Immunological Quantification by High-Affinity Antibodies of OÂ ... · immunological procedures for the quantification of DNA com ponents structurally modified by N-nitroso carcinogens,

[CANCER RESEARCH 40, 887-896, March 1980]0008-5412/80/0040-0000$02.00

Immunological Quantification by High-Affinity Antibodies ofOÂ°-Ethyldeoxyguanosinein DNA Exposed to N-Ethyl-N-nitrosourea

RoIf MUller and Manfred F. Rajewsky2

Institut fÃ¼rZellbiologie (Tumorforschung), UniversitÃ tEssen (GH), Hufelandstrasse 55, D-4300 Essen 1, Federal Republic of Germany

a potent pulse-carcinogen with a pronounced neurooncogeniceffect in rats (20, 35), leads to a high 06-EtdGuo:7-EtdGuomolar ratio in DNA [â€œ-0.6(i 5)], while the corresponding value,e.g. , for the very weakly carcinogenic diethyl sulfate, is about200 times lower (42). Furthermore, contrary to other ethylationproducts, O6-EtdGuo persists in the DNA of rat brain (hightumonigenic effect of EtNU) but is rapidly eliminated enzymatically from the DNA of other â€˜â€˜low-risk'â€˜rat tissues (15, 35). Asimilar selective persistence of O6-EtdGuo has been observedin EtNU-treated human xeroderma pigmentosum fibroblasts incontrast to their normal counterparts (i , 3, i 6).

The specific detection of carcinogen-modified DNA components and the kinetic analysis of their enzymatic eliminationfrom (or persistence in) DNA require highly sensitive methods.This will be even more important if such studies are now to beextended to very low levels of DNA modification (i.e. , the effectsof low doses of carcinogens) and/or small numbers of cells.Conventional radio-chromatographic techniques are, however,limited (a) by the specific radioactivity of radiolabeled carcinogens and (b) by the need to restrict the analyses to laboratoryanimals or cultured cells exposed to radiolabeled carcinogens.These shortcomings can possibly be circumvented by immunological detection methods, due to the high specificity ofantibodies in recognizing minor alterations of molecular structure. As part of studies concerned with the development ofimmunological procedures for the quantification of DNA components structurally modified by N-nitroso carcinogens, wehave recently described a RIA for 06-EtdGuo (27, 28). In anextension of this work, we now report on 3 highly sensitiveimmunological assays for O6-EtdGuo, using antibodies with anincreased affinity and specificity.

MATERIALS AND METHODS

AnimalsI This work was supportad by Grant 1RO1 CA2001 7 awarded by the National

Cancer Institute, Department of Health, Education, and Welfare, and by theDeutsche Forschungsgameinschaft (Schwerpunkt â€˜â€˜Biochamiades Narvensystamsâ€•;Re 19/7).

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: EtNU, N-athyl-N-nitrosouraa; O6-EtdGuo, O6@

ethyl-2'-deoxyguanosine; 7-EtdGuo, 7-athyl-2'-daoxyguanosine; RIA, radioimmunoassay; 06-EtGua, 06-ethylguanlne; O6-EtGuo, OÂ°-ethylguanosina;O6@MeGuo, 06-methylguanosine; 7-EtGua, 7-ethylguanina; 7-EtGuo, 7-ethylguanosine; dGuo, 2'-deoxyguanosina; O6-EtGMP, 06-ethylguanosina 5'-monophosphata; 06-EtdGMP, 06-athyl-2'-deoxyguanosina 5'-monophosphata; O6-BuGuo,06-butylguanosine; AP, alkaline phosphatasa; RSA, rabbit serum albumin; BSA,bovine serum albumin; KLH, keyhole limpet hemocyanin; PBS, phosphatebuffered saline (140 mMNaCI:2.7 mMKCI:8.1 mMNa2HPO4:1.5 mMKH2PO4,pH7.2); i.c., intracutaneously;AbAP, antibody-alkalinephosphatasaconjugate;TBS, Tris-bufferedsaline(140 mM NaCI:20 mM Tris-HCI, pH 7.5:3 mM NaN3);ELISA enzyme-linkedimmunosorbentassay; RIST, radioimmunosorbenttechnique; ADA, adenosina deaminase; dAdo, 2'-deoxyadanosina; dlno, 2'-daoxyinosina; 55, single-stranded; DS, double-stranded.

Received September 23, 1979; accepted November 30, 1979.

BDIX rats (8) were used for in vivo application of EtNU.Antisera were produced in rabbits (Graue Riesen; Kaninchenfarm H. Schriever, Iselersheim, Germany).

Ethylnitrosourea

EtNU (Roth, Karlsruhe, Germany), twice recrystallized frommethanol, was dissolved in citric acid:disodium phosphatebuffer, pH 6.0 (24), to give a 0.1 Msolution, immediately beforei.v. injection into a tail or hind leg vein.

Synthesis of Alkylated Nucleic Acid Components

06-EtGua, 06-EtGuo, 06-EtdGuo, 06-MeGuo, and 7-EtGuawere synthesized according to published methods (2, 5, i i,

MARCH1980 887

ABSTRACT

Three immunological methods [radioimmunoassay (RIA), enzyme-linked immunosorbent assay, and radioimmunosorbenttechnique] were established for quantification of the potentiallymutagenic 06-ethyldeoxyguanosine (O6-EtdGuo) in DNAtreated with the carcinogen ethylnitrosourea in vivo or in vitro.To obtain high-affinity antibodies for specific detection of lowlevels of O6-EtdGuo in small amounts of DNA (cells), differentschemes were applied for immunization of rabbits with thehapten 06-ethylguanosine coupled to various carrier proteins(matserum albumin, bovine serum albumin, keyhole limpethemocyanin).

Low-dose immunization with the hapten-keyhole limpet hemocyanin conjugate resulted in antibodies with an affinity constant of 1 to 2 x 1010liters/mol and very low levels of crossreactivity with normal as well as other alkylated DNA components. The AlA (the most sensitive of the three assays) detects0.05 pmol of 06-EtdGuo at 50% inhibition of tracer (Q6@ethyl[8,5'-3H]-2'-deoxyguanosine)-antibody binding. This permits quantification by RIA of 06-EtdGuo at an O6-EtdGuo:2'-deoxyguanosine molar ratio of â€”3x 1O@ in a hydrolysate of100 @sgof ethylated DNA. By chromatographic separation ofO6-EtdGuo prior to the RIA, this value can be lowered to <5x i0'8.

INTRODUCTION

Among the various reaction products of alkylating carcinogens with target cell DNA, the potentially mutagenic 06-alkyldeoxyguanosine deserves particular attention (21 , 22, 29, 35,37, 38). The extent of 06-alkyldeoxyguanosine formation inDNA by different compounds seems to correlate with theirrelative camcinogenicity(15, 21 , 29). Thus, exposure to EtNU,3

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R. MÃ¼!Ierand M. F. Rajewsky

i 4) as previously described (27). 7-EtGuo and 7-EtdGuo wereobtained by reacting equal amounts (w/w) of guanosine ordGuo, respectively, with ethyl iodide in dimethylacetamide for72 hr at 50Â°.These products were purified by thin-layer chromatography on silica gel (developed with n-butyl alcohol:glacialacetic acid:H2O, 3:i :i ). O6-EtGMPand 06-EtdGMP were synthesized according to a published procedure (44) as describedpreviously (27). Nucleoside 3'-monophosphates and nucleoside 3',5'-diphosphates were destroyed by treatment of theproducts with Nuclease P1 (Boehninger, Mannheim, Germany;10 @og/ml)in 15 mM sodium acetate buffer (pH 7.2) containing0.4 m@ZnSO4 for 30 mm at 37Â°.The nucleoside 5'-monophosphates were purified on a column of DEAE-Sephadex A-25 (Pharmacia, Uppsala, Sweden), eluted with a linear gradientof 0 to 0.5 M tniethylammonium bicarbonate (pH 8.0). O6@BuGuo was prepared by Dr. R. Saffhill (Manchester, England).All synthesized products showed >99% purity when analyzedby high-performance liquid chromatography on Aminex A6(BioRad, Munich, Germany), eluted with 0.45 M ammoniumformate, pH 4.5 (6). They were stored in liquid N2 either inlyophilized form or dissolved in methanol. Natural nucleic acidcomponents were purchased from Sigma-Chemie (Munich,Germany).

O6-Et[8,5'-3HJdGuo(specific activity, 14 Ci/mmol), the tracerin the RIA (see below), was synthesized from [8,5'-3H]dGTP(New England Nuclear; specific activity, 29.8 Ci/mmol). Forhydrolysis of the PP1linkages, [8,5-3H]dGTP was incubated in0.oi N HCI for 30 mm at 37Â°.After neutralization, the a-phosphate was removed by incubation with i 00 jzg of AP (EC3.1 .3.1 ; Grade I; Boehringer) pen ml for 30 mm at 37Â°.Theenzyme was removed by adsorbtion to DE-52 cellulose (Whatman, Ferniens,France). Following evaporation, the labeled nucleoside was dissolved in absolute methanol and ethylated withdiazoethane at 0Â°for 2 hr (11), and the product was purified

CD

0a,

EC

UI')

â€˜U

on a Sephadex G-10 (Pharmacia) column eluted with H20(Chart 1).

Nucleoside-Protein Conjugates

O6-EtGuo was coupled to the carrier proteins RSA (Calbiochem, Marbung, Germany), BSA (99% purity; Semva,Heidelberg, Germany), onKLH (Calbiochem), respectively, accordingto the procedure or Enlangerand Beiser (10). One hundred mgof 06-EtGuo were stirred in i 0 ml of 0.1 M NaIO4for 15 mm atroom temperature. The reaction was stopped by 20 ,sl ofethylene glycol, and the mixture was then added dropwise to5 ml of the cannierprotein solution (20 mg/mI). The pH wasadjusted to 9.5 with 2 M Na2CO3and was kept at this value for45 mm. The O6-EtGuo-pnotein complex (Schiff's base) wasstabilized by 2.5 ml of a sodium bomohydnidesolution (30 mg/ml). After 3 hr at 4Â°,i drop of n-octanol was added to avoidfoam formation, and the pH was adjusted to 6 to 7. The reactionmixture was dialyzed against PBS overnight and purified on aSephadex G-50 (Phanmacia)column (30 x 1.5 cm) eluted withPBS (conjugate in the void volume). The nucleoside:pmoteinmolar ratios, calculated from difference spectra (19), were â€œ@â€˜20for RSA and BSA and -.-200 for KLH [assuming a molecularweight of 800,000 for KLH (41)].

Immunization Procedures

Procedure I . Three rabbits were immunized with nucleoside-protein conjugate (5 mg/animal) in 2.5 ml of PBS emulsified in 2.5 ml of complete Fneund's adjuvant (Behning, Manburg, Germany), by injections into the hind foot pads and 5 toi 0 other sites (i.m. and s.c.). Beginning 4 weeks later, the@rabbits were boosted by i.v. injection of 500 @gof conjugatepen animal in 1 ml of PBS at 2-week intervals. Sera werecollected at 10 days after the second booster.

0@03â€˜I

a40 @â€˜

x

3o@

20

10

10 20 30 40 50

Fraction NumberChart 1. Chromatographic separation of enzymatically hydrolyzed (Chart 2) DNA, (12 mg; deoxynucleosidas, ADA treated) on a Sephadax G-i 0 column (1.6 x 85

cm) aluted with H2O.06-Et[8,5'-3H)dGuo (â€”2x 106dpm) was added to the DNAhydrolysate. Flow rate, 0.6 mI/mm. Fraction volume, â€”9ml. Separation characteristicsremain unchanged over a pH range of 6 to 8. In a hydrolysata not treated with ADA, dAdo would appear in Fractions 31 to 35. Relative absorbanca and 3Hradioactivity per fraction are plotted against the fraction number. dCyd, 2'-daoxycytidina; dThd, 2'-deoxythymidine.

888 CANCERRESEARCHVOL. 40



!mmuno!ogica! Quantification of 06-EtdGuo

Procedure 2. A mixture of 500 @gof nucleoside-proteinconjugate in 0.5 ml of PBS and 0.5 ml of aluminum hydroxide(Alugel S; Serva) was stirred for i hr at 4Â°and emulsified in 1ml of complete Freund's adjuvant. Three rabbits were immunized by injections into the hind foot pads and into about 50i.c. sites (neck and axillae). Eight weeks later, the animals wereboosted by the same procedure. After another 8-week interval,the rabbits received a second booster by i.m. injection of 500,Lgof conjugate in 1 ml of PBS emulsified in 1 ml of incompleteFreund's adjuvant (Behning). The semawere collected 2 weekslater.

Antibody Concentrations and Affinity Constants

The serum concentrations of antibodies directed against O6@EtdGuo and their affinity constants for O6-EtdGuo were determined by the method of Stewart and Petty (39). Binding curveswere established for an antiserum dilution of @5x 10@ mol ofspecific antibody-binding sites per liter, over a range of 5 to500 x 10-'Â° mol of hapten (tracer) per liter.

Isolation of O@-EtdGuo-specificAntibodies

Thirty mg of O6-EtdGuo were incubated with 1.5 mg ofepoxy-activated Sepharose 6B (Pharmacia), in 3 ml of 0.Oi NNaOH for 20 hr at 50Â°.The gel was washed with H20 and 0.iM borate buffer (pH 8.5) and incubated in i 0 ml of i M

ethanolamine (pH 9.0) for 12 hr at room temperature, in orderto block remaining active groups. The immunosorbent was thenwashed with PBS and incubated in 5 ml of anti-O6-EtGuo-KLHserum for 2 hr at room temperature, with gentle shaking. Aftercentrifugation, the supennatant was removed, and the gel was

.washed extensively with PBS until the absorption (280 nm) of

the supemnatantdecreased below 0.02. The antibodies werethen eluted by incubating the gel in 2.5 ml of 1 M acetic acidfor 3 x 5 mm at room temperature. The combined supemnatantswere dialyzed against PBS overnight. Recovery of antibodieswas >50%. The antibodies were stored at _200.

AbAP Conjugate

AP (type VII; Sigma-Chemie) was linked to O6-EtdGuo-specific antibodies according to a published procedure (9). Briefly,I .5 mg of AP (calf intestine, type VII; Sigma-Chemie) weredissolved in 0.5 ml of a solution containing 1 mg of antibodiesper ml of PBS and dialyzed against PBS (5 liters) for 2 hr.Glutaraldehyde (electron microscopy grade; Sigma-Chemie)was added to a final concentration of 0.2%. The reactionmixture was stirred for 2 hr at room temperature and thenseparated by elution with PBS on a Sephadex G-i 00 (Phanmacia) column (30 x 1.5 cm). The AbAP appeared in the voidvolume and was stoned at 4Â°after dilution to a final proteinconcentration of 3 pg/mI with TBS containing 1% BSA.

Isolation of Specific Goat Anti-Rabbit IgG Antibodies

Rabbit IgG was isolated from normal rabbit sera as previouslydescribed (27). One hundred mg of lgG were dissolved in 0.1M sodium acetate buffer (pH 5.0) at a concentration of i 5 mg/ml. After addition of 2 ml of a 2.5% glutanaldehyde solution(electron microscopy grade; Sigma-Chemie), the mixture wasgently stirred at room temperature overnight. The polymenizedgel was homogenized (Polytron homogenizer; Kinematica, Lu

cerne, Switzerland), incubated in a solution of 1 M glycine in0.1 M borate buffer (pH 8.5) for i 0 mm, and finally washed 5times with PBS. The isolation of specific antibodies from goatanti-rabbit lgG serum (Behning) was carried out as describedfor the isolation of 06-EtdGuo-specific antibodies with theexception that 0.i M glycine-HCI buffer was used instead of iM acetic acid for the elution of antibodies.

1251 Labeling of Antibodies

After evaporation of the benzene solvent, i mCi of 1251..labeled Bolton-Hunter reagent (specific activity, 1,500 Ci/mmol; New England Nuclear, Dreieichenhain, Germany) wasdissolved in i 0 @Iof a solution of specific goat anti-rabbit lgGantibodies in 0.1 M borate buffer (pH 8.5) and incubated for 3hr at 0Â°.After addition of 500 .tl of a solution of 0.2 M glycinein borate buffer and â€˜-â€˜-1mg of phenol red, the mixture wasseparated on a Sephadex G-50 column (i x i 2 cm) eluted withPBS. The iodinated antibodies were found in the first peak(void volume), while free 1251appeared in the second, phenolred-containing peak. About 95% of the radioactivity in theprotein peak were precipitable with 5% tnichloroacetic acid.About 20% of 1251was incorporated into the protein; the specificradioactivity of the iodinated antibodies was @2,000Ci/mmol.

Enzyme-linked Immunosorbent Assay

The competitive ELISA was performed in 96-well microtiterplates (type M 129 A; Greiner, NUrtingen, Germany). Wellswere filled with 200 jsl each of coating solution (2 @zgofnucleoside-protein conjugate per ml of TBS) and incubated for30 mm at room temperature. The solution was removed, andthe wells were washed twice with TBS. Remaining free bindingsites on the plastic surface were saturated with a solution of1% BSA (w/v) in TBS for 30 mm at room temperature. Theprotein solution was removed, and the wells were incubatedfor 1 hr at 37Â°with 200 @leach of the AbAP conjugate (3 jsg/ml TBS containing 0.i % BSA), premixed with either TBS or theinhibitor (i.e. , a calibration standard or test sample). Thereafter,the AbAP solution was removed, the wells were washed 4 timeswith TBS, and 200 @slof a solution of 10 mM p-nitnophenylphosphate (phosphatase substrate; Sigma-Chemie) in 50 mMsodium carbonate buffer (pH 9.5):2 mt@iMgCI2 were pipettedinto each well. After incubation for 1 hr at 37Â°,the reactionwas stopped by addition of 100 ,zI of 1 N NaOH, and theabsorption at 405 nm was determined using either a flowthrough cuvet in a Gilford Model 250 spectrophotometer or aMultiscan spectrophotometen (Flow, Meckenheim , Germany).The degree of inhibition of AbAP binding to the solid phase wascalculated by the formula::@ :@@@ oo=inhibition(%)where E1 is absorption of inhibitor-containing well; E2 is absorption of well without inhibitor (0% inhibition); and E3 isabsorption of well without nucleoside-protein coat (100% inhibition). Samples were generally run as tniplicates, and theinhibition values were within Â±5%(S.D.) of the mean.

Radloimmunosorbent Technique

The competitive RIST was carried out in microtiter plateswith 96 disposable wells (type M 74 of M i 74; Gneiner).Coating

MARCH1980 889



R. MÃ¼!!erand M. F. Rajewsky

of wells and saturation of free binding sites were performed asdescribed for the ELISA. Fifty @dof a solution of O6-EtdGuospecific antibodies (10 ng/well) in 0. 1% BSA in TBS or inhibitor(calibration standard or test sample) were pipetted into eachwell. After incubation for 2 hr at 37Â°,the antibody solution wasremoved, and the wells were washed 5 times with TBS andfilled each with 100@ of a solution of â€˜251-labeledspecific goatanti-rabbit lgG antibodies (-â€˜@50,0O0dpm/weIl) in 0.1 % BSA inTBS. After incubation at 4Â°for -@-i5 hr, the antibody solutionwas removed, the wells were washed 5 times with TBS, andthe remaining radioactivity was measured in a Packard Model3375 gamma spectrometer. The degree of inhibition of antibody binding to the 06-EtGuo-BSA on the plastic solid phasewas calculated by the formula

dpm2â€” dpm1 x 100 = inhibition(%)dpm2 â€”dpm3

where dpm1 is dpm in inhibitor-containing well, dpm2 is dpm inwells without inhibitor (0% inhibition), and dpm3 is dpm in wellswithout O6-EtGuo-BSA coat (100% inhibition). Samples weregenerally run as duplicates, and the inhibition values werewithin Â±5%of the mean.

Radioimmunoassay

In the competitive RIA [modified Farr assay (i 2)], the radiolabeled nucleoside (tracer) competes for the antibodies withthe unlabeled nucleoside of the calibration standard or testsample (inhibitor). Each sample contained in a total volume of100 ILl of buffer [TBS with i % BSA (w/v) and 0. 1% normalrabbit IgG (w/v)], i O@dpm of tracer (06-Et[8,5'-3H]dGuo),antinucleoside serum at a dilution giving 50% binding of tracerin the absence of inhibitor, plus varying amounts of inhibitor.After incubation for 2 hr at room temperature (equilibrium), 100@dof a saturated ammonium sulfate solution (pH 7.0) wereadded. Ten minutes later, the samples were centrifuged for 3mm at 10,000 x g. The radioactivity in 1 50 @sIof supernatant

was measured in 10 ml of a toluene-based scintillation fluid(Rotiszint 22; Roth) in a Searle-Nuclear Chicago Mark II liquidscintillation spectrometer. The degree of inhibition of tracerantibody binding was calculated by the formula

dpm1 â€”dpm2 . . ..x 100=inhibition(%)dpm3 â€”dpm2

where dpm1 is dpm in the supernatant of inhibitor-containingsample, dpm2 is dpm in the supemnatantof sample withoutinhibitor (0% inhibition), and dpm3 is dpm in the supernatant ofsample without antibodies (100% inhibition). Samples were runas duplicates, and inhibition values were within Â±2%of themean.

Isolation and Enzymatic Hydrolysis of DNA

DNA of rat brain and liver was isolated using a modificationof the method of Meinke et a!. (25). The liquid N2-frozen tissuewas thawed in a solution of 1% sodium dodecyl sulfate (i 0 ml/g tissue) and homogenized with a Polytron homogenizer (speedcontrol Position 4) for 30 sec. After addition of proteinase K(Merck AG, Darmstadt, Germany) to a final concentration of500 @sgof enzyme per ml, the homogenate was incubated for30 mm at 37Â°.A 10-fold volume of urea buffer (5 M urea:2 M

NaCI:0.i 8 M sodium phosphate, pH 6.8) was added, and themixture was extracted with an equal volume of chloroform:isoamyl alcohol (24:1 ). Hydroxylapatite (Bio-GeI HTP, DNAgrade; BioRad Laboratories, Munich, Germany) was washedwith urea buffer and added to the aqueous phase (3 g/g oftissue). The suspension was incubated with gentle shaking for30 mm at room temperature. After centnifugation (â€”10 sec at500 x g), the supernatant was withdrawn, and the hydroxylapatite was washed 3 times with urea buffer and finally with imM sodium phosphate, pH 6.8 (20 ml of buffer pen g of

hydroxylapatite). The DNA was eluted by incubating the hydroxylapatite in 0.5 M sodium phosphate buffer (pH 6.8) for 5mm at room temperature (3 ml of buffer per g of hydmoxylapatite). The elution was repeated twice, and the supemnatantswere combined, dialyzed against a 100-fold volume of H2Ofor5 x 12 hr at 4Â°,and lyophilized. The average yield of DNA was72% of the theoretical value, and the contaminations with RNA(7) and protein (protein assay kit; BioRad) were <0. 1 and<0.5%, respectively. The ratios of E2@&E2soand E2so:E2sowereâ€”0.42and â€”i.8, respectively. The isolated DNA had a molecular weight of@ 10@,as estimated by aganose:gel electrophoresis.

For enzymatic hydrolysis, the DNA (@i mg/mI) was firstdigested with DNase I (EC 3.1 .4.5; Grade II; Boehninger; 100pg/mI) in a buffer containing i 0 m@Tnis-HCI (pH 7.0):5 mMMgCl2 for 0 mm at 37Â°.The pH was then raised to 8.0 byaddition of 1 M Tnis-HCI buffer (pH 9.0). Snake venom phosphodiesterase (EC 3.1 .4.5; Boehninger; 100 @g/ml)and AP(Grade I; Boehninger; 400 units/mg; i 00 pg/mI) were added.Hydrolysis was complete after 45 mm at 37Â°(Chart 2), asmeasured by determination of free phosphate (i 3). In general,ADA (EC 3.5.4.4; Boehninger;0.3 unit/mI) was used to convertdAdo to dlno in the DNA hydrolysates, prior to chromatographicseparation (Chart i ) or immunological analysis. The reactionwas complete after 5 mm at room temperature, as indicated bythe decrease of absorption at 265 nm. This procedure doesnot lead to measurable deethylation of 06-EtdGuo. dGuo concentrations in the samples were determined UV spectrophotometrically as described previously (27). After chromatographicseparation of the DNA hydrolysate, dGuo concentrations werecalculated from the UV absorption values of the respectivefractions (Chart 1), based on a molar absorption coefficient atneutral pH of â‚¬253 i 3,700 for dGuo.

RESULTS

Evaluation of Immunization Procedures and Characteristics of Antibodies. To obtain anti-06-EtdGuo antibodies of highaffinity and high specificity (i.e. , low cross-reactivity with nonmal DNA components and other ethylation products in DNA),2 different immunization procedures (Procedures 1 and 2; seeâ€â€˜Materials and Methods' â€˜) were compared, as well as 3 carrier

proteins (RSA, BSA, KLH) of different molecular weight andphylogenetic resemblance to rabbit proteins. The results areshown in Table i . Immunization Procedure 2 was superior toProcedure 1 with respect to the affinity of the antibodies for06-EtdGuo, while the concentration of O6-EtdGuo-specific antibodies was higher in the antiserum obtained by Procedure 1.The phylogenetically most dissimilar carrier (KLH) producedthe antibodies with the highest affinity (antibody associationconstant for 06-EtdGuo, 1 to 2 x 1010 liters/mol) and the

890 CANCER RESEARCH VOL. 40



Table1Propertiesof anti-OÂ°-EtdGuoantisera obtained with the use of different carrier proteins for the hapten 06-EtGuo, and by different immunizationproceduresRabbits

ware Immunized with conjugates of O6-EtGuo and various carriers (RSA, BSA, KLH), using Immunization Procedures 1 and 2 (sea â€˜â€˜MaterialsandMethodsâ€•).Antibody titers in the antisera are expressed as final serum dilutions giving 50% tracer binding in the RIA. Relative antibody reactivities for O6-EtdGuoascompared

with dGuo and dAdo were calculated from the respective inhibition values in the competitiveAlA.Concentration

of Antibody affinity Relative antibody reactivityImmunization No. of rabbits spacific antibodies constant, K (liter/

Immunogen procedure immunized Antibody titer (lgG) (mg/mi) mol) O6-EtdGuo:dGuoO6-EtdGuo:dAdoO6-EtGuo-RSA

1 4 <1:3 NDa ND NDND06-EtGuo-BSA1 4 1:20,000-1 :40,000 1.9-4.3 6-8 x 108 1.5 x 106:1 3 Xi0@:i06-EtGuo-BSA2 2 1:60,000 1.1 2-3 X i09 NDND06-EtGuo-KLH2 3 1:50,000-1:200,000 0.9-1.5 1-2 X io'@ 5 X 106:1 1 xi0@:ia

ND, not done.

!mmuno!ogiCa! Quantification of 06-EtdGuo

@0N>1

0

@0>1

I

z

0C0

C.)CDU-

Chart 2. Kinetics of enzymatic DNA hydrolysis todeoxynucleosidas. Calf thymus DNA was digestedwith DNaseI and then treated with phosphodiesterasaand AP (see â€œMaterialsand Methodsâ€•).Extant ofhydrolysis determined by measurement of the liberated phosphate (13) (100% value calculated frominput DNA).

lowest degree of cross-reactivity with dGuo (06-EtdGuo:dGuo,5 x I 0@)(Tables i and 2). Of the antisera thus far producedby the different immunization schemes, 3 (El , E2, and E3)were analyzed in detail by competitive RIA. As can be seenfrom the data shown in Table 2, Antiserum E3 exhibited themost favorable properties for quantification of O6-EtdGuo inDNA hydrolysates.

Sensitivity and Specificity of the RIA. As expected, O6@EtdGuo was the strongest inhibitor in the RIA. With AntiserumE3, 0.05 pmol of O6-EtdGuo was sufficient to inhibit tracerantibody binding by 50% (Table 2; Chart 3). Using a probabilitygrid to linearize the sigmoid inhibition curve over the rangebetween I 0 and 90% inhibition, the O6-EtdGuo concentrationrequired to inhibit tracer-antibody binding by, e.g. , 20% canbe determined, thus lowering the detection limit for O6-EtdGuoto @â€˜0.01pmol (Chart 4).

With Antiserum E3, the 50% inhibition value for 06-EtdGuoremained unaltered up to concentrations of -â€˜-300@gof hydrolyzed DNS (ADA treated) per ml in the test samples (Chart 5),thus obviating correction for nonspecific inhibition in this rangeof DNA concentrations. In the presence of higher amounts of

hydrolyzed DNA, 06-EtdGuo concentrations can be determinedby comparing the inhibition values with calibration curves constructed for the respective DNA concentrations (Chart 6). In100 ,sgof DNA (equivalent to the DNA content of â€˜â€”1.6 X 1@diploid cells), 06-EtdGuo can thus be measured at an O6@EtdGuo:dGuo molar ratio of â€œ-3X i0@ (i.e., @-70006-EtdGuomolecules per diploid genome).

The degree of inhibition by 06-EtdGuo of tracer-antibodybinding was comparatively measured with Antiserum E3 in 55,DS, and hydrolyzed DNA (nucleosides; ADA treated), ethylatedby EtNU in vitro (O6-EtdGuo:dGuo, 2.5 X 10@). The followingratios were obtained (relative 06-EtdGuo concentrations required for 50% inhibition of tracer-antibody binding): DNAhydrolysate:SS, 1:13; SS:DS, 1:20; DNA hydrolysate:DS [S@nuclease (Boehninger)-treated], 1:250 (Chart ).

Sensitivity and Specificity of ELISA and RIST. Both theRIST and the ELISA can be carried out in a microtiten systemand are, therefore, preferable to the RIA in case of large seriesof measurements. In both assays, free 06-EtdGuo in the testsample on in calibration standards, respectively, inhibits thebinding of O6-EtdGuo-specific antibodies to an O6-EtGuo-BSA

MARCH 1980 891

Time (mm)



Inhibition of tracer-antibody binding by various alkylated and natural nucleic acid components inthecompetitiveRIA: comparison of three antisera (El, E2, and E3) directed against06-EtdGuoEl

: immunogan, O'-EtGuo-BSA; Immunization Procedure 1, 2 booster injections; E2: immunogen,0'-EtGuo-KLH;Immunization Procedure 2, 1 booster injection; E3: immunogen, OÂ°-EtGuo-KLH;ImmunizationProcedure

2, 2 booster injections.Amount

required for 50% inhibition of tracer-antibodybindingAntiserum

E3Antiserum

El Antiserum E2 Multiple of O@Compound (pmol) (pmol) pmolEtdGuoOÂ°-EtdGuo

0.6 0.12 0.051OÂ°-EtGuo3.6 0.7 0.1530'-EtdGMP

20 1.8 0.714O'-EtGua60 6 1.428O'-BuGuoNT' NT 240O'-MaGuo276 30 122407-EtdGuoNT 420 17034007-EtGuaâ€”4x 104b@ x io@ 4 x i0@ 8 xio@dGuoâ€”1x 105C ,_@x 1056 3 x 104C@ x 10'

dAdo â€”8x 104b@@ iO4b â€”6x 104C@@ i0@dlnoâ€”1.5x 105C@ x 105c â€”6x 104C@ x i0@

2'-Daoxycytidine â€”5x 1o@d@ x i o5d@ x i o@d >@@@ o72'-Daoxythymidina â€”5x 10â€•' â€œ1x 105d ,,@x i0@' >3 x io@DNA hydrolysate (ADA- â€”1x iO@ -â€˜1x 105C ,,@X 104C@ xi0@treated)


Table 2

a NT, not tested.b 20% inhibition at this concentration.C 10% inhibition at this concentration.d <@ 0% inhibition at this concentration.

Chart 3. Inhibition of tracer-antibody binding by various alkylatad and natural nucleic acid components in the competitive AlA. Antiserum E3 (see Table 2). Theenzymatic DNA hydrolysata (deoxynucleosidas; sea Chart 2) was treated with ADA. Immunogan, O'-EtGuo-KLH; tracer, O'-Et[8,5'-3HJ-2'-dGuo.

01C@0C

â€˜I@02C4

0

0C0

.0-CC

Inhibitor (Picomol)

conjugate on the plastic surface of the reaction vessels. In theRIST, antibody binding is measured via the radioactivity of 125l.labeled goat anti-rabbit IgG antibodies, while the same panameter in the ELISA is measured via the cleavage of p-nitnophenylphosphate by the AP covalently linked to the antibodies (i.e.,by the color reaction resulting from the formation of dinitrophenol).

Using antibodies purified from Antiserum E2 in the RIST andthe ELISA, 1 or 0.25 pmol, respectively, of 06-EtdGuo wassufficient to inhibit antibody binding by 50% (Table 3; Chart 8).O6-EtGuoand 7-EtdGuo exhibited considerably lower degreesof inhibition. As in the RIA (Table 2; Chart 3), the naturally

occurring nucleosides showed inhibition of antibody bindingonly at very high concentrations. dAdo exhibited the companatively highest degree of inhibition but could easily be converted to dlno by addition of ADA to the DNA hydnolysates priorto the assay. The inhibitory effect of dlno is 5 and 20 timeslower than that of dAdo in the RIST and in the ELISA, nespectively. In the ELISA, the inhibition value for O6-EtdGuoremaineduninfluenced over a concentration mangeof up to -@350 @gofADA-treated hydrolyzed DNA per ml in the test sample. Foranalysis of O6-EtdGuo in the presence of higher amounts ofDNA, calibration curves were constructed for the respectiveDNA concentrations (350 to 1500 @sg/ml).In an amount of




Immuno!ogica! Quantification of 06-EtdGuo

01C

@0C

>1

-g.0

C4

0Chart 4. R1Acalibration curve for O'-EtdGuo. Inhibition of tracerantibody binding by 0'-EtdGuo plotted against 0'-EtdGuo (inhibitor)concentration in a probability grid. Reading at 20% inhibition givesan0â€¢-EtdGuovalueofâ€”0.01pmol.

O6@EtdGuo (Picomol)

C 100VC

a

V0

C

â€˜@ 50@ 20mg DNA/mI

@ Â£ 10mg DNA/mI

i@ - 05mg DNA/mI

,@ . 00mg DNA/mIC0

;@@ _1_ i I 1111111 1 I 1111111@ OnI 0.1 1

@6EtdGuo (Picomol)

Chart 6. AlA calibration curves for O'-EtdGuo in the presence of ADA-treatedDNA hydrolysata.Inhibitionof tracer-antibodybindingis shownas a functionofthe O'-EtdGuo concentration in the presence of different amounts of hydrolyzedDNA in the test samples.

oration of the solvent (H2O),prior to the AlA. By this procedure,06-EtdGuo:dGuo molar ratios in DNA of <5 x 10_8 can beeasily quantitated (Chart 9). Chart 10 indicates that the molarcontent of O6-EtdGuo in the DNA of brain and liver is approximately identical and linearly dependent on the dose of EtNUovena dose range of 2.5 to 100 @tgof EtNU per g body weight,when measured by AlA at 1 hr after an i.v. EtNU pulse to 5-week-old BDIX rats.

DISCUSSION

The present and previous studies (27, 28) on antibodiesspecifically directed against O6-EtdGuo demonstrate that immunological methods represent powerful tools for overcoming

$5

GC

@7o

@;L @o!2o!3o!5Ã E@ DNAâ€”Concsntration

chart5. InfluenceoftheDNAhydrolysataconcentrationinthetestsampleonthe Inhibition of tracer-antibody binding by 0'-EtdGuo in the AlA. Samplescontaineda constantamountof 0'-EtdGuo(giving50%inhibitionof tracerantibody bindIng in the absence of DNA hydrolysata) and varying amounts ofenzymaticallyhydrolyzedDNA.Inhibitionof tracer-antibodybindingpiottedagainst DNA (hydrolysate) concentration In the test sample.

.,.,100 iLgof DNA, 06-EtdGuo could thus be determined at a O6@EtdGuo:dGuo molar ratio of @â€˜-3x 10_6(i.e. , @700OO6-EtdGuomolecules per diploid genome).

Determination by RIA of OÂ°-EtdGuoin DNA. Charts 9 and10 show examples of the application of the AlA (Antiserum E3)for determination of the molar concentration of O6-EtdGuo inDNA ethylated by EtNU in vitro or in vivo. In order to measurethe 0-EtdGuo content of DNA at very low molar concentrations(i.e., after exposure of DNA to low levels of the carcinogen),O6-EtdGuowas quantitatively separated and concentrated fromthe remaining nucleosides in the DNA hydmolysateby chromatography on Sephadex G-10 (Chart 1) and subsequent evap

MARCH 1980 893



Inhibition of antibody binding by ethylated and natural nucleosides in the RISTand the ELISA forO'-EtdGuoAntiserum

E2 was used (sea Table2).Amount

required for 50% inhibition of antibodybindingRIST

ELISAMultiple

of Multiple ofCompound pmol O'-EtdGuo pmolO'-EtdGuoO'-EtdGuo

1 1 0.25 1O'-EtGuo 3 3 1 47-EtdGuo 100a >1 o@ 100 400dAdo 3 x 10' a â€”2x 1o@ -â€˜2x 1o5 -.8 x 1o@dGuo 10@a X i0@ 105a â€”2x 106dlno 105a ,_@X i05 >i0@>i0@a

20% inhibition at this concentration.


8001 ss.@ DNA Hy&oI@saâ€¢ Os lOS' SI)

1:@2:

@ i02 10_i 100 i0@ 102

I- 06â€”EtdGuo (Picomol)

Chart 7. Inhibition of tracer-antibody binding in the AlA by O'-EtdGuo inhydrolyzed (daoxynucleosides), SS, and DS, EtNU-treatad calf thymus DNA.TheDNA was digested with Si nuclease in order to eliminate SS regions and had anO'-EtdGuo/dGuo molar ratio of 2.5 x i0@ and a molecular weight of 2 to 5magadaltons. SS DNA was obtained by boiling the DNA for 3 mm at 100Â°andcooling rapidly in ice. This treatment did not lead to a measurable loss of O@EtdGuo from DNA. Control DNA(not ethylated) did not cause significant inhibitionof tracer-antibody binding over the same range of DNA concentrations.

Table 3

100 io2

Inhibitor (Picomol)

as

2'

â€˜2

>1â€˜O

2C4

0C0

â€˜aâ€˜CC

Chart 8. Inhibition of antibody binding to OÂ°-EtGuo-BSAattached to theplastic solid phase by various alkylated and natural nucleic acid components asa function of the inhibitor concentration in the RIST and the ELISA, respectively.

and low-specificity antibodies. In contrast, Procedure 2 couldstimulate a more limited number of immunocytes preferentiallycarrying high-specificity and high-affinity receptors for O6@EtdGuo. The antiserum (E3) with the highest association constant for 06-EtdGuo (1 to 2 x 1010liters/mol) and the lowestdegree of cross-reactivity with normal DNA constituents (Table2; Chart 3) were indeed obtained by Immunization Scheme 2,using KLH as the carrier protein for O6-EtGuo (i.e. , a carrierphylogenetically very distinct from the proteins of the immunized animal species, with a molecular weight of about800,000).

The RIA for O6-EtdGuo established with Antiserum E3 ishighly sensitive (50% inhibition of tracer-antibody binding at0.05 pmol of 06-EtdGuo) and specific. The normal nucIeoside@dGuo, dAdo, and dlno (produced by deamination of dAdo byADA during DNA hydrolysis) and the deoxypynimidines reactedwith the antibodies about 107-fold less than did O6-EtdGuo.The RIA will thus detect â€”700O6-EtdGuomolecules per diploidgenome in a DNA sample of 100 @g(equivalent to â€œ-1.6 x 10@diploid cells). In combination with chromatognaphic separationof O6-EtdGuofrom the DNA hydnolysate prior to the RIA (Chart2), O6-EtdGuo:dGuo molar ratios of <5 x 10_8 can be measured without difficulty. Recently published RIA's for 06-methyldeoxyguanosine (4)4 show considerably lower levels of sensitivity and specificity than does the present RIA for O6-EtdGuo.This may be ascribed to the higher affinity and higher specificityof the antibodies obtained with Immunization Procedure 2 andthe use of a high-specific-activity (14 Ci/mmol) 3H-Iabeledtracer. Furthermore, the ethyl group in the 06-position ofguanine may represent a more antigenic site than did thecorresponding methyl substitution. In this respect, it is interesting to note that in the present RIA Antiserum E3 showed ahigher degree of cross-reactivity with the butyl- (1:40) thanwith the methyl-substituted O@(1:240) of guanosine (Table 2).

As demonstrated by the RIA, the antibodies of Antisera El,

some of the shortcomings of conventional radiochromatographic techniques for the specific quantification of carcinogen-induced, structural modifications of DNA. Similar, althoughless sensitive and less specific assays have been reported forimmunological detection of 2-acetylaminofluorene-DNA adducts (17, 31 , 32, 34), benzo(a)pyrene-DNA adducts (33), o6@methyldeoxyguanosine (4),4 7-methylguanine (36), and N6(methylnitroso)adenosine (42). The pioneering work of Erlanger, Beiser, and their coworkers (10, 23, 26) on the productionof antibodies specific for normal and modified minor bases inDNA has provided important guidelines for this work.

The sensitivity of an immunological assay for a specific,structurally modified DNA component mainly depends on theaffinity and specificity of the antibodies directed against therespective purified product. Therefore, phylogenetically different carrier proteins for the hapten O6-EtGuo with varying molecular weights (RSA, BSA, KLH), as well as 2 different immunization procedures, were compared with respect to the properties of the resulting antibodies. In Immunization Procedure 1,high amounts of conjugate (5 mg/rabbit) were administered atrather short intervals (i.e. , 2 to 4 weeks) by routes (i.m., s.c.)permitting fast release of the immunogen into the lymphaticsystem. In Procedure 2, rabbits were immunized i.c. with a 10-fold-lower amount of conjugate at 8-week intervals, the immunogen being adsorbed to aluminum hydroxide to ensure slowrelease. Procedure 1 should produce high serum antibodyconcentrations, with a considerable proportion of low-affinity

4 5. A. Kyrtopoulos and P. F. Swann, personal communication.




4

C

SC,

10 100

Des. ef EtNU I @.igIgBedyWeight IChart 10. InItialguanina06-ethylationin liver and brain DNA of 6-week-old

BDIX rats as a functionof the EtNU dose (measuredat i hr after iv. injection).RIA.

E2, and E3 have greater affinity for O6-EtdGuo than for thecorresponding base, deoxynucleotide, or nibonucleoside (Tables 2 and 3; Charts 3 and 8). Therefore, the antibodiesrecognize not only the 06-ethylated punine but also the sugarmoiety. The lower affinity of the antibodies for O6-EtGuo canbe explained by the structure of the O6-EtGuo-pmoteinconjugate used for immunization. 06-EtGuo is oxidized by pemiodate,thus yielding 2 cambonylgroups at the 2',3'-nibose positions.These react spontaneously with the free amino groups in thecarrier protein, especially with the â‚¬-aminogroup of lysine. The

!mmuno!ogica! Quantification of 06-EtdGuo

iO@

0 RIA. Sephodsx G10+RIA

iO@@

10@S

Chart 9. 0-Ethylation of guanine in double-stranded calfthymus DNAin vitro, as a function of EtNUconcentration. Thereaction was carried out for 1 hr at 37Â°in 0.1 1 PANa2HPO4:0.01 Mcitricacid buffer,pH 7.2, at a DNAconcentrationof 2mg/mI. RIA.

â€” I I I0.01 0.1 1 10 100

EtNU Concsntration(gig/mI)1000

resulting unstable Schiff's base is stabilized by reduction withborohydnide. Therefore, the immunogen structurally resemblesmore closely the deoxynibonucleoside than the nibonucleoside.

Tracer-antibody binding in the RIA is inhibited by O6-EtdGuocontaining denatured DNAto the same extent as by 06-EtdGMP[06-EtdGuo:O6-EtdGMP, 1:14 (Table 2); hydrolyzed DNA (nucleosides):SS = 1:13 (Chart 7)]. The antibodies thus seem torecognize 06-EtdGuo contained in 55 DNA equally well as thefree modified nucleotide. The antibodies react with O6-EtdGuoin denatured DNA about 20 times better than with OÂ°-EtdGuoin DS DNA. This indicates that due to steric hindrance only afraction of the O6-EtdGuo in native DNA is recognized by theantibodies. Antibodies to other naturally occurring modified(18, 26, 30) and carcinogen-modified (34)4 nucleosides alsorecognize these residues preferentially in denatured ratherthan in native DNA.

A comparison of the 3 immunological assays for O6-EtdGuo(RIA, RIST, and ELISA) shows that the ELISA and the RIST aresomewhat less sensitive than is the RIA (Tables 2 and 3; Charts3 and 8). On the other hand, the ELISA has the advantage ofbeing carried out more readily in large-scale experiments.Furthermore, since the ELISA does not require a radiolabeledtracer, it can be performed with DNA samples radioactivelyprelabeled for other purposes.

The measurements by RIA of the molar content of 06-EtdGuoin DNA after ethylation by EtNU in vivo or in vitro (Charts 9 and10) show the potential of immunological detection methods foranalyses of carcinogen-modified, potentially mutagenic DNAcomponents at low levels of carcinogen exposure. These techniques will also permit studies on the persistence (or rate ofelimination) of DNA lesions in small numbers of cells or indefined fractions of chromatin.

I 10 100

50

xlC

SC,

SC,

1

1

LIVER

. BRAIN

MARCH1980 895



R. MÃ¼I!erand M. F. Rajewsky

22. Loveless, A. Possible relevance of 06-alkylation of daoxyguanosine to themutagenicity and carinoganicity of nitrosaminas and nitrosamidas. Nature(Lond.), 223: 206â€”207,1969.

23. Lubit, B. W., Pham,T. D., Miller, 0. J., and Erlanger, B. F. Localization of 5-mathylcytosine in human metaphase chromosomes by immunoelactron microscopy. Cell, 9: 530-509, 1976.

24. Mcllvaine, T. C. A buffer solution for colorimatric comparison. J. Biol. Cham.,49:183-186,1921.

25. Meinke, W., Goldstein, D. A., and Hall, M. A. Rapid isolation of mouse DNAfrom cells In tissue culture. Anal. Biochem., 58: 82â€”88,1974.

26. Miller, 0. J., Schnadl, W., Allan, J., and Erlanger, B. 5-Methylcytosinelocalized in mammalian constitutive haterochromatin. Nature (Lond.), 251:636â€”637,1974.

27. MUller, A., and Rajewsky, M. F. Sensitive radioimmunoassay for detectionof 0Â°-ethyldaoxyguanosinein DNA exposed to the carcinogen athylnitrosourea in vivo or in vitro. Z. Naturforsch., 33c: 897â€”901, 1978.

28. Muller, A., and Rajewsky, M. F. Immunological detection of ethylationproducts in DNA exposed to athylnitrosourea in vivo or in vitro. In: L. Santiand S. Parodi (ads.), Short Term Tests for Prescreening of Potential Carcinogens, pp. 70â€”75.Genoa, Italy: Istituto Sciantifico par 10Studio a Ia Curs@deiTumori, 1979.

29. Pagg, A. E. Formation and metabolism of alkylated nucleosidas: possiblerole in carcinoganasis by nitroso compounds and alkylating agents. Adv.Cancer Res., 25: 195â€”269,1977.

30. Plescia, 0. J., Braun, W., and Palczuk, N. C. Production of antibodies todanaturated deoxyribonucleic acid (DNA). Proc. NatI. Acad. Sd. U. S. A.,52: 279â€”285,1964.

31. Poiriar, M. C., and Connor, A. J. A radloimmunoassay for acatylaminofluorane-DNA adducts. In: H. Van Vunakis and J. J. Langone (eds.), Immune.chemical Techniques (A volume of Methods in Enzymology), Vol. 2. NewYork: Academic Press, Inc., in press, 1980.

32. Poirier, M. C., Dubin, M. A., and Yuspa, S. M. Formation and removal ofspecific acatylaminofluorane-DNA adducts in mouse and human cells massured by radioimmunoassay. Cancer Res., 39: 1377â€”1381 , 1979.

33. Poirier, M. C., Grunbergar, D., Weinstein, I. B., and Yuspa, S. M. Antibodiesspecific for DNA modified with a diol oxide of banzo(a)pyrana (BP). Proc.Am. Assoc. Cancer Ras., 19: 102, 1979.

34. Poiriar, M. C., Yuspa, S. M., Weinstein, I. B., and Blobstain, S. Detection ofcarcinogen-DNA adducts by radioimmunoassay. Nature (Lond.), 2 70: 186â€”188, 1977.

35. Rajawsky, M. F., Auganlicht, L. H., Biassmann, H., Goth, A., HUlser, D. F.,Laerum, 0. D., and Lomakina, L. V. Nervous system-specific carcinoganasisby ethylnitrosourea in the rat: molecular and cellular mechanisms. ColdSpring Harbor Conf. Cell Proliferation, 4: 709â€”726,1977.

36. Sawicki, D. L., Beiser, S. M., Srinivasan, D., and Srinivasan, P. R. Immunochemical detection of 7-methylguanina residues in nucleic acids. Arch.Biocham. Biophys., 176: 457â€”464,1976.

37. Singer, B. N-Nitroso alkylating agents: Formation and persistence of alkylderivatives in mammalian nucleic acids as contributing factors in carcinogenesis. J. NatI. Cancer Inst., 62: 1329â€”1339, 1979.

38. Singer, B., Bodall,W. J., Cleaver, J. E., Thomas,G. H., Rajawsky,M. F.,and Thon, W. Oxygans in DNA are main targets for ethylnitrosourea innormal and xarodarma pigmantosum fibroblasts and fatal rat brain cells.Nature (Lond.), 276: 85â€”88,1978.

39. Stewart, M. W., and Petty, A. E. The antigen-binding characteristics ofantibody pools of different relative affinity. Immunology, 23: 881â€”887,1972.

40. Stollar, B. D., and Borel, V. Nucleoside specificity in the carrier lgG-dapendant induction of tolerance. J. Immunol., 117: 1308â€”1313, 1976.

41. Stollar, B. D., and Borel, V. Nucleoside-specific tolerance suppresses antinucleoside antibody forming calls. Nature (Lond.), 267: 158â€”160, 1977.

42. Sun, L., and Singer, B. The specificity of different classes of ethylatingagents towards various sites of HeLa cell DNA in vitro and in vivo. Biochemistry, 14: 1795â€”1802,1975.

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896 CANCER RESEARCH VOL. 40

ACKNOWLEDGMENTS

The authors express their appreciation to Ing. grad. W. Drosdziok for thesynthesis of alkylated compounds, to B. Wirth for expert technical assistance,and to Drs. J. Adamkiewicz, W. Fraist and Professors F. Cramer, H. Druckrey, D.Hammer, W. Pflaiderar, and K. Rajawsky for valuable methodological advice.

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1980;40:887-896. Cancer Res Rolf Müller and Manfred F. Rajewsky

-nitrosoureaN-Ethyl-N-Ethyldeoxyguanosine in DNA Exposed to 6OImmunological Quantification by High-Affinity Antibodies of

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Immunological Quantification by High-Affinity Antibodies of OÂ ... · immunological procedures for the quantification of DNA com ponents structurally modified by N-nitroso carcinogens,