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Advances in immunoassay technology
Advances in immunoassay technology Colin H Self and David B Cook Major developments continue to be reported in key areas of immunoassay technology. Following the development of excellent signal generation methods, attention has shifted to the development of immunochemical methods and instrumentation to provide convenient systems of high performance. Important advances have been made in the design of immunochemical approaches that permit the replacement of competitive format assays for small molecules, such as drugs, metabolites and pollutants, with non-competitive formats, bringing advantages previously seen only with large molecular analytes. Bispecific antibodies and recombinant proteins are also beginning to impact immunodiagnostics, with the promise of even more highly specified reagents. Improvements in automation have brought the facility of homogeneous systems to high-throughput and high-performance heterogeneous systems. Similarly, 'point of need' testing continues to progress. Through all of these advances, systems are evolving according to the needs of users in terms of operator convenience, accuracy, specificity, speed, robustness, and sensitivity. Address Department of Clinical Biochemistry, Medical School, Framlington Place, University of Newcastle upon Tyne, NE2 4HH, UK
chemiluminescent compounds) have been used in place of radioisotopes. A great deal of ingenuity has gone into devising homogeneous assay systems, which do not require the separation of bound and free moieties before a signal is measured. Yet, heterogeneous assays (where antibody-bound and antibody-unbound analyte are separated before the signal is measured) retain advantages, particularly in an age of automation, in which the necessary separation and washing procedures are less tedious for an operator (who may not even be aware they are taking place). Thus, this review describes some of the more recently developed and applied methods of generating signals for quantification of immunoassays, novel developments in immunochemistry (particularly those that have extended the immunometric approach to small molecules), the application of recombinant DNA technology to immunoassays, and developments in assay formats. 'Point of need' testing is highlighted as an area of growing importance. Finally, we refer to the increasing use of fully automated instrumentation in immunoassay laboratories, which is profoundly affecting diagnostic practice.
Current Opinion in Biotechnology 1996, 7:60-65 © Current Biology Lid ISSN 0958-1669 Abbreviations ALP alkalinephosphatase EA enzymeacceptor ED enzymedonor GOD glucoseoxidase HRP horseradishperoxidase
Introduction The introduction of labelled antibody technology in the late 1960s, in place of the labelled antigen (competitive) approach, led to many benefits in diagnostic technology, notably in the development of more sensitive, rapid, specific and robust assays. T h e non-competitive labelled antibody (immunometric) approach has, for all practical purposes, resulted in the application of two-site 'sandwich' assays, where the analyte is of sufficient size to be trapped between 'capture' and labelled 'detector' antibody, determining the fraction of capture antibody that has bound analyte. For smaller analytes, such as drugs, metabolites and pollutants, the swing away from competitive immunoassays that measure 'analyte-unbound' sites has come more slowly. Even so, methods have now been developed to bring the advantages of non-competitive im,munoassays, outlined above, to this large group of key analytes. A large variety of labels (e.g. enzymes as well as fluorescent and
Developments in s i g n a l g e n e r a t i o n Following earlier demonstrations that the performance of enzyme labels could be dramatically improved by employing enzyme amplification cycling reactions [1], further refinements have been made by development of a fluorescence read-out [2] as well as an excellent demonstration of the power of the colorimetric system [3]. Time-resolution fluorimetry and chemiluminescent labels have also continued to be applied and developed. For example, a highly sensitive chemiluminescent method developed recently employs as a label the calciumactivated photoprotein aequorin. Originally extracted from the jellyfish Aequorea victoria, it is now produced by recombinant DNA technology and marketed as Aqualite. Aequorin has been applied to several hormone assays now available in kit form, notably thyrotrophin, lutrophin, follitrophin and chorionic gonadotrophin [4], where the signal is produced by the introduction of calcium ions.
Highly sensitive procedures have been described previously for the chemiluminescent detection of alkaline phosphatase (ALP) and, more recently, for horseradish peroxidase (HRP). Albrecht et al. [5] have described indoxyl phosphate as an alternative ALP substrate, yielding indoxyl, a substance exhibiting strong chemiluminescence in the presence of oxalate and H202. In another study, Akhavan-Tafti et al. [6°] report a peroxidase system that
Advancesin immunoassaytechnologySelf and Cook
produces a chemiluminescent acridinium ester, giving rise to a longer duration of light generation than that found when the esters are used as labels directly. Increasing interest in the use of electrochemical detection promises the possibility of highly convenient immunoassays. The design of surfaces and their fabrication is particularly important in this area. A recent publication [7] reports an assay system in which a microporous nylon membrane is coated with a thin layer of gold, which serves as an electrode and solid phase to which capture antibody is bound. Prostate-specific antigen was incubated with the gold surface and excess ALP-labelled detector antibody, forming a sandwich at the gold solid phase. Detection of the label was achieved by the enzyme substrate passing through the membrane to give rise to an electrochemically active product measured at the gold electrode. Several strategies can be employed to estimate different analytes simultaneously. However, a problem typical to biosensors was encountered; each assay required a fresh sensor. Nonetheless, electrochemical methods have been developed towards practical diagnostic application. A new amperometric assay depending on the detection of ALP by enzyme amplification cyclingemploys thermostable NADH oxidase to oxidize NADH with concomitant production of H20 z, which is detected by oxidation at a platinum electrode [8°]. Although the sensitivity of this assay has not yet matched that of photometric procedures, the dynamic range is reported to be greater. In another enzyme cycling approach, Suzawa et al. [9] describe a homogeneous system based on glucose oxidase (GOD). Digoxin-conjugated GOD was modified with ferrocene. After binding to antibody, the ferrocene-GOD-digoxin conjugate exhibited less electrochemical activity because of steric hindrance. Analyte prevents this and can thus be detected. Enzyme channelling has also been adapted to the biosensor field [10]. In this approach, two enzymes are brought into close proximity at the surface of an electrode. The product of the first acts as substrate for the second before it diffuses into the surrounding solution. In a model system, HRP is fixed to the surface of an electrode as well as a specific binding agent. The label used for analyte is GOD, producing H202 from glucose substrate, which is detected at the HRP electrode. An equivalent system detecting ALP label has also been described [11], where the phosphatase substrate resulting in HzO 2 production is bromo-chloro-indolyl phosphate. A potentially important feature is that the background signal appears to be low. Such approaches hold the capacity for the development of diagnostic homogeneous assays with simple practical steps. So great is the interest in ultra-sensitivity that even the polymerase chain reaction has been adapted to detect DNA-labelled antibodies. The resulting assay has been reported to have a sensitivity of < 1 zeptomole in an immunoassay of albumin [12]. Recently, it has been
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used for simultaneous ultrasensitive detection of multiple analytes [13°], giving a sensitivity for thyrotrophin of l×10-19mol, which is comparable to that achieved by enzyme amplification [1]. Although the adoption of nucleic acid probes is very interesting, concerns that must be overcome relate to the degree of complexity introduced and the potential for contamination leading to false signals.
D e v e l o p m e n t s in i m m u n o c h e m i s t r y Over the past two decades, the competitive assay format for large molecular analytes has given way to two-site sandwich assays, in which independent epitopes are bound by different antibodies, a concept initiated by Miles and Hales. The two-site assay provides the advantages the user seeks: speed (through the use of excess reagent), sensitivity (through correct analyte-bound binding site measurement), and specificity (through multiple site recognition). The problem has been that such an approach has not been directly applicable to the important area of small molecular weight analytes, such as therapeutic and abused drugs, steroids, thyroid hormones, metabolites and pollutants [14,15], which are not large enough to bind simultaneously more than one antibody independently. Competitive-format assays have, therefore, continued to be used for small molecular analytes in spite of the fact that in their usual applications they determine how much analyte is not present through the measurement of analyte-unbound sites.
To overcome this problem, non-competitive methods for small molecules, such as the anti-immune complex assay [1,161 and selective antibody methodology [1], have been devised and introduced. Our group [17*°] has described such a non-competitive immunometric assay for a small molecule (digoxin) that utilizes a monoclonal antibody specific for the immune complex formed by digoxin and an antibody specific for digoxin itself. The method produces a particularly convenient, very rapid and highly sensitive and specific technique for the estimation of small molecules. Towbin et al. [18°1 have developed an assay for angiotensin II based on the same principle. The selective antibody methodology has also been termed 'idiometric' [19"*]. In this approach, the analyte bound capture antibody sites are determined by the addition of a reagent that binds analyte unbound sites (e.g. analyte analogue or anti-idiotypic antibody) and that prevents them binding a secondary antibody, which thus only selectively binds the analyte-bound sites. Mares et al. [19°°] have employed this approach with a hybrid monoclonal antibody produced by a rat/mouse heterohybridoma, permitting the use of ALP-labelled anti-rat F(ab') 2 for signalling. Their assay for oestradiol was appropriate for development into a dip-stick format for 'point of need' testing.
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The importance of determining analyte-bound sites, rather than unbound sites, has led to other, possibly relatively complex, approaches. These include a chemiluminescent method [20] that depends on binding with acridinium ester labelled antibody. Subsequently, unbound antibody is saturated with an analyte analogue-bound microparticle immunoabsorbent that sterically hinders its luminescence, whereas analyte-bound antibody binds with anti-acridinium ester antibody bound to paramagnetic particles in a manner that allows it to subsequently luminesce. Typical of such approaches is the requirement that the immunoabsorbent removes antibody unbound to analyte, but does not disturb the fraction bound to analyte. Such considerations are obviated by the more complex approach of immunocapturing analyte on a microtitre plate, subsequent covalent linking, denaturation, and quantification of the covalently bound analyte with the same antibody labelled with enzyme [21°]. The advantages inherent in non-competitive reagent excess immunometric methods [22] and the current intensity of activity in this area makes it likely that immunodiagnostic methods for small molecular analytes will develop towards non-competitive technologies, as for large molecular analytes. Present immunoassays for small molecular analytes, such as steroids, often have a requirement for enhanced specificity. This has been met by means of the multiple binding assay system [P1], in which the small molecular analyte is sequentially bound by antibodies having different cross-reactivity profiles, the overall specificity of the system being the product of these. Developments involving hybrid and bispecific antibodies demonstrate the importance of monoclonal antibodies in modern immunoassay technology. Auriol et al. [23°] have created a mouse hybrid hybridoma by fusing a hybridoma producing monoclonal antibody against acetyl-aminofluorene with a hybridoma producing antibody against ALP. The resulting tetradoma secreted IgG with parental and bispecific binding characteristics and was used for one-step immunodetection of non-radioactive DNA and RNA probes. Using heterobifunctionai cross-linkers [24], monoclonai antibodies have been produced in which the binding sites are directed against different epitopes of the same target, for example, against the MM and MB forms of creatine kinase and against opposite ends of the 39 aminoacid sequence of adrenocorticotropin. The production of certain bispecific antibody combinations enhances the formation of matrices, thereby conferring higher affinities than are attainable with antibody mixtures. Kuroki et al. [25] have employed a labelled chimeric human/mouse monoclonal antibody directed against carcinoembryonic antigen in a solid-phase sandwich assay, with a mouse monoclonal antibody as capture antibody. The chimeric antibody was reported to give fewer false positives resulting from interference from heterophilic antibodies
and was more effective than the existing standard method (i.e. using normal mouse serum or purified mouse IgG) in circumventing this troublesome practical problem.
Recombinant DNA technology Recombinant DNA technology is being exploited increasingly in a number of areas. For example, an enzyme assay for rat prolactin [26 °'] has been reported in which ALP-labelled prolactin is prepared using genetic engineering techniques. Recombinant antibody-ALP conjugates permit application of this principle to immunometric procedures [27]. Here, a fusion protein, consisting of a dimer of bacterial ALP and fragments of a monoclonal antibody against human IgG, is produced in the periplasm of Escherichia call Crude periplasmic extracts were effective in an ELISA. Genetically engineered lipid tagged antibody has also been produced [28] and used to construct a fluorescence assay with europium chelate loaded liposomes.
D e v e l o p m e n t s in assay formats Heterogeneous phase separation tests continue to demonstrate the excellent performance that immunoassays can achieve. The use of plastic microtitre plates to which antibody has been adsorbed continues to be popular. It has been reported, however, that by linking antibodies covalently to the plates, improved results are obtained. Recent papers describe Xenobind ® plates [29] with chemical groups on the surface that react with amino groups of proteins and 'hydrocoating' [30] in which peptides are immobilized onto microtitre plates via covalent bonds to an activated hydrophilic polymer (dextran). In both cases, superior results were obtained compared with either passive adsorption or existing techniques for covalent linkage. For many yeats, technologists have sought to devise homogeneous assays that avoid the need for the separation of antibody-bound and free moieties. The history of these attempts demonstrates how difficult this is. Although many techniques have been interesting, they have, to a large extent, demonstrated what important steps phase-separation and washing really are in ridding the system of unwanted interferences. The goal is a very simple system in which all that happens is the addition of sample followed by read-out of the result. The very strong advances in automation and dip-stick technologies have meant that phase-separation and washing can take place very effectively without the operator even knowing they are happening. Thus, homogeneous systems have increasingly difficult competition to match, despite important advances that have occurred. One of these developments, cloned enzyme donor immunoassay (CEDIA), is an early application of recombinant DNA technology to immunoassay, and practical
Advances in immunoassay technology Self and Cook
homogeneous diagnostic procedures are now appearing that utilize this technology. The technique depends on the preparation of two inactive fragments of I]-galactosidase - - enzyme donor (ED) and enzyme acceptor (EA)mwhich, when mixed together, spontaneously associate to form the active enzyme. ED is labelled with hapten, and the assay works by the sample hapten binding an anti-hapten antibody, which would otherwise bind the ED-hapten complex and thus inhibit its combination with EA to form active enzyme. Recently, this approach has been employed for the estimation of small molecules such as drugs of abuse [31]. Though the technique results in increased signal with increased analyte concentration, the method remains a competitive method depending on the measurement of unbound antibody sites and, consequently, retains the limitations of competitive assay formats [22].
'Point of need' immunochemical testing is becoming increasingly important, particularly for the general public, and includes applications such as pregnancy tests (chorionic gonadotrophin) and fertility monitoring (lutrophin). Methodology applicable to blood samples will enable the expansion of immunochemical methods in 'near patient' testing. A serum method (FlexSure®), which is similar to established techniques for urine samples in that it depends on immunochromatography, has also been described [32]. Detection is achieved with colloidal gold particles conjugated to antibody, which are captured by antigen on the strip. The method can be used either for detecting antibodies or, in sandwich format, for detecting antigens. The technique is also being developed to detect human haemoglobin in faecal samples so that it will be highly specific for faecal occult blood.
A different interesting procedure for whole-blood samples [331 employs a dry film in a test card reaction chamber. This method depends on the use of thrombin as a label that is inhibited by a p-amidinophenyl ester of cinnamate when covalently bound to the active site. Thus, inhibited thrombin cannot take part in the cascade of clotting reactions. On exposure to UV light, photoisomerization of the inhibitor causes it to be released, restoring the clotting activity. A monoclonal antibody to the thrombin-labelled ligand sterically hinders the thrombin activity of the ligand when bound and thus keeps it inactive, even after release of the inhibitor by UV light. Thus, the amount of antibody binding can be determined by coagulation activity. When the reaction is triggered by UV light, only the thrombin not bound to antibody is active. Before triggering, the thrombin not yet reacted with antibody is kept inactive by the inhibitor. The reading method is already in use for near patient testing of blood coagulation. This technique represents an ingenious application of an existing point-of-need chemistry, which extends it for use with immunological techniques, and applications of this
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sort are certain to expand. Liquid circuit immunoassay devices represent another such format where capillary flow has been exploited to determine timing of reactions and sample volume [P2",P3]. They are simple to operate by lay persons, but nonetheless, deliver accurate volumes and timing for complex immunochemical procedures. The multi-analyte microspot assay developed by Ekins and Chu [34] uses arrays of microspots on a sensor, each of which is directed against a different analyte. This approach has the potential for rapid simultaneous measurement of thousands of a great many different substances in a small sample. Diagnostic laboratory practice in the 1990s demands the use of fully automated random access analyzers delivering rapid and accurate results. A great variety of instruments are already in use. Published assessments of the performance of these instruments suggest, for the most part, they are functioning well (e.g. the Bayer Immuno 1 [Bayer, Basingstoke, UK] [35], the Abbott IMxTM [Abbott, Maidenhead, UK] [36], Boehringer-Mannheim ES 300 [Boehringer-Mannheim GmbH, Penzberg, Germany] [37], Ciba Coming ACS-180 [Ciba Coming, Norwood, USA] [38], Tosoh AIA-1200 [Tosoh, Tokyo, Japan] [39], Vidas [Vidas-bioM6rieux, Marcy-rl~toile, France] [40], Sanofi Access [Sanofi Pasteur, Chaska, USA] [41] and the Bio-Rad Radias [Bio-Rad, Benicia, USA] [ES Cooper, abstract, National Neonatal Screening Symposium, Corpus Christi, Texas, September 1995]). Roche (Basel, Switzerland) have devised a method (OnLine) depending on the kinetic interaction of microparticles in solution (KIMS), which has been applied to automated instruments [42,43]. Similarly, Syva (San Jose, USA) have introduced EMIT II, the newest formulation of their established homogeneous enzyme immunoassay system [42]. Although it is clear that from so large a pack there will be losers, the successful instruments will not only win a great deal, but certainly consolidate the position of immunodiagnostics as a dominant technology in areas of clinical biochemistry, haematology, microbiology and virology, as well as changing the boundaries and natures of the specialities themselves.
Conclusions Immunodiagnostics remains a vibrant area, with important advances having been made in signal generation, immunochemistry and assay formats. Non-competitive approaches should allow the small-molecule field to catch up with high-performance assay systems for large molecular analytes. The introduction of high-precision auto-analyzers will serve both to stimulate the development and introduction of new reagent systems and to break down the compartmentation of specialities within laboratory medicine.
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References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •*
Self CH: The impact of new diagnostic technologies, In Rapid Methods and Automation in Microbiology and Immunology. Edited by Spencer RC, Wright EP, Newsom SWB. Andover, UK: Intercept; 1994:185-190.
2.
Cook DB, Self CH: Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatasa: application In an Immunoassay of prolnsulln. Clin Chem 1993, 39:965-971.
3.
Bates DL: Enzyme ampllficetlon: the quest for the xeptomole. Intl Labmate 1995, 20:11-13.
4.
Rigl T, Rivers HN, Petal MT, Ball RT, Stults NL, Smith DF: Blolumlnescenca immunoasseys for human endocrine hormones based on aquellte, a calcium-activated photoproteln. Clin Chem 1995, 41:1363-1384. Albrecht S, Brandl H, St•ink• M, Freidt T: Chemllumlnescent enzyme immunoassey of prostate-specific antigen based on Indoxyl phosphate substrata. C/in Chem 1994, 40:1970-1971.
6. •
Akhavan-Tafti H, Sugioka K, Arghavani Z, DeSilva R, Handley RS, Sugioka Y, Eickholt RA, Perkins MP, Schapp AP: Chemllumlneacent detection of horseradish peroxides• by enzymatic generation of acrldinium esters. Clin Chem 1995, 41:1368-1369. This paper describes the application of acridinium esters for signalling, not as labels themselves, but when produced enzymatically by HRR Thus, the advantage of using an enzyme label is combined with the chemiluminescence of acridinium esters, which produce a more long-lived signal because they are generated during detection. 7.
Meyerhoff ME, Duan C, Meuasl M: Novel nonseparetion sandwich-type electrochemical enzyme Immunoassay system for detecting marker proteins In undiluted blood. Clin Chem 1995, 41:1378-1384.
8. •
Athey D, McNeil C J: Amplified electrochemical Immuncassay for thyrotropln using thermophillc ~-NADH oxides•. J Immunol Methods 1994, 176:153-162. The enzyme amplification system for ALP detection, which exploits alchohol dehydrogenase as an amplification step, is applied to electrochemical detection. NADH oxidase is incorporated into the cycling reaction in order to generate H202, which is detected amperometrioally at a platinum electrode. The immunosensor has a large working range. 9.
Suzawa T, Ikariyama Y, Aizawa M: Multilabelling of ferrocenes to a glucose oxldase-digoxln conjugate for the development of a homogeneous electroenzymstic Immunoassay. Anal Chem 1994, 66:3889-3894.
10.
Wright JD, Rawson KM, Ho WO, Athey D, McNeil C J: Specific binding assay for biotin based on enzyme channelling with direct electron transfer electrochemical detection using horseradish peroxldase. Biosens Bioelectron 1995, 10:495-500.
11.
Ho WH, Athey D, McNeil C J: Amperometrlc detection of alkaline phosphatase activity at a horseradish peroxides• enzyme electrode based on activated carbon: potential application to electrochemical Immunoassey. Biosens Bioelectron 1995, 10:683-691.
1 2.
16.
of special interest of outstanding interest
1.
5.
for the presence of strezlne, metolachlor and 2,4-D. J Environ Sci Health 1993, 28:577-598.
Sano T, Smith CL, Cantor CR: Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 1992, 258:120-122.
Hendrickson ER, Haffield-Truby TM, Jcerger RD, Majarian WR, Ebersole RC: High sensitivity multlanalyte Immunoessey using covalent DNA-labelled antibodies and polymerese chain reaction. Nucleic Acids Res 1995, 23:522-529. DNA is employed as an antibody label in an ELISA assay. The PCR is exploited for amplicetion of the assay response, resulting in a highly sensitive detection system.
17. **
Self CH, Deasi JL, Winger LA: High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience demonstrated with • noncompetitive Immunornetrlc anti-immune complex assay system for digoxln. Clin Chem 1994, 40:2035-2041. Describes a high-performance anti-immune complex assay that extends the advantages of non-competitive immunometric assays to produce a rapid, sensitive, robust and operationally simple assay system for small molecular weight substances in serum. Digoxin is used as an example. 18. •
Chakraborti P, Hatcher FM, Blake RC, Ladd PA, Blake DA: Enzyme Immunoassay to determine heavy metals using antibodies to specific metaI-EDTA complexes: optimization and validation of an Immunoassey for soluble Indium. Anal Biochem 1994, 217:70-75.
15.
Hall JC, Van Deynze TD, Struger J, Chan CH: Enzyme Immunoassay based survey of precipitation and surface water
Towbin H, Motz J, Oroszlan P, Zingel O: Sandwich Immunoassey for the hapten angiotensin II. A novel assay pdndple based on antibodies against Immune complexes. J Immunol Methods 1995, 181:167-176.
Another application of anti-immune complex technology (see [17"]), in this case, for the small peptide angiotensin II. The sandwich immuncesey is shown to have high sensitivity. 19. *•
Mares A, De Boever J, Osher J, Quiroga S, Bernard G, Kohen F: A direct non-competitive Idlomebtc enzyme Immunoassay for serum oestredioL J Immuno/Methods 1995, 181:83-90. Reports an assay system based on the selective-antibody non-competitive approach technology approach to the assay of small molecules in serum. This 'idiometric' approach is demonstrated to provide a high-performance assay system for a steroid, that is appropriate for development as a dipstick method. 20.
Piran U, Riordan WJ, Livehin LA: New noncompetitive Immunoasseys of small analytes. C/in Chem 1995, 41:985-990.
21. •
Etienne E, Cn~minon C, Lamourette P, Grassi J, PradeHes P: Enzyme Immunometrlc assay for L-thyroxine using direct ultraviolet Irradiation. Anal Biochem 1995, 225:34-38. L-thyroxine is captured onto antibody-co•ted plates, and subsequent exposure to UV light is then used to cause the formation of a covalent link. The covalently linked L-thyroxine is then quantified with the same antibody, which is now labelled with enzyme. Thus, the technique exploits UV irradiation to provide another approach to providing non-competitive assays for small molecules. 22.
Cook DB, Self CH: Monoclonal antibodies In diagnostic immuncessays. In Monoclonal Antibodies. Edited by Ritter MA, Ladyman HM. Cambridge: Cambridge University Press; 1995:180-208.
23. •
Auriol J, Guasdon JL, Mazie JC, Nato F: Development of a blspecific monoclonal antibody for use In molecular hybrldisetlon. J Immuno/Methods 1994, 169:123-133. Describes production in a tetradoma of a monoclonal antibody bispecific for antigen and alkaline phosphetase. The antibody has been used in immunospot assays, dot-blot hybridization assays, and Southern blot analysis (but not in conventional ELISA). In this paper, the authors demonstrate the utility of bispecific monoclonal antibodies, avoiding drawbacks of chemical coupling, reducing background and using a crude Ig fraction. Immunodetection employs small amounts of unmodified enzyme; enzyme conjugated antibody is consequently not required. 24.
Cook AG, Wood PJ: Chemical synthesis of blspecific monoclonel antibodies; potential advantages In Immunoassey systems, J Immunol Methods 1994, 171:227-237.
25.
Kuroki M, Matsumcto Y, Arakawa F, Haruno M, Murakemi M, Kuwahars M, Ozaki H, Senba T, Mats,Joke Y: Reducing Interference fl'om heterophillc antibodies in 8 two-site immunoassey for carcinoembryonlc antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer. J Immunol Methods 1995, 180:81-91.
13. •
14.
UIImanEF, Milburn G, Jeleako J, Radika K, Pirio M, Kemp• T, Skold C: Anti-Immune complex antibodies enhance affinity and specificity of primary antibodies, Proc Natl Acad Sci USA 1993, 90:1184-1189.
26. **
Ezan E, Ducancel F, Gllet D, Drevet P, Manez A, Grognet JM, Boulaln JC: Recombinant technology In the preparation of Immunogen and enzymatic tracer. Application to the development of an enzyme Immuncessey for rat prolactin. J Immunol Methods 1994, 169:205-211. These authors produce a recombinant enzymatic tracer by inserting the rat prolactin DNA sequence into the amino-terminal end of the Escherichia coli ALP gene. In addition, recombinant immunogen, consisting of rat prolactin fused with two synthetic IgG binding domains derived from protein A, is used to raise polyclonal antibodies. Large amounts of fusion proteins are extracted from the culture media (see also [27]).
Advances in immunoassay technology Self and Cook
27.
28.
Carrier A, Ducancel F, Settiawan NB, Cattolico L, Maillere B, Leonetti M, Dr•vet P, Menez A, Boulain JC: Recombinant antibody-alkaline phosphatsse conjugates for diagnosis of human IgGs: application to antI-HBsAg detection. J Imrnunol Methods 1995, 181:177-186. Laukkanen M-J, Or•liana A, Keiniinen K: Usa of genetically engineered Ilpld-tsgged antibody to generate functional europium chelate loaded Ilposornes. Application In fluoroimmunoassay. J Immunol Methods 1995, 185:95-102.
29.
Douglas AS, Monteith CA: Improvements to Immunoassays by use of covalent binding assay plates. C/in Chem 1994, 40:1833-1837.
30.
Gregorius K, Mouritsen S, Eisner HI: Hydrocoating: • new method for coupling blomolecules to solid phases. J Immunol Methods 1995, 181:65-73.
31.
Armbruster DA, Hubster EC, Kaufman MS, Ramon MK: Cloned enzyme donor Immunoassay (CEDIA) for drugs-of-abuse screening. C/in Chem 1995, 41:92-98.
32.
Bradshaw P, Fitzgerald D, Stephens L, Baddam S, Doe J, Hue J, Chandler H: FlexSure test device: qualitstive Immunochromatographl¢ test format. C/in Chem 1995, 41:1360-1363.
33.
Immunoassay system. A multi(entre evaluation. Eur J Clin Chem Clin Biochern 1994, 32:395-407. 39.
Costongs GM, Janson PC: Comparison of the automated random access Immunoassay analysars, ACS-180 (Cltm Coming) and AIA-1200 (Tosoh). Eur J Clin Chem Clin Biochem 1993, 31:701-706.
40.
Azevedo-Pereira JM, Lourenco MH, Barin F, Cisterns R, Denis F, Moncharmont P, Grillo R, Santo-Ferreira MO: Multlcenter evaluation of a fully automated screening test, VIDAS HIV 1 + 2, for antibodies to human Immunodefidency virus types 1 and 2. J Clin Microbiol 1994, 32:2559-2563.
41.
Patterson W, Wemeas P, Payne WJ, Matsson P, Leflar C, Melander T, Quest S, Stejakal J, Carlac A, Macera M, Schubert F~V: Random and continuous-access Immunoasaays with chemilumlnesoant detection by Access automated analyzer. Clin Chem 1994, 40:2042-2045.
42.
Armbruster DA, Schwarzhoff RH, Hubster EC, Liserio MK: Enzyme Immunoassay, klneffc mlcropartlde Immunoassay, radlolmmunoassay, and fluorescence polarization Immunoassay compared for drugs-of-abusa screening. C/in Chem 1993, 39:2137-2146. Ruiz R, Borque L, Soria AG, Cordova MA, Asolo B: Evaluation of an Immunoturbidometrlc assay of serum dlgoxln without sample pretreatment. Eur J C/in Chem Clin Biochem 1995,
43.
Merenbloom BK, Oberhardt BJ: Homogeneous Immunoassay of whole-blood samples. C/in Chem 1995, 41:1385-1390.
34.
Ekins RP, Chu F: Mulflanalyte testing. C/in Chem 1993, 39:369-3?0.
35.
Abubaker MA, Rlos DY, Petersen JR: Four automated randomaccess immunoassay analysers evaluated for thyroid function testing. C/in Chem 1995, 41:1538-1540.
36.
Kamariah K, Lopez JB, Satgunasingam N: Immunoassay of gonsdotroplns using a fully-automated benchtop snslysar. Brit J Biomed Sci 1994, 51:296-298.
37.
D'Costa M, Feld R, Laxdal V, Trundle D, Collineworth WL: A mulflcenter evaluation of the Boehrlnger-Mannhelm ES 300 immunoassay system. Clin Biochem 1993, 26:51-57.
38.
Romer M, Haeckel R, Cap•Ill M, Rocipon J: The analytical performance of the Clba Coming ACS-180 automated
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33:171-175.
Patents • of special interest • * of outstanding interest P1.
Self CH: Determination of haptens. 17101/92 GB993/00100.
P2. ";hie
Bunce RA, Starsmore SJ, Thorpe GH: Liquid transfer device useful in diagnostic tests. 31103194 GB 2276940. patent and [P3] disclose dipstick devices that enable complex interactions to take place without user involvement. Capillary flow is exploited to determine volumes introduced into reaction areas of the devices without the use of pipettes by an operator. Other channels are used to introduce diluent and reagent whereby capillary flow is employed to time the sequence of reagent addition. P3.
Bunce RA: Liquid transfer device. 25/11194 GB 2284479.
chemiluminescent compounds) have been used in place of radioisotopes. A great deal of ingenuity has gone into devising homogeneous assay systems, which do not require the separation of bound and free moieties before a signal is measured. Yet, heterogeneous assays (where antibody-bound and antibody-unbound analyte are separated before the signal is measured) retain advantages, particularly in an age of automation, in which the necessary separation and washing procedures are less tedious for an operator (who may not even be aware they are taking place). Thus, this review describes some of the more recently developed and applied methods of generating signals for quantification of immunoassays, novel developments in immunochemistry (particularly those that have extended the immunometric approach to small molecules), the application of recombinant DNA technology to immunoassays, and developments in assay formats. 'Point of need' testing is highlighted as an area of growing importance. Finally, we refer to the increasing use of fully automated instrumentation in immunoassay laboratories, which is profoundly affecting diagnostic practice.
Current Opinion in Biotechnology 1996, 7:60-65 © Current Biology Lid ISSN 0958-1669 Abbreviations ALP alkalinephosphatase EA enzymeacceptor ED enzymedonor GOD glucoseoxidase HRP horseradishperoxidase
Introduction The introduction of labelled antibody technology in the late 1960s, in place of the labelled antigen (competitive) approach, led to many benefits in diagnostic technology, notably in the development of more sensitive, rapid, specific and robust assays. T h e non-competitive labelled antibody (immunometric) approach has, for all practical purposes, resulted in the application of two-site 'sandwich' assays, where the analyte is of sufficient size to be trapped between 'capture' and labelled 'detector' antibody, determining the fraction of capture antibody that has bound analyte. For smaller analytes, such as drugs, metabolites and pollutants, the swing away from competitive immunoassays that measure 'analyte-unbound' sites has come more slowly. Even so, methods have now been developed to bring the advantages of non-competitive im,munoassays, outlined above, to this large group of key analytes. A large variety of labels (e.g. enzymes as well as fluorescent and
Developments in s i g n a l g e n e r a t i o n Following earlier demonstrations that the performance of enzyme labels could be dramatically improved by employing enzyme amplification cycling reactions [1], further refinements have been made by development of a fluorescence read-out [2] as well as an excellent demonstration of the power of the colorimetric system [3]. Time-resolution fluorimetry and chemiluminescent labels have also continued to be applied and developed. For example, a highly sensitive chemiluminescent method developed recently employs as a label the calciumactivated photoprotein aequorin. Originally extracted from the jellyfish Aequorea victoria, it is now produced by recombinant DNA technology and marketed as Aqualite. Aequorin has been applied to several hormone assays now available in kit form, notably thyrotrophin, lutrophin, follitrophin and chorionic gonadotrophin [4], where the signal is produced by the introduction of calcium ions.
Highly sensitive procedures have been described previously for the chemiluminescent detection of alkaline phosphatase (ALP) and, more recently, for horseradish peroxidase (HRP). Albrecht et al. [5] have described indoxyl phosphate as an alternative ALP substrate, yielding indoxyl, a substance exhibiting strong chemiluminescence in the presence of oxalate and H202. In another study, Akhavan-Tafti et al. [6°] report a peroxidase system that
Advancesin immunoassaytechnologySelf and Cook
produces a chemiluminescent acridinium ester, giving rise to a longer duration of light generation than that found when the esters are used as labels directly. Increasing interest in the use of electrochemical detection promises the possibility of highly convenient immunoassays. The design of surfaces and their fabrication is particularly important in this area. A recent publication [7] reports an assay system in which a microporous nylon membrane is coated with a thin layer of gold, which serves as an electrode and solid phase to which capture antibody is bound. Prostate-specific antigen was incubated with the gold surface and excess ALP-labelled detector antibody, forming a sandwich at the gold solid phase. Detection of the label was achieved by the enzyme substrate passing through the membrane to give rise to an electrochemically active product measured at the gold electrode. Several strategies can be employed to estimate different analytes simultaneously. However, a problem typical to biosensors was encountered; each assay required a fresh sensor. Nonetheless, electrochemical methods have been developed towards practical diagnostic application. A new amperometric assay depending on the detection of ALP by enzyme amplification cyclingemploys thermostable NADH oxidase to oxidize NADH with concomitant production of H20 z, which is detected by oxidation at a platinum electrode [8°]. Although the sensitivity of this assay has not yet matched that of photometric procedures, the dynamic range is reported to be greater. In another enzyme cycling approach, Suzawa et al. [9] describe a homogeneous system based on glucose oxidase (GOD). Digoxin-conjugated GOD was modified with ferrocene. After binding to antibody, the ferrocene-GOD-digoxin conjugate exhibited less electrochemical activity because of steric hindrance. Analyte prevents this and can thus be detected. Enzyme channelling has also been adapted to the biosensor field [10]. In this approach, two enzymes are brought into close proximity at the surface of an electrode. The product of the first acts as substrate for the second before it diffuses into the surrounding solution. In a model system, HRP is fixed to the surface of an electrode as well as a specific binding agent. The label used for analyte is GOD, producing H202 from glucose substrate, which is detected at the HRP electrode. An equivalent system detecting ALP label has also been described [11], where the phosphatase substrate resulting in HzO 2 production is bromo-chloro-indolyl phosphate. A potentially important feature is that the background signal appears to be low. Such approaches hold the capacity for the development of diagnostic homogeneous assays with simple practical steps. So great is the interest in ultra-sensitivity that even the polymerase chain reaction has been adapted to detect DNA-labelled antibodies. The resulting assay has been reported to have a sensitivity of < 1 zeptomole in an immunoassay of albumin [12]. Recently, it has been
61
used for simultaneous ultrasensitive detection of multiple analytes [13°], giving a sensitivity for thyrotrophin of l×10-19mol, which is comparable to that achieved by enzyme amplification [1]. Although the adoption of nucleic acid probes is very interesting, concerns that must be overcome relate to the degree of complexity introduced and the potential for contamination leading to false signals.
D e v e l o p m e n t s in i m m u n o c h e m i s t r y Over the past two decades, the competitive assay format for large molecular analytes has given way to two-site sandwich assays, in which independent epitopes are bound by different antibodies, a concept initiated by Miles and Hales. The two-site assay provides the advantages the user seeks: speed (through the use of excess reagent), sensitivity (through correct analyte-bound binding site measurement), and specificity (through multiple site recognition). The problem has been that such an approach has not been directly applicable to the important area of small molecular weight analytes, such as therapeutic and abused drugs, steroids, thyroid hormones, metabolites and pollutants [14,15], which are not large enough to bind simultaneously more than one antibody independently. Competitive-format assays have, therefore, continued to be used for small molecular analytes in spite of the fact that in their usual applications they determine how much analyte is not present through the measurement of analyte-unbound sites.
To overcome this problem, non-competitive methods for small molecules, such as the anti-immune complex assay [1,161 and selective antibody methodology [1], have been devised and introduced. Our group [17*°] has described such a non-competitive immunometric assay for a small molecule (digoxin) that utilizes a monoclonal antibody specific for the immune complex formed by digoxin and an antibody specific for digoxin itself. The method produces a particularly convenient, very rapid and highly sensitive and specific technique for the estimation of small molecules. Towbin et al. [18°1 have developed an assay for angiotensin II based on the same principle. The selective antibody methodology has also been termed 'idiometric' [19"*]. In this approach, the analyte bound capture antibody sites are determined by the addition of a reagent that binds analyte unbound sites (e.g. analyte analogue or anti-idiotypic antibody) and that prevents them binding a secondary antibody, which thus only selectively binds the analyte-bound sites. Mares et al. [19°°] have employed this approach with a hybrid monoclonal antibody produced by a rat/mouse heterohybridoma, permitting the use of ALP-labelled anti-rat F(ab') 2 for signalling. Their assay for oestradiol was appropriate for development into a dip-stick format for 'point of need' testing.
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The importance of determining analyte-bound sites, rather than unbound sites, has led to other, possibly relatively complex, approaches. These include a chemiluminescent method [20] that depends on binding with acridinium ester labelled antibody. Subsequently, unbound antibody is saturated with an analyte analogue-bound microparticle immunoabsorbent that sterically hinders its luminescence, whereas analyte-bound antibody binds with anti-acridinium ester antibody bound to paramagnetic particles in a manner that allows it to subsequently luminesce. Typical of such approaches is the requirement that the immunoabsorbent removes antibody unbound to analyte, but does not disturb the fraction bound to analyte. Such considerations are obviated by the more complex approach of immunocapturing analyte on a microtitre plate, subsequent covalent linking, denaturation, and quantification of the covalently bound analyte with the same antibody labelled with enzyme [21°]. The advantages inherent in non-competitive reagent excess immunometric methods [22] and the current intensity of activity in this area makes it likely that immunodiagnostic methods for small molecular analytes will develop towards non-competitive technologies, as for large molecular analytes. Present immunoassays for small molecular analytes, such as steroids, often have a requirement for enhanced specificity. This has been met by means of the multiple binding assay system [P1], in which the small molecular analyte is sequentially bound by antibodies having different cross-reactivity profiles, the overall specificity of the system being the product of these. Developments involving hybrid and bispecific antibodies demonstrate the importance of monoclonal antibodies in modern immunoassay technology. Auriol et al. [23°] have created a mouse hybrid hybridoma by fusing a hybridoma producing monoclonal antibody against acetyl-aminofluorene with a hybridoma producing antibody against ALP. The resulting tetradoma secreted IgG with parental and bispecific binding characteristics and was used for one-step immunodetection of non-radioactive DNA and RNA probes. Using heterobifunctionai cross-linkers [24], monoclonai antibodies have been produced in which the binding sites are directed against different epitopes of the same target, for example, against the MM and MB forms of creatine kinase and against opposite ends of the 39 aminoacid sequence of adrenocorticotropin. The production of certain bispecific antibody combinations enhances the formation of matrices, thereby conferring higher affinities than are attainable with antibody mixtures. Kuroki et al. [25] have employed a labelled chimeric human/mouse monoclonal antibody directed against carcinoembryonic antigen in a solid-phase sandwich assay, with a mouse monoclonal antibody as capture antibody. The chimeric antibody was reported to give fewer false positives resulting from interference from heterophilic antibodies
and was more effective than the existing standard method (i.e. using normal mouse serum or purified mouse IgG) in circumventing this troublesome practical problem.
Recombinant DNA technology Recombinant DNA technology is being exploited increasingly in a number of areas. For example, an enzyme assay for rat prolactin [26 °'] has been reported in which ALP-labelled prolactin is prepared using genetic engineering techniques. Recombinant antibody-ALP conjugates permit application of this principle to immunometric procedures [27]. Here, a fusion protein, consisting of a dimer of bacterial ALP and fragments of a monoclonal antibody against human IgG, is produced in the periplasm of Escherichia call Crude periplasmic extracts were effective in an ELISA. Genetically engineered lipid tagged antibody has also been produced [28] and used to construct a fluorescence assay with europium chelate loaded liposomes.
D e v e l o p m e n t s in assay formats Heterogeneous phase separation tests continue to demonstrate the excellent performance that immunoassays can achieve. The use of plastic microtitre plates to which antibody has been adsorbed continues to be popular. It has been reported, however, that by linking antibodies covalently to the plates, improved results are obtained. Recent papers describe Xenobind ® plates [29] with chemical groups on the surface that react with amino groups of proteins and 'hydrocoating' [30] in which peptides are immobilized onto microtitre plates via covalent bonds to an activated hydrophilic polymer (dextran). In both cases, superior results were obtained compared with either passive adsorption or existing techniques for covalent linkage. For many yeats, technologists have sought to devise homogeneous assays that avoid the need for the separation of antibody-bound and free moieties. The history of these attempts demonstrates how difficult this is. Although many techniques have been interesting, they have, to a large extent, demonstrated what important steps phase-separation and washing really are in ridding the system of unwanted interferences. The goal is a very simple system in which all that happens is the addition of sample followed by read-out of the result. The very strong advances in automation and dip-stick technologies have meant that phase-separation and washing can take place very effectively without the operator even knowing they are happening. Thus, homogeneous systems have increasingly difficult competition to match, despite important advances that have occurred. One of these developments, cloned enzyme donor immunoassay (CEDIA), is an early application of recombinant DNA technology to immunoassay, and practical
Advances in immunoassay technology Self and Cook
homogeneous diagnostic procedures are now appearing that utilize this technology. The technique depends on the preparation of two inactive fragments of I]-galactosidase - - enzyme donor (ED) and enzyme acceptor (EA)mwhich, when mixed together, spontaneously associate to form the active enzyme. ED is labelled with hapten, and the assay works by the sample hapten binding an anti-hapten antibody, which would otherwise bind the ED-hapten complex and thus inhibit its combination with EA to form active enzyme. Recently, this approach has been employed for the estimation of small molecules such as drugs of abuse [31]. Though the technique results in increased signal with increased analyte concentration, the method remains a competitive method depending on the measurement of unbound antibody sites and, consequently, retains the limitations of competitive assay formats [22].
'Point of need' immunochemical testing is becoming increasingly important, particularly for the general public, and includes applications such as pregnancy tests (chorionic gonadotrophin) and fertility monitoring (lutrophin). Methodology applicable to blood samples will enable the expansion of immunochemical methods in 'near patient' testing. A serum method (FlexSure®), which is similar to established techniques for urine samples in that it depends on immunochromatography, has also been described [32]. Detection is achieved with colloidal gold particles conjugated to antibody, which are captured by antigen on the strip. The method can be used either for detecting antibodies or, in sandwich format, for detecting antigens. The technique is also being developed to detect human haemoglobin in faecal samples so that it will be highly specific for faecal occult blood.
A different interesting procedure for whole-blood samples [331 employs a dry film in a test card reaction chamber. This method depends on the use of thrombin as a label that is inhibited by a p-amidinophenyl ester of cinnamate when covalently bound to the active site. Thus, inhibited thrombin cannot take part in the cascade of clotting reactions. On exposure to UV light, photoisomerization of the inhibitor causes it to be released, restoring the clotting activity. A monoclonal antibody to the thrombin-labelled ligand sterically hinders the thrombin activity of the ligand when bound and thus keeps it inactive, even after release of the inhibitor by UV light. Thus, the amount of antibody binding can be determined by coagulation activity. When the reaction is triggered by UV light, only the thrombin not bound to antibody is active. Before triggering, the thrombin not yet reacted with antibody is kept inactive by the inhibitor. The reading method is already in use for near patient testing of blood coagulation. This technique represents an ingenious application of an existing point-of-need chemistry, which extends it for use with immunological techniques, and applications of this
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sort are certain to expand. Liquid circuit immunoassay devices represent another such format where capillary flow has been exploited to determine timing of reactions and sample volume [P2",P3]. They are simple to operate by lay persons, but nonetheless, deliver accurate volumes and timing for complex immunochemical procedures. The multi-analyte microspot assay developed by Ekins and Chu [34] uses arrays of microspots on a sensor, each of which is directed against a different analyte. This approach has the potential for rapid simultaneous measurement of thousands of a great many different substances in a small sample. Diagnostic laboratory practice in the 1990s demands the use of fully automated random access analyzers delivering rapid and accurate results. A great variety of instruments are already in use. Published assessments of the performance of these instruments suggest, for the most part, they are functioning well (e.g. the Bayer Immuno 1 [Bayer, Basingstoke, UK] [35], the Abbott IMxTM [Abbott, Maidenhead, UK] [36], Boehringer-Mannheim ES 300 [Boehringer-Mannheim GmbH, Penzberg, Germany] [37], Ciba Coming ACS-180 [Ciba Coming, Norwood, USA] [38], Tosoh AIA-1200 [Tosoh, Tokyo, Japan] [39], Vidas [Vidas-bioM6rieux, Marcy-rl~toile, France] [40], Sanofi Access [Sanofi Pasteur, Chaska, USA] [41] and the Bio-Rad Radias [Bio-Rad, Benicia, USA] [ES Cooper, abstract, National Neonatal Screening Symposium, Corpus Christi, Texas, September 1995]). Roche (Basel, Switzerland) have devised a method (OnLine) depending on the kinetic interaction of microparticles in solution (KIMS), which has been applied to automated instruments [42,43]. Similarly, Syva (San Jose, USA) have introduced EMIT II, the newest formulation of their established homogeneous enzyme immunoassay system [42]. Although it is clear that from so large a pack there will be losers, the successful instruments will not only win a great deal, but certainly consolidate the position of immunodiagnostics as a dominant technology in areas of clinical biochemistry, haematology, microbiology and virology, as well as changing the boundaries and natures of the specialities themselves.
Conclusions Immunodiagnostics remains a vibrant area, with important advances having been made in signal generation, immunochemistry and assay formats. Non-competitive approaches should allow the small-molecule field to catch up with high-performance assay systems for large molecular analytes. The introduction of high-precision auto-analyzers will serve both to stimulate the development and introduction of new reagent systems and to break down the compartmentation of specialities within laboratory medicine.
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References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •*
Self CH: The impact of new diagnostic technologies, In Rapid Methods and Automation in Microbiology and Immunology. Edited by Spencer RC, Wright EP, Newsom SWB. Andover, UK: Intercept; 1994:185-190.
2.
Cook DB, Self CH: Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatasa: application In an Immunoassay of prolnsulln. Clin Chem 1993, 39:965-971.
3.
Bates DL: Enzyme ampllficetlon: the quest for the xeptomole. Intl Labmate 1995, 20:11-13.
4.
Rigl T, Rivers HN, Petal MT, Ball RT, Stults NL, Smith DF: Blolumlnescenca immunoasseys for human endocrine hormones based on aquellte, a calcium-activated photoproteln. Clin Chem 1995, 41:1363-1384. Albrecht S, Brandl H, St•ink• M, Freidt T: Chemllumlnescent enzyme immunoassey of prostate-specific antigen based on Indoxyl phosphate substrata. C/in Chem 1994, 40:1970-1971.
6. •
Akhavan-Tafti H, Sugioka K, Arghavani Z, DeSilva R, Handley RS, Sugioka Y, Eickholt RA, Perkins MP, Schapp AP: Chemllumlneacent detection of horseradish peroxides• by enzymatic generation of acrldinium esters. Clin Chem 1995, 41:1368-1369. This paper describes the application of acridinium esters for signalling, not as labels themselves, but when produced enzymatically by HRR Thus, the advantage of using an enzyme label is combined with the chemiluminescence of acridinium esters, which produce a more long-lived signal because they are generated during detection. 7.
Meyerhoff ME, Duan C, Meuasl M: Novel nonseparetion sandwich-type electrochemical enzyme Immunoassay system for detecting marker proteins In undiluted blood. Clin Chem 1995, 41:1378-1384.
8. •
Athey D, McNeil C J: Amplified electrochemical Immuncassay for thyrotropln using thermophillc ~-NADH oxides•. J Immunol Methods 1994, 176:153-162. The enzyme amplification system for ALP detection, which exploits alchohol dehydrogenase as an amplification step, is applied to electrochemical detection. NADH oxidase is incorporated into the cycling reaction in order to generate H202, which is detected amperometrioally at a platinum electrode. The immunosensor has a large working range. 9.
Suzawa T, Ikariyama Y, Aizawa M: Multilabelling of ferrocenes to a glucose oxldase-digoxln conjugate for the development of a homogeneous electroenzymstic Immunoassay. Anal Chem 1994, 66:3889-3894.
10.
Wright JD, Rawson KM, Ho WO, Athey D, McNeil C J: Specific binding assay for biotin based on enzyme channelling with direct electron transfer electrochemical detection using horseradish peroxldase. Biosens Bioelectron 1995, 10:495-500.
11.
Ho WH, Athey D, McNeil C J: Amperometrlc detection of alkaline phosphatase activity at a horseradish peroxides• enzyme electrode based on activated carbon: potential application to electrochemical Immunoassey. Biosens Bioelectron 1995, 10:683-691.
1 2.
16.
of special interest of outstanding interest
1.
5.
for the presence of strezlne, metolachlor and 2,4-D. J Environ Sci Health 1993, 28:577-598.
Sano T, Smith CL, Cantor CR: Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 1992, 258:120-122.
Hendrickson ER, Haffield-Truby TM, Jcerger RD, Majarian WR, Ebersole RC: High sensitivity multlanalyte Immunoessey using covalent DNA-labelled antibodies and polymerese chain reaction. Nucleic Acids Res 1995, 23:522-529. DNA is employed as an antibody label in an ELISA assay. The PCR is exploited for amplicetion of the assay response, resulting in a highly sensitive detection system.
17. **
Self CH, Deasi JL, Winger LA: High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience demonstrated with • noncompetitive Immunornetrlc anti-immune complex assay system for digoxln. Clin Chem 1994, 40:2035-2041. Describes a high-performance anti-immune complex assay that extends the advantages of non-competitive immunometric assays to produce a rapid, sensitive, robust and operationally simple assay system for small molecular weight substances in serum. Digoxin is used as an example. 18. •
Chakraborti P, Hatcher FM, Blake RC, Ladd PA, Blake DA: Enzyme Immunoassay to determine heavy metals using antibodies to specific metaI-EDTA complexes: optimization and validation of an Immunoassey for soluble Indium. Anal Biochem 1994, 217:70-75.
15.
Hall JC, Van Deynze TD, Struger J, Chan CH: Enzyme Immunoassay based survey of precipitation and surface water
Towbin H, Motz J, Oroszlan P, Zingel O: Sandwich Immunoassey for the hapten angiotensin II. A novel assay pdndple based on antibodies against Immune complexes. J Immunol Methods 1995, 181:167-176.
Another application of anti-immune complex technology (see [17"]), in this case, for the small peptide angiotensin II. The sandwich immuncesey is shown to have high sensitivity. 19. *•
Mares A, De Boever J, Osher J, Quiroga S, Bernard G, Kohen F: A direct non-competitive Idlomebtc enzyme Immunoassay for serum oestredioL J Immuno/Methods 1995, 181:83-90. Reports an assay system based on the selective-antibody non-competitive approach technology approach to the assay of small molecules in serum. This 'idiometric' approach is demonstrated to provide a high-performance assay system for a steroid, that is appropriate for development as a dipstick method. 20.
Piran U, Riordan WJ, Livehin LA: New noncompetitive Immunoasseys of small analytes. C/in Chem 1995, 41:985-990.
21. •
Etienne E, Cn~minon C, Lamourette P, Grassi J, PradeHes P: Enzyme Immunometrlc assay for L-thyroxine using direct ultraviolet Irradiation. Anal Biochem 1995, 225:34-38. L-thyroxine is captured onto antibody-co•ted plates, and subsequent exposure to UV light is then used to cause the formation of a covalent link. The covalently linked L-thyroxine is then quantified with the same antibody, which is now labelled with enzyme. Thus, the technique exploits UV irradiation to provide another approach to providing non-competitive assays for small molecules. 22.
Cook DB, Self CH: Monoclonal antibodies In diagnostic immuncessays. In Monoclonal Antibodies. Edited by Ritter MA, Ladyman HM. Cambridge: Cambridge University Press; 1995:180-208.
23. •
Auriol J, Guasdon JL, Mazie JC, Nato F: Development of a blspecific monoclonal antibody for use In molecular hybrldisetlon. J Immuno/Methods 1994, 169:123-133. Describes production in a tetradoma of a monoclonal antibody bispecific for antigen and alkaline phosphetase. The antibody has been used in immunospot assays, dot-blot hybridization assays, and Southern blot analysis (but not in conventional ELISA). In this paper, the authors demonstrate the utility of bispecific monoclonal antibodies, avoiding drawbacks of chemical coupling, reducing background and using a crude Ig fraction. Immunodetection employs small amounts of unmodified enzyme; enzyme conjugated antibody is consequently not required. 24.
Cook AG, Wood PJ: Chemical synthesis of blspecific monoclonel antibodies; potential advantages In Immunoassey systems, J Immunol Methods 1994, 171:227-237.
25.
Kuroki M, Matsumcto Y, Arakawa F, Haruno M, Murakemi M, Kuwahars M, Ozaki H, Senba T, Mats,Joke Y: Reducing Interference fl'om heterophillc antibodies in 8 two-site immunoassey for carcinoembryonlc antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer. J Immunol Methods 1995, 180:81-91.
13. •
14.
UIImanEF, Milburn G, Jeleako J, Radika K, Pirio M, Kemp• T, Skold C: Anti-Immune complex antibodies enhance affinity and specificity of primary antibodies, Proc Natl Acad Sci USA 1993, 90:1184-1189.
26. **
Ezan E, Ducancel F, Gllet D, Drevet P, Manez A, Grognet JM, Boulaln JC: Recombinant technology In the preparation of Immunogen and enzymatic tracer. Application to the development of an enzyme Immuncessey for rat prolactin. J Immunol Methods 1994, 169:205-211. These authors produce a recombinant enzymatic tracer by inserting the rat prolactin DNA sequence into the amino-terminal end of the Escherichia coli ALP gene. In addition, recombinant immunogen, consisting of rat prolactin fused with two synthetic IgG binding domains derived from protein A, is used to raise polyclonal antibodies. Large amounts of fusion proteins are extracted from the culture media (see also [27]).
Advances in immunoassay technology Self and Cook
27.
28.
Carrier A, Ducancel F, Settiawan NB, Cattolico L, Maillere B, Leonetti M, Dr•vet P, Menez A, Boulain JC: Recombinant antibody-alkaline phosphatsse conjugates for diagnosis of human IgGs: application to antI-HBsAg detection. J Imrnunol Methods 1995, 181:177-186. Laukkanen M-J, Or•liana A, Keiniinen K: Usa of genetically engineered Ilpld-tsgged antibody to generate functional europium chelate loaded Ilposornes. Application In fluoroimmunoassay. J Immunol Methods 1995, 185:95-102.
29.
Douglas AS, Monteith CA: Improvements to Immunoassays by use of covalent binding assay plates. C/in Chem 1994, 40:1833-1837.
30.
Gregorius K, Mouritsen S, Eisner HI: Hydrocoating: • new method for coupling blomolecules to solid phases. J Immunol Methods 1995, 181:65-73.
31.
Armbruster DA, Hubster EC, Kaufman MS, Ramon MK: Cloned enzyme donor Immunoassay (CEDIA) for drugs-of-abuse screening. C/in Chem 1995, 41:92-98.
32.
Bradshaw P, Fitzgerald D, Stephens L, Baddam S, Doe J, Hue J, Chandler H: FlexSure test device: qualitstive Immunochromatographl¢ test format. C/in Chem 1995, 41:1360-1363.
33.
Immunoassay system. A multi(entre evaluation. Eur J Clin Chem Clin Biochern 1994, 32:395-407. 39.
Costongs GM, Janson PC: Comparison of the automated random access Immunoassay analysars, ACS-180 (Cltm Coming) and AIA-1200 (Tosoh). Eur J Clin Chem Clin Biochem 1993, 31:701-706.
40.
Azevedo-Pereira JM, Lourenco MH, Barin F, Cisterns R, Denis F, Moncharmont P, Grillo R, Santo-Ferreira MO: Multlcenter evaluation of a fully automated screening test, VIDAS HIV 1 + 2, for antibodies to human Immunodefidency virus types 1 and 2. J Clin Microbiol 1994, 32:2559-2563.
41.
Patterson W, Wemeas P, Payne WJ, Matsson P, Leflar C, Melander T, Quest S, Stejakal J, Carlac A, Macera M, Schubert F~V: Random and continuous-access Immunoasaays with chemilumlnesoant detection by Access automated analyzer. Clin Chem 1994, 40:2042-2045.
42.
Armbruster DA, Schwarzhoff RH, Hubster EC, Liserio MK: Enzyme Immunoassay, klneffc mlcropartlde Immunoassay, radlolmmunoassay, and fluorescence polarization Immunoassay compared for drugs-of-abusa screening. C/in Chem 1993, 39:2137-2146. Ruiz R, Borque L, Soria AG, Cordova MA, Asolo B: Evaluation of an Immunoturbidometrlc assay of serum dlgoxln without sample pretreatment. Eur J C/in Chem Clin Biochem 1995,
43.
Merenbloom BK, Oberhardt BJ: Homogeneous Immunoassay of whole-blood samples. C/in Chem 1995, 41:1385-1390.
34.
Ekins RP, Chu F: Mulflanalyte testing. C/in Chem 1993, 39:369-3?0.
35.
Abubaker MA, Rlos DY, Petersen JR: Four automated randomaccess immunoassay analysers evaluated for thyroid function testing. C/in Chem 1995, 41:1538-1540.
36.
Kamariah K, Lopez JB, Satgunasingam N: Immunoassay of gonsdotroplns using a fully-automated benchtop snslysar. Brit J Biomed Sci 1994, 51:296-298.
37.
D'Costa M, Feld R, Laxdal V, Trundle D, Collineworth WL: A mulflcenter evaluation of the Boehrlnger-Mannhelm ES 300 immunoassay system. Clin Biochem 1993, 26:51-57.
38.
Romer M, Haeckel R, Cap•Ill M, Rocipon J: The analytical performance of the Clba Coming ACS-180 automated
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33:171-175.
Patents • of special interest • * of outstanding interest P1.
Self CH: Determination of haptens. 17101/92 GB993/00100.
P2. ";hie
Bunce RA, Starsmore SJ, Thorpe GH: Liquid transfer device useful in diagnostic tests. 31103194 GB 2276940. patent and [P3] disclose dipstick devices that enable complex interactions to take place without user involvement. Capillary flow is exploited to determine volumes introduced into reaction areas of the devices without the use of pipettes by an operator. Other channels are used to introduce diluent and reagent whereby capillary flow is employed to time the sequence of reagent addition. P3.
Bunce RA: Liquid transfer device. 25/11194 GB 2284479.