Mutation Research, DN,4 Repair. 273 (19921 253-261

253

,~5 1992 Elsevier Science P h .sher~ B.V. All rigi~ir, reserx,ed 0921-8777/92/$05.~)0

MUTDNA 06475

Properties of a monoclonal antibody for the detection of abasic sites, a c o m m o n D N A lesion * B i - X i n g C h e n a, K i h e i K u b o b,:~, H i r o s h i I d e b., * ~.. B e r n a r d F. E r l a n g e r ~, S u s a n S. W a l l a c e b a n d Y o k e W . K o w b "Department of Mierobiology, Columbia Unit'ersiO,, College of Physicians and Surgeons. New York, N Y 10032. ~' Department of Microbioio~' and Molecular Genetics, Unicersity o]" Vermont, Burlington, VT 05405 ( U. S.A.) (Received 1 March 1991) (Revision received 6 June t99I) (Accepted 18 July 199i;

Keywords: Oxidative DNA damage; DNA damage, oxidative: AP sites; Monoclonal antibody; Antibody. monoclonal: DNA repair

Summary The abasic site is one of the most frequent changes occurring in D N A and has been shown to be lethal and mutagenic. An abasic site in D N A can be tagged by reaction with O-4-nitroben~qhydrox3'lamine (NBHA), resulting in the formation of an oxime linkage between the abasic site and the NBHA moiety. In order to measure NBHA-tagged abasic sites, a monoclonal antibody was elicited against a 5'-phosphodeoxyribosyl O-4-nitroben,:yl hydroxylamine-BSA conjugate. The antibody was specific for the NBHA residue as demonstrated by hapten inhibition, with ICs~ values for 5'-phosphodeoxyribosylNBHA, deoxyribosyl-NBHA, ribosyl-NBHA and NBHA of 0.3 #M, 5/xM, 5 /xM and 7/.tM, respectively. Other haptens examined, including benzythydroxylamine, 5'-phosphodeox3'ribosyl-, deox3'ribosyl-, and ribosyl-benzylhydroxylamine, showed no inhibition even at 1 raM. The antibody showed high specificity for NBHA-modified AP sites in D N A and exhibited no cross reactivity with normal D N A bases, otherwise-modified D N A bases or unmodified a ~ sites. Using a direct ELISA assay, the antibody detected 1 AP site (after NBHA-modification) per 10000 base-pairs or approximately 10 femtomoles of AP sites in DNA. D N A lesions were detectable in 6~Co y-irradiated D N A at a dose as low as 10 rad (0.t Gy) and the production of antibody detectable sites was proportional to the v-ray dose. Since NBHA

Correspondence: Dr. Susan S Wa!lace, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington. VT 05405 (U.S.A.). * This work was supported by grant DE-FG02-88ER60742 from the U.S. Department of Energy (lo SSW) and NIH grant NS-15581 (to BFE).

** Present address: Departrnent of Veterinary Radiology, School of VeterinaD, Medicine, College of Agriculture. University of Osaka Prefecture, Mozuume-cho-4-804. Sakai, Osaka 591 (Japan).

*** Present address: Department of Polymer Science and Engineering. Kyoto Institute of Technolog3'. Matsugasaki. Kyoto 6!)6 (Japan).

Ah#,reciations: AP. apyrimidinic/apurinic: BHA. O-benzylhydro-%:!amine: NBHA. b-nitrobenzylhydrox3"lamine: NBHAdRp, 5'-phosphodeo~'ribosyl O-4-nitrobenzylhydroxylamine.

254 reacts with lesions containing an aldehyde group, the simplicity and sensitivity of the antibody assay should provide a useful method for the quantitation of AP sites or other DNA lesions conta-ning an aldehyde group.

The apyrimidinic/apurinic (AP) site is one of the most frequent changes occurring in DNA (Loeb and Preston, 1986). AP sites are formed as a result of cleavage of the N-glycosylic bond between the base and the deoxyribose moiety and are produced spontaneously in cells, after insult by chemical agents such as alkylating agents (Loeb and Preston, 1986) and bleomycin (Rabow et al., 1986), by free radicals produced endogenously and by physical agents such as ~onizing radiation (Von Sontag, !987). AP sites are also formed by the action of DNA N-glycosylases, enzymes that initiate the base excision-repair pathway for the removal of damaged DNA bases (Freidberg, 1985; Weiss and Grossman, 1987; Wallace, 1988). Currently, AP sites in DNA can be quantified by a number of methods including alkali elution (Brent et al., 1978), DNA unwinding (Birnboim and Jevcak 1981; Kohn et al., 1981), 32P-postlabeling (Weinfeld et al., 1990), [t4C]methoxyamine labeling (Talpaert-Borle and Liuzzi, 1983; Liuzzi and Talpaert-Borle, t988) and more recently, using the aldehyde reactive probe reagent (Kubo et aI., in preparation). All these procedures detect AP sites in DNA at the femtomole level. However, due to the chemical instability of AP sites, lengthy procedures involved in the preparation of DNA samples can lead to strand breaks (Weiss and Grossman, !987) and the possible oxidation of the aldehyde group, processes which could potentially reduce the number of measurable AP sites in the DNA, We previously demonstrated that NBHA reacts with AP sites to produce O-NBHA residues at the AP site (Kow, 1989). This reaction stabilizes the phosphodiester bond 3' to the original AP site (Liuzzi and Talpaert-Borle, 1988; Kow, 1989). Furthermore, NBHA-modified AP sites should resist further oxidation since the atdehydc group is converted to an oxime upon NBHA modification. To detect such modified AP sites, monoclonal antibodies were raised against 5'phosphodeoxyribosyl O-4-nitrobenzyl hydroxyl-

amine (NBHA-dRp) conjugated to BSA. The present publication describes some of the properties of one of these antibodies as well as its application for quanti:ation Gf the number of AP sites in DNA. Materials and methods

Chemicals. Deoxyribose 5-phosphate, deox3'ribose, ribose, O-4-nitrobenzyl-hydroxylamine and O-benzylhydroxylarnine were purchased from Aldrich. Nucleic acids. PM2 DNA was routinely prepared in the laboratory according to Katcher and Wallace (1983) and Kow (1989) and fl-K12 DNA was routinely prepared according to Messing (1983). The host for fl, SIvlH77, was grown at 37°C with vigorous shaking in glucose-M9 medium (Kow and Wallace, 1987) with necessary amino acids (arginine, histidine and threonine at 20 /xg/ml), FeC12 (i0 /xM), and thiamine (1 /,g/mt). At a cell density of 2 x 10s/rot, fi-K12 phage stock was added to the medium at a multiplicity of infection of 5. The phage DNA was then isolated and purieied according to Messing (I983). Phenol extracted calf-thymus DNA was obtained from Pharmacia. DNA containing large numbers of AP sites was prepared by alk3,1ation followed by heat-induced depurination (Paquette et al., 1972). Calfthymus DNA was atk3,1ated with 0.3 M methyl methanesulfonate at 37°C for I h. After exhaustive dialysis against Tris-HCl, pH 7.5, t m M EDTA, the DNA was partially depurinated by heating at 50°C for an ars,propriate amount of time to produce DNA containing approximately 200 AP sites per 10000 bp (Paquette et al., 1972). PM2, calf-thymus or fl-K12 DNA, containing 2-16 AP sites per 10000 bp, was prepared according to Lindahl and Nyberg (1972). The DNA (100/xg/mt) was dissolved in 0.1 M NaCI, 0.01 M sodium citrate, pH 5.0 at 70°C for 15, 30, 60 or

2~5

120 min produce approximately 2, 4, 8 or 16 AP sites per 10000 bp respectively (Lindahl and Nyberg, 1972). The average number of AP sites in calf tilymus and fl DNA was correlated to the average number of AP sites in PM2 DNA, which was quantitated by the number of endonuclease IV-sensitive sites as described earlier (Kow and Wallace, 1987). Since the enzymatic assay is only accurate up to 2 sites per PM2 molecule or 2 AP sites per t0000 base pairs, the number of AP sites for DNA molecules containing more than 2 per molecule was estimated by extrapolation. This is reasonable, since the rate of depurination is linearly proportional to the time of depurination (Lindahl and Nyberg, 1972). Depurinated DNA preparations were modified by reaction with 5 mM NBHA in phosphate buffer, pH 7.2 at room temperature for 2 h. The product was then ethanol precipitated in zhe presence of 2.5 M sodium acetate (Maniatis et al., 1982). The precipitated DNA was washed once with 75% ethanol and redissolved in 10 mM Tris-HC1, pH 7.5, 1 mM EDTA. The DNA solution was precipitated with ethanol again to remove traces of NBHA. The precipitated DNA was redissolved in 10 mM Tris-HC1, pH 7.5, 1 mM EDTA. Alternatively, NBHA-treated DNA was dialyzed extensively against 10 mM Tris-HC1, pH 7.5, 1 mM EDTA to remove excess NBHA. Undamaged calf-thymus DNA was treated similarly with NB!L,~ to serve as the control. Calf-thymus DNA containing thymine glycols, urea residues or metho~'arnine residues was prepared as described previously (Kow and Wallace, 1987).

similarly without further purification. To prepare the N B H A - d R p - B S A conjugate for immunization, N B H A - d R p was coupled to BSA through the 5'-phosphate by the carbodiimide procedcre as described earlier (ttalloran and Parker, 1966; Hubbard et al., 1989b).

Haptens and antigen. NBHA-dRp was prepared by incubating NBHA (10 mM solution at pH 7) with 15 mM deox~ribose 5-phosphate overnight at room temperature. Since the reaction of O-alk2A hydrox3qamine with aldehyde was shown to be quantitative (Talpaert-Borle and Liuzzi, 1983; Liuzzi and Ta!paert-Borle, 1988; Kc-w. 1989; Kow and Wallace, 1987), the solution was used for the hapten inhibition studies without further purification. Solutions (10 mM) of 5' phosphodeox3'ribosyl O-benzythydrox3'lamine, deoxyribosyl O-4-nitrobenzs'thydroxylamine and deoxyribosyl O-benzylhydroxylamine were prepared

Direct-binding assay. Direct-binding assays were carried out as previously described (Hubbard et at., 1989). Polys~-rene 96 wells mic qtLer plates (Coming 25855 or Immulon t from Dynatech Laboratories, Inc.) were UV-irradiated overnight (Zouali and Stoltar, t980) and coated with 200 gl of NBHA-modified caff-thymus DNA in phosphate-buffered saline (PBS, t50 mM NaC1, I0 mM N a 2 H P O J N a H : P O 4, pH 7.4) overnight at 4°C. Alternatively, microtiter plate wefts were coated with NBHA-modified DNA for 2 h at 37°C. After blocking the plate with 1% horse serum in PBS-Tween (PBS buffer + 0.5% Tween

Monoclonal antibody. Immunization of B A L B / c mice was performed according to Holmdah! et al. (1985). The N B H A - d R p - B S A conjugate was dissolved in 0.05 mM sodium phosphate buffer, pH 7.5 to a concentration of 2 mg/mi. The solution was emulsified with an equal volume of complete Freund's adjuvant (Gibco). An aliquot of the emulsion (50 p.t) was injected subcutaneously into each hind foot pad. 9 days after immunization, lymphocytes were isolated from the draining popiiteal lymph nodes and fused with mouse myeloma P3 × 6.~-AG.~3-6~ (Sharon et al.. 1979). Screening of antibody production was done by Elisa using N B H A - d R p - R S A conjugate as the antigen. Of 440 v, eiis tested, l0 were positive. The positive supernatants were subsequently assayed by Elisa using calf-thymus DNA containing NBHA modified AP sites (200 sites per 100000 bp). Of the t0 positive lines, t reacted equally wetI with undamaged ca!f-thymus DNA as with NBI-La~-modified AP DNA and 2 clones bound specifica!ly to calf-thymus DNA containing NBHA-modified AP sites. The latter were subcloned twice by limiting dilution. 4 monoclonal antibedy clones were obtained and t of them, 3 - t 2 G - 1 2 H - t 2 H , was chosen for further study. The antibody was IgM, kappa as determined by Ouchterlony get diffusion.

25¢~

20) for 1 h at room temperature, the monoclonal ~,ntibody, diluted in PBS-Tween, was added to the coated wells and incubated at 37°C for 2 h. After washing with PBS-Tween, bound antibody was detected with horse radish peroxidase-conjugated goat-anti-mouse lgG + IgM (1:3000 dilution, T A G O Inc.) by the addition of H 2 0 2 and O-phenylenediamine. The absorbance at 490 nm was taken after stopping the reaction with 50 ¢ I of 8 N H 2 S O 4 a t appropriate times.

Inhibition assay. Competitive enzyme

immunoassay by haptens or calf-thymus D N A containing NBHA-modified AP sites was performed with microtiter plates which were precoated with calf-thymus D N A containing NBHA-modified AP sites (200 sites per 10000 bp, 270 n g / m l ) according to procedures previously described (Hubbard et al., 1989).

X- and ~°Co-irradiation. Cal.-thymus and fl D N A were X-irradiated at a concentration of 30 / x g / m l in 10 mM potassium phosphate buffer, pH 7.5 using a Philips X-ray generator with b - ~ l l i u m window Machlett tube operated at 50kVp and 2 m A (9.6 Gy per min). The dose rate was determined by gricke ferrous sulfate dosimet~, and phage T4 su:Mval, mCo-irradiation was performed with a Thcratron Junior (~°Co-irradiator (Atomic Energy of Canada Limited). The dose rate was adjusted to 12 G y / h . Results

Selection of monoclonal antibody clones for study. Clones that were reactive with the N B H A - d R p - R S A conjugate were then screened for their reactivity with NBHA-modified AP DNA. Positive reactive ciones were subcloned and 4 monoclonaI antibody ctones were obtained. These were then examined for their reactivities with NBHA-modified AP sites using a competitive enryme immunoassay. The results are shown in Table 1. Clone 3 - 1 2 G - 1 2 H - 1 2 H was consistently more reactive with competing NBHA-modified AP D N A at all D N A concentrations tested; at 0.08 g g / m l of competing NBHA-modified AP DNA, 2 I % inhibition was observed for clone 3 - 1 2 G - 1 2 H - 1 2 H as compared to 12%, 0% and

TABLE 1 REACTIVITY OF VARIOUS NBHA P R E P A R A T I O N S

MONOCLONAL

ANTI-

Microtiter wells were coated with calf-thymus D N A containins 40 NBHA-modified A P sites per 100011 bp. 100 t~l of the NBHA-modified DNA at various concentrations was incubated with 100 #I of each of the an|ibody preparations for i h al 37 ° C. Aliquots were removed and added to the individual wells containing the adsorbed NBHA-modified A P DNA. Elisa assays were performzd using horse-radish peroxidase as the indicator enzyme Monoc!onal clone

c2 Inhibition of antibody reactivity (inhibitc, r concentration, g g / m l ) 5

3-12G-12H-t2H

-

2.5

1.25 0.625

(L3t2 0.16 0.08

64

55

44

33

31

21

4-8H-2D-12G

52

5l

48

43

28

21

12

4-SH-3H-6H

46

48

4t

43

15

12

0

4-SH-12H-9A

29

23

14

13

t2

(1

(~

0% for clones 4 - 8 H - 2 D - 12G, 4 - 8 H - 3 H - 6 H and 4 - 8 H - 1 2 H - 9 A , respectively. Based on these data, clone 3 - 1 2 G - i 2 H - ! 2 H was selected for further characterization.

Amibody specificio,. In o r d e r to examine antibody specificity, various haptens were used as competitors of the binding of antibody to NBHA-modified A P DNA. The results are shown in Fig, 1 and Table 2. 5'-Deox~'ribosyl-NBHA was about 20 times more effective as a competitor (IC~,: = 0.3 /xM) than N B H A deoxyribc,s y l N B H A and r i b o s y i - N B H A , which gave ICse' vatues of 5, 5 and 7 # M respectively (Table 2). Haptens lacking a nitro group such as benzyl-hydroxylamine, ribosyl-, deoxyribosyl- and 5'-phosphoribosyl-benzylhydrox-ylamine showed no inhibition up to 1 mM. Deoxyribose, ribose and deoEvribose 5-phosphate showed no inhibition even up to 10 mM (Table 2). Thus the nitro group appears to play a major role in antibody specificity. When D N A containing NBHA-modified A P sites was used to inhibit antibody reactivity, inhibition occurred with D N A containing as few as 2 NBHA-modified AP sites per 10 kitobase pairs at a concentration of 5 / x g / m l of D N A (Fig. 2). Fig. 3 shows that inhibition was proportional to the

25 / I00

100

8O

80

B

tl

e,,

6o I

60

-r

..-2.

40

4o

20

2o

.l 1 0 "~

10 °

101

tO

[Hapten] gM Fig. 1. Hapten inhibition of anli-NBHA-dRp antibody reactNity. UV-irradiated microtiter plates were previously adsorbed with calf-thymus DNA (27f} ng/well) containing 200 NBHA-modified AP sites per 1000q b~. Serial dilutions of haptens were incubated with l:100fl dih~don of antibody ((NH,DzSOa-precipitated supernatant) at 37"C for 1 h. 200 p.l of the bapten-antibody reaction mix was added to each well mr Elisa. After washing with PBS-Tween. the amount of bound antibody was determined with horse radish pcroxidase-conjugated goat anti-mouse IgG+ IgM (I :300 dilution} in PBS-Tween. Haptens ,:sed in the experiments were as follows: 5"-phosphoribosyt-NBHA ~II ). deoxTribosyl-NBHA ( z L ribosyl-NBHA ( ~ ~. ° ~BHA (*).

.1

.0!

102

1

I0

tDNA} ggimL Fig. 2. Inhibition of anti-NBHA antibody reactivity by calfthymus DNA containing NBHA-medifled AP sites, UVirradiated microtJter p!ates v,ere adsorbed with 2(~) /xt of calf-thymus DNA (t60 ng/ml/:or panel A and 270 n g / m l for panel B) containing NBHA-modified AP si~es ~'200 sites per !0(~-fl bp). Dilutions of caif-thymus DNA containing various amounts of NBHA-modified AP sites were incubated with a 1: lfffD dilutkm of the ae,~ibody preparation. Panel A" calf".hymus DNA containing {~ (A). 2 {~). 4 ( ~ ) 8 (e), 16 ( I ) NBHA-modified AP sites per 10{g~ bp. Panel B: calf-thymus DNA containing 25 (" k 50 {~J. lg}~)(o) or 20f} (11!) NBHAmodified AP sites per H~fRI0hp N}0 p.l of the NBHA-treated DNA/antibody reacfion~, ".,,-ere added to each welt and the bound antibody v.as detected with horse-radish peroxidaseconjugated anti-mouse igM diluted 1:30~g) in PBS-Tween as described in Materials and Methods,

TABLE 2 HAPTEN INHIBITION OF ANTi-NBHA-dRp ANTIBODY REACTIVITY WITH NB~'A-MGDIFIED AP SITES IN CALF-THYMUS DNA Hapten

~C~,, a

5'-Phosphoribosyl-NBHA Deoxyribosyl-NBHA RibosyI-NBHA

IL3 # M 5/.e M 5#M

NBHA 5'-Phosphoribosyt-BHA Deox3~ribosyl_BHA Ribosyl-BHA

7 ,a M '~ h >

BHA Deox3'ribose 5-phosphate Ribose Deox3'ribose

'~

too i r-i

Q

t &

~' IC5o is the concentration of inhibitor to effect ~ 50G inhibition of the antibody response. ~' No inhibition at 1 raM. c No inhibition al 10 raM.

O

t 2o [-[ o

o

.

.1

D

.

.

.

.

.

.

,~

1

,

,

r

r ~ f l l l

,

t0

. . . . . . .

100

Picomoles of AP sites

Fig. 3. Inhibition of antbNBHA-antibody reactivity by NBHA residues in DNA, The data were calculated from Fig. 2B. Calf-~hymus DNA containing 25 (5), 50 ( ~ 1~ I00 (®) and 200 ( D } NBttA-modified AP sites per 100¢g}bp.

258

total number of AP sites regardlcss of the extent of depurination of DNA used. Using a direct Elisa assay with native calfthymus DNA, the antibody reacted only with NBHA-modified AP sites but not with DNA containing AP sites, reduced AP sites or thymine glycols (Fig. 4) as well as fl DNA containing 5% uracil residues (data not shown). The immunoreactivity of the antibody with NBHA-modified AP sites was about 2-fold higher with heat denatured calf-thymus DNA than with native D N A (Fig. 5A). This preference was confirmed by comparing antibody reactivity with fl (single stranded) to that of calf-thymus (double-stranded) DNA (Fig. 5B). Here the signal obtained with NBHA-modified fl AP DNA was 2-fo~d higher than NBHAmodified calf-thymus DNA, again suggesting that the antibody reacts more effectively with the lesion in single-stranded D N A than doublestranded DNA.

.1.5 HI

1.0

6

0

2

4

6

D a m a g e s p e r 10,000 n u c l e o t i d e s Fig. 4. Specificity of the anti-NBHA-dRp antibody. UVirradiated microtiter plates were coated with 200 /.t[ of DNA (10 ,ttg/ml) contahling AP sites (~), reduced AP sites ([]). NBHA-modifled AP sites ([]) and thymine glycols (o). After blocking the microtiter plate with 1% calf serum in PBSTween. the monoclor~al antibody (1:250 dilution of the ascites) was added to the wells and the bound antibody was detected with horse-radish peroxidase-conjugated goat antimouse lgG + tgM at a 1:3000 dilution as described in Materials and Methods.

1.4

A

•

B

1.2 1.0

[]

0.8 0.6

6 0.4 0.2

0.0

, 5

i l0

15

DNA [ng per well]

5

I 10

f 15

20

A P S i t e s / 1 0 , 0 0 0 nucleotides

Fig. 5. Reactivity of anti-NBHA antibody towards singlestranded and duplex DNA. Panel A: Native ( m ) or heat-denatured (o) calf-thymus DNA containing NBHA-modified AP sites was adsorbed to microtiter platcs. Bound antibody ~.::~ detected with the horse-radish pcroxidase-conjugated goat anti-mouse IgM secondai.'y antibody as described in Materials and Methods. Panel B: 2(10 ~tl of calf-thymus (double-stranded. e) and fl (single-stranded. • ) DNA containing NBHA-modified AP sites (10 ~ g / m l ) was adsorbed onto the UV-irradiated microtiter piate. The number of detectable NBHA residues was measured with the anti-NBHA-dRp monoclonal antibody.

Antibody sensitit'ity. Calf-thymus D N A containing on the average 2 NBHA-modified AP sites per 10000 bp gave an O.D. of 0.65 after a 2-min reaction time with substrate (Fig. 4). It appeared, therefore, that the antibody should be able to detect 1 AP site per 10000 nucleotides. In order to test this, fl D N A containing 1-8 AP sites per 10 000 nucleotides was prepared. As Fig. 6 shows, the antibody signal with fl D N A was found to be linearly proportional to the number of AP sites. Further, the anti-NBHA antibody was able to detect 1 AP site per 10000 bases (Fig. 6). Since 70-100 ng of D N A was adsorbed to each well, the antibody was abie to detect approximately 10 femtomoles of AP sites. Reaction of the antibody with damaged f l DNA. A major class of lesions produced by ionizing radiation is the so called alkali-labile lesions or abasic sites (Von Sontag, 1987). In order to measure antibody reactivity with irradiated DNA, fl D N A was irradiated in phosphate buffer ~nd

259 1.5

_=

1.0

6

0.5

//

0.0 0

2

I

I

4

6

A P siles per 10,000 nucleotides Fig. 6. Sensitivity of anti-NBHA-antibody towards singlestranded ft DNA containing NBHA-modified AP sites, fl (single-stranded) DNA containing NBHA-modified AP sites ( I - 8 sites per 10000 nucteotides, 10 g g / m l ) was adsorbed onto the UV-irradiated microtiter plate. The reactivity of anti-NBHA was measured as described in Materials and Methods.

an inhibitor than its corresponding nucleoside or free base ('Fable 2). No cross reactivity of the antibody was observed with DNA containing unmodified AP sites, thymine glycols or uracil (Fig. 4). However, once the AP site was modified with NBHA, the monoclonal antibody easity detected femtemole levels of AP sites in DNA. This sensitivity is comparable to that observed with other antibodies elicited against modified DNA bases (Haugen et al., 1981; Hubbard et at., t989a,b; for review see Poirer, !984). Since the detection of AP sites involved the reaction of AP sites with NBHA, the antibody should be able to detect any tesions that contain aldehyae groups, as predicted by the chemistry, of the NBHA. Therefore, it is likely that the number of antibody-reactive sites detected in X-irradiated DNA includes not only AP sites (alkali-labile lesions), but also strand breaks containing AP sites at one terminus, as well as formamidopyrimidine. AP sites and strafed breaks that do not contain an aldehyde group (Von Sontag, 1987;

1.2

modified with NBHA. The estimated detectable limit of antibody reactive sites was obtained with an X-ray dose of approximate!y !).5 Gy (Fig. 7). Since the X-ray source was not amenable to low dose studies, calf-thymus DNA was irradiated with 6°Co y-rays in order to push the sensitivi~, of the anti-NBHA antibody reaction. The antibody signal produced was linearly proportional to the dose of 6°Co y-rays and AP sites were measurable at a dose as low as 0.1 Gy (Fig. 8).

1.0

m

"J"

0.6

O

0.4

Discussion

The monoclonal antibody elicited against the NBHA-13SA conjugate is highly specific for the NBHA residues as measured by either hapten inhibition (Fig. 1 and Table 2) or by inhibition using DNA containing NBHA-modified AP sites (Figs. 2 and 3)° The antibody appears to be specific for the nitro group since no inhibition was observed with benzyl hydro~lamine (Tabte 1). Additional determinants appear also to be present on the DNA backbone since the 5'-phosphorylated hapten was 20-fold more effective as

1

2

3

X r a y Dose (G y) Fig. 7. Production of antibody-reactive sites in X-irradiated fl DNA. Single-stranded ft DNA was X-irradiated in phosphate buffer at a concentration of 30 ~ g / m t at the indicated X-ray dose. The X-irradiated DNA was treated with 10 mM NBHA for 2 h at rcmm temperature, and excess NBHA was removed by ethanol precipitation. The DNA was ~ben adsorbed onto microtiter wells and the reactivity of anti-NBHA-antibody towards the X-irradiated fl DNA was determined as described in Materials and Methods.

260

thus reducing the lo~s of labile AP sites during purification of D N A f'om cells. This method, therefore should have broad applicability for the detection of abasic site,, in D . . .Y. A ,~*-,~r,,,~,,,J' fiofit damaged cells.

8 ¢v

7

f-

6

s

Acknowledgements

4

The authors like to thank Dr. Robe; Melamede for helpful discussions as well as the technical assistance of Ms. Grace Sterling and Lisa Rayburn.

,m

3

~a

2

/

References

o 0.2

0.4

0.6

o.g

1,0

6 ° C o Dose (Gyt Fig. 8. Production of antibody-reactive sites in ~"Co y-rayirradiated calf-thymus DNA, Calf-thymus DNA was iradiated with ~"Co y-rays in phosphate buffer at the indicated dose. The irradiated DNA was treated with 10 mM NBHA for 2 b at room temperature and the number of anti-NBHA reactive sites was determined as described in Materials and Methods.

Povirk and Steighner. 1989) would not be detected. The fact that more than one type of X-ray damage can be measured by this assay suggests that it could provide a simple and s.?nsitivz method for the detection of X-ray damage or, perhaps, a general method for monitoring X-ray exposure. The antibody assay is equal in sensitivity to other procedures that measure AP sites, including alkali elution (Brent et al., 1978), D N A unwinding (Birnboim and Jevcak, 1981; Kohn et al., 1981), 3Zp-posttabeling (Weinfeld et al., 1990) as well as an aldehyde-reactive probe assay (Kubo et al., in preparation). Some of these methods involve laborious procedures (Brent et al., 1978; Birnboim and Jevcak 1981; Kohn et al., 1981; Weinfeld et al., 1990) and others require the use of radioactive materials (Talpaert-Borle and Liuzzi, t983; Liuzzi and Talpaert-Borle, 1988; Weinfeld et al., 1990). On the other hand, the antibody assay is simple and does not require radioactive isotopes. An additional advantage of this assay is that AP sites are stabilized by N B H A

Birnboim, H.C.. and J.J. Jevcak (1981) C!uorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation. Cancer Res., 4I. 1889-1892. Brent. T.P, G.W, Te~-bor and N.J. Duker (t978) Lesions in alkylated DNA determined by susceptibility to alkali, apurinic endonuclease or N-glyeosidase. in: P,C. Hanawalt. E.C. Friedberg and C.F. Fox (Eds.), DNA Repair Mechanisms, Academic Press. New York. pp. 19-22. Friedberg, E,C. (1985) DNA Repair. Freeman. New York. Haltoram M.J.. and C.W. Parker (t966) The preparation of nucleotide-protein conjugates: Carbodiimides as coupling agents. J. lmmunoL, 96. 373-378. Haugen. A,. J.D. Groopman. I.C. Hsu. G.R. G,3odrich. G.R. Wogan and C.C. Harris (1981) ?'donoclonal antibody to aflatoxin Bl-modifled DNA detected by enzyr~e immunoassay, Proc. Na;J, Acad. Sci, (U.S.A.). 78. 4t24-4t28. HolmdahL R.. T. Moran and M. Andersson (~985) A rapid and efficient immunization protocol for production of monoelonal antibodies reactive with autoantigens. J. !mmunol, Methods. 83. 379-384. Hubbard, K.. H. Huang. M.F. Laspia, H. Ide. B.F. Erlanger and S.S. Wallace (19SPa) Immunochemical quantitation of thymine glycol in oxidized and X-irradiated DNA. Radiat. Res.. 118, 257-268. Hubbard. K., H. Ide. B.F. Erlanger and S.S. W~llace (1989b) Characterization of antibodies to dihydrothymine, a radiolysis product of DNA, Biochemist~'. 28. 4392-4387. Katcher. H.L.. and S.S. Wallace Q983) Characterization of the Escherichia coli X-ray endonaclease, endonuclease III, Biochemistry, 22, 4071-4081. Kohn, K.W., R.A.G. Ewig, LC. Erickson and L.A. Zwelting (t981) Measurement of strand breaks and cross-links by alkaline elution, in: E.C. Friedberg and P. Hanawalt (Eds.), DNA Repair: A Laborato~" Manual of Research Procedures, Vot. 1, Part B, Marcel Dekker, New York, pp. 379-401. Kow, ~,W. (1989) Mechanism of action of Escherichia coil exonuc!ease It!, Biochemist~', 28, 3280-3287.

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Kow, Y.W., and S.S. Wallace 1!987) Mechanism of action of Escherichia coli endonuclease tll, Biochemistry. 26. 82008206. LindahL T.. and B. Nyberg 11972) Rate of depurinatioe of native deoxyribonucleic acid, Biochemistry, 11, 3610-3618. Liuzzi, M.. and M. Talpaert-Borle 11988) Characterizatior~ of damage in gamma-irradiated and OsO4-treated DNA using methoxyamine, Int. J, Radiat. Biol., 54, 709-722. Loeb, L.A., and B.D. Preston 11986) Mutagenesis by apurinic/apyrimidinic sites, Annu. Rev. Genet., 20, 20t230. Maniatis, T., E.F. Fritsch and J. Sambrook 11982~ Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 461 pp. Messing, J. 11983) New MI3 vectors for cloning, Methods EnzymoL, 101, 20-79. Paquette, Y., P. Crine and W.G. Verly (t972~ Properties of the endonuclease for depnrinated DNA from Esckerichia coli. Can. J_ Bioehem., 50, t199-t209. Poirer, M.C. 1t984) The use of carcinogen--.DNA adduct antisera for quantitation and localization of genomic damage in animal models and the human population. Environ. Mutagen.. 6, 879-887. Povirk, LF., and RJ. Steighner 11989) Oxidized apurinic/apyrimidinic sites formed in DNA by oxidation. Mutation Res.. 214. 13-22. Rabow. J.. J. Stubbe, J.W. Kozarich and J.A. Gerlt (1986}

Identification of lht- alkaline-labile product accompanyiag cytosine release during Neomycin-mediated degrada~.L'm of d(CGCGCG), J. Am. Chem. Soc.. 1!:t8. 7t30-7131. Sharon. J., S.L. Morrison and E. Kabat 11979) Detection of specific hybridoma clones by replica immunoa&-(~rp~5~m,A their secreted antibodies, Proc- r":.,:L Acad. Sci. ~U.S.A.), 76. 1420-t424. Talpaert-Borle. M., and M. Liuzzi (19831 Reaction of apurinic/apyrimidinic site', with [14Clmethox3"amine: A method for the quantitative assay of AP si~.es in DNA. Biochim. Biophvs. Acta, 740. 410-4t6, Von Sontag. C. 11987) The Chemical Basis of Radiation Bielogy. Taylor and Francis. Ltmdon. Wallace. S.S. 11988) Detection and repair of DNA base damages produced by ionizing radiation, En,,dron. MoL Mutagen., 12. 431-477. Weinfetd. M., M. Liuzzi and M.C, Paterscm 1t99,9) Response of ; hage T4 polynuc!eotide kinase ~owards dinucleotides containing apurinic sites: Design of a ~-~P-postlabefing assay for apurinie sites in DNA, Biochemist~'. 29, I7371743. Weiss. B., and L. Grossman 119S7,~ Phosphodiesterases involved in DNA repair, Adv. En~¢moL. 60, 1-34. Zouali, M.. and B.D. Stoilar 1!9~il} A rapid ELISA far measurement of antibodies to ~meieic acid antigens using UVtreated polystyrene micr~'pia~_es, J. Immanol. Methods. 90, i05- i 10.