminepentaacetic acid
  • EPTA ethylenediaminetetraacetic acid
  • the antigen or antibody reagents useful in the present invention also can be detectably labeled by coupling to a chemiluminescent compound.
  • a chemiluminescent- tagged antibody or antigen is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antigen or antibody reagents useful in the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Petection of the detectably labeled reagent according to the present invention may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer or colorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by colorimetric methods which employ a substrate for the enzyme.
  • Petection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • the antibody specific for an oxidized PNA base which is detected by the assay of this invention may be present in a biological sample. Any sample containing such an antibody can be used. However, one of the benefits of the present diagnostic invention is that invasive tissue removal may be avoided. Therefore, preferably, the sample is a biological solution such as, for example, blood, serum, saliva, urine, cerebrospinal fluid, amniotic fluid, lymph and the like. However, the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.
  • the binding molecules useful in the methods of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • an immunometric assay also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested (i.e., blood, CSF, amniotic fluid, tissue homogenate, etc.) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody- antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule”) . After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
  • This type of forward sandwich assay may be a simple "yes/no” assay to determine whether antigen is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample- containing known quantities of antigen.
  • Such "two-site” or “sandwich” assays are described by Wide, Radioimmune Assay Method. Kirkham et al.. Eds., E. & S. Livingstone, Edinburgh, 1970, pp 199-206.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
  • agglutination assays An alternative to the RIA and EIA is provided by various types of agglutination assays. Both direct and indirect agglutination immunoassays are well known in the art. In these assays, the agglutination of particles to which an antigen or an antibody is bound is used to indicate the presence or absence of the corresponding antibody or antigen. A variety of particles, including particles of latex, charcoal, kaolinite, or bentonite, as well as microbial cells or red blood cells, may be used as agglutinable carriers (Mochida, US 4,308,026; Gupta et al.. J. Immunol. Meth. 80:177-187 (1985); Castelan et al.. J. Clin.
  • the present invention provides methods to detect and enumerate cells secreting an antibody specific for an oxidized PNA base.
  • any of a number of plaque or spot assays may be used.
  • a sample containing lymphoid cells such as peripheral blood lymphocytes
  • a reagent containing the antigen of interest As the antibody secreting cells of the sample secrete their antibodies, the antibodies react with the antigen, and the reaction is visualized in such a way that the number of antibody secreting cells (or plaque forming cells) may be determined.
  • the antigen such as oxidized PNA bases in the present invention, may be coupled to indicator particles, such as erythrocytes, preferably sheep erythrocytes, arranged in a layer.
  • indicator particles such as erythrocytes, preferably sheep erythrocytes, arranged in a layer.
  • erythrocytes preferably sheep erythrocytes
  • antibodies attach to the surrounding antigen-bearing erythrocytes.
  • complement components By adding complement components, lysis of the erythrocytes to which the antibodies have attached is achieved, resulting in a "hole” or "plaque” in the erythrocyte layer.
  • Each plaque corresponds to a single antibody-secreting cell.
  • the sample containing antibody-secreting cells is added to a surface coated with an antigen-bearing reagent, for example, an oxidized PNA base conjugated to bovine serum albumin, attached to polystyrene.
  • an antigen-bearing reagent for example, an oxidized PNA base conjugated to bovine serum albumin
  • the cells are gently washed away.
  • the presence of a colored "spot" of bound antibody, surrounding the site where the cell had been, can be revealed using modified EIA or other staining methods well-known in the art.
  • IgM antibodies reactive with PNA in addition to or instead of IgG antibodies reactive with PNA, as determined by IIF and other assays
  • IgA antibodies specific for oxidized dsPNA have been found in SLE (Blount et al.. supra) .
  • compositions and methods of the present invention are useful for detecting antibodies to oxidized DNA bases in any of a number of conditions wherein oxidative damage occurs.
  • oxidative damage is known to occur in response to a number of exogenous stimuli.
  • prolonged exposure to ionizing radiation, UV-irradiation, or cancer chemotherapeutic agents, such as adriamycin and bleomycin, is known to be associated with oxidative damage.
  • a phototherapy regimen for skin disease such as with the psoralens
  • a phototherapy regimen for skin disease such as with the psoralens
  • due to accidental exposure to low level radiation from nuclear power plants or the testing of nuclear devices may respond to the production of oxidized DNA bases by developing antibodies to these bases.
  • compositions and methods of the present invention are useful for monitoring such individuals for the presence of such antibodies. Not only may this permit appropriate actions to avoid the pathogenic potential of these antibodies, but the detection serves in itself as a sensitive measure of ongoing oxidative damage. Thus, the detection of such antibodies may be used as the basis for modifying or terminating certain therapies or avoiding certain exposure risks. Furthermore, in the development of new chemotherapeutic or radiotherapeutic agents or regimens wd decreased side effects, it is useful to be able to assess oxida..ve damage in vivo. The compositions and methods of the present invention are therefore useful as screening tools in pre-clinical or clinical trials.
  • Serum samples were obtained from patients tested for the presence of antinuclear antibodies (ANA) , known to be associated with SLE, and from healthy people (normal sera, NS) . Tr * were tested for the presence of antibodies that reco -ji, * ; 5 hydroxymethyl uracil (HMU) , one of the chemically stable oxiu f.d PNA bases (Frenkel, 1989, supra: G. W. Teebor et al.. Proc. Natl. Acad. Sci. USA 81. 318 (1984) ; K. Frenkel et al.. Biochemistry 24. 4527 (1985)).
  • ANA antinuclear antibodies
  • HMU 5 hydroxymethyl uracil
  • ELISA was used as the assay system.
  • Antigen was prepared by periodate coupling of HMU riboside to bovine serum albumin (BSA), according to the method of B. F. Erlanger et al.. Proc. Natl. Acad. Sci. ⁇ 2:68 (1964) , and followed by reduction with boro ⁇ hydride. This procedure led to the formation of a conjugate of HMU 2' -deoxyribonucleoside (HMdU) with lysine residues of BSA. The conjugate is designated herein HMdU-BSA.
  • dT-BSA conjugates in which thymine riboside was coupled to BSA
  • M-BSA "mock coupled" bovine serum albumin
  • the antibodies were conjugated with the enzyme, horseradish peroxidase (HRPO) . After adding the labeled antibody, wells were again washed 3 times.
  • the chromogenic substrate, o-phenylenediamine was used for E ⁇ 0 2 oxidation mediated by HRPO bound to the wells through the anti- human Ig antibodies.
  • the color reaction measured as absorbance at 490 nm (A 490 ) at acid pH in an ELIPA-5 Microplate Reader (Physica Inc.) was proportional to the amount of human antibodies bound to the coated plates.
  • HMdU is Recognized by Antibodies Present in ANA-Positive Sera
  • thymidine and its oxidation product HMdU are shown in Figure 8. These moieties, when coupled to BSA and coated onto wells, provided the antigens in assays for the presence of antibodies in human sera.
  • HMdU was found to be recognized by antibodies present in patients' sera.
  • anti-IgM reacted with HMdU-bound human antibodies ( Figure IB) .
  • Figure 1C anti-human IgG had only marginal binding.
  • the anti-IgA was totally ineffective.
  • Sera from healthy human donors also contain detectable levels of antibodies that bind to HMdU-BSA (group NS in Figure 5) .
  • Such antibodies are presumably normal, and may subserve a protective role from damage caused by the ongoing basal oxidative processes that are constantly occurring in the body.
  • Such antibodies would provide an additional line of defense against oxidative DNA damage, after the repair enzymes which remove oxidized bases or excise damaged PNA fragments (reviewed in G. W. Teebor et al.. Adv. Cancer Res. 38. 23 (1983); Int. J. Radiation Biol.. supra) .
  • the oxidized bases 5-hydroxymethyl uracil, thymine glycol and 8-hydroxyguanine, and some of their nucleosides, are known to be excreted in urine by humans and animals (R. L. Saul et al.. in: Mechanism of PNA Pamage and Repair. M. G. Simic et al.. Eds., Plenum Publishing Corp., New York, NY, 1986, pp. 529-535; K. C. Cundy et al.. in: Oxygen Radicals in Biology and Medicine. M. G. Simic et al.. Eds., Plenum Publishing Corp., New York, NY, 1988, pp. 479-482.).
  • the remainder of the subjects did not fit into any of the above groups because of multiple cutaneous problems or because they had a dermatologic problem such as acne, rosacea or dermatophytosis, which did not fit into any of the above categories.
  • the number of such patients was too low for separate categorization.
  • the immune complex diseases represented in the above patientpopulation includedleukocytoclasticvasculitis, urticarial vasculitis, Raynaud's disease as well as SLE.
  • the neoplastic disease group included patients with a history of a benign or malignant neoplasms of the cutaneous, female reproductive or respiratory systems, or were so diagnosed upon examination for another complaint.
  • HMdU or dT was coupled to BSA as described above, and ELISA assays performed according to the procedure detailed above. The results are shown in Table 3.
  • Anti-HMdU antibody titers were markedly elevated in patients with psoriasis, immune complex disease or a history of neoplasia. The reactivity of sera from patients undergoing systemic treatment with antiinflammatory or cytotoxic drug, including prednisone, methotrexate, plaquenil, etretinate and dapsone, was significantly decreased compared to patients not receiving systemic therapy.
  • Antibody titers of patients with venous leg ulcers were significantly lower than controls.
  • Neoplastic Piseases p ⁇ 0.001
  • the scattergram in Figure 9 shows the distribution of the actual absorbance mean values/ ⁇ l of sera calculated for the individual patients.
  • the venous leg ulcer group is contained within the absorbance distribution of the controls, while the drug treated group has only a few individual values above control levels. More than half of the values in the psoriasis group (11 of 18) were above the control mean. In groups of patients with immune complex diseases or a history of neoplasia, only one point in each group fell below the control mean. Sera of over 85% of the individuals had anti-HMdU antibody titers above the mean control value.
  • Figure 10 shows a scattergram of the individual mean values of sera incubated in dT-BSA-coated wells.
  • a comparison of the values listed in Tables 3 and 4 shows that avidities of antibody binding to dT-BSA (Figure 10) are on average two-fold lower than those binding to HMdU-BSA ( Figure 9) .
  • pretreatment of the serum with HMdU-BSA, dT-BSA, or a mixture of the two, binding to HMdU-coated wells was decreased more markedly than binding to dT-coated wells ( Figure 3) .
  • the large variation in anti-HMdU titers in psoriasis patients may reflect the state of the disease process, such as whether it was stable, improving or flaring.
  • the percentage of body surface covered with lesions and/or the severity of the lesions likely modulate the immune response, thereby affecting the anti-HMdU titers.

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Abstract

Compositions, méthodes de titrage immunologique et équipements permettant de détecter des anticorps spécifiques à certaines bases d'ADN oxydé, y compris de diagnostiquer des maladies inflammatoires et des états pathologiques connexes ainsi que de suivre l'évolution ou le traitement de ces maladies ou états pathologiques.
PCT/US1992/001419 1991-02-27 1992-02-27 Titrage immunologique pour anticorps contre l'adn oxyde et utilisations WO1992015707A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038588A1 (fr) * 1995-05-31 1996-12-05 University Of Leicester Agents analytiques et therapeutiques
WO1999063349A1 (fr) * 1998-06-02 1999-12-09 Diasorin International Inc. Methode de detection d'anticorps specifiques a des phases d'infection
US6187551B1 (en) * 1995-09-19 2001-02-13 Cytochem, Inc. Detection and quantitation of 8-OH-Adenine using monoclonal antibodies

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458006A (en) * 1982-06-21 1984-07-03 Hoechst Aktiengesellschaft Photopolymerizable mixture and photopolymerizable copying material prepared therewith

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458006A (en) * 1982-06-21 1984-07-03 Hoechst Aktiengesellschaft Photopolymerizable mixture and photopolymerizable copying material prepared therewith

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PROCEEDINGS OF THE AMERICAN ASSOCIATION OF CANCER RESEARCH ANNUAL MEETING, Volume 31, issued 23 May 1990, E. KIM, "Antibodies present in lupus erythematosus sera recognized oxidized thymines", page 147, see title and authorship. BIOSIS ABSTRACT 39033413. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038588A1 (fr) * 1995-05-31 1996-12-05 University Of Leicester Agents analytiques et therapeutiques
AU704928B2 (en) * 1995-05-31 1999-05-06 University Of Leicester Analytical and therapeutic agents
JP3510267B2 (ja) 1995-05-31 2004-03-22 ユニヴァーシティ オブ レイセスター 分析および治療用薬剤
US6187551B1 (en) * 1995-09-19 2001-02-13 Cytochem, Inc. Detection and quantitation of 8-OH-Adenine using monoclonal antibodies
US6900291B2 (en) 1995-09-19 2005-05-31 Cytochem, Inc. Detection and quantitation of 8-OH-adenine using monoclonal antibodies
WO1999063349A1 (fr) * 1998-06-02 1999-12-09 Diasorin International Inc. Methode de detection d'anticorps specifiques a des phases d'infection

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