Journal of Immunological Methods, 150 (1992) 33-49


© 1992 ElsevierSciencePublishers B.V. All rights reserved 0022-1759/92/$05.00


Immunoenzymatic techniques applied to the specific detection of nucleic acids A review Jean-Luc Guesdon Laboratoire des Sondes Froides, Institut Pasteur, 75724 Paris Cedex 15, France

(Accepted 12 February 1992)

Numerous enzymatic and chemical methods are now available for the preparation of non-radioactive nucleic acid probes. Labels, such as enzymes, fluorophores, lumiphores can be attached to the nucleic acid probe either by covalent bonds (direct labelling) or by biospecific recognition after hybridization (indirect labelling). The principle of the latter method is based on the use of a hapten-labelled nucleic acid probe which is generally detected by an immunoenzymatic assay. Indirect labelling has several advantages: this procedure uses muitienzyme complexes to increase the number of enzyme molecules associated with hybridization and hence provides an increase in detectability; moreover, haptens (biotin, dinitrophenoi, acetylaminofluorene analogues, digoxigenin, brominated or sulphonylated pyrimidines) used to label nucleic acid probes are not sensitive to elevated temperatures (42-80°C), ex-tended incubation times (several hours), detergents and organic solvents currently required in hybridization techniques. The application of the immunoenzymatic and related techniques to nucleic acid probing is reviewed, focussing on the strategies of non-radioactive hybridization, hapten-labelling of nucleic acids and methods for the immunodetection of the hybrids. Key words: Molecular hybridization; Non-radioactiveprobe

Introduction Since its development 25 years ago, the immunoenzymatic methods have been extensively applied to the detection and the quantitation of various antigens and antibodies. More recently, immunoenzymatic techniques have been success-

Correspondence to: J.-L. Guesdon, Laboratoire des Sondes Froides, lnstitut Pasteur, 75724 Paris Cedex 15, France (Fax: 33-1-

fully applied to molecular hybridization, leading to the new technology of non-radioactive probes (cold probes). Nucleic acid hybridization technology has been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The introduction of immt~noenzymatie or related techniques into the repertoire of molecular hybridization methods enables the routine use of this method outside the specialized molecular biology laboratory. The nucleic acid hybridization protocol most familiar to molecular biologists involves the de-

34 tection by radioactively labelled probes of target nucleic acids that have been immobilized on nitrocellulose or nylon filters. This protocol was derived from the work of Nygaard and Hall (1963, 1964), who were the first to immobilize D N A on nitrocellulose paper, and Gillespie and Spiegelman (1965) and Denhardt (1966), who detected such fixed nucleic acids with radioactive probes. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in a biological fluid or tissue. Hybridization leads to the formation of a doublestranded molecule called a hybrid or duplex, which can be detected with great sensitivity by a high-energy radioisotope. However, the use of radioisotope-labelled probes is limited by the short half-life of the isotope, radiolysis of the probe and the dangers of radioactivity. In order to overcome the long time required for autoradiography and the drawbacks of techniques using radioisotopes, non-radioactive labels have been devised. The non-radioactive molecule used for nucleic acid labelling should fulfil the following criteria: (1) the label molecule must be detected with high sensitivity and must function satisfactorily after labelling; (2) the label must be stable under the hybridization conditions of temperature, ionic strength and pH; (3) no measurable effect on hybridization quality or kinetics should be observed; and (4) the label must be inexpensive and easy to use. In addition, test formats should involve only a few and simple manipulations and the results should be obtained within 1 day to make the hybridization techniques suitable for routine use. It is indeed difficult to combine all these requirements in a single protocol. For example, results could be rapidly obtained using an enzyme-labelled oligonucleotide, but not all enzymes are able to withstand the conditions required for hybridization; therefore, the choice of the enzyme will be limited to the most resistant ones. Generally, a non-radioactive hybridization assay requires the use of a modified probe. This modification can lower the melting temperature of the probe, thus disturb the probe's hybridization properties. Moreover, long hybridization and detection times are usually required to obtain

sufficient sensitivity. Non-radioactive detection systems are not as sensitive as isotopes, and the assay procedure is more complicated than with isotopically labelled hybridization probes. Immunoenzymatic and related techniques have been proposed as alternative methods to detect and localize specific nucleic acid sequences (Kourilsky et al., 1978). The major difficulty encountered in the use of immunoenzymatic techniques in nucleic acid probing lies in setting up a procedure which yields the highest signal-to-noise ratio (specific signal versus background signal). The strength of the specific signal depends mainly upon the specific activity of the probe, the accessibility of the probe to the target, the stability of the duplex, and the accessibility of the immunoenzymatic detection system to the duplex. The non-specific signal depends upon the interaction of the components of the sample with the entire detection system (i.e., nucleic acids and their contaminant molecules, on the one hand, labelled probe and immunoenzymatic system on the other). Other than this difficulty, immunoenzymatic techniques possess several advantages over other methods used to detect non-radioactive nucleic acid probes. The main advantages are the stability of the reagents, the intrinsic amplification, and the different ways available to measure enzyme activity. Indeed, these techniques make use of small, stable molecules as markers, highly specific antibodies and a label enzyme which can be detected with high resolution by a variety of sensitive methods (colorimetric, fluorescent or luminescent reactions). This review does not attempt to provide a complete overview of the various nucleic acid non-radioactive labelling methods, but rather, it is limited to the applications of the immunoenzymatic and related techniques to the specific detection of nucleic acid hybrids. Applications, specific protocols for use of nucleic acid probes and non-immunological methods for the detection of nucleic acid hybrids are outside the scope of this review. For further information, readers are referred to reviews and books by Lowe (1986), Cooper and Schmidtke (1986, 1987), Symons (1989), Tenover (1989) and Macario and Conway de Macario (1990).

When applying the immunoenzymatic method to the specific detection of nucleic acid sequences, one should bear in mind that the choice of the basic parameters which are involved in this type of assay is of great importance. The characteristics of these parameters will influence the sensitivity, the reproducibility and the practicability of the assay. In order to obtain accurate and reproducible results in any hybridization assay based on the immunoenzymatic method, it is necessary to ensure that all reagents and a~.~ay conditions are optimized. The three main parameters discussed below and involved in immunoenzymatic-based hybridization assay are: (1) the assay format of the hybridization; (2) the hapten labelling of the nucleic acid used as a probe; (3) the non-radioactive system for detection of the hybrid molecules.

Nucleic acid hybridization strategies A method to distinguish between or separate formed nucleic acid hybrids from single-stranded molecules is an essential part of a hybridization experiment. Different properties of the nucleic acids can be exploited for this purpose: hypochromic effect, hydroxyapatite chromatography, equilibrium density gradient centrifugation, specific enzyme degradation of unhybridized molecules. However, the most utilized method consists of performing hybridization using a probe labelled with a marker that enables detection and quantitation of the formed hybrids. The number of hybridization assay formats has considerably increased with the development of the non-radioactive hybridization technology (Matthews and Kricka, 1988), and can be artificially classified into two main categories: solid support-immobilized nucleic acid hybridization and free-solution hybridization.

Solid support-immobilized nucleic acid hybridization Solid phase-based hybridization offers the most convenient method for separation of nucleic acid hybrids (Meinkoth and Wahl, 1984). One of the reacting nucleic acids is immobilized on a solid support and, after the hybridization reaction, the



targetnt,~lalc ~it:J~ ~

Fig. 1. Hybridization procedure based on the use of an immobilized target nucleic acid and a probe nucleic acid that has been labelled with a marker molecule.

formed hybrids are separated from unhybridized molecules by simply washing the solid support. There are a number of alternatives available to carry out solid phase-based hybridization assays. From a practical point of view, solid phase can be used either to immobilize the target nucleic acid to be analysed or to immobilize a capture probe.

Hybridization procedures using an immobilized target nucleic acid. The most familiar nucleic acid hybridization protocols (dot-blot, spot-blot, slot-blot) involve the immobilization of target nucleic acids to be analysed on nitrocellulose or nylon filters and the detection of such fixed nucleic acids with labelled probes (Fig. 1). This protocol is not practical in a clinical laboratory because the filters are fragile and not suited to automation. A number of porous or non-porous solid supports are used in nucleic acid hybridization assays. Since the solid phase is the component which limits the success of any particular investigation, it is strongly recommended that attention be devoted to optimizing this aspect of the solid phase hybridization system. Polystyrene surfaces, which are widely used as solid supports in enzyme immunoassays, radioimmunoassays and in the latex method to quantify various antigens and antibodies, were proposed for nucleic acid hybridization assays as solid supports to immobilize target D N A by passive adsorption. Various D N A sequences were immobilized on microplates, namely h. D N A (Nagata et ai., 1985), hepatitis B virus (HBV)-specific recombinant D N A (Oser and Valet, 1988), total chromosomal DNA extracted from various bacterial species (Ezaki et al., 1988, 1989) or a polymerase chain reaction (PCR)-amplified viral D N A fragment (Inouye and Hondo, 1990). Recently, we reported the use of polystyrene microwells as a solid support for target DNA in the detection of

36 point mutations by hybridization with biotinylated hcptadecanucleotide probes and an enzymofluorometric revelation (Tham and Guesdon, 1992). Presently, a standard protocol for immobilizing nucleic acid on microtitration plates is not available. Using MicroFluor microtiter wells (Dynatech), Nagata et al. (1985) have shown that the addition of MgCI 2 (0.1 M) to phosphate-buffered saline (PBS) was necessary to dramatically enhance the adsorption of DNA onto the wells; moreover, they found that Ca 2÷ is as effective as Mg 2÷ and that Na + has little effect on adsorption. To secure the immobilization of DNA, these authors used UV irradiation. Using M13 D N A to coat microtiter wells, Keller et al. (1990) obtained results indicating that UV irradiation does not improve the performance of the well strips (Costar). In another study, lnouye and Hondo (1990) examined optimal ionic conditions for the adsorption of an amplified 642 bp sequence of DNA onto Maxisorp microplate wells (Nunc) and demonstrated that 1.5 M NaCI or 0.5 M ammonium sulphate were necessary to obtain strong hybridization signals. Determining the optimal conditions of 4.3 kb pBR322 plasmid adsorption onto Maxisorp F16 microwells (Nune), we found that varying the NaCI concentration had no influence on the hybridizable adsorption and that addition of 0.1 M MgCI 2 to saline sodium citrate buffer did not significantly facilitate the plasmid D N A adsorption onto this kind of microwell. Moreover, we observed a zone phenomenon ('hook effect') when the amount of D N A for adsorption ranged from 3 p.g to 92 pg. Optimal adsorption was obtained with about 312 ng of DNA; increasing this concentration resulted in a decrease in the amount of bound DNA. When 3 p.g of D N A were added to the microwelis, only 10-20% of the maximal adsorption was reached. In addition, we observed that incubation steps and washes during the test procedure eluted about 40% of the coated plasmid D N A at the maximal adsorption concentration. These experiments show that maximal hybridizable adsorption in microwell hybridization was obtained with an input of 312 ng of plasmid per well and only 7 ng (2.6 fmol) of DNA participating in the reaction and that polystyrene surface can bind 28 attomoles of pBR322 per mm 2 in a stable manner.

Theretore, in contrast to other solid supports namely nylon or nitrocellulose sheets, it is important to note that if supraoptimal amounts of target D N A are used to coat polystyrene microwells, the hybridization efficiency is, in fact, decreased. The practical consequence of this finding is that it is necessary to determine the amount of DNA affording maximal reactivity in the microwell hybridization assay. Moreover, the low binding capacity of polystyrene surfaces and variations in binding between lots, batches and commercial sources (Shekarchi et al., 1984) limit the use of this kind of solid support in hybridization assays. Simple covalent attachment of nucleic acid to microwells should be an interesting alternative to passive adsorption and an important improvement in the microweil hybridization assay.

Hybridization procedures using immobilized capture probes. Regardless of the solid support used, hybridization procedures using immobilized target nucleic acids require the purification of the nucleic acids before immobilization, in order to avoid background signals from the unspecific adsorption of the detection system onto biological material in the sample. Nucleic acid purification techniques necessitate the use of various reagents, such as enzymes and organic solvents, and are often time consuming. The sandwich hybridization technique was designed to simplify the analysis of large numbers of biological samples without D N A purification (Ranki et al., 1983). In this method (Fig. 2) the immobilized probe is used as a capturing reagent to collect the target DNA, which is then detected using a labelled second probe. Recently developed techniques enable capture of target D N A from a sample by a synthetic oligonucleotide (which has sequences complementary to the target DNA) immobilized on Sepharose (Polsky-Cynkin et ai., 1985), latex (Kremsky et al., 1987; Wolf et al., 1987; Urdea et al., 1988) or another inert particle. In one format, the capture oligonucleotide has a poly (dA) tail which is captured by a poly (dT) coating on a polystyrene dipstick.

Free-solution hybridization The time taken to complete a hybridization assay on a solid support is usually determined by

the hybridization kinetics, with overnight incubation generally being required. Instead of carrying out hybridization reactions with the DNA immobilized on a solid matrix where the kinetics of hybridization are not optimal, free-solution hybridization procedures have been devised to accelerate hybridization reactions (Wolf et al., 1987). Two main approaches are available: homogeneous or heterogeneous systems. Homogeneous system. The principle of the homogeneous hybridization assay is based on the use of two interacting molecules that emit "a signal when brought into the correct alignment. Among the homogeneous hybridization assays, one exploiting the enzyme-channelling principle has been described (Albareila et al., 1985). In this assay, a pair of enzyme labels is bound to an antibody where the two enzymes are related by the fact that the product of one serves as the substrate of the second. Fig. 3 illustrates an application of this type of assay to detect nucleic acid hybrids; anti-hybrid antibodies are labelled with glucose oxidase and horseradish peroxidase. The hydrogen peroxide generated by the action of the glucose oxidase-coupled antibody on glucose is utilized efficiently by peroxidase-coupled antibody only when the two labelled antibodies are

marker-labelled probe

target nucleic acid

probenucleicadd f





~OD ~



2 ------~,:otou~i ~ a

Fig. 3. Homogeneous hybridization assay based on an enzyme-channelling immunoassay. Y=anti-hybrid antibodies; GOD = glucoseoxidase;POD = peroxidase. bound to the same nucleic acid hybrid molecule, The method produced a rate difference be,~veen bound and free enzyme-antibody conjugates thus enabling direct quantitation of hybridization without the need to separate hybridized and unhybridized probes. Homogeneous hybridization methods offer significant advantages to the clinical laboratory due to their speed and simplicity, but these methods suffer from two main disadvantages: in general their sensitivity is lower than that obtained with the non-radioactive hybridization assays using solid supports, and they are sensitive to interfering compounds, which, whenever present in the sample, may alter enzymatic activity.

Heterogeneous system: affinity-based hybrid collection procedure. A prerequisite for solution hy-

Fig. 2. Hybridization procedure based on the use of an immobilized capture probe and a second probe that has been labelled with a detectable marker molecule.

bridization formats using a non homogeneous system is that a simple method to separate free and hybridized probe be available. In an affinitybased hybrid collection procedure, the target nucleic acid is allowed to hybridize with a pair of probes in solution. One of the probes has been modified with a ligand, by which the hybrids are collected on an affinity matrix after the hybridiza-

marker*labelled probe

target nucleic acid hapten-modified probe

affinitymal~x Fig. 4. Solution hybridizationwith two probes, the capturing probe is labelled with a hapten and the detecting probe is labelled with a marker. The target nucleic acid binds both probes. The hybrids are collectedon an affinitymatrix which can be either an antibody-or an avidin-coatedsolid support.

tion step. The other probe carries a detectable marker molecule for the measurement of the hybrids (Fig. 4). In this method, biotin in conjunction with streptavidin immobilized on agarose beads or a hapten with an antibody immobilized in microtitration wells were used as the affinity pair (Syv~inen and Korpela, 1986; Syv~inen et al., 1986). Alternatively, in a method described more recently (Yehle et ai., 1987; Coutlee et al., 1989a,b), the hybridization of R N A with a biotinylated D N A probe is carried out in solution and then the resultant hybrids are allowed to bind simultaneously to avidin immobilized on latex and to an enzyme-labelled monoclonal antibody specific to the D N A : R N A duplex.

Hapten-labeiling of nucleic acid for hybridization The first efficient non-radioactive marker of nucleic acid probes to be used was biotin (vitamin H). In the first papers reporting the use of biotin/avidin technology in hybridization assays, Drosophila ribosomal R N A was coupled to biotin, hybridized in situ to salivary gland chromosomes

and detected by scanning electron microscopy with avidin coupled to polymethacrylate microspheres (Manning et al., 1975). Then, Kourilsky et al. (1978, 1986) applied the biotin/avidin interaction to design the first molecular hybridization assay using an enzyme as the label to detect quantitatively nucleic a~:id hybrids. In these earliest applications, biotin's affinity for avidin, streptavidin or anti-biotin antibodies was used to detect biotinylated probes. In hybridization to crude cellular extracts, such as those generally used for the detection of pathogens in animal or plant tissues, biotinylated nucleic acid probes can possibly generate unworkably high background noise due the presence of biotin-binding proteins or biotin-carrying proteins (Zwadyk et al., 1986; Bialkowska-Hobrzanska, 1987). In order to overcome this major drawback which could account for the limited use of non-radioactive probes, other immunoehemically detectable haptens, such as dinitrophenol (DNP) (Vincent et al., 1982), 5-bromo-2'-deoxyuridine (5-BrdU) (Traincard et al., 1983), acetylaminofluorene (AAF) (Tchen et al., 1984), sulphone groups (Cyt-SO 3) (Lebacq et al., 1988), and digoxigenin (DIG) (Kessler et al., 1989) have been proposed as alternatives to biotin as markers for hybridization probes. Non-radioactive-labelling of nucleic acid is currently approached in two ways: the label molecule can be introduced into the nucleic acid probe by either a chemical or an enzyme reaction.

Chemical labelling Hapten markers have been introduced chemically into nucleic acids by direct modification with activated haptens. Among the haptens available to chemically prepare non-radioactive probes, aeetylaminofluorene (AAF) or its 7-iodo derivative has been used by a number of investigators to label both D N A and RNA. At neutral pH, nucleic acids react readily in vitro with N-acetoxy-N-2-acetylaminofluorene in a simple reaction (1 h at 37°C); covalent coupling of 2-acetylaminofluorene groups takes place, mainly at the C8 position of guanine residues, thereby yielding about 5-10% modified bases (Miller et al., 1966; Kriek et al., 1967). After hybridization, the modified probe is de-

tected by using either polyclonal (Tchen et al., 1984) or monoclonal (Masse et al., 1985) antibodies directed against guanosyl-acetylaminofluorene and an immunoenzymatic technique. AAF-modified nucleic acids have been widely used for in situ (Landegent et al., 1984, 1985; Cremers et al., 1987), dot-blot (Tchen et al., 1984; Masse et al., 1985; Larzul et al., 1987; Sakamoto et al., 1988; Chevrier et al., 1989), Southern, colony-and plaque hybridizations (Masse et al., 1985) as well as for the detection of PCR products (Larzul et al., 1989). The ability to label RNA and DNA, double-stranded as well as single-stranded nucleic acids, extends the applicability of the AAF method. Probes prepared in this way can be detected at the pg level and are stable for at least 2 years. The hybridization test using AAF-labelled genomic DNA probes was sensitive enough to enable the detection of as few as l0 s Campylobacter cells (Chevrier et al., 1989). In a recent report (Grimont et al., 1989), we described the use of AAF-labelled Escherichia coil 16S + 23S rRNA as a probe for DNA fingerprinting in molecular epidemiology. Many methods have been proposed for chemically biotinylating the nucleic acid probes. Some of them make use of biotinylated-cytochrome C (Manning et al., 1975; Sodja and Davidson, 1978), biotinylated-histone (Renz, 1983) or biotinylated, single-strand binding protein (SSB) of E. coli linked to M13 single-stranded DNA (Syv~inen et al., 1985) as the DNA-binding component. These biotinylated proteins were bound to RNA or DNA and then cross-linked to the nucleic acids by formaldehyde or glutaraldehyde in order to stabilize the complex. In a further variation of this general approach, using various basic proteins and cross-linking reagents, AI-Hakim and Hull (1986) have bound biotin to single-stranded phage M13 DNA. Forster et ai. (1985) developed a photoactive reagent for direct biotin-labelling of nucleic acid probes. The reagent (Photobiotin) consists of biotin attached by a linker arm to a photoactivable azido group. Strong visible light for 10-20 min is used to convert the azido group into a highly reactive nitrene, which forms stable linkages with the nucleic acid (RNA or single- or doublestranded DNA). The sites of coupling have not

been determined, but an average incorporation of one biotin per 100-150 bases is obtained. Another photolabelling procedure for direct biotin-labelling of nucleic acid probes was developed by Sheldon et ai. (1986). It involves the use of single-stranded circular DNA of the bacteriophage M13 labelled with a biotinylated psoralen derivative, N-biotinyi-N'-(4'-methylene trioxsalen)-3,6,9-trioxaundecane-l,ll-diamine, as the probe. Using this reagent, 5-10 biotinylated psoralen moieties per 100 bases could be bound under UV irradiation. Viscidi et al. (i986) have developed a procedure involving the transamination of the 4-amino group of nucleic acid cytosine residues with a bifunetional amine, ethylene diamine, in the presence of sodium bisuiphite. The aliphatic primary amino groups on the cytosine derivatives are then coupled to biotinyi-(6-amino-hexanoyi)-N-hydroxysuccinimide ester to yield the biotinylated nucleic acid probe. DNAs modified by this method have 3-4% of their cytosine residues biotinylated. Cytosine residues can also be biotinylated by a bisulphite-catalysed transamination reaction between DNA and biotin hydrazide (Reisfeid et al., 1987). Recently, a novel bioanalytical indicator based on the digoxigenin:anti-digoxigenin interaction has been proposed for the detection of macromolecules (Kessler, 1991). This biospecific interaction has been found to be efficient in non-radioactive hybridization. As for biotin, the cardenolide digoxigenin can be chemically coupled to nucleic acid molecules both with a photoreactive azid~', derivative (Miihlegger et a1.,1990) or with digoxigenin-N-hydroxysuccinimide ester (Zischler et ai., 1989). A non-radioactive labelling technique based on the introduction of a mercury atom at the C5 position of the pyrimidine bases in nucleic acids has been described by Hopman et al. (1986a). Unlike the other non-radioactive hybridization methods, the hapten required for the hybrid detection is bound after the hybridization step; the mercurated hybridized probe is coupled to a hapten having a sulphydryl group. Verdlov et al. (1974) showed that the combined action of bisulphite and methyl-hydroxylamine upon cytosine residues in oligonucleotides

or denatured DNA results in stable derivatives of N-4-methoxy-5-6-dihydrocytosine-6-sulphonate. This reaction forms the basis of a non-radioactive DNA-labelling system (Lebacq et al., 1988), after hybridization; the sulphonated probe is detected by a monoclonal antibody. Oligonucleotides are almost always labelled with haptens at the 5' or 3' terminus rather than internally because internal modification of oligonucleotides can drastically lower melting temperature (Sanford and Krugh, 1985). The more general chemical methods of labelling oligonucleotides are based on reactions depending upon the presence of a unique functional group at the terminus of the oligomer. This functional group must be more reactive toward the activated hapten than the other reactive groups, such as phosphates or aromatic amino groups that are normally present in oligonucleotides. Generally, this functional group is an aliphatic primary amine (Coull et al., 1986; Wachter et al., 1986; Connolly, 1987) or thiol group (Connolly and Rider, 1985) that is introduced during the synthesis of the oligomer via an appropriately protected phosphoramidite. A variety of chemical methods have been developed to achieve this oligonucleotide modification (Mclnnes and Symons, 1989). Various haptens having an N-hydroxysuccinimide ester function or maleimide group can be easily bound to the 5' or 3' functionalized oligonucleotides. Alternatively, purified and deproteeted oligonucleotides can be derivatized via the free 5'-hydroxyl (Chu et al., 1983; Chollet and Kawashima, 1985; Chu and Orgel, 1985). According to this method, the oligonucleotide is first phosphoryiated using polynucleotide kinase, followed by treatment with a water-soluble carbodiimide and imidazole. The resulting phosphorimidazolidate is incubated with an excess of a diamine providing the phosphoramidate of this diamine, which is then ~.~sedto bind an activated hapten. Chemical modification reactions are advantageous: they are generally less expensive than enzymatic labelling and they can be controlled more easily than enzymatic reactions to yield probes in large amounts with optimal modification levels and unaltered hybridization properties. However', to achieve chemical modification, large quantities

of nucleic acids are needed, therefore, enzymatic labelling is preferable when only a small amount of aucleie aeid is available to prepare the probe.

Enzymatic labelling Hapten-modified nucleotide analogues, which are accepted as substrates by various enzymes (Klenow polymerase, E. coli DNA polymerase, reverse transcriptase, SP6/T7/T3 RNA polymerases, Taq DNA polymerase) can be introduced into nucleic acids (DNA or RNA) using the nick-translation technique (Rigby et al., 1977), by synthesis of the probe molecule on a template (Hu and Messing, 1982; Feinberg and Vogelstein, 1983, 1984; Melton et al., 1984; Studencki and Wallace, 1984) or during PCR (Lo et al., 1988, Seibl et al., 1990). The first enzymatic method for labelling DNA with biotin was described in 1981 by Langer et al. (1981), who used biotinylated analogues of thymidine triphosphate (biotinyl-dUTP). Although the incorporation rate was significantly lower than that of thymidine 5'-triphosphate (TI'P), nick translation with biotinyl-dUTP instead of "ITP still proceeded effectively, with a final incorporation generally in the range of 5 biotinylated bases per 100 bases of DNA. In addition to biotinyldUTP analogues, several biotinyl-dCTP and dATP analogues have also been synthesised and incorporated by various enzymes to obtain biotinylated DNA. Similarly, biotinylated ribonucleoside triphosphates can be incorporated into RNA by T7 or SP6 RNA polymerases (Langer et al., 1981; Luehrsen and Baum, 1987). An analogue of ATP, 8-[N(2,4-dinitrophenyi)6-aminohexyl] amino-ATP (DNP-rATP), is a substrate for terminal deoxynucleotidyitransferase and polyrnerases and, therefore, can be incorporated into DNA by 3' end labelling or nick translation (Vincent et al., 1982). Alternatively, the DNP group can be chemically bound to nucleic acids that have been enzymatically modified with 8-aminohexyl-ATP (Vincent et al., 1982). 5-bromo-2'-deoxyuridine (5-BrdU) can be incorporated into plasmid DNA using in vitro enzymatic labelling (Traincard et al., 1983) or into M13 DNA by growing the thymidine-requiring strain of E. coli in the presence of 5-BrdU using in vivo enzymatic labelling (Sakamoto et al., 1987).

M o r e recently, digoxigenin-modified deoxyribo- or ribonucleoside triphosphates have been synthesized to be enzymatically incorporated into D N A or R N A probes (H6Itke and Kessler, 1990; H~ltke et al., 1990; Kessler et al., 1990). When random-primed D N A synthesis, nick translation, eDNA synthesis or in vitro transcription is used for nucleic acid labelling in the presence of D I G - d U T P or DIG-UTP, an average of one digoxigenin molecule per 25-36 nucleotides is incorporated. Synthetic oligonucleotide probes are endlabelled using terminal deoxyribonucleotide transferase (Riley et al., 1986), T4 R N A ligase (Cosstiek et al., 1984) or a primer extension reaction witia E. coli D N A polymerase (Murasugi and Wallace, 1984). End-labelling should avoid any effect that internal modified bases may have on the stability of the oligonucleotide probe hybridized to the target sequence.

lmmunoenzymatic detection techniques for nucleic acid probing applications A variety of methods are available to detect hybridized non-radioactive probes. Detection systems can be classified into two main categories


(Fig. 5): (1) direct detection systems; and (2) indirect detection systems. In the direct detection systems (Fig. 5A) the signal-emitting molecule (label molecule) is attached directly to the nucleic acid probe. Various substances, such as enzymes (peroxidase, phosphatase, luciferase), fluorescent groups (fluorescein, rhodamine, europium, bimane) or chemiluminescent groups (acridinium esters) have been used as direct labels. Hybridized probe molecules are deteeted in one step by the addition of the appropriate substrate, chemicals or catalyser and by visualizing the color change, the fluorescence emission or the flash of light produced. In the indirect detection method, the probe cannot be detected alone; rather, it requires the use of an immunoenzymatic technique or a re-" lated affinity method. The simplest indirect procedure makes use of a specific antibody to D N A : R N A (Rudkin and Stoilar, 1977; Van Prooijen-Knegt et al., 1982; Boguslawski et al., 1986; Stollar and Rashtchian, 1987; Yehle et al., 1987) or D N A : D N A duplexes (Mantero et al., 1991). The duplex: antibody complex is then detected by using an.immunoehemicai method (Fig. 5B). Alternatively, in a more general procedure, the label is attached to a ligand specific to the marker molecule which is bound to the probe.


C D A 13 Fig. 5. Schematic representation of different detection systems. A: direct detect!on systemwith a probe that has been coupled to a label able to emit a measurable signal. B: indirect detection system using hybrid-specificproteins that have been coupled to a label able to emit a measurable signal. C: indirect detection systemwith a marker-modified probe and a marker-specificprotein that has been coupled to a label able to emit a measurable signal. D: amplified indirect detection systemwith a marker-modifiedprobe and a marker-specificprotein that is detected using a second labelled affinityprotein {antibodies,avidin,lectins... ).

The marker molecule is chemically or enzymatically incorporated into the nucleic acid probe (see above), and detected after hybridization with the aid of a recognizing molecule (antibody or other specific protein) carrying one of the available labels. The label molecule can be directly attached to the recognizing molecule (Fig. 5C) or alternatively, to an indirect detection system (Fig. 5D). Compared to the direct procedures, indirect procedures require additional steps after hybridization but they have several advantages: one of the various amplification systems available for enzyme immunoassay (Guesdon, 1988) can be introduced into the assay, hapten-labelled or unmodified probes are more stable than enzymelabelled probes, indirect procedures are more flexible since one given labelled probe can be detected with various techniques. We developed two procedures using avidinbiotin interacfio~ in a quantitative enzyme immunoa~say (Gue~0on et al., 1979). In the labelled avidin-biotin (LAB) technique, biotin-labelled antibody and enzyme-labelled avidin are used sequentially. In the bridged avidin-biotin (BRAD) technique, avidin acts as a bridge between biotin-labelled antibody and biotin-labelled enzymes. An indirect bridged avidin-biotin (IBRAD) technique using a second antibody has been developed by Hsu et al. (1981) to localize antigens in formalin-fixed, paraffin-embedded tissues. The avidin-biotin system associated with enzymatic detection was introduced by Kourilsky et al. in 1978 to specifically detect nucleic acid hybrids. After the hybridization, the biotinylated probe can be detected with avidin, streptavidin or antibodies. Although anti-biotin antibodies were used with some success (Megret et al., 1987) avidin or streptavidin have generally proved superior for non-radioactive hybridization assays (Leary et al., 1983). Hapten-labelled probes can be detected by either monoclonal or polyclonal antibodies. Comparison of the signal-to-noise ratio of the immunodetection system shows a clear advantage of the monoclonal versus the polyclonal antibody. The polyclonal antibodies generally yield a higher non-specific signal. This observation may be related to the presence in antisera of naturally

occurring anti-DNA antibodies. Moreover, the monoclonal antibodies enable the reproducible isolation, with good yield, of a homogeneous, highly specific and purified affinity protein, whereas such a purification cannot be obtained with polyclonai antibodies. I n immunoenzymatic techniques, the enzyme is generally cross-linked with the antibodies through covalent bonds using various organic compounds (Avrameas et a!.,1978; Ishikawa et al., 1983). It should always be considered that chemical modification of an enzyme affects its activity to a certain extent. For example, the covalent linkage between an enzyme and an antibody can decrease the enzyme's activity due to steric hindrance and modification of the net charge or the charge distribution of the enzyme protein. Moreover, a severe diminution of the antibody binding capacity has been observed during conjugate preparation, and the product of a coupling reaction may often form a heterogeneous molecular population. In order to overcome the drawbacks of techniques based on covalent coupling, several methods involving a biospecific interaction between the enzyme and the antibodies have been devised (Avrameas et al., 1983). These methods include a lectin-immunotest and a chimera antibody technique which make use of lectin-antibody conjugates (Guesdon and Avrameas, 1980) or chimera antibodies of double specificities (Guesdon et al., 1983). One novel detection system uses a monoclonai antibody with dual specificities directed against a biotinyl group and peroxidase (Leong et al., 1986). This bispecific monoclonal antibody was produced by an anti-peroxidase/anti-biotin hybrid-hybridoma rat cell line. The use of such antibodies and free enzymes is an interesting alternative to obviate the need for a second antibody and coupling procedures. The assay detection limit depends largely upon the detection limit of the label. Therefore, a probe assay based on a label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). One can easily imagine that if greater sensitivity were required, the enzyme would be allowed to operate for a longer time.

43 Although many enzymes have been suggested as labels, most currently used hybridization assays employ either calf intestine alkaline phosphatase or horseradish peroxidase. /3-galactosidase from Escherichia coli is also used, but to a lesser extent. Perhaps the greatest advantage of enzyme labels, and the above-cited enzymes in particular, is that they offer the choice of the detection method. Indeed, enzyme activity can be determined by reactions giving either insoluble products or coloured, fluorescent or luminescent soluble products.

Enzyme reactions giving insoluble products Generally, the enzyme label is used to catalyse the formation of visible, coloured spots on a hybridization filter. Today, the most sensitive detection which leads to the formation of insoluble precipitates is obtained using alkaline phosphatase and a mixture of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue-tetrazolium salt (NBT) as the substrate solution (McGadey, 1970). First, the reaction involves the dephosphorylation of BCIP by the enzyme, then the hydroxyl group undergoes tautomerization forming a ketone which oxidizes and dimerizes to produce a blue precipitate (5,5'-dibromo-4,4'-dichloro-indigo). During dimerization, hydrogen ions are released and these reduce the NBT which precipitates, forming purple diformazan. Reactions giving insoluble products can also be obtained using peroxidase or /3-galactosidase and the corresponding substrates, such as 3,3'-diaminobenzidine (DAB) or 3,3',5,5'-tetramethyibenzidine (TMB) for peroxidase and 5-bromo-4chloro-3-indolyl-/3-galactopyranoside (BCIG) for /3-galactosidase (Bondi et a1.,1982). For in situ hybridization with analysis at the light and electron microscope levels, the peroxidase label is widely used. In this approach, electron-dense detection is provided by gold/silver enhancement of the coloured product of the peroxidase reaction with DAB. The non-radioactive hybridization method applied for in ~itu analysis possesses a definite advantage over radioactive hybridization. Indeed, the former allows the bicolour detection and, thus, two different nucleic acid sequences can be detected and localized on the same sample (Hopman et al., 1986b).

The enzyme reaction giving insoluble products is a very simple and convenient method to detect nucleic acid hybrids on a membrane; it does not require any special equipment and a visual readout is possible. The main disadvantage is that the insoluble coloured product is very difficult to remove, and hence the filters can only be probed once.

Enzyme reactions giving soluble products Colorimetric detection. The well-known chromogenic substrates, p-nitrophenylphosphate (pNPP) for phosphatase, hydrogene peroxide with o-phenylenediamine (OPD) for peroxidase and o-nitrophenyl-/3-o-galactopyranoside (ONPG) for fl-galactosidase, form soluble, spectrophometricaUy measurable products and are extensively used in enzyme immunoassays. The detection methods using these chromogenic substrates are not sensitive enough to be utilized in most hybridization assays. The highly sensitive method based on an enzyme amplification reaction, described by Self (1985) and applied to enzyme immunoassay (Stanley et al., 1985; Johannsson et ai., 1986), has also been proposed to probe DNA (Gatley, 1985). In this method, alkaline phosphatase is used as the label. The substrate nicotinamide adenine dinucleotide phosphate (NADP +) is dephosphorylated by phosphatase to produce NAD ÷. NAD ÷, in turn, activates a seeondary enzyme system which comprises a redox cycle driven by two enzymes, namely, alcohol dehydrogenase and diaphorase. During each round of this cycle one molecule of a tetrazolium salt, iodonitrotetrazolium violet, is reduced to an intensely coloured soluble formazan dye. Absorbance is measured at 492 rim. This detection method is capable of measuring as little as 0.01 amol (approx. 6600 molecules) of alkaline phosphatase (Johannsson et al., 1986). Fluorescence detection. Fluorescence-amplified enzyme immunoassays appear to be more sensitive than the corresponding enzyme immunoassay performed with a chromogenic substrate. Fluorogenie substrates have been used in enzyme immunoassay to measure peroxidase (Zaitsu and Ohkura, 1980), alkaline phosphatase (Shalev et al., 1980) or/3-galactosidase (Ishikawa and Kato, 1978; Labrousse et al., 1982). The best

signal amplification is observed when using fl-galactosidase and its fluorogenic substrate 4-methylum belliferyl-/3-o-galactopyranoside (MUG). Indeed, it is noteworthy that fluorescence detection of peroxidase is less sensitive than fluorescence detection of alkaline phosphatase or fl-galactosidase, and that 4-methylumbelliferyl-phosphate gives a higher reagent blank than MUG and shows significant spontaneous hydrolysis, limiting the sensitivity of alkaline phosphatase determination. Nagata et al. (1985) and Yokota et al. (1986) have detected picogram levels of specific DNA using M U G as the substrate for fl-galactosidase. Luminescence detection. Luminescence detection techniques have become available to detect small amounts of enzyme. In these techniques, the light emission from the luminescent reaction is quantified either in a luminometer, or recorded on instant photographic or X ray film directly from the hybridization filter. Using the glycoprotein Pholas dactylus luciferin as the substrate in a bioluminescence technique, Puget et al. (1977) were able to estimate extremely low amounts of peroxidase: as little as 5 fg could be detected. A number of chemiluminescence assays for peroxidase have been developed. Among them, the assay combining luminol, hydrogen peroxide and an enhancer (e.g., certain phenols; naphthols, aromatic amines or benzothiazoles) was proven to be highly sensitive and to have some advantages over the other chemiluminescence assays for peroxidase (for review, see Kricka et a1.,1991). This peroxidase-catalysed enhancement of chemiluminescence has been applied in the final detection of nucleic acid hybrids in E'NA probing assays performed on filters and on beads (Matthews et al., 1985; Figueiredo and Malcolm, 1986; Urdea et al., 1987, 1988) with detection limits for DNA in the attomole range: A bioluminescence assay for alkaline phosphatase has been described using a firefly Dluciferin-O-phosphate substrate. The enzyme cleaves the phosphate group to generate firefly D-luciferin which reacts with firefly luciferase to produce light. This bioluminescence detection system has been used in hybridization experiments enabling the detection of 19 ng of target DNA (Hauber and Geiger, 1988). Recently, a

chemiluminescent substrate for alkaline phosphatase (adamantyl-l,2-dioxetane phenyl phosphate) has been developed (Schaap et ai., 1987; Bronstein and McGrath, 1989). This stable 1,2-dioxetane derivative is converted by alkaline phosphatase into labile 1,2-dioxetane and, as a result, an almost instantaneous chemiluminescence is observed following the kinetics of the enzyme reaction. As few as 100 molecules of alkaline phosphatase can be detected using this substrate. D N A hybridization experiments using this substrate have been performed successfully (Bronstein et al., 1989; Urdea et al., 1989). A similar chemiluminescent compound (adamantyl-l,2-dioxetane phenyl-/3-o-galactopyranoside) is available for/3-galactosidase detection. A bioluminescent enzyme-channelling reaction, coupling glucose-6-phosphate dehydrogenase to bacterial luciferase and an oxidoreductase system has been applied to DNA detection (Balaguer et al., 1989,1991). The bioluminescent reaction is triggered by the NADH produced by the glucose-6-phosphate dehydrogenase linked to the oligonucleotide probe and the light emitted at 495 nm can be detected with a cooled-charge collection device (CCD) camera or a luminometer. The main advantage of the luminescent detection techniques is that they allow reprobing. Indeed the probe can be completely washed off the solid support and a second probe can be used on the same set of samples. The luminescent detection reagents for peroxidase, alkaline phosphatase and fl-galactosidase are now commercially available in kits.

Concluding remarks It is now clear that non-isotopic detection systems are successfully replacing radioisotopes in the field of molecular hybridization. Numerous non-radioactive methods for labelling and detecting nucleic acids have been described over the past 10-15 years. However, relatively few of them have been routinely used to detect hybridization events, lmmunoenzymatic or related techniques have proven to be useful for the detection as well as the quantitation of DNA and R N A sequences

and, therefore, they are being extensively applied to nucleic acid probing. U n d o u b t e d l y , f u t u r e detection s y s t e m s will be b a s e d o n amplifications: amplification of the target molecule ( P C R ) a n d / o r amplification of t h e signal g e n e r a t e d by a m a r k e r molecule. However, it is w o r t h noting that detection limit improvem e n t is s e l d o m achieved simply by u s i n g a highly sensitive d e t e c t i o n s y s t e m i n s t e a d of t h e classical colorimetric m e t h o d . I n d e e d , a significant increase in detection system sensitivity generally results in a b a c k g r o u n d noise increase as well, therefore, t h e signal-to-noise ratio is n o t improved. C o u t l e e et al. (1989c)demonstr,'-,-ed that colorimetric d e t e c t i o n can be r e n d e r e d as sensitive as t h e e n z y m o f l u o r e s c e n t detection or an e n z y m a t i c amplification cycling system simply by p r o l o n g i n g t h e d e t e c t i o n time. Clearly, major g a i n s will d e p e n d u p o n r e m o v i n g t h e b a c k g r o u n d signal. Since t h e initial r e p o r t s o f t h e i m m u n o e n z y m a t t e m e t h o d - b a s e d hybridization assays, d a t a c o n c e r n i n g t h e s e t e c h n i q u e s have gradually accum u l a t e d a n d it is now a p p a r e n t that this system is applicable to a wide variety o f tests which are utilized in various fields o f biology, including genetics, bacteriology, virology, mycology, oncology. A f u t u r e a n d potentially m a j o r application of non-radioactive D N A p r o b e technology would be testing o f water, food, soil a n d plant s a m p l e s for t h e p r e s e n c e of p a t h o g e n i c m i c r o o r g a n i s m s . In m y opinion, t h e versatility a n d flexibility of t h e hybridization assay b a s e d on t h e i m m u n o e n zymatic m e t h o d are p e r h a p s its m o s t attractive features. F u r t h e r m o r e , chemical a n d e n z y m e labelling m e t h o d s , several labels a n d m a n y detection systems, a variety of solid p h a s e s a n d formats, a n d several i n s t r u m e n t a l a p p r o a c h e s are available.

Acknowledgements 1 would like to t h a n k t h e following colleagues, D. Chevrier, F. Girard, T h a m T o N a m a n d D. T h i e r r y for their cooperation in this study a n d for useful discussions. 1 also wish to t h a n k V. S t o n n e t for help in reviewing t h e m a n u s c r i p t .

T h e a u t h o r is grateful for the financial support of the Minist~re de I'Agriculture ct dc la For~t (grant R 9 1 / 2 8 ) .

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Immunoenzymatic techniques applied to the specific detection of nucleic acids. A review.

Numerous enzymatic and chemical methods are now available for the preparation of non-radioactive nucleic acid probes. Labels, such as enzymes, fluorop...
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