Biocatalysis in Agricultural Biotechnology - American Chemical Society

Chapter 16

Enzyme-Probe Conjugates as Analytical Tools in Diagnostics Charles A. Dangler and Bennie I. Osburn

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch016

Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 Applications of enzymatic detection systems in the context of diagnostics are presented in this article. An overview of detection systems, including those based on non-enzymatic markers such as radioactive, fluorescent and particulate labels, is discussed i n i t i a l l y . Direct and indirect strategies for applying detection systems are also presented with respect to the identification of immunoglobulin and nucleic acid probe molecules. Enzymatic detection systems are discussed with an emphasis on chromogenic enzyme reactions. Methods of designing enzymatic assays often involve using molecular bridges between enzyme molecules and the probe species; immunodetection is discussed in this context as well as procedures for attaching immunodetectable moieties to probe molecules. Finally, current and potential developments which may improve the application of enzymatic detection systems are briefly outlined. In this presentation we will discuss analytical detection systems composed of enzyme and probe molecular components working in tandem, with the enzymes serving the role of the signal-generating mechanism linked to ligand-specific biomolecular probes. In general, we will draw a distinction between the enzyme and the probe components. These two components may be applied simultaneously as a single conjugated reagent or in series with the enzymatic component following the application of the probe. In this design, the enzyme molecules offer the means for evaluating the result of an assay, and in large part affect the sensitivity of the assay in question by the degree of substrate metabolism and the signal to background ratio; however, the probe species, which will be more fully described later, directs the specificity of the assay by virtue of its particular binding relationship with the defined target ligands. Since the broad topic of the agricultural application of enzymes is being addressed and since some of the recent work of the 0097-6156/89/0389-0230$06.00/0 « 1989 American Chemical Society

Whitaker and Sonnet; Biocatalysis in Agricultural Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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Enzyme-Probe Conjugates in Diagnostics

authors has been concentrated on diagnostic applications of molecular probes for analyzing b i o l o g i c a l specimens for the presence of infectious pathogens, c h i e f l y of veterinary i n t e r e s t , the applications to be discussed w i l l be i n large part from the point of view of a diagnostic laboratory, rather than an a n a l y t i c a l research laboratory. While maximum s p e c i f i c i t y and s e n s i t i v i t y are desirable i n both laboratory situations, the p o t e n t i a l hazards, expense, r a p i d i t y of execution, and labor requirements of an assay technique are factors which might require greater consideration when adapting an assay system for p r a c t i c a l application i n a diagnostic laboratory. The l a t t e r concerns, i n general, support an i n t e r e s t i n enzyme-based detection systems. Before l i m i t i n g the discussion to the use of enzyme-based detection methods, we w i l l present an overview of detection scheme, including non-enzymatic techniques. The probe systems currently used for analyzing the macromolecular composition of b i o l o g i c a l specimens are t y p i c a l l y composed of a s p e c i f i c probe molecule linked i n some fashion to an, oftentimes, multipurpose detection system. The two major probe groups we w i l l discuss w i l l be antibodies and nucleic a c i d probes. Antibody probes, of either polyclonal or monoclonal o r i g i n , are used for the i d e n t i f i c a t i o n of s p e c i f i c antigenic determinants or epitopes; nucleic acid probes, commonly developed nowadays through recombinant cloning techniques, are used for i d e n t i f y i n g s p e c i f i c genetic sequences. Selection of the probe system i s dependent on the target molecules available for detection. As an example, a polyclonal antibody reagent may prove superior to a more s p e c i f i c monoclonal antibody reagent i n diagnosing the presence of a v i r u s i n an infected c l i n i c a l specimen because of the a b i l i t y of the former reagent to detect multiple target epitopes on the v i r a l p a r t i c l e s , whereas the l a t t e r reagent may detect only a s o l i t a r y epitope subset within the sample, thereby l i m i t i n g the s e n s i t i v i t y . The polyclonal reagent may s a c r i f i c e assay s p e c i f i c i t y , however, by cross-reacting with other related viruses that bear a few common epitopes. In the case of a latent v i r a l i n f e c t i o n though, both the polyclonal and monoclonal antibody reagents might prove useless because i n s u f f i c i e n t amounts of v i r a l peptides are synthesized by the infected tissue. In this instance nucleic a c i d probes might detect the v i r a l genome within the specimens more r e a d i l y ; t h i s i s the case with the provirus form of the human immunodeficiency v i r u s and other retroviruses. Following s e l e c t i o n of the probe system, the detection system i s chosen to complement the needs of the user and to o f f e r appropriate and acceptable l e v e l s of s e n s i t i v i t y . Detection systems may be divided into radioactive/nonradioactive (Table I) and d i r e c t / i n d i r e c t detection systems. Radioactive/Non-radioactive Detection Systems. Radioisotopic detection methods frequently employ molecules containing radionuclides of hydrogen, s u l f u r and phosphorus, and less frequently iodine and chromium. Oftentimes, the probe molecules themselves are d i r e c t l y radiolabeled for immediate detection. Nucleic acid probes are commonly labeled by enzymatic incorporation of radiolabeled nucleotides or enzymatic addition of radiolabeled phosphate groups to the nucleic a c i d chain. Proteins, i n p a r t i c u l a r immunoglobulins, are labeled commonly by d i r e c t


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radioiodination which achieves a high s p e c i f i c a c t i v i t y and may be accomplished by chemical means using chloramine Τ ( 1 ) , Nsuccinimidyl 3-(4-hydroxy,-5-[^^1]iodophenyl) propionate ( 2 ) , or l,3,4,6-tetrachloro-3oc,6oc-diphenyl g l y c o l u r i l ( 3 ) . Radioiodination of proteins may also be accomplished by enzymatic means by lactoperoxidase-catalyzed iodination ( 4 ) . Proteins may also be t r i t i a t e d by reductive methylation (5.) or radiolabeled with a v a r i e t y of isotopes by biosynthetic incorporation of labeled amino acids. Radioisotopic labels may also be linked to probe molecules through a series of bridge molecules, which w i l l be presented under i n d i r e c t detection methods. The s e n s i t i v i t y of r a d i o i s o t o p i c assays i s frequently excellent; however, low s i g n a l to background noise r a t i o s may impair assay i n t e r p r e t a t i o n . The l i m i t i n g factors i n the a p p l i c a t i o n of radioisotopic techniques i n diagnostic laboratories are the b i o l o g i c a l hazard, p o t e n t i a l contamination problems, and short h a l f - l i f e of labeled probes. Despite the drawbacks, radiolabeled probes are used frequently i n nucleic a c i d b l o t and i n s i t u h y b r i d i z a t i o n assays, and i n radioimmunoassays. Current non-radioactive detection methods generally only approach the s e n s i t i v i t y of radioisotopic assays; however, reduced hazards and enhanced reagent s t a b i l i t y are v a l i d considerations i n t h e i r defense. While enzymatic detection systems that y i e l d colorimetric r e s u l t s , which we w i l l discuss more f u l l y l a t e r , are perhaps the most popular of the non-radioactive methods, a v a r i e t y of other techniques for detecting probe molecules e x i s t , as well as a v a r i e t y of techniques for l i n k i n g detection systems to probe molecules. Other common detection systems are based on (a) i n e r t materials, t y p i c a l l y p a r t i c u l a t e , such as latex, f e r r i t i n and c o l l o i d a l gold ( 6 - 8 ) , and (b) fluorescent compounds ( 9 ) . The p a r t i c u l a t e markers are t y p i c a l l y used by coating them with a material that can bind them to target-probe complexes. Latex p a r t i c l e s are commonly used i n agglutination assays. F e r r i t i n and c o l l o i d a l gold more recently have been used as markers i n electron microscopic studies because of t h e i r electron dense properties ( 8 ) . C o l l o i d a l gold i s also being used i n tandem with s i l v e r coating enhancement for l i g h t microscopic studies. Fluorescent compounds tagged onto immunoglobulins have long been used i n fluorescent antibody techniques for i n s i t u v i s u a l i z a t i o n of antigens and are now being incorporated into r e l a t i v e l y new areas such as flow cytometry ( 1 0 ) and nucleic a c i d sequencing. Less common, and at t h i s point esoteric from the standpoint of a g r i c u l t u r a l applications, are chemiluminescent ( 1 1 - 1 3 ) and electrochemical ( 1 4 ) detection methods. These l a t t e r detection systems because of purported enhanced s e n s i t i v i t y may f i n d applications i n c l i n i c a l chemistry. Direct/Indirect Detection Systems. Direct detection of molecular probes requires physical attachment of the detection system component to the probe, such that binding of the probe to i t s target molecule r e s u l t s i n immediate attachment of the detection system to the target-probe complex. In contrast, i n d i r e c t detection requires that further separate steps be performed to i d e n t i f y target-probe complexes a f t e r they are formed. Direct and i n d i r e c t target-probe-



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enzyme interactions as they are used i n immunoassays are b r i e f l y i l l u s t r a t e d i n Table I I . Radioisotopes are an obvious example of a d i r e c t l a b e l i n g system. Countless biomolecules have been synthesized and are commercially available f o r d i f f e r e n t radioisotopic assays. As noted above, there i s a trend away from radioactive assays which has l e d to the design of d i f f e r e n t methods of d i r e c t l y l i n k i n g detectable moieties, such as fluorescent compounds or enzymes, to molecular probes, l i k e immunoglobulins and nucleic acid hybridization probes. The d i r e c t method has the obvious advantage of requiring fewer steps to i d e n t i f y target-probe complexes; however, this technique does require the s p e c i f i c synthesis of probe-detector reagents f o r each target assayed. Indirect detection does require more steps, but oftentimes y i e l d s amplified signals r e l a t i v e to d i r e c t methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. I t i s probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the s e n s i t i v i t y of radiolabeled systems. The use of these i n d i r e c t methods reduces s t e r i c problems that might arise from having enzyme molecules d i r e c t l y bound to probe molecules. Acceptable bridging molecule systems have been developed which have also s i m p l i f i e d the u t i l i z a t i o n of d i f f e r e n t detection systems. To i l l u s t r a t e t h i s point, a researcher who has developed a unique monoclonal antibody (a primary antibody) i n the mouse may select from a v a r i e t y of commercially available products consisting of d i f f e r e n t detection systems (e.g. fluorescein, alkaline phosphatase, c o l l o i d a l gold) attached to an immunoglobulin that w i l l s p e c i f i c a l l y bind to mouse antibodies (a secondary antibody). In this way the researcher may readily obtain and test a number of detection methods for v i s u a l i z i n g target-probe interactions without having to d i r e c t l y l a b e l the monoclonal antibody probe. For nucleic acid probes, which i n themselves are not r e a d i l y immunodetectable, i t i s useful to incorporate or attach detectable moieties to the nucleotides. B i o t i n has served this purpose well i n both nucleic acid and antibody probe systems. As well as being e a s i l y detected with immunoglobulins s p e c i f i c f o r b i o t i n , b i o t i n may also be detected non-immunologically with avidin or streptavidin, two proteins which share a marked, highly s p e c i f i c a f f i n i t y f o r b i o t i n . The a f f i n i t y constant f o r a v i d i n - b i o t i n interactions i s approximately 1 0 ^ liters/mole, much higher than the range f o r antigen-antibody interactions which are commonly between 10^-10^ liters/mole. Consequently, a vast number of detection complexes composed of avidin or streptavidin bound to a detection system are commercially available (e.g. streptavidin-alkaline phosphatase). Two r e l a t i v e l y new immunodetectable moieties have recently been developed f o r nucleic acid probe systems which allow us to i l l u s t r a t e two d i f f e r e n t methods of incorporating labels into nucleic acids. Both methods have been employed f o r b i o t i n labeling. O r i g i n a l l y nucleic acid probes were labeled by enzymatic incorporation of pre-labeled nucleotides, radiolabeled nucleotides, biotin-UTP/dUTP, 5-bromodeoxyuridine (15), or most recently a s t e r o i d hapten linked nucleotide analogue, digoxigenin-dUTP (16).


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Table I. Detection Systems


Detection Label Radioactive

Non-radioactive

Signal types

Radioisotopic

Particulate Fluorescent Enzymatic : Chromogenie Chemiluminescent Electrochemical

Advantages

High s e n s i t i v i t y Direct detection

Reduced hazard Stable s h e l f - l i f e

Disadvantages

B i o l o g i c a l hazard T y p i c a l l y used by Potential contaminants i n d i r e c t detectionShort s h e l f - l i f e time consuming Less sensitive, although potential for enhancement exists

Table I I .

Immunoassays: Target-Probe-Enzyme Interactions

Direct Detection Ag - Abi*Enzyme Indirect Detection Ag - Abi - Ab2*Enzyme Ag - Ab]*B - SA - B*Enzyme Ag - Abj - Ab2*B - SA - B*Enzyme

Ag Ab^Ab2Β SA - *

target antigen primary antibody secondary antibody biotin streptavidin non-covalent interaction - covalent i n t e r a c t i o n



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The l a t t e r i s detected using a proprietary enzyme-linked immunoassay. A new development appears to be post-synthesis labeling, i n which subsequent to nucleic a c i d probe synthesis or p u r i f i c a t i o n , the unlabeled nucleic acid probe i s subjected to a non-enzymatic, chemical process that modifies the o r i g i n a l probe molecule. Chemical methods for attaching b i o t i n to macromolecules using succinimide esters containing b i o t i n y l a t e d moieties have been described (17.18). B i o t i n y l a t i o n occurs under mild conditions by reaction of the succinimide esters with amino groups i n the proteins or on transaminated nucleotides. A r e l a t i v e l y new reagent, photobiotin, i s a photoactivatable compound that apparently achieves s i g n i f i c a n t l a b e l i n g of nucleic acids with b i o t i n upon b r i e f exposure to an intense sunlamp source (19). Another method of generating chemically modified, immunodetectable nucleic a c i d probes employs a rather toxic chemical N-acetoxy-N-2-acetylaminofluorene (10.20). A newer system with reduced p o t e n t i a l hazard i s available which chemically modifies nucleic acids, s p e c i f i c a l l y forming sulfonated cytidine residues (21) using b i s u l f i t e ions and 0methylhydroxylamine. DNA probes containing the sulfonated cytidine residues are detected by a proprietary system consisting of a mouse monoclonal antibody to sulfonated DNA, which i n turn i s detected by a secondary antibody-enzyme complex composed of anti-mouse antibodies covalently linked to a l k a l i n e phosphatase. This l a s t case probably best exemplifies the rather furious manner i n which new techniques may be p i l e d upon one another to achieve a goal or a product. Enzyme-Probe Conjugates While enzymes may be covalently attached d i r e c t l y to primary probe molecules, as noted above for reasons of reagent v e r s a t i l i t y , s t e r i c factors, and p o t e n t i a l signal amplification, i n d i r e c t detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are t y p i c a l l y complexes of a desired enzyme marker and a secondary l e v e l probe; that i s , a probe molecule that can s p e c i f i c a l l y i d e n t i f y a primary l e v e l probe molecule, such as an a l k a l i n e phosphatase-streptavidin conjugate can i d e n t i f y a b i o t i n y l a t e d nucleic acid probe by v i r t u e of the binding a f f i n i t y between s t r e p t a v i d i n and b i o t i n . Other examples of enzyme-probe systems are given i n the preceding section on d i r e c t and i n d i r e c t detection systems. Enzyme markers and associated substrates are selected for several properties, including s t a b i l i t y and a low detection l i m i t . A low detection l i m i t d i r e c t l y influences the s e n s i t i v i t y of the enzyme-based assay. The f i n a l enzyme-substrate i n t e r a c t i o n must y i e l d an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors' experiences have been c h i e f l y with enzymatic detection systems which culminate i n a v i s i b l e chromogenic reaction (e.g. a l k a l i n e phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). Results are assessed either by d i r e c t v i s u a l observation or spectrophotometrically. For the sake of completeness, i t should be mentioned i n this section that enzyme detection systems have been described which are monitored by alternative methods. These


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techniques, as noted e a r l i e r , seem esoteric with respect to most diagnostic assay procedures at t h i s time. Rather than standard colorimetrie monitoring, these other methods involve enzyme catalyzed generation of luminescent (11-13) or electrochemically (14) detectable end products which are subsequently monitored by s p e c i a l i z e d equipment. A v a r i e t y of enzymes have become popular f o r use i n nonradioactive detection systems. The most popular enzymes, as judged by what i s commercially marketed, are horseradish peroxidase, alkaline phosphatase, and /?-galactosidase. As an aside, the gene for 0-galactosidase i s also commonly used i n molecular biology as a marker f o r gene transfer into /3-galactosidase d e f i c i e n t bacteria. B a c t e r i a l colonies grown i n the presence of substrate-laden agar generate a blue pigment i f the gene has been successfully transferred and translated. In general, these enzymes are s i m i l a r i n s e n s i t i v i t y , although some authors have indicated higher signals on s o l i d phase systems with alkaline phosphatase r e l a t i v e to horseradish peroxidase. Selection of enzyme i s often based on the samples being analyzed and the reaction environment. Some c l i n i c a l specimens or other samples may have a high endogenous l e v e l of a given enzyme, which might y i e l d a high background signal i n enzymebased assays. /?-Galactosidase-based assays are subject to less interference f o r this reason because this enzyme i s not present i n mammalian systems. In the case of high endogenous a c t i v i t y , methods for selective i n a c t i v a t i o n of endogenous enzymes have been devised. The presence of i n t e r f e r i n g compounds, such as preservatives (e.g. sodium azide), might also influence the s e l e c t i o n of enzyme. In the case of sodium azide, horseradish peroxidase would be i n h i b i t e d and, therefore, of l i m i t e d value. Enzyme-linked Immunoassays. Enzyme-linked immunoassays (EIA) are, by the most general d e f i n i t i o n , systems which use an enzymeimmunoglobulin conjugate to detect the presence and amount of a given antigenic compound. L i k e l y antigens may include infectious microorganisms, drugs, hormones, tumor antigens, and even other immunoglobulins. In veterinary medicine EIA have been developed to detect a v a r i e t y of exogenous agents such as bacteria, viruses, metazoan parasites, and mycotoxins, and host factors such as progesterone. The technical subdivisions of EIA are covered elsewhere (22). In general, immunodetection i s usually accomplished by binding the antigen to a s o l i d matrix p r i o r to application of the enzymeantibody conjugate. The interaction between antigen and s o l i d matrix i s a variable factor depending on the nature of the assay format and the o r i g i n a l state of the antigen. I f the antigen i s i n solution i t may be applied d i r e c t l y to a s o l i d matrix, such as p l a s t i c multi-well plates (enzyme-linked immunosorbent assay [ELISA] p l a t e s ) , and n i t r o c e l l u l o s e or nylon sheets. Oftentimes, these media are s p e c i a l l y modified or coated to enhance the binding of macromolecules, t y p i c a l l y proteins. The interactions between the antigenic material and binding matrices i n these cases are not s p e c i f i c or selective f o r the desired antigen. An antigen solution may also be subjected to electrophoresis p r i o r to b l o t t i n g to a n i t r o c e l l u l o s e or nylon sheet (western b l o t s ) , allowing some non-



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immunologic d i f f e r e n t i a t i o n of the antigen from other materials i n the solution. S p e c i f i c antigen trapping to a s o l i d matrix may be accomplished by f i r s t coating the matrix with antibodies that are s p e c i f i c for the desired antigen. In t h i s fashion when the antigen solution i s exposed to the s p e c i a l l y prepared matrix, the antigen w i l l be s e l e c t i v e l y bound to the matrix by v i r t u e of the antibody coating. Antigens not i n solution, as i n a piece of biopsied tissue, may obviously be l i b e r a t e d to form an antigen solution; however, frequently i t i s desirable to v i s u a l i z e the antigens i n s i t u . In the case of infectious microorganisms the relationship of a pathogen's antigens with respect to infected host tissues may reveal c r i t i c a l information about disease processes. In the instance of i n s i t u antigen detection the antigen-bearing material i s usually preserved by methods that hopefully w i l l not modify the antigen. The detection of antigens i n s i t u often follows s i m i l a r procedures to those used on antigens bound to a r t i f i c i a l media. Regardless of the d i f f e r e n t methods of preparing antigens, enzyme-linked immunodetection techniques are reasonably s i m i l a r . As noted above, detection may proceed d i r e c t l y or i n d i r e c t l y . In the former case only a primary layer of antibodies are used. These antibodies are s p e c i f i c for the desired antigen and are d i r e c t l y conjugated to molecules of a selected enzyme. For i n d i r e c t detection the primary antibody i s not d i r e c t l y conjugated to an enzyme. Instead, the enzyme i s bridged to the primary antibodies by a layer of enzyme-conjugated, secondary antibodies usually directed to the species-specific, xenospecific epitopes on the primary antibodies. A l t e r n a t i v e l y and commonly, b i o t i n i s employed for i n d i r e c t detection. The primary or secondary layer of antibodies are chemically b i o t i n y l a t e d before use and a f t e r a p p l i c a t i o n are followed by a b i o t i n - s p e c i f i c immunoglobulin or avidin/streptavidin molecules conjugated to the detection enzyme, which i d e n t i f y the antigen-biotinylated antibody complexes. After establishing a molecular bridge between antigen and the detection enzyme by means of s p e c i f i c antigen-antibody reactions, the antigen-antibody-enzyme complexes are v i s u a l i z e d t y p i c a l l y by chromogenic reactions. A v a r i e t y of enzyme-substrate systems have been defined which y i e l d soluble or insoluble, pigmented end products of d i f f e r e n t colors based on the needs of the researcher. It should be noted that while the enzymes and bridging molecules are r e l a t i v e l y harmless, the substrate chemicals may vary from harmless, to unknown biohazard p o t e n t i a l , or to recognized carcinogenic/mutagenic compounds. An i n t e r e s t i n g twist has been added to the basic ELISA format using a l k a l i n e phosphatase as the detection enzyme. Rather than the usual system of measuring the attachment of enzyme molecules by following immediately with a chromogenic substrate mixture, a system has been reported which generally amplifies the enzyme signal 30-50f o l d (23) by layering multiple enzyme systems. Application of the technique for assaying thyroid stimulating hormone resulted i n a 70f o l d increase i n s e n s i t i v i t y (24). The amplification of the a l k a l i n e phosphatase signal i s accomplished by incubating a NADP solution i n the presence of a l k a l i n e phosphatase s p e c i f i c a l l y bound to the surface of an ELISA well and following this incubation with +


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the addition of an amplifier mixture containing alcohol dehydrogenase, diaphorase, ethanol, and a chromogenic substrate, piodonitro-tetrazolium v i o l e t . NAD , i n i t i a l l y formed by dephosphorylation, activates a redox cycle driven by alcohol dehydrogenase and diaphorase. The NAD i s reduced by the alcohol dehydrogenase, and the resultant NADH i n turn reduces the tetrazolium s a l t substrate to form a formazan dye . The amount of dye formed i s proportional to the o r i g i n a l amount of NAD formed by the bound alkaline phosphatase. The design of t h i s system l i m i t s i t s application at t h i s time to assays which are c a r r i e d out i n solution phase; the technique has not been adapted to b l o t or i n s i t u assay formats. +

+


+

Nucleic Acid Hybridization. The potential diagnostic uses of nucleic a c i d hybridization assays (NAHA) have been discussed (26.27), usually with respect to i d e n t i f y i n g pathogenic organisms by the presence of t h e i r genetic material. Some infectious agents of interest to the livestock industry for which nucleic a c i d probes have been reported include infectious bovine r h i n o t r a c h e i t i s (28), bluetongue (29.30), pseudorabies (31), and Anaplasma marginale (32). Hybridization assays have also been developed for detecting the b a c t e r i a l contaminants Salmonella and Campylobacter i n food (33). In addition, nucleic acid probes have been applied for the i d e n t i f i c a t i o n of host genetic factors associated with the occurrence of hereditary diseases (34). Nucleic a c i d hybridization may also be applied with DNA f i n g e r p r i n t i n g techniques for animal i d e n t i f i c a t i o n and paternity testing (35). NAHA d i f f e r from EIA at the l e v e l of the s p e c i f i c target-probe i n t e r a c t i o n . In EIA the target-probe complex i s formed by the union of antigen and s p e c i f i c antibody, whereas i n NAHA the target-probe complex i s formed by the hybridization between complementary target and probe nucleic a c i d strands with the intent of i d e n t i f y i n g s p e c i f i c genetic sequences rather than s p e c i f i c antigens. Despite t h i s rather d i s t i n c t fundamental difference, the methods of enzymatic detection of antigen-antibody or nucleic acid target-probe complexes are e s s e n t i a l l y the same, so a lengthy discussion on enzymatic detection i n hybridization assays w i l l be not be made here. Nucleic acids are not p a r t i c u l a r l y immunogenic under normal conditions and antibodies to nucleic acid strands generally do not d i s t i n g u i s h between the nucleotide sequence of the probe strand or any other nucleic acid. By incorporating unique nucleotide analogues or chemically modifying the nucleic acid probe, i t i s possible to make nucleic acid probe molecules d i s t i n c t l y immunodetectable. In the case of b i o t i n moieties, again these may be detected non-immunologically with avidin or streptavidin. Once the nucleic acid probe i s made unique r e l a t i v e to the target or any other nucleic acids, i t i s possible to u t i l i z e any of the enzymatic detection sequences outlined above i n the section on enzyme-linked immunoassays, subsequent to the s p e c i f i c hybridization reaction. As i n the case of EIA, t y p i c a l l y i t i s necessary to l i n k nucleic a c i d target-probe complexes to a s o l i d matrix for v i s u a l i z a t i o n . NAHA may be performed on samples applied d i r e c t l y to n i t r o c e l l u l o s e or nylon sheets (dot b l o t assays) or samples may be e l e c t r o p h o r e t i c a l l y separated p r i o r to application to the sheets (northern [RNA] (36) or



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Enzyme-Probe Conjugates in Diagnostics 239

Southern [DNA] (32) b l o t s ) . Recently, a technique has been described f o r treating nucleic acid target-probe complexes as antigens i n an antigen capture system performed i n p l a s t i c ELISA plates (38)· The l a t t e r technique i s of interest because i t allows automated equipment designed f o r performing standard ELISA to be used f o r nucleic acid hybridization techniques. A new enzymatic technique currently being explored enhances the s e n s i t i v i t y of nucleic acid assays by amplifying target sequences p r i o r to reaction with probe molecules. This method i s unique because v i r t u a l l y a l l p r i o r attempts to enhance the s e n s i t i v i t y of NAHA have focused on amplifying the assay signal a f t e r formation of the target-probe complexes. The new technique, dubbed polymerase chain reaction (PCR), employs a DNA polymerase, o r i g i n a l l y the Klenow fragment of DNA polymerase I, and predetermined oligonucleotide primer sequences (39). The technique requires p r i o r knowledge of a nucleotide sequence s p e c i f i c f o r the target being probed, f o r instance the human immunodeficiency v i r u s (40) and human papilloma v i r u s (41). Oligonucleotide primers sequences which flank the target sequence, generally between 15-30 nucleotides long, are synthesized. The DNA sample being assayed i s heated to 95°C to heat denature the double-stranded DNA target, followed by a lower temperature incubation to allow the primer sequences to anneal to the complementary regions within the denatured target DNA sample. Addition of the DNA polymerase and nucleotide substrate generates new copies of the primed DNA sequences by primer extension. Repeated cycles of heat denaturation, primer annealing, and enzymatic synthesis s p e c i f i c a l l y increase the amount of target sequences up to 10^-fold after approximately 20-40 cycles. A recent modification of the technique has incorporated the DNA polymerase of the thermophilic bacterium, Thermus aquaticus. which r e s i s t s heat i n a c t i v a t i o n during the denaturation cycles (42). The PCR technique, although quite simple i n theory, w i l l undoubtedly improve the potential s e n s i t i v i t y of NAHA f o r a v a r i e t y of pathogens. The Future Non-radioactive detection methods, p a r t i c u l a r l y enzymatic systems, w i l l increase i n popularity as the l e v e l of s e n s i t i v i t y of these methods are enhanced. Already some enzymatic detection techniques allow the i d e n t i f i c a t i o n of subpicogram amounts of nucleic acid target and nanogram levels of protein i n NAHA b l o t and immunoblot assays, respectively. Applications employing multiple enzyme systems, such as the amplified ELISA technique, as well as the PCR technique f o r amplifying the amount of nucleic a c i d target molecules within samples, w i l l greatly enhance the s e n s i t i v i t y of current assays i n the near future. I f the enzymatic marker systems become more potent to enhance s e n s i t i v i t y , with the a i d of molecular linkers to reduce s t e r i c hindrance between enzyme and probe molecules, i t may be possible to return to d i r e c t detection methods. The steps required to employ bridging molecules w i l l be removed, r e s u l t i n g i n time and labor reductions per assay. Automated systems w i l l also reduce time and labor, while improving performance consistency. Design of less toxic substrates and more stable reagents would also enhance adoption of enzymatic detection systems.


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Finally, to s o l i d employed from the

development of new systems which l i n k chromogenic reactions phase detection of target molecules, s i m i l a r to those i n dipsticks f o r c l i n i c a l chemistry would help move assays laboratory into the f i e l d , and f a c i l i t a t e rapid diagnoses.

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Biocatalysis in Agricultural Biotechnology - American Chemical Society

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