APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2698-27050099-2240/93/082698-08$02.00/0Copyright © 1993, American Society for Microbiology

Production and Characterization of Anti-DNA-RNAMonoclonal Antibodies and Their Application in

Listeria DetectionISMAIL FLISS,1 M. ST. LAURENT,2'3 E. EMOND,1 R. LEMIEUX,2'3

R. E. SIMARD,1 A. ET1RIKI,1 AND S. PANDIAN1*

Departement de Sciences et Technologie des Aliments, Centre de Recherche en Science et Technologie duLait, Pavillon Paul-Comtois, 1 and Departement de Biochimie, Faculte de Sciences et Genie,3

Universite Laval, Sainte-Foy, Quebec, Canada GlK 7P4, and Blood Services,Canadian Red Cross, Quebec City Center, Quebec, Canada G6V 4M32

Received 19 November 1992/Accepted 23 May 1993

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with twodifferent heteropolymers as antigens, cDNA-mRNA and +X174 DNA-RNA heteroduplexes. The former wassimpler to prepare. Both had shown similar immunogenicities. Two different immunoglobulin M MAbs wereisolated. The 20D3 MAb, generated with the +X174 DNA-RNA hybrid, showed association constants of 1.05x 1o12, 2.12 x 1010, and 1.68 x 107 for the antigens +X174 DNA-RNA, cDNA-mRNA, and poly(rA)-poly(dT),respectively. The 6B5 MAb, obtained with the cDNA-mRNA hybrid, showed association constants of 1.59 xl0S, 5 x 1012, and 7.1 x 108 for the above-described antigens, respectively. With the 20D3 MAb, animmunoassay was developed for the detection ofListeria DNA-RNA hybrids. In brief, a biotinylated rRNA geneprobe specific for the genus Listeria was hybridized with rRNA in the solution phase. The hybrids thus formedwere then captured in microtiter plate wells precoated with the purified 20D3 MAb, and the probe-targethybrids were detected with a streptavidin-alkaline phosphatase conjugate. This assay was shown to be specificfor the genus Listeria and highly sensitive, allowing the detection of as little as 2.5 pg of target rRNA.

Nucleic acid hybridization is a powerful analytical tool forthe detection of infectious agents in biological samples. Insuch assays, liquid-phase hybridization, as opposed to solid-phase hybridization, provides many advantages. For exam-ple, it enhances the rate of hybridization, minimizes theinterference from extraneous materials, and ensures thecomplete accessibility of target sequences, resulting ingreater sensitivity. However, the separation of hybridizedfrom unhybridized probes represents a critical step in detec-tion assays with liquid-phase hybridization. Methods cur-rently used, such as hydroxyapatite chromatography (3) andendonuclease digestion (13), do not efficiently separate hy-bridized from unhybridized molecules. In the absence ofsatisfactory separation, the assay could lead to false-positiveresults.The development of antibodies against a specific confor-

mation of nucleic acids could provide an attractive andreliable method for detecting annealed probes. Much workdone previously on nucleic acid-specific antibodies focusedon pathogenic double-stranded DNA (dsDNA)-specific anti-bodies produced in several autoimmune diseases (5). In-duced nucleic acid-specific antibodies have been studiedmuch less, probably because of the poor immune response toinjected nucleic acids (9, 15).

Purified anti-nucleic acid antibodies were successfullyproduced earlier by Wollack and Erlanger (21) and byTraincard et al. (20). Antibodies recognizing the character-istic helical shapes of nucleic acids (A, B, and Z) also havebecome available (15). However, the production of antibod-ies to dsDNA (helical B-DNA) is not easily achieved. Manysuch antibodies produced do not adequately distinguish

* Corresponding author.

between native and denaturated DNAs (16). However, im-munization with the helical A conformation (RNA-DNA orRNA-RNA) yielded backbone-specific antibodies (4, 10),with little dependence on the base sequence (17). Anti-RNA-DNA (anti-HNA, for antibody to heteroduplex nucleic acid)antibodies do not recognize dsDNA, mainly because of thedifferences in conformation between the two types of helices(vertical rise per base pair, helical diameter, and rotation perbase pair).

Synthetic nucleic acid hybrids have been used widely asimmunogens for the induction of anti-HNA antibodies.These antigens can be prepared in amounts suitable forimmunization. However, the antiserum produced by suchsynthetic antigens usually results in significant cross-reac-tions with dsDNA and/or RNA because of the unmatchedcopolymers in the synthetic immunogens (10, 12). Twopolymers frequently used in the production of anti-HNApolyclonal antibodies are poly(A)-poly(dT) (15) and poly(I)-poly(dC) (10). Of the two, poly(A)-poly(dT) was found to bemuch more suitable, having certain similarities to naturallyoccurring DNA-RNA hybrids. The antibodies producedwere successfully used to detect heteroduplex nucleic acids(HNAs) after in situ hybridization with an RNA probe (15).Antibodies raised against synthetic polymer nucleotidesoften were found to be dependent on repetitive sequences.DNA-RNA heteropolymers synthesized from naturally

occurring sequences also have been used as immunogens,particularly to obtain antibodies that are sequence indepen-dent. HNA antigens prepared by in vitro transcription ofphage 4X174 single-stranded DNA (ssDNA) with RNApolymerase are the only antigens from naturally occurringsequences that have been used in raising anti-DNA-RNAantibodies. This antigen group has been shown to be moreantigenic than synthetic polymers. This result was confirmed

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by Nakazato (11, 12), who showed that antibodies topoly(A)-poly(dT) duplexes had only a weak affinity forDNA-RNA heteropolymer duplexes. The major disadvan-tages of immunization with natural HNA antigens is relatedto the large amount of ssDNA required by the transcriptionreaction. In an in vitro transcription reaction, the OX174viral DNA is usually incompletely copied (because of itslarge size), resulting in the generation of cross-reactingantibodies, especially anti-ssDNA antibodies. The screeningof hybridoma clones producing HNA-specific antibodies istherefore critical.

In this study, we prepared a full-length DNA-RNA hybridby synthesizing cDNA over globin mRNA with murineleukemia virus reverse transcriptase. The hybrid generatedwas used in an immunization protocol from which anti-HNAmonoclonal antibodies (MAbs) were obtained.The immunogenicity of this antigen was compared with

that of an antigen prepared by transcription of ssDNA withDNA-dependent RNA polymerase. The immunogenicity ofthe antigen, as influenced by its polymer length, also wasexamined.

Specific anti-DNA-RNA MAbs against antigens obtainedby both methods were characterized and used to measurethe hybridization of Listeria rRNA with a biotinylated DNAprobe. Hybridization was carried out in the liquid phase, andthe resulting HNAs were estimated in an enzyme-linkedimmunosorbent assay (ELISA) with the antibodies (HNA-ELISA). The DNA probe had been developed previously inour laboratory (6).

MATERIALS AND METHODS

For avoiding the degradation of nucleic acids, all glass-ware and plasticware coming into contact with nucleic acidswas washed thoroughly and rinsed with distilled waterpreviously treated with a 0.1% solution of diethylpyrocar-bonate (Sigma Chemical Co., St. Louis, Mo.) as describedby Sambrook et al. (14).

Preparation of the cDNA-mRNA antigen. cDNA-mRNAhybrids were synthesized by use of a first-strand cDNAsynthesis kit (Pharmacia, Uppsala, Sweden). In accordancewith the manufacturer's protocol, the reaction was expectedto result in the full-length transcription of mRNAs. Commer-cial rabbit globin mRNAs of 600 and 650 bases (GIBCO/Bethesda Research Laboratories, Burlington, Ontario, Can-ada) were used as templates. In brief, 5 ,ug of mRNA wasplaced in a microcentrifuge tube, and the volume wascompleted with water to 20 ,ul. RNA was heat denatured at65°C for 10 min and then chilled on ice. To this RNA wereadded 11 ,u1 of the bulk first-strand reaction mixture, 1 p.1 ofdithiothreitol solution, and 1 ,u1 of pd(N)6 primer. Murineleukemia virus reverse transcriptase was added, and themixture was incubated for 1 h at 37°C (murine reversetranscriptase was preferred over avian reverse transcriptasebecause of its lower endonuclease activity [11]). The first-strand cDNA reaction product was extracted with phenol-chloroform and then precipitated twice in 2.5 M ammoniumacetate. These two sequential precipitations resulted in theremoval of over 99% of the free deoxynucleoside triphos-phates remaining in solution (13). The pellet was thenwashed twice with 70% ethanol and resuspended in 1 ml of0.1x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate).The material produced in the reaction was analyzed by

carrying out a small-scale parallel reaction in which[32P]dCTP was incorporated. The amount of cDNA synthe-

sized was calculated by the following formula (13): p.g ofcDNA synthesized = (cpm incorporated x d)/total cpm,where d is the amount of cDNA, in micrograms, that thereaction is capable of generating. The yield of this reactionwas usually close to 50% of the weight of the RNA used inthe reaction mixture.

Preparation of the +X174 DNA-RNA antigen. XX174DNA-RNA hybrids were synthesized by in vitro transcrip-tion of 4X174 circular ssDNA (GIBCO/Bethesda ResearchLaboratories) with Escherichia coli DNA-dependent RNApolymerase (Pharmacia). This procedure was adapted fromthe method of Nakazato (12) and modified as follows. Theincubation mixture contained 40 mM Tris HCl buffer (pH8.0), 150 mM KCl, 0.1 mM dithiothreitol, 10 mM MgCl2, 0.1mM EDTA, 0.5 mg of RNase-DNase-free, bovine serumalbumin (BSA), 70 U of RNA polymerase, and 400 p.g of4oX174 ssDNA. The reaction was initiated by adding the fourribonucleoside triphosphates (final concentration of each,800 p.M). After 6 h at 35°C, the reaction was stopped byadding 0.25 volume of 20x SSC, and the reaction mixturewas dialyzed overnight at 4°C against 4 liters of TNE buffer(30 mM Tris HCl, 50 mM NaCl, 5 mM EDTA). Incubationwas continued for another 30 min in the presence of 10 p.g ofproteinase K (GIBCO/BRL) per ml. One volume of TESbuffer (0.02 M Tris HCl [pH 7.4], 1 mM EDTA, 0.5% sodiumdodecyl sulfate [SDS]) was added before phenol-chloroformextraction. The HNAs were then treated and analyzed asdescribed above (for the cDNA-mRNA antigen) and storedat -20°C until use.

Preparation of the antibodies. Female BALB/c mice(Charles River, St. Constant, Quebec, Canada) were immu-nized intraperitoneally by standard immunization protocolswith a BSA-DNA-RNA complex. In brief, 25 p.l of 1%methylated BSA (mBSA; Sigma) was mixed at room tem-perature with 0.5 ml of DNA-RNA solution (1 A260 unit perml) under the conditions described by Nakazato (11). Theresulting hapten-carrier complex formed was emulsified inan equal volume of complete Freund adjuvant for the firstinjection and incomplete Freund adjuvant for the subsequentinjections. The immunization protocol consisted of twointraperitoneal injections of 10 p.g of the HNA-mBSA com-plex antigen at a 2-week interval. Eleven days following thesecond injection, the mice were boosted once intraperito-neally with 20 p.g of the HNA-mBSA complex. The micewere splenectomized 3 days later. The spleen cells werefused with SP2/0 myeloma cells in the presence of polyeth-ylene glycol 4000 (Merck). The fused cells were suspendedin Iscove medium (Sigma) containing 20% fetal bovine serum(GIBCO Laboratories, Grand Island, N.Y.), antibiotics (50U of penicillin and 50 p.g of streptomycin per ml), 100 p.Mhypoxanthine-0.4 p.M aminopterin-16 p.M thymidine, and 15U of macrophage-derived hybridoma growth factor (inter-leukin-6) per ml. The suspension was dispensed (200 p.l) into96-well microplates (Costar, Cambridge, Mass.). The mi-croplates were incubated at 37°C for 12 days. The cultureswere screened by adding 50 p.l of culture supernatant towells of 96-well Immulon II microplates (Dynatech Labora-tories, Alexandria, Va.) previously coated (for 12 h at 4°C)with HNA (1 p.g/ml in 0.1 M carbonate buffer [pH 9.6]).Following incubation for 30 min at 37°C, the plates werewashed and the presence of anti-HNA antibodies was as-sessed with polyspecific goat anti-mouse immunoglobulin(Ig)-peroxidase conjugates (Sigma). The enzyme conjugateswere diluted in a phosphate-buffered saline-1% BSA solu-tion as suggested by the supplier and were revealed with theorthophenylenediamine substrate (OPD) (Abbott Laborato-

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ries, North Chicago, Ill.). Optical densities were read on amicrotiter plate reader (Dynatech) at 490 nm. The contentsof wells showing an optical density of >0.7 were cloned bylimiting dilution culturing, and the isotypes of the MAbswere determined with isotype-specific goat anti-mouse Ig-peroxidase conjugates as described above. Two such clones,20D3 and 6B5, were identified and used in this study.

Purification of the MAbs. Supernatants were concentratedby ultrafiltration and then purified by affinity chromatogra-phy with an ImmunoPure immunoglobulin M (IgM) purifica-tion kit (Pierce, Rockford, Ill.). The purity of the MAbs wasthen determined by electrophoresis in an SDS-12.5% poly-acrylamide gel.

Specificity of selected clones. The specificity of the MAbsproduced was further characterized by competitive bindingassays. Inhibition tests were performed in the presence of 50ng of antibody per ml and various potentially competitiveantigens. The antigens used were 4X174 DNA-RNA,cDNA-globin mRNA, poly(rA)-poly(dT) (Pharmacia) 4X174ssDNA, X dsDNA, E. coli single-stranded RNA (ssRNA),and viral double-stranded RNA (dsRNA) (reovirus). Theantigens (20 p,g/ml) were incubated overnight at 4°C witheither 20D3 or 6B5 MAb, added to HNA-coated plates (5,ug/ml), and incubated for 30 min at 37°C. Peroxidase-conjugated goat anti-mouse IgM antibody (1:5,000) wasadded, and bound anti-HNA antibodies were detected by theaddition of OPD. Inhibition was expressed as the antigenconcentration resulting in a 50% decrease in optical density.Measurement of association constants. The procedure used

for the measurement of association constants was describedby Friguet et al. (8). Inhibition tests were performed with thethree types of HNA antigens as described above. Thedissociation constants were determined by measuring theslope of the regression curve obtained by plotting the recip-rocal of the fraction of bound antibody versus the reciprocalof the molar concentration of the antigen present in thesolution. The association constants were then deduced bycalculating the reciprocal of the slope of the linear regressioncurve.Development of an HNA-ELISA for the detection of liste-

riae. (i) Production of biotinylated probes by a nick translationreaction. A ribosomal DNA probe already developed byEmond et al. (6) was nick translated in the presence ofbiotin-14-dATP with a BIONICK labeling kit (BethesdaResearch Laboratories) in accordance with a standard pro-tocol (19) recommended by the manufacturer. The reactionwas performed at 15°C for 60 min. Unincorporated nucle-otides were separated from the biotinylated probe by pre-cipitation with 95% ethanol at -70°C for 2 h in the presenceof 0.3 M sodium acetate. After centrifugation at 10,000 x gfor 15 min at 4°C, the pellet was washed with 70% ethanol,air dried, and suspended in 100 ,ul of Tris HCI (50 mM; pH7.2)-EDTA (2 mM).

(ii) Liquid-phase hybridization assay. The liquid-phasehybridization assay is based on a hybridization reactionbetween target rRNA prepared from a lysate of Listeriamonocytogenes (7) and the biotinylated DNA probe. Serialdilutions of L. monocytogenes rRNA ranging from 2.5 pg to20 ng were prepared in diethylpyrocarbonate-treated water(25 pl). RNAs were denatured either by heating at 65°C for5 min in the presence of 3 volumes (75 pl) of 1.3x morpho-linepropanesulfonic acid (MOPS) (10x MOPS is 0.2 MMOPS, 0.05 M sodium acetate, and 0.01 M EDTA)-8%formaldehyde-66% formamide or by heating at 90°C for 3min and then chilling in ice. The biotinylated probe was firstdiluted to 500 ng/ml in a buffer containing 8x SSC (pH 7.0),

FIG. 1. First-strand cDNA synthesis from rabbit globin mRNA.The cDNA-mRNA hybrid was electrophoresed through a 1.5%agarose gel. Lanes: 1, rabbit globin; 2, cDNA-mRNA hybrid.

40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid(HEPES) (pH 7.4), 4 mM EDTA, and 20 mM vanadyl-ribonucleoside complex. One hundred microliters of theprobe thus prepared was added to an equal volume of RNAsolution, and the hybridization reaction was carried out at70°C for 1 h.

(iui) HNA-ELISA. For the HNA-ELISA, the hybridizationreaction mixture (200 ,ul containing HNA) was added to thewells of microtiter plates previously coated with 100 ,ul ofanti-HNA MAbs (5 ,ug/ml) and incubated at 37°C for 60 minto capture the biotinylated DNA-RNA hybrid. The plateswere then washed, and 100 ,ul of a streptavidin-alkalinephosphatase (SA-AP) conjugate (1 ,ug/ml) was added to eachwell. After incubation for 30 min at 37°C, the plates werewashed and 100 ,ul of p-nitrophenyl phosphate (4 mg ofp-nitrophenyl phosphate in 10% diethanolamine-0.5 mMMgCl2 [pH 9.8]) was added. After 15 min of incubation, thecolor generated was measured at 410 nm.The optimal concentrations of capture antibodies and

SA-AP conjugates used in this assay were determined asfollows. The microtiter plates were coated with serial two-fold dilutions of capture antibodies starting at 5 ,ug/ml andending at 1.25 pg/ml. A constant amount of HNA antigenwas applied to each microtiter plate (either 25 or 0.05 ng/ml).SA-AP conjugates were then added as serial twofold dilu-tions starting at 1.6 ,ug/ml and ending at 50 ng/ml. Aftersubstrate addition, the absorbance of each well was mea-sured and the parameters yielding the highest signal/noiseratio were chosen.

RESULTS AND DISCUSSION

The formation of a DNA-RNA hybrid during the reversetranscriptase reaction was monitored by carrying out aparallel reaction in which radioactive nucleotides were in-corporated during polymerization. An analysis of the reac-tion products on a 1% agarose gel (Fig. 1) showed theefficiency of the transcription reaction. From the figure, weestimated that the cDNA synthesized was equal in quantityto the mRNA template and therefore concluded that full-length first-strand cDNA synthesis was achieved, as expectedtheoretically (14). The formation of such a DNA-RNA hybridis imperative for preparing specific anti-DNA-RNA MAbs toavoid inducing nonspecific antibodies related to the nontran-scribed fragment in the single-stranded template.Two antigens were used to immunize groups of mice. In a

total of six fusion experiments (three for each antigen; 7,200culture wells), about 30 wells were positive in an ELISA

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measurements. The two antibodies were incubated separately with X174 DNA-RNA, cDNA-mRNA, poly(rA)-poly(dT), 4X174 ssDNA,dsDNA, or E. coli ssRNA. The mixture was then added to HNA-coated plates, and the bound anti-HNA antibodies were detected by use ofperoxidase-conjugated anti-mouse IgM.

with wells coated with antigens. Of these, the 20D3 and 6B5wells were retained for additional work. Hybridoma celllines were established by limiting dilution culturing (twice).The secreted antibodies were of the IgM class.

Epitope specificity of MAbs. Six competing species ofnucleic acid molecules (ssDNA, ssRNA, dsDNA, DNA-RNA hybrids) and mBSA were used to verify the abilities ofboth antibodies to detect HNAs specifically. The results ofthe inhibition tests are shown in Fig. 2. Percentages ofinhibition were calculated by use of the amount of theantibody bound to the original antigen used for eliciting theMAbs.The reactivity of the 20D3 MAb was completely inhibited

by the two HNA antigens and by poly(rA)-poly(dT). Nosignificant inhibition was obtained with ssDNA or dsDNA.However, significant (40%) inhibition was observed withssRNA. A similar inhibition was also observed with reovirusdsRNA (data not shown).The level of inhibition at high concentrations of ssRNA

was not higher than 40%, indicating that the avidity of 20D3for DNA-RNA is much higher than that for ssRNA. Thecross-reactivity with RNA is probably due to the presence ofsecondary structures in the RNA molecules. It is known thatthe conformations of dsRNA and HNA molecules are in factvery similar (12). This characteristic does not pose anyproblem for the detection of a DNA-RNA duplex (see

below). The 6B5 MAb was significantly inhibited only byHNAs formed by synthetic DNA-RNA and weakly inhibitedby those formed by cDNA-mRNA. The reactivity of the 6B5MAb against these molecules indicates that the poly(A)sequence in both poly(rA)-poly(dT) and cDNA-mRNA hy-brids is probably important for the recognition of the targetantigen. The inhibition observed with mRNA (ssRNA in Fig.2) corroborates this hypothesis. The short stretch ofpoly(rA)-poly(dT) could serve as an epitope because of theparticular helical structure associated with this region (16)and the conformational flexibility associated with such re-gions in antigenic nucleic acids (1).The results demonstrate that the 20D3 MAb recognizes

the helical conformation of the HNA duplex with no depen-dence on the nucleotide sequence. dsDNA was not recog-nized by both antibodies, probably because of the differencein the helical conformation. Results obtained with the 6B5MAb agree with those of Stollar et al. (17, 18), who showedthat the presence of unusual base sequences (like repetitivesequences) in a hybrid molecule could lead to base se-

quence-dependent reactivity.Measurement of MAb avidity. Avidity is an important

parameter in studying the selectivity of an MAb. This aviditycan determine the detection limit of the immunoassay. Twoexperiments were performed to evaluate the avidity of thetwo MAbs for three HNA antigens. First, the association

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2702 FLISS ET AL.

y = 0,26 + 0,94x RA2 = 0,968

y = 3,36 + 6,28x RA2 = 0,988

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FIG. 3. Association constants of the 20D3 and 6B5 MAbs measured with three HNA antigens [+X174 DNA-RNA, cDNA-mRNA, andpoly(rA)-poly(dT)] and determined by linear regression with the Klotz representation.x axis: 1/molar concentration of the total antigen tested;y axis: 1/fraction of bound antibody. (a, b, and c) Klotz plots of the binding of 20D3 to +X174 DNA-RNA, cDNA-mRNA, andpoly(rA)-poly(dT), respectively. (d, e, and f) Klotz plots of the binding of 6B5 to the same antigens.

constants were measured for the 20D3 and 6B5 MAbs. In thesecond experiment, the kinetics of MAb binding to HNAhybrids were determined.

(i) Determination of the association constants. The associ-ation constants of the antibodies raised against HNA hybridswere determined by use of an ELISA with data from theinhibition tests. Figure 3 shows the Klotz representation ofthe competition assay, and the association constants calcu-lated from the linear regression equation are presented inTable 1. The results confirmed those obtained in studies ofepitope specificity. The 20D3 MAb had the highest avidityfor natural HNA antigens; it recognized 4X174 DNA-RNAand cDNA-mRNA with avidities 107 and 100 times higherthan those of the 6B5 MAb, respectively. The 6B5 MAb hadthe highest avidity for synthetic HNA antigens and recog-nized poly(rA)-poly(dT) with a 105-times-higher avidity thanthat of the 20D3 MAb.

Variations in association constants showed a small depen-dence on antigen size, probably because of the multivalent

TABLE 1. Association constants of MAbs 20D3 and 6B5 asdetermined by an ELISA inhibition test

Association constant (M-1) for the following antigen:Antibody 4)X174 cDNA- poly(rA)-

DNA-RNA mRNA poly(dT)

20D3 1.05 x 1012 2.12 x 1010 1.68 x 1076B5 1.59 x 105 7.1 x 108 5 x 1012

antigenic structure of HNA molecules and the number ofbinding sites along the double helix. Results obtained byBallard et al. (1, 2) for the epitopes presented to theanti-dsDNA antibodies indicated that nucleotide lengths of 3to 10 bases could represent the binding sites for suchantibodies. The antigenic nucleic acids (dsDNA and HNAs,etc.), being multivalent, could present n epitopes, dependingon their total length. Thus, more antibodies were bound to4X174 DNA-RNA (5,386 bp) than to cDNA-mRNA (650bp), explaining the difference in association constants deter-mined for the 20D3 MAb. The 6B5 MAb recognizedpoly(rA)-poly(dT) (1,110 bp) 104 times better than cDNA-mRNA, in which the poly(rA)-poly(dT) region (which isprobably important in the presentation of the epitope; see

above) was present in a single copy per molecule. This resultcould also explain the difference in association constantsbetween 6B5 and 20D3 for cDNA-mRNA.

(ii) Time course of MAb binding to various HNA antigens.The kinetics of signal formation were studied with the twoMAbs. For these experiments, 0.5 ,ug of each MAb per mlwas incubated with 5 ,g ofHNA antigens per ml for differenttimes. The amount of bound MAb was quantitated in anELISA with peroxidase-conjugated anti-mouse IgM. Figure4 shows the dose-response curves obtained with MAbs 20D3and 6B5. The signal obtained with the 20D3 MAb increasedrapidly when this MAb was incubated with 4X174 DNA-RNA or cDNA-mRNA. The maximum signal was reachedafter 5 or 10 min of incubation. However, the 6B5 MAbsignal remained very low even after 60 min. When poly(rA)-

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ANTI-DNA-RNA ANTIBODIES FOR LISTERIA DETECTION 2703

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Incubation time (min)FIG. 4. Time course of MAb binding to the antigens cDNA-

mRNA (A), poly(rA)-poly(dT) (B), and 4X174 DNA-RNA (C). EachMAb was incubated separately for different times with constantamounts of the HNA antigens. The amount of bound MAb was thenquantitated in an ELISA with peroxidase-conjugated anti-mouseIgM. Symbols: 0, 20D3 antibody; 0, 6B5 antibody.

poly(dT) was used as the antigen, the maximum signal wasobtained with the 6B5 MAb.The intensity of the signal generated was verified by

studying the effect of the concentration of the 20D3 MAbapplied to each well and the concentration of the targetantigen 4X174 DNA-RNA. Figure 5 indicates that the doseresponse with each MAb concentration increased between 0and 300 ng of target antigen. The maximum signal producedwas constant for higher antigen concentrations. The signalalso depended on the MAb concentration used.

Application of anti-HNA antibodies to the detection of liste-riae. Since the specificity and the ability of the 20D3 MAb todetect DNA-RNA hybrids have been confirmed, we studiedits ability to detect Listeria rRNA-probe DNA hybrids. Asshown in Fig. 6, the assay consisted of two steps: in the firststep, a biotinylated DNA probe was allowed to hybridize withListeria rRNA in the liquid phase. In the second step, the

0 200 400 600 800 1000

Amount of 0X174 DNA:RNA antigen (pg)

FIG. 5. Binding of the 20D3 MAb to the antigen 4X174 DNA-RNA. Various amounts of the antigen were coated on the surface ofthe wells and reacted with various quantities of the antibody (3.20[Cl], 1.60 [0], 0.80 [A], 0.40 [0], 0.10 [A], 0.05 [ffl], and 0.025 [A]ng/ml). The amount of bound MAb was determined with peroxidase-conjugated anti-mouse Ig as described in the text. Data are averagesof triplicate measurements.

HNA specifically bound to the previously immobilized MAbwas measured with SA-AP conjugate.

(i) Development of the liquid-phase hybridization assay.Four parameters were studied to define the optimal condi-tions under which the hybridization assay had to be per-formed. These were (i) hybridization temperature, (ii)method of denaturation, (iii) hybridization time, and (iv)probe concentration. Emond et al. (6) observed that follow-ing hybridization with the 782-bp probe on a membrane, theexcess probe had to be washed at 65°C to obtain signalsspecific for Listeria spp. Under our conditions (solution-phase hybridization), the same specificity was obtainedwhen hybridization was carried out at 70°C, and no-crosshybridization was obtained, even with closely related bacte-ria (data not shown). The best results were obtained whenthe hybridization reaction was carried out for 60 min withformaldehyde-denaturated RNA in the presence of 250 ng ofbiotinylated probe per ml (data not shown).

(ii) Optimization of the immunoassay. The concentrations ofthe 20D3 MAb and the SA-AP conjugate were optimized toachieve the best detection sensitivity and to eliminate thebackground signal which was obtained when microtiter plateswere coated with 1.25 ,ug of 20D3 per ml and when the SA-APconjugate was used at 0.3 ,ug/,l~. This combination yielded thebest signal/noise ratio (29:1). Different saturating agents, suchas gelatin (0.5%), casein (0.25%), Tween 80 (0.05%), and BSA(1%), were tested. The best results were obtained with caseineand gelatin; the other agents, especially BSA, yielded a higherbackground (data not shown).The optimal conditions defined above were used to eval-

uate the performance of the method developed for detectingListeria rRNA at various concentrations. The results shownin Fig. 7 validate the assay in quantifying the amount ofHNA formed during hybridization. As shown in Fig. 7, aslittle as 2.5 pg of rRNA could be detected, yielding a signalsignificantly higher than that of the background. The signalgenerated was linear between 2.5 and 500 pg of rRNA (R2 =

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I111TFfl

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1 - Hybridization between biotinylatedDNA probe and listeria rRNA

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wash to removeunhybridized probe

wash to remove unboundconjugates

PNP

4 - addition of alkaline phosphatasesubstrate

30 min at 37°C

3- addition of SA-AP conjugates

30 min at 37°CFIG. 6. Schematic diagram of the HNA-ELISA. After the biotinylated DNA probe was hybridized with the target RNA, the hybrids

formed were captured with the 20D3 MAb in the precoated wells. The captured hybrids were then visualized with SA-AP conjugate. B, biotin;PNP, p-nitrophenyl phosphate.

0.951), indicating that the signal was closely correlated to theamount of target rRNA. Above this concentration of rRNA,saturation occurred in the colorimetric assay. No signal wasobtained with the contaminating bacteria tested, includingBrochothrix thermosphacta, a bacterium phylogeneticallyclosely related to bacteria of the genus Listena, emphasizingthe specificity of the assay. This specificity, of course, isrelated to the DNA probe used in the hybridization step (6).

The advantage of the antibodies described here is that theycan be used for capturing all HNA molecules, irrespective ofthe nucleotide sequence. The system described here is moresensitive than that described by Coultee et al. (4), in whichan antibiotin antibody was used to coat microtiter plates andthus capture the biotinylated hybrid formed; this procedureis particularly useless for samples such as dairy products,which contain biotin.

<SA-AP

9F

2704 FLISS ET AL.

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ANTI-DNA-RNA ANTIBODIES FOR LISTERIA DETECTION 2705

2

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6

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O K II

0 1 2 3 4 50 10 10 10 10 10

Target RNA concentration (pg)FIG. 7. Estimation of rRNA from L. monocytogenes by an HNA-ELISA with a biotinylated DNA probe and the 20D3 MAb.

The development of the 20D3 MAb provides an interestingtool for the nonisotopic detection of gene probes in aliquid-phase hybridization assay. The immunoassay proce-dure developed was successfully used to measure ListeriaHNA formed in solution. This procedure could be used inthe detection of any bacterial rRNA on the condition that thetarget molecule and its specific probe form an HNA.

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1984. Specificity and kinetics defining the interaction between amurine monoclonal autoantibody and DNA. J. Biol. Chem.259:3492-3498.

2. Ballard, D. W., and E. W. Voss, Jr. 1985. Base specificity andidiotype of anti-DNA autoantibodies with synthetic nucleicacids. J. Immunol. 135:3372-3386.

3. Bernardi, G. 1971. Chromatography of nucleic acids on hy-droxyapatite columns. Methods Enzymol. 21:95-147.

4. Coult6e, F., L. Bobo, K. Mayur, R H. Yolken, and R. P. Viscidi.1989. Immunodetection ofDNA with biotinylated RNA probes:a study of reactivity of a monoclonal antibody to DNA:RNAhybrids. Anal. Biochem. 181:96-105.

5. Diamond, B., J. B. Katz, E. Paul, C. Aranow, D. Lustgarten, andD. M. Scarf. 1992. The role of somatic mutation in the patho-genic anti-DNA response. Annu. Rev. Immunol. 10:731.

6. Emond, E., I. Fliss, and S. Pandian. 1993. A ribosomal DNAfragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay. Appl.Environ. Microbiol. 59:2690-2697.

7. Fliss, I., E. Emond, R E. Simard, and S. Pandian. 1991. A rapidand efficient method of lysis of Listeria and other gram-positivebacteria using mutanolysin. BioTechniques 11:453-457.

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16. Stollar, B. D. 1973. Nucleic acid antigen, p. 1. In M. Sela (ed.),The antigens, vol. I. Academic Press, New York.

17. Stollar, B. D., and A. Rashtchian. 1987. Immunochemical ap-proaches to gene probe assays. Anal. Biochem. 161:387-394.

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