immunoassays using capillary electrophoresis laser induced fluorescence detection for dna adducts

Analytica Chimica Acta 500 (2003) 13–20

Immunoassays using capillary electrophoresis laser inducedfluorescence detection for DNA adducts

Hailin Wanga, Meiling Lua, Nan Meib, Jane Leeb, Michael Weinfeldb, X. Chris Lea,∗a Environmental Health Science Program, Department of Public Health Sciences, University of Alberta,

10-102 Clinical Sciences Building, Edmonton, Alta., Canada T6G 2G3b Experimental Oncology, Cross Cancer Institute, Edmonton, Alta., Canada T6G 1Z2

Received 29 April 2003; received in revised form 20 May 2003; accepted 20 May 2003

Abstract

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage.The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assaysfor DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillaryelectrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assaysusing CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescentlylabeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay.Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes betweenthe MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing bindingstoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay,a fluorescently labeled secondary antibody fragment F(ab′)2 was used as an affinity probe to recognize a primary antibodythat was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies wasseparated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assaywas used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA fromA549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1�M) showed a clear dose–responserelationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentiallyuseful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.© 2003 Elsevier B.V. All rights reserved.

Keywords: DNA adducts; Immunoassays; Capillary electrophoresis; Laser induced fluorescence; Benzo[a]pyrene; Affinity interaction;Binding stoichiometry; Environmental carcinogen; DNA repair

1. Introduction

Capillary electrophoresis (CE) is a high-efficiencyanalytical separation technique[1] that has found

∗ Corresponding author. Tel.:+1-780-492-6416;fax: +1-780-492-7800.E-mail address: [email protected] (X.C. Le).

tremendous applications in many areas of chemical,biological, environmental, pharmaceutical, and healthresearch (see[2–6] for reviews). Laser induced flu-orescence (LIF) offers extremely sensitive detectionfor CE [7–12], leading to a single molecule detectionlimit [9–12]. However, covalent labeling of trace lev-els of analyte in the presence of sample matrix is verydifficult because of non-quantitative derivatization

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0003-2670(03)00631-7

14 H. Wang et al. / Analytica Chimica Acta 500 (2003) 13–20

and matrix interference on the labeling reaction, lead-ing to potential problems in real sample analysis usinglaser induced fluorescence.

Immunoassays have evolved from clinical analysisto a wide variety of biochemical and environmental ap-plications[13,14]. Immunoassays are usually specificbecause antibodies generally possess high specificity,targeting unique structural elements (epitopes) in anti-gens. Innovative combinations of immunoassays withcapillary electrophoresis/chromatography separationhave been shown to be extremely useful for bioanal-ysis. For example, a number of research groups havecombined immunoassays with CE/LIF for the deter-mination of peptides and proteins[15–30], therapeuticdrugs [31–39], natural toxins[40–42], and bindingprocesses[43–46], to name a few. These techniquestook advantage of high sensitivity of LIF detectionand good selectivity by immuno-recognition and CEseparation. Thus, they could potentially circumventproblems that otherwise would be encountered whenimmunoassays and CE/LIF would be used separately.

The objective of this paper is to describe immunoas-says using CE/LIF for DNA damage analysis. Wehave developed both competitive and non-competitiveimmunoassays, and have demonstrated their appli-cations to the determination of DNA adducts ofbenzo[a]pyrene diol epoxide (BPDE). BPDE is areactive metabolite from benzo[a]pyrene, a commonenvironmental carcinogen that is generated from in-complete combustion of organic materials. BPDE isknown to form DNA adducts through electrophilicreaction with guanine. Adduct formation at particularsites in DNA can lead to the inappropriate activation ofoncogenes and to the inactivation of tumor-suppressorgenes. The critical role played by DNA damagemakes it essential to develop sensitive and reliableassays for DNA damage. Such assays should assistus to further our understanding of the biological con-sequences of DNA modification and enzymatic repairpathways.

2. Experimental

2.1. Instrumentation

A CE/LIF system, built in this laboratory, isschematically shown inFig. 1. A high voltage power

Fig. 1. Schematic diagram showing a lab-built capillary elec-trophoresis laser induced fluorescence system. CE stands for cap-illary electrophoresis and PMT is photomultiplier tube. A greenHeNe laser (543.5 nm) was used in this work.

supply (Model 1000R, Spellman, Plainview, NY) wasused for the electrophoresis through a fused-silicacapillary. The detection end of the capillary wasinserted into a quartz sheath flow cuvette (NSG Pre-cision Cells, Farmingdale, NY). Fluorescence wasexcited using a green (543.5 nm) HeNe laser (MellesGriot, Irvine, CA). Fluorescence was collected at 90◦with respect to the laser beam using a 60× micro-scope objective (0.7 NA, Universe Kogaku, OysterBay, NY). It was filtered through a 580DF40 band-pass filter (Newport, Irvine, CA), passed through apinhole, and detected with a photomultiplier tube(Model R1477, Hamamatsu, Japan). A Power Mac-intosh computer equipped with a NI-DAQ board andapplication software written in LabView (NationalInstruments, Austin, TX) was used to control theinstrument and to acquire data.

Fused-silica capillaries (30 cm long, 20�m i.d.,150�m o.d.) from Polymicro (Phoenix, AZ) wereused for separation. Samples were electrokineticallyinjected into the capillary using an injection voltageof 15 kV for 5 s. The separation was carried out atroom temperature with an electric field of 500 V/cm(separation voltage of 15 kV). The running buffer was1× Tris–glycine (25 mM Tris, 192 mM glycine, pH8.3). The capillary was regularly washed after threesample injections with 0.02 M NaOH electrophoret-ically (15 kV for 7 min) followed by electrophoresisusing water and the running buffer for 7 min. Allcapillary electrophoresis data were analyzed usingIgorPro software (WaveMetrics, Lake Oswega, OR).

H. Wang et al. / Analytica Chimica Acta 500 (2003) 13–20 15

2.2. Reagents and standards

Three primary antibodies, 8E11, 5D11, and E5,recognizing BPDE-DNA adducts, were tested. Mousemonoclonal antibodies 8E11 and 5D11 were pur-chased from BD PharMingen (San Diego, CA), and E5was kindly provided by Dr. William Watson (SyngentaCTL, Cheshire, UK)[47]. Fluorescently labeled goatanti-mouse IgG secondary antibodies were obtainedfrom Molecular Probes (Eugene, OR) and Calbiochem(La Jolla, CA). Polyclonal rabbit IgG antibody waspurchased from Calbiochem. Solvents and other bio-chemicals were obtained from Sigma (St. Louis,MO), Fisher Scientific (Pittsburgh, PA), and VWRCanlab (Mississauga, Ontario, Canada). (±)-r-7,t-8-Dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] was supplied by the NationalCancer Institute Chemical Carcinogen ReferenceStandard Repository (Midwest Research Institute,Kansas City, MO).

Caution: BPDE is carcinogenic. Appropriate careshould be exercised when handling BPDE.

Two 16mer oligonucleotides with the sequence5′-CCCATTATGCATAACC-3′ were synthesized byUniversity Core DNA Services (University of Calgary,AB, Canada). They were purified by sequencing poly-acrylamide gel electrophoresis prior to use. One of the16mer oligonucleotides was labeled with tetramethyl-rhodamine (TMR) at the 5′ end, and the other wasnot labeled with a dye. Both 16mer oligonucleotideswere reacted with BPDE to yield the BPDE-N2 de-oxyguanosine (dG) adduct as described previously[48]. Briefly, each 16mer oligonucleotide was di-luted in 20 mM phosphate buffer (pH 11) containing1.5% triethylamine, to a concentration of 60�M ina volume of 400�l. To the oligonucleotide solutionwas added 40�l, 3 mM BPDE in DMSO. This cor-responded to a BPDE:oligonucleotide molar ratio of5:1. The reaction was carried out at room temperaturefor 20 h, in the dark with gentle shaking. The majorproduct was thetrans-(+)-BPDE-dG adduct, whichwas separated using reversed-phase HPLC[48]. Thefraction corresponding to thetrans-(+)-BPDE-dGadduct was collected for subsequent use. The concen-trations of the BPDE-16mer and TMR-BPDE-16merwere estimated using absorbance at 260 nm. Purity ofthe modified oligonucleotides was confirmed by gelelectrophoresis and32P post-labeling.

2.3. Cell treatment and DNA samples

Cellular DNA containing BPDE-DNA adducts wasobtained from A549 human lung carcinoma cells incu-bated with BPDE. Prior to incubation with BPDE, thecells were maintained in DMEM/F12 medium (GibcoBRL, Gaithersburg, MD) supplemented with 10% fe-tal bovine serum. The cells were seeded at 1× 105

cells per plate and maintained at 95% humidity and5% CO2 for 20 h prior to the addition of BPDE. Themedium containing BPDE was then added, and thecells were further incubated for 2 h. The cells weresubsequently washed with phosphate buffered saline(PBS) prior to the addition of DNAzol lysis reagent(Gibco BRL) to facilitate cell lysis and DNA extrac-tion. DNA was precipitated with an ice cold 99.9%ethanol and washed with a cold 70% ethanol. The fi-nal DNA pellet was dissolved in distilled deionizedwater and DNA concentration was measured with ab-sorbance at 260 nm. An aliquot of the DNA samplewas heat-denatured at 95◦C followed by cooling onice. An aliquot of the denatured DNA was used forthe analysis of BPDE-DNA adducts.

2.4. Antibody binding stoichiometry

Binding stoichiometry was studied by using mousemonoclonal antibody (MAb) 8E11 (33 nM) andTMR-labeled BPDE-16mer oligonucleotide (35 nM).They were incubated in 20�l of Tris–glycine buffer(pH 8.3) at room temperature for 30 min. An aliquotwas subjected to CE/LIF analysis. The running bufferwas 1× Tris–glycine (25 mM Tris, 192 mM glycine,pH 8.3). The 1:1 and 1:2 complexes were separatedfrom each other and from the unbound 16mer oligonu-cleotide using CE and detected with LIF. Furtherexperiments also involved the addition of unlabeledBPDE-16mer oligonucleotide (50 nM–5�M) to studycompetitive binding.

2.5. Competitive immunoassay

Competitive immunoassays were carried out asdescribed previously[48,49]. The TMR-labeledBPDE-16mer oligonucleotide was used as the probe(tracer). The oligonucleotide probe (60 nM), rab-bit IgG (0.7�M), mouse monoclonal antibody8E11 (2.7 nM), and DNA samples (e.g. 80�g/ml of


Fig. 2. Schematic diagram showing non-competitive immunoassay for DNA damage. A primary antibody binds to the specific DNA lesionand is itself then bound by a fluorescently labeled secondary antibody.

DNA from A549 cells) were incubated in 20�l ofTris–glycine buffer (pH 8.3) at room temperaturefor 30 min. Rabbit IgG did not have specific bindingwith DNA and it was added to enhance the formationand stability of the immuno-complexes[50]. Sampleswere subjected to CE/LIF analysis to detect both freeand antibody-bound fluorescent BPDE-16mer probe.

2.6. Non-competitive immunoassay

A schematic of non-competitive assay for DNAdamage is shown inFig. 2. An aliquot of a DNAsample was heat-denatured at 95◦C followed bycooling on ice. The DNA sample was diluted in a1× Tris–glycine buffer (pH 7.5). Mouse anti-BPDEantibody 8E11 was used as the primary antibody,and the Alexa Fluor® 546 labeled fragment F(ab′)2of goat anti-mouse IgG (Molecular Probes) was usedas the secondary antibody. The denatured DNA sam-ple was mixed with human IgG, primary antibodyand secondary antibody in sequence. The mixturecontained∼80�g/ml DNA, 2.0�g/ml primary an-tibody, 2.0�g/ml secondary antibody fragment, and10.0�g/ml human IgG in 1× Tris–glycine buffer (pH7.5). Addition of human IgG was to stabilize the an-tibody and enhance the stability and formation of the

ternary complex between the antibodies and the DNAadduct. The sample was incubated at room tempera-ture for 30 min prior to CE/LIF analysis. The runningbuffer was 1× Tris–glycine (25 mM Tris, 192 mMglycine, pH 8.3).

3. Results and discussion

3.1. Competitive immunoassay using CE/LIF

Competitive immunoassays are based on competi-tive binding of two antigens (or haptens) to a limitingamount of antibody. Typically, one antigen is labeledand is used as a detection probe (or tracer). The otherantigen (usually the target analyte) is not labeled, andcan be indirectly determined by monitoring the rel-ative intensity of signals produced from the labeledfree (Ag∗) and the antibody-bound (AbAg∗) antigen,as indicated in the following equilibrium.

Ab + Ag∗ + Ag = AbAg∗ + AbAg (1)

where Ab is unlabeled antibody, Ag∗ is labeled anti-gen, Ag is unlabeled antigen, and AbAg∗ and AbAgare antibody complexes with labeled and unlabeledantigen, respectively.


The commonly used primary antibody for im-munoassays is immunoglobulin G (IgG), which isbidentate, capable of binding with two antigens.However, few studies have been able to reveal de-tailed binding stoichiometry because the multiplecomplexes between an antibody and an antigen areusually difficult to differentiate. This is especially thecase with small analyte molecules (e.g.<1000 Da)that are usually analyzed by competitive immunoas-says. The separation of the unbound fluorescentprobe (e.g. 1000 Da) from the antibody-bound probe(∼151,000 Da) is relatively easy using CE becauseof the large mobility differences between the freeand antibody-bound probes. However, the separa-tion between the 1:1 (∼151,000 Da) and the 1:2(∼152,000 Da) complexes is more difficult.

To separate the 1:1 and 1:2 complexes of verysimilar size, we made use of a highly charged probeto bind with the antibody. Because both the size andcharge of the molecules contribute to CE separa-tion, additional charges introduced to the complexdue to binding make the separation of the multiplecomplexes possible. We have designed fluorescentoligonucleotide probes that contain a single BPDEadduct which can be recognized by an antibody.

The 1:1 and 1:2 complexes between antibody andthe TMR-BPDE-16mer oligonucleotide can be sep-arated because of the additional charges introducedby the oligonucleotide. This provided a convenientapproach for studying the binding stoichiometry.Fig. 3A shows a typical electropherogram fromCE/LIF analysis of a mixture containing 33 nM anti-body and 35 nM TMR-BPDE-16mer (16mer∗). Theantibody complexes with one 16mer∗ (1:1 complex,peak 1) and two 16mer∗ (1:2 complex, peak 2) arebaseline resolved, clearly demonstrating the bindingstoichiometry.

Results in Fig. 3B–D further show competitivebinding behavior between an antibody and the com-peting ligands, one fluorescently labeled (16mer∗ ata constant 35 nM concentration) and the other un-labeled (16mer, at varying concentrations of 0.05,0.5 and 5.0�M). In the absence of the unlabeledBPDE-16mer, the 1:1 complex [Ab(16mer∗), peak 1]dominates (Fig. 3A). With increasing concentrationsof the competing unlabeled 16mer, the 1:1 complex(peak 1) decreases, a typical competitive immunoas-say behavior. However, the 1:2 complex (peak 2) does

Fig. 3. Typical electropherograms from CE/LIF analyses of mix-tures containing 35 nM TMR-BPDE-16mer∗, 33 nM (5�g/ml) an-tibody, and increasing concentrations of unlabeled BPDE-16mer(0, 0.05, 0.5, and 5�M). CE separation was carried out us-ing a fused-silica capillary (30 cm long, 20�m i.d. and 150�mo.d.) and a Tris–glycine buffer (pH 8.3). Fluorescence was ex-cited at 543.5 nm and detected at 580 nm. Peak 1 corresponds toAb(16mer∗) complex (1:1 stoichiometry), peak 2 corresponds toAb(16mer∗)2 and Ab(16mer∗)(16mer) complexes (1:2 stoichiom-etry), and peak 3 corresponds to the unbound 16mer∗.

not follow the conventional competitive immunoas-say pattern. At a low concentration (0.05�M) of thecompeting unlabeled 16mer, the 1:2 complex (peak2) does not decrease. Instead, it increases initially(Fig. 3B). This behavior is uniquely observed. It isdue to the formation of Ab(16mer∗)(16mer) com-plex, co-binding of both the labeled and unlabeledligands with the same antibody[51,52]. This obser-vation is different from that of traditional competitiveimmunoassays where only the mixture of antibodycomplexes are commonly detected and the separationof the 1:1 and 1:2 complexes is not available for ex-amination. The use of BPDE-16mer as a probe, asdescribed in this study, provided a unique ability tomonitor both the 1:1 and 1:2 complexes in the system.

Having examined the competitive binding andco-binding of the BPDE-oligonucleotide probes with


the antibody, we have further applied these findingsto performing competitive assays for BPDE-DNAadducts. We have successfully demonstrated an appli-cation of a competitive assay for the determination ofBPDE-DNA adducts in A549 human lung carcinomacells that were incubated with varying concentra-tions of BPDE (1–10�M) and have achieved a cleardose–response relationship[48]. The 1:1 and 1:2complexes were baseline resolved, and their fluores-cence intensities were used for the quantitation ofBPDE-DNA adducts in DNA samples isolated fromA549 cells.

3.2. Non-competitive immunoassay for BPDE-DNAadduct in cellular DNA

The competitive assay described above used an an-tibody and a labeled probe to determine unlabeledDNA adduct in cell samples. Competitive assays areusually limited by background and are less sensitivethan direct assays of non-competitive format. Thus,we have further developed a non-competitive assayand demonstrated its application to the determinationof BPDE-DNA adducts in cells. The non-competitiveimmunoassay uses two antibodies, one of which is flu-orescently labeled secondary antibody and the otheris unlabeled primary antibody 8E11 that is specificfor BPDE damage (Fig. 2). Appropriate antibodies arecrucial in order to achieve high sensitivity and speci-ficity of an assay.

We have evaluated a number of fluorescently la-beled secondary antibodies (Table 1) and primaryanti-BPDE antibodies with regards to relative fluores-cence intensity and binding specificity. A comparisonof six fluorescently labeled, goat anti-mouse anti-

Table 1Comparison of fluorescently labeled secondary antibodies as affinity probes for non-competitive immunoassay of DNA adduct

Secondary antibody Cat. No. Source Relative fluorescenceresponse (%)

Alexa Fluor® 546 F(ab′)2 fragment of goat anti-mouse IgG(H+ L) A-11018 Molecular Probes 100Alexa Fluor® 546 goat anti-mouse IgG(H+ L) A-11003 Molecular Probes 52Alexa Fluor® 546 goat anti-mouse IgG(H+ L), highly cross-adsorbed A-11030 Molecular Probes 45Alexa Fluor® 546 goat anti-mouse IgG1(�1) A-21123 Molecular Probes 44Tetramethylrhodamine goat anti-mouse IgG(H+ L) T-2762 Molecular Probes 15Tetramethylrhodamine goat anti-mouse IgG 401217 Calbiochem 12

Relative fluorescence intensities of the ternary complex composed of secondary antibody, primary antibody (8E11), and BPDE-DNAadducts were compared. BPDE-DNA adducts were isolated from A549 cells incubated with 1�M BPDE for 2 h.

bodies shows that the highest fluorescence responsewas obtained when the Alexa Fluor® labeled, F(ab′)2fragment of goat anti-mouse antibody, was used asthe secondary antibody to perform the immunoassay.Therefore, we choose this antibody fragment for thenon-competitive assay.

We have also compared three available mouseanti-BPDE primary antibodies, E5, 5D11, and 8E11.Although both E5 and 5D11 antibodies recognizedand bound with BPDE-DNA adducts in cellularDNA from BPDE-treated A549 cells, they alsoshowed cross-reaction with control DNA from A549cells that were not exposed to BPDE. The antibodycross-reaction could result in elevated background,not desirable for trace analysis. Antibody 8E11, how-ever, had very little cross-reaction with the controlDNA, while it specifically recognized the BPDE-DNAadducts in cellular DNA. Because of its higher affinityand specificity for BPDE-DNA adducts, mouse mon-oclonal antibody 8E11 was chosen for immunoassaysdescribed in this study.

Fig. 4 shows a series of electropherograms fromthe analysis of BPDE-DNA adducts in A549 cells thatwere exposed to BPDE (0.032–1.0�M) for 2 h. Thecomplex between DNA adduct and antibodies (peak2) is well separated from the antibodies (peak 1). Thesecondary antibody and its complex with the primaryantibody are not resolved and appear overlapped (peak1). This is not a problem because the assay for DNAadducts is based on the complex (peak 2) that is wellresolved.

It is evident fromFig. 4 that the signal of the DNAadducts (peak 2) increases with increasing concentra-tion of BPDE that was incubated with the A549 cellsfor 2 h, demonstrating a clear dose–response relation-


Fig. 4. Representative electropherograms showing analysis ofBPDE-DNA adducts in A549 cells. The A549 cells were incu-bated with 0.032–1.0�M BPDE for 2 h before the cellular DNAwas extracted for analysis. A 30 cm long, 20�m i.d., 150�m o.d.,fused-silica capillary was used for separation with an electrophore-sis buffer containing 25 mM Tris and 192 mM glycine at pH 8.3.Separation was performed with an electric field of 500 V/cm. Flu-orescence was excited at 543.5 nm and detected at 580 nm. Peak 2corresponds to the antibody complex with the BPDE-DNA adductsand peak 1 corresponds to the excess antibodies.

ship. The signal from the unexposed DNA is verylow, indicating very little non-specific binding (littlecross-reaction).

With the high sensitivity, we were able to detectthe BPDE-DNA adducts in A549 cells incubatedwith as little as 32 nM BPDE for 2 h. The detec-tion limit, based on a signal-to-noise ratio of 3, is∼3 BPDE-DNA adducts per 109 nucleotides. This iscomparable to the most sensitive method availablefor detecting DNA damage, namely32P post-labelingassay[53,54]. The 32P post-labeling assay requiresthe digestion of DNA and the use of radioactive ma-terial. The non-competitive immunoassay describedhere measures DNA damage more directly and doesnot require digestion of the cellular DNA, therebyreducing any artifacts that may be produced duringDNA digestion and treatment procedures. It is spe-cific for the DNA lesion of interest. This assay will

be useful for studying cellular repair of DNA damageand for monitoring human exposure to environmentalcarcinogens.

4. Conclusion

Both competitive and non-competitive immunoas-says can be used for the determination of specific DNAlesions using capillary electrophoresis with laser in-duced fluorescence detection (CE/LIF). Using a flu-orescently labeled 16mer oligonucelotide containinga single BPDE adduct as the probe, we were able tostudy the binding stoichiometry of this oligonucelotidewith a monoclonal antibody. Capillary electrophoresisenabled the baseline resolution of the 1:1 and 1:2 com-plexes of the antibody and the oligonucleotide probe.Laser induced fluorescence provided detection of theantibody-bound and unbound oligonucleotide probe.Other oligonucleotides containing specific DNA le-sions can be fluorescently labeled and used as tracers(fluorescent probes) for competitive assays of otherDNA lesions.

The combination of non-competitive immunoas-says with CE/LIF can further improve the detectionlimits for DNA damage analysis. For the determi-nation of BPDE-DNA adducts, a mouse monoclonalantibody was used as the primary antibody to bindwith BPDE-DNA adducts. A fluorescently labeledsecondary antibody fragment F(ab′)2 recognizing theprimary antibody served as a fluorescent probe for de-tection. The ternary complex composed of the DNAadducts and the primary and secondary antibodieswas separated from the excess antibodies by CE anddetected by LIF. This approach combined good selec-tivity (antibody specificity and CE separation) withhigh sensitivity LIF detection. The methodology canbe applied to assays for other DNA lesions providedthat antibodies to the specific lesions are available.The affinity and specificity of an antibody are impor-tant attributes to the determination of minute amountsof specific DNA lesions.

Acknowledgements

This work was supported by grants from the NaturalSciences and Engineering Research Council of Canada


(NSERC), National Institutes of Health, National Can-cer Institute of Canada, the Canada Research ChairsProgram, and the Canadian Water Network NCE.

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