- Email: [email protected]
Immunosensors: Antibody-based biosensors
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Immunosensors: antibody-based biosensors
detection of a wide range of analytes. Development of a wide product range would appear possible - an attractive opportunity for manufacturers. On the other hand, efficient transduction of the antibody-antigen binding event into a usable signal turns out to be rather difficult. The problem is that, unlike enzymes (which catalyse the generation of new molecular species) antibodies merely attach to their analyte. Without newly formed molecules, protons or electrons to measure, the demands on transducer design are severe. Before discussing the variety of approaches which are being undertaken to overcome this hurdle, it is pertinent to consider the features that successful immunosensors must embody. For application in medical and veterinary immunodiagnostics, immunosensors might be offered as fast (1-3 min), inexpensive, easily-used
John R. North T h e challenges in developing practical i m m u n o s e n s o r s lie in converting (without added reagents) the binding event into an electrical or optical signal, and in creating fully reversible s y s t e m s capable of m o n i t o r i n g both increases and decreases in analyte concentration. N u m e r o u s approaches are under investigation, s o m e of which should lead to c o m m e r c i a l products within a couple of years; their strengths and limitations are r e v i e w e d . The range of potential uses for probelike devices which continuously and specifically measure the concentration of biological molecules is very broad, including the diagnosis of human disease, monitoring of body functions during intensive care, measurement of food freshness and contamination, effluent monitoring, and fermentation process control. For this reason, considerable attention is now being given to the development ofbiosensors. In essence, biosensors comprise a biological "receptor' element - chosen for its ability to bind to a very narrow range of analytes, plus a transducing element which detects the fact that analyte and biological receptor have combined. Electrical or optical signals generated by the transducer require amplification and processing to provide a comprehensible output. While the bio-receptor and transducer must be in intimate contact, signal processing may be performed at a distance. An operating device may therefore have two separate parts - 'probe' and 'readout instrument'. Whereas the overall field ofbiosensor research and development has been discussed in these pages 1 and elsewhere, the present review will be entirely concerned with those biosensors which embody antibodies as their selective binding components: immunosensors.
gross molecular architecture and behaviour of Mabs is fairly constant, differences in antigen binding between Mabs resulting from alterations in a small, and predictable, section of the molecule. Consequently, sensors embodying a range of Mabs and using a common transduction mechanism could, in principle, be made for i
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Fig. I. Enzyme immunosensors. These are made by attaching antibody to the surface af a Clarke oxygen electrode. An enzyme such as catalase or glucose dehydrogenase is attached either to a second antibody (i) or to antigen (ii) giving modes of sensor operation equivalent to conventional
John R. North is Consultant in Biotechnology at PA Technology, Melbourn, Nr Royston, Herts, SG8 6DP, UK.
immunoassays in the "two-site' and 'competition' configurations, respectively. In use, enzyme becomes either attached to (i) or displaced from (ii) the electrode in the presence of the antigen. After washing, a substrate is added leading to a local change in 02 concentration which in turn causes an altered current through the electrode.
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Trends in Biotechnology, VoL 3, No. 7, 1985
disposables for the physician's office, the veterinarian's case, and for certain home healthcare and farmyard applications. For other applications in the medical and veterinary sector an alternative specification would provide even faster response, full reversibility and long life - forming the key component of high throughput, continuous flow immunoanalysers and thereby replacing the 'batched tube' mode of immunoassay currently practised. Probes which offer continuous measurement would also find wide application in bioprocess control, while continuously reading, sterilizable and miniaturized probes may have a role as in vivo sensors for medical use.
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182 gonadotropin (hCG), alpha-fetoprotein and hepatitis B surface antigen (Fig. 1). In an alternative configuration, the enzymic activity of an enzymeimmunosensor can be detected by measurement of its heat of reaction 6'7, but sensitivities are low due to the small heat output and the need to shield from the relatively large temperature fluctuations of a laboratory environment (unshielded signal:noise ratio of 1:1000). A related system making use of a concept developed for pH and glucose sensors 8'9, would involve an optical fibre, near the tip of which is attached the antibody (Fig. 2). Light of the appropriate excitation wavelength would be passed down the fibre, which would also be used for inspection of the emitted light. Devices could be designed in 'two site' or 'competitive' configurations, requiring reagent additions (Fig. 2 i) or reagent-independent (Fig. 2 ii). Such arrangements are essentially' alternative forms of established heterogeneous immunoassay principles, adapted to a probe format. They are unlikely to be the direct forbearers of reagent independent and reversible immunosensors. Direct-acting sensors Very much more effort has been committed to developing transducers which rely upon direct direction of antigen becoming attached to an antibodycoated surface (or vice versa). These transducers have in common their fundamental non-specificity- any molecule bound to the surface affects the measured parameter to some extent. The specificity of analyte detection therefore relies entirely on achieving high ratios of specific to nonspecific binding; in the context of low concentrations of an analyte in blood, this can represent a formidable problem. Measurable surface parameters which are modified by the presence of bound molecules include local changes in density and dielectric constant. These effects can be monitored using electromechanical or optical sensing techniques and have formed the basis for experimental immunosensors. Piezoelectric systems Electromechanical sensing has been carried out using piezoelectric transducers and surface acoustic wave
Trends in Biotechnology, VoL 3, No. 7, 1985
devices. The piezoelectric effect describes the ability of quartz crystals and certain plastics to generate a small electrical charge in response to mechanical stress. Alternatively, a voltage
propagation of acoustic waves across the surface of a crystal. Surface acoustic wave (SAW) devices are widely used as radiofrequency filters in consumer electronics and are already manufactured in high volume at low unit cost and, / t3~XTEELECTRODE therefore, attractive as potential transducers for low-cost immunoprobes. Surface deposits on the crystal modify the acoustic wave velocity and attenuation, thereby altering the frequency of an oscillator circuit controlled by the device. By covalently attaching antibodies to SAW devices, Roederer and Bastiaans 12 constructed immunosensors for human IgG and for influenza type A virus. The C~IN ~ T ~ crystals respond quickly (within 20 s) Fig. 3. Insulated gate field effect transistor. The and detect nanogram quantities of antisource-to-drain current (Io) is determined by gen ~3, but it is claimed that their full the gate potential and the source--drain voltage potential has yet to be realized. These difference. devices function in liquid - the resonances and wave velocities being applied to such a material results in a damped in a characteristic manner small mechanical deformation. By determined largely by the viscosity of choice of device structure, an electrical the medium. In an early report 12, signal can be used to induce a mechan- specificity was obtained by comparison ical resonance in the device. Such a of SAW crystals with and without transducer can also be used to launch a antibody pre-coating. Later studies surface acoustic wave (SAW) along the involved comparing devices coated surface of the crystal, which can be with antibodies of different specificity. detected by a second transducer a short Given these observations and the low distance away. cost of piezoelectric devices, the The interaction between the attached prospects for this approach to immunomass and these piezoelectric systems sensing are exciting. can be detected in two disntict ways, which may be conveniently categorized as 'intrinsic' and 'extrinsic'. In the ~,ATEVOLTAG~ ~ OATFELECTROD~ 'intrinsic', bulk crystal mode, the change in characteristic resonant frequency of a crystal in an oscillator cricuit is a function of the attached mass Si O-z INSULATOR (in much the same way as a weight on a spring). It is usual to compare matched pairs of crystals to avoid spurious signals arising from temperature and electronic fluctuations. Piezoelectric crystals operated in this mode are very sensitive; a crystal with a characteristic frequency of 15 MHz will display a shift of approximately 2 600 Hz per microgram of attached material - Fig. 4. Antibody-coated field effect transistor (immunoFET). Analogous devices have been giving a theoretical detection limit of constructed with antigen-coated membranes. about 10 12 g (Ref. 10). A number of highly sensitive gas, metal and vapour sensors have been developed using this Potentiometric systems principle 1°'12. Similarly, Karube et al. Since biological macromolecules measured cell concentrations in a carry a number of both positive and fermentation broth using paired negative charges, a layer of such molepiezoelectric crystals H. cules attached to an electrode surface is The 'extrinsic' mode of operation of expected to be detectable as an altered piezoelectric mass sensors involves potential. Using both glass electrodes detection of changes in the velocity of enveloped in antibody-coated mere-
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Trends in Biotechnology, Vol. 3, No. 7, 1985
branes and antibody-coated amino silanized titanium elec- i trodes, a number of authors reported between 1977 and 1980 that such direct potentiometric measurement of antigen is feasible ~4-17.Thus, for example, hCG could be detected in the urine of pregnant women 15. It is likely that the detected potential is a mixed potential resulting from the double layer of buffer ions and ionized groups of the analyte and antibody molecules at the electrode surface. Owing to differential ion permeation across the thin metal oxide insulating surfaces of these electrodes, the detected potential is subject to change with alterations in buffer strength, pH and ionic composition. The consequent dependence of potential readings on factors unrelated to analyte concentration may have some bearing on ii the lack of reported progress in studies of this type over the intervening years 16.
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Another reason for the shortage of data on direct potentiometric immunoelectrodes is that an equivalent, and far more elegant, measurement can be made using an antibody-coated field effect transistor - immunoFET. Field effect transistors (FETs) are devices in which the conductivity of the 'channel' region (Fig. 3) - measured by application of a voltage between 'source' and 'drain' electrodes - is controlled by the strength of the electrical field generated by the 'gate', thus providing a miniaturizable Fig. 5. Evanescent light. This occurs when light is totally reflected at a reamplifier. Conventional FET devices have an insulating fractive index interface, such as that between a waveguide and the surlayer - usually of SiO2 - over the doped silicon channel rounding medium. (i) A light wave is a rapidly oscillating electromagnetic between source and drain, which is overlaid with a metal field (like a radio wave). When totally reflected, the wave induces a correselectrode (gate electrode) for applying the controlling ponding field which decreases exponentially at the other side of the intervoltage. By replacing the gate electrode with a layer of face. This short-range field is called the evanescent wave. (ii) When large antigen and exposing this surface to a buffer or antibody molecules adhere to the interface, the evanescent field is disturbed and solution, immunoFET devices have been made (Fig. 4) total reflection breaks down allowing light to escape from the waveguide. which appear to be sensitive to anti-albumin or anti-syphilis Detectors based on this system are consequently sensitive to the number and size of surface-adherent particles. antibodies 1s,19. While showing enormous promise in their early stages of development, effective, reliable and analyte-selective immunoFET sensors are yet reason to believe that the theoretical to be constructed. A number of their problems are shared with other FET-based predictions of sensitivity for immunobiosensors such as ion-sensitive FETs and enzyme-FETs; in general these shared FET sensors (10 mV signal for 10 -7problems can now be overcome. For example, the SiO2 insulating layer can be 10 -11 M analyte)22 are misleading; protected from pinhole generation upon exposure to aqueous buffer using a layer immunoFET probes may yet become of Si3N4 (Ref. 18), and silicon hydration can be prevented by encapsulating the commercially successful. whole device in multiple layers of photosetting polymers 2° or using sapphire substrates (silicon on sapphire)2L A hurdle peculiar to the immunoFET concept is the need to provide a very thin Optical s y s t e m s The changes in local dielectric probut fully insulating layer (membrane) between the antigen or antibody coating and the semiconductor surface. Such a membrane must be thin enough to allow a small perties associated with the binding of charge redistribution - occurring as a result of analyte (antibody or antigen) antigens to a surface can also be binding - to exert a detectable change in electrical field. However, it must also detected by a variety of optical measureprovide adequate insulation to prevent dissipation of the field by leakage of ions or ments; refractive index and dielectric by electron tunnelling effects. After some years of study, Janata recently constant being closely related indices of concluded that manufacture of satisfactory membranes will require the use of new the interaction between materials and technologies, as yet undeveloped 22. Pending this breakthrough, the development electromagnetic energy. Fourier transform infra-red spectroscopy (FTIR) has ofimmunoFET sensors appears to be stagnant. Even assuming that the ideal insulating membrane can be developed, a further been applied to inspect thin aqueous hurdle may need to be overcome. Surface charges and hydrogen-bonding sites of layers (a few micrometers) near the surproteins cause a counter-ion shell (double-layer) and structured water shells to face of IR-transparent crystals. The use surround the molecules; these regions of structured charge will inevitably of F T I R for measuring antibodycontribute to the electrical field affecting the FET gate. It is likely that the size of, antigen reactions has been extremely and charge density within, such regions of structured charge will be affected by the limited and requires sophisticated and species and concentration of buffer ions. Nevertheless, there is no persuasive expensive instruments.
Trends in Biotechnology, FoL 3, No. 7~ 1985
184 Ellipsometric measurements also reveal dielectric changes - seen as alterations in optical polarization phenomena - but their use for monitoring antibody- antigen reactions at a useful range of sensitivity also demands large, complex and expensive intruments. Neither FTIR nor ellipsometry appear to be amenable to the miniaturization and low cost implied in the concept of immunosensor probes.
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molar range, although this will be affected by a number of variables, including developments in the attachment of antibodies to the waveguide surface, analyte molecular weights and the efficiency of any fluorescent components used. Surface plasmon resonance
Compared with the evanescent wave approach, surface plasmon systems ii At the resonance may provide immunoprobe devices which are more readily manufactured at low Evanescent wave unit cost4'24. While making In contrast, both evanuse of a similar electroescent wave and plasmon magnetic field configuraresonance systems have tion to probe the molecular been conceived in probe layer, plasmon resonance format. An immunosensor devices do not involve evansystem has been investigated in which molecules escent light waves. Both gas sensors and dye detection bound to the external Hi surface of a waveguide (e.g. systems have been conoptical fibre) interact with Fig. 6. Surface plasmon structed using this Finresonance. A surface light propagating through plasmon is a collective ciple2~-2!. the waveguide2L Such light motion of electrons in the The particular optical is carried by multiple total surface of a metal conducconditions required for internal reflections and, at tor, excited by the impact generation and use of each reflection, generates an of light of appropriate surface plasmons for evanescent light wave wavelength at a particular immunosensing applicawhich exponentially decays angle, Op. The exciting tions have been generated into the external medium light could be evanescent using metallized diffraction Angle of incidence gratings 24 and using the (Fig. 5 i). Since the dis- light from a waveguide or if the metal is flat, or tance of penetration of the prism metallized surface of glass a collimated beam iraNo bound material evanescent wave is of the pinging on a metallized difprisms 4. For chemical ..... Bound protein same order of magnitude as stability in aqueous buffer fraction grating. For a given wavelength of light a surface plasmon effect is the molecular dimensions of observed as a sharp minimum in light reflectance when the angle of inci- environment, gold is the antibody-antigen com- dence is varied (compare i with ii). This is known as a "Woods anomaly" preferred metal. Model plexes, it is only surface- when observed on metallized diffraction gratings (iii). The critical angle is immunoprobes have been bound molecules which in- very sensitive to the dielectric constant of the medium adjacent to the metal constructed using antiteract with the light. surface and is therefore affected by analytes binding to that surface. human IgG, anti-human Probes embodying this albumin and other macroprinciple can therefore dismolecular systems, curtinguish between bound and free com- must have a characteristic A ~ (unique rently showing sensitivities in the low ponents in the medium surrounding within the sample mixture) and (b) in its nanogram range and with indications the waveguide. Reaction kinetics have sensitivity - the absorbed light is only a that these can be improved markedly. been studied and devices have been very small % of the total transmitted In general, surface plasmon systems constructed for both light absorption, light. respond to the average optical thickness scattering and fluorescence systems, These problems can be overcome by of the layer of bound molecules, this For absorption, a narrow frequency adding antibody-linked fluorophores to being related to molecular volume as band of incident light corresponding to the bound antibody-antigen complex well as concentration. the absorption max (A~,,) of the analyte as in conventional fluorescent immunoComparison of diffraction grating of interest is used. Binding of the assays. A requirement for external and prism systems indicates that the analyte by the antibodies on the surface reagents is, however, an obvious dis- performance of the latter is heavily is detected as a change in transmitted advantage in designing biosensor con- dependent upon the deposition of very light intensity. The absorption configu- figurations. thin (~30 nm) and highly reproducible ration is limited (a) in the range of Claimed sensitivities of evanescent metal layers. Volume manufacture to analytes that can be measured - each wave systems are at present in the nano- these tolerances is a significant hurdle.
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On the other hand, the reproducibility of grating systems depends upon the quality of the diffraction grating - a hurdle similar to that already overcome during the manufacture of compact audio discs. As the optical requirements for inspection of surface plasmort resonance effects can be met using inexpensive instrumentation, and volume manufacture of the appropriate antibody-sensitized metallized surfaces appears feasible, the prospects for commercially viable plasmon immunoprobes are exciting.
Conformation-sensitive transducers The approaches to direct acting immunosensors described above have in common their use of antibodies merely to provide selective binding of analyte to a surface; some property of that surface being inspected by the transducing element. A limitation to this approach is that no fundamental distinction can be made by the transducer between specifically and nonspecifically bound molecules. Such a distinction could be made if a transducer were sensitive to changes in the antibody binding site conformation or changes in the electron probability distribution around the binding site, which occur when antigen binds. All available immunochemical evidence indicates that conformational transitions in individual binding sites are not transmitted past the hinge region of antibody molecules to the Fc region; interaction between transducer and antibody is, therefore, required in the Fab or, preferably, Fv regions (for recent review of antibody structure, see Ref. 28). A number of speculative concepts, which provide for transduction of local events in the binding site, involve the insertion of reporter groups close to the binding site. For example, the activity of an ionophore attached to antibody might be modulated by antigen binding 29,just as the activity of antigenconjugated ionophore has been shown to be modified by antibody binding 3°. Quantifying the ionophore function would require the incorporation of the complex into a stable plastics 3° or lipid bilayer 3~membranes and the provision of buffers and circuitry to measure ion flux. Construction of stable and reproducible devices of this type will depend on advances in membrane biochemistry and in conjugation procedures.
Akernative reporter groups, particularly sensitive to subtle changes in their electronic environment, are fluorochromes and luminophores. Ideally, antigen binding would lead to reversal of the quenching of a binding-site associated fluorochrome or luminophore. An immunoprobe incorporating such a fluorescent conjugate might well embody evanescent wave or surface plasmon optics for transmission of excited and emitted light, while a luminescent conjugate could be excited by exposure to a pulsed electrical field. The routine construction of such devices, specific for a wide range of antigens, will depend upon advances in our detailed understanding of the various alterations in electron distribution which can follow antibody-antigen combination. Furthermore, directed covalent attachment of reporter groups to well-defined sidechains close to the antigen-binding site will become essential. There are exciting challenges here.
repulsions, etc., will prevent incorrect analyte engaging sufficiently closely with the binding site and will thereby provide specificity. Naturally, screening for highly selective but low affinity monoclonal antibodies which perform in this way will be carried out on a different basis from screening for high affinity antibodies. Further specificity in such systems might be achieved by the use of panels of immunosensors, each with different selectivity in low affinity antigen binding, where the 'fingerprint' of the binding characteristics of the desired analyte would be known. Whichever routes to the ideal immunosensor turn out to be the most technologically elegant and commercially viable, their development will necessarily involve close cooperation between scientists and engineers from different parts of the technological spectrum; a truly multidisciplinary opportunity,
Reversibility
I wish to thank my colleagues Nick Higgins and Craig Sawyers for numerous invaluable discussions during the preparation of this manuscript, Sheila Patterson for patient retyping, and PA Technology for making time and resources available to me.
Once binding-site reporter systems become available for commercially important antibody-antigen systems, there will be an opportunity for creation of truly reversible, or continuously reading, immunosensors. In most systems of analysis, the functional specificity of an antibody results from its higher affinity for one antigen over the others present in the sample. When the antigen of relevance is present at low concentration relative to other components in the sample, as is commonly the case, good functional specificity relies upon high affinity. Since the affinity of antibody-antigen interaction is largely controlled by the dissociation rate rather than the association rate, high affinity binding of antigen is essentially irreversible without alterations in pH and/or ionic strength of the buffer. Consequently, most immunosensors of high specificity will be irreversible unless pulsed with protein denaturing conditions. Where a close interaction between antigen and the combining-site of the antibody can be monitored using reporter-groups, the necessity for very long antigen residence times (high affinity) diminishes. Antibody specificity can be determined by whether the analyte sufficiently modifies electron distributions to affect reporter group function. Steric hindrance, charge
Acknowledgements
References 1 Lowe,C. R. (1984) Trends Biotechnol. 2, 59-65 2 Kfhler, G. and Milstein, C. (1975) Nature 256, 495-497 3 Gronow, M. (1984) Trends Biochem. Sci. 9, 336-340 4 Liedberg, B., Nylander, C. and LundstrSm, I. (1983) Sensors and Actuators 4, 299-304 5 Matsuoka,H., Karube, I. and Suzuki, I. (1983) International Symposium on Electroanalysis, Proc. Roy. Soc. Chem. 6 Aizawa,M. (1983) in Proceedings of the International Meeting on Chemical Sensors., pp. 683-692, Kodansha
7 Mattiasson, B., Danielsson, B. and Mosbach, K. (1980)Food Process Eng. 2, 59-68 8 Peterson, J. I., Goldstein, S. R. and Fitzgerald, R.V. (1980)Anal Chem. 52, 864-869 9 Schultz,J., Mansouri, S. and Goldstein, I. J. (1982)Diabetes Care 5, 245-253 10 Guilbault, G. G. (1980) Ion-selective Electrode Rev. 2, 17-71 11 Ishimori, Y., Karube, I. and Suzuki, S. (1981) Appl. Environ. Microbiol. 42, 632-636 12 Roederer, J. E. and Bastiaans, G. J. (1983) Anal Chem. 55, 2333-2336 13 Chem. Eng. News (1984)April, pp. 18 19 14 Aizawa, M., Kato, S. and Suzuki, S. (1977)J. Membr. Sci. 2, 125-132
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69, 4001-4011 21 Kuriyama,T., Kimura, J. and Kawana, Y. (1984) Announcement by NEC 26 Weber, W. H. and Eagen, C. F. (1979) Optics Lett. 4, 236-238 Corporation at International Conference on Solid State Devices and Materials, 27 Nylander, C., Liedberg, B. and Lind, T. of the International Meeting on Chemical (1982/83) Sensors and Actuators 3, 79-88 Kobe City, Japan Sensors, pp. 699-704, Kodansha 22 Janata, J. and Blackburn, G. F. (1984) 28 Burton, D. R. (1985) Mol. Immunol. 22, See bibliography in Ref. 22 Ann. N Y Acad. Sci. 286-292 161-206 Janata,J. and Huber, R. J. (1980) in: Ionsensitive Electrodes in Analytical Chemis- 23 Carter, T., Dahne, C. and Place, J. F. 29 Petty-Saphon,S. (1983) UK patent appli(1983) Patent number WO83/01112, cation 8325267, assigned to PA try (Freiser, H., ed.), 2, 107-174, Plenum assigned to the Battelle Memorial Consulting Services,Ltd Press 30 Keating, M. Y. and Rechnitz, G. A. Institute Collins, S. and Janata, J. (1982) Anal, (1984) Anal Chem. 56, 801-806 24 Pettigrew, R. M. et al. (1984) Patent Chim. Aaa 136, 93-99 number WO84/02578, assigned to Com- 31 Thompson, M., Krull, U. J. and Sibbald, A., Whalley, P. D. and Worsfold, P. J. (1980)Anal. Chim. Acta. tech Research Unit Ltd Covington, A. K. (1984) Anal Chim. 117, 121-132 25 Pockrand, I. et al. (1978)J. Chem. Phys, Acta 159, 47-62
15 Yamarnoto,N. et al. (1980) Clin. Chem. 26, 1569-1572 16 Yamamoto,N. et aL (1983) in Proceedings 17 18
19 20
There are no standard procedures RECOMBINANT DNA METHODOLOGY b y J. R. Dillon, A. Nasim and E. R. Nestmann, John Wiley & Sons, 1985. £22.95 (xx + 219 pages) 1 S B N 0 471 89851 1 Recombinant D N A Methodology is an
introductory laboratory manual for students and research workers venturing into the field for the first time, and not an encyclopaedic compilation of methods. In this respect, its scope is more limited than that of the indispensable Molecular Cloning by Maniatis et al. How well does the book by DiUon et al. fulfil its aims? Books giving practical details of techniques pose several problems for reviewers. How should such books be criticized? Can it be done by simply examining the procedures and comparing them with the methods used in one's own work? This is clearly unacceptable. There are no 'standard' procedures, for these are always modified, and the number of variations in use must equal the number of laboratories using the procedure! There can be very few methods that are beyond improvement, but procedures enshrined in manuals may become fossilized and used uncritically. The habit of referring to such manuals as 'bibles', with the implication that they contain commandments to be followed unquestioningly, is a reflection of this attitude. Dillon et al. have made a valiant attempt to minimize this danger by adding a commentary to the practical procedures. The pages are divided vertically into halves, the left column
containing the steps of the method while the right column contains comments on the individual steps. These comments may give the rationale for the step, or give hints as to how to perform it, or suggest alternatives that have been used by others. This gives the reader a grasp of what is going on and will perhaps encourage a more critical approach. One consequence of this split page format is that the layout looks rather confused. I frequently found that my eyes were drawn to the comments rather than the method and it would have been better to have made them more distinct by using different typefaces. Major sections and subsections should begin on a new page, although this would make a longer book. The size of the book makes it easy to handle; at 219 pages it is about 215 the size of Maniatis. I hope that the binding is sufficiently strong to withstand rough treatment at the bench.
There are some unfortunate omissions for a book intended to be a reference manual for recombinant DNA methodology. Detailed maps and restriction enzyme sites are not provided for the common vectors, nor is a table giving restriction enzyme recognition sequences. These are admittedly readily available elsewhere but they should be included in a text of this sort. They could have been put in the space given to a list of suppliers, surely unnecessary when companies go to great lengths to make sure their wares are known. Overall, I think that Dillon et al. have achieved their aims. The book cannot replace that by Maniatis et al. as the reference work for a research laboratory, but it should find wide acceptance as a laboratory manual for undergraduates and postgraduates new to the field. I recommend it:
JAN WITKOWSKI
Imperial Cancer Research Fund Laborawries, St Bartholomew's Hospital, Dominion House, Bartholomew Close, London E C I A 7BE, UK.
Introducing enzymes ENZYMES IN ORGANIC SYNTHESIS. (C1BAFOUNDATION SYMPOSIUMIII) edited by Ruth Porter and Sarah Clark, Pitman, 1985. £2Z95 (viii + 248 pages) 1 S B N 0 272 79785 5
When a 'real' organic chemist reads such a title, he or she will very likely ask: 'Why should I want to use enzymes as catalysts in my syntheses?' This is a highly pertinent point given the stateof-the-art in the field. Factors such as limited availability, instability, single
batch-use and high costs continue to restrict the use of enzymes and the fact is that most organic chemists are unfamiliar with the application of enzymes. In the last two decades, however, several new techniques, especially genetic engineering, immobilization of biocatalysts, and affinity chromatography have strongly stimulated the application of enzymes, not only in organic chemistry. Assessment, discussion and criticism of the topic
Trends in Biotechnology, VoL 3, No. 7, 1985
Immunosensors: antibody-based biosensors
detection of a wide range of analytes. Development of a wide product range would appear possible - an attractive opportunity for manufacturers. On the other hand, efficient transduction of the antibody-antigen binding event into a usable signal turns out to be rather difficult. The problem is that, unlike enzymes (which catalyse the generation of new molecular species) antibodies merely attach to their analyte. Without newly formed molecules, protons or electrons to measure, the demands on transducer design are severe. Before discussing the variety of approaches which are being undertaken to overcome this hurdle, it is pertinent to consider the features that successful immunosensors must embody. For application in medical and veterinary immunodiagnostics, immunosensors might be offered as fast (1-3 min), inexpensive, easily-used
John R. North T h e challenges in developing practical i m m u n o s e n s o r s lie in converting (without added reagents) the binding event into an electrical or optical signal, and in creating fully reversible s y s t e m s capable of m o n i t o r i n g both increases and decreases in analyte concentration. N u m e r o u s approaches are under investigation, s o m e of which should lead to c o m m e r c i a l products within a couple of years; their strengths and limitations are r e v i e w e d . The range of potential uses for probelike devices which continuously and specifically measure the concentration of biological molecules is very broad, including the diagnosis of human disease, monitoring of body functions during intensive care, measurement of food freshness and contamination, effluent monitoring, and fermentation process control. For this reason, considerable attention is now being given to the development ofbiosensors. In essence, biosensors comprise a biological "receptor' element - chosen for its ability to bind to a very narrow range of analytes, plus a transducing element which detects the fact that analyte and biological receptor have combined. Electrical or optical signals generated by the transducer require amplification and processing to provide a comprehensible output. While the bio-receptor and transducer must be in intimate contact, signal processing may be performed at a distance. An operating device may therefore have two separate parts - 'probe' and 'readout instrument'. Whereas the overall field ofbiosensor research and development has been discussed in these pages 1 and elsewhere, the present review will be entirely concerned with those biosensors which embody antibodies as their selective binding components: immunosensors.
gross molecular architecture and behaviour of Mabs is fairly constant, differences in antigen binding between Mabs resulting from alterations in a small, and predictable, section of the molecule. Consequently, sensors embodying a range of Mabs and using a common transduction mechanism could, in principle, be made for i
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Fig. I. Enzyme immunosensors. These are made by attaching antibody to the surface af a Clarke oxygen electrode. An enzyme such as catalase or glucose dehydrogenase is attached either to a second antibody (i) or to antigen (ii) giving modes of sensor operation equivalent to conventional
John R. North is Consultant in Biotechnology at PA Technology, Melbourn, Nr Royston, Herts, SG8 6DP, UK.
immunoassays in the "two-site' and 'competition' configurations, respectively. In use, enzyme becomes either attached to (i) or displaced from (ii) the electrode in the presence of the antigen. After washing, a substrate is added leading to a local change in 02 concentration which in turn causes an altered current through the electrode.
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Trends in Biotechnology, VoL 3, No. 7, 1985
disposables for the physician's office, the veterinarian's case, and for certain home healthcare and farmyard applications. For other applications in the medical and veterinary sector an alternative specification would provide even faster response, full reversibility and long life - forming the key component of high throughput, continuous flow immunoanalysers and thereby replacing the 'batched tube' mode of immunoassay currently practised. Probes which offer continuous measurement would also find wide application in bioprocess control, while continuously reading, sterilizable and miniaturized probes may have a role as in vivo sensors for medical use.
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182 gonadotropin (hCG), alpha-fetoprotein and hepatitis B surface antigen (Fig. 1). In an alternative configuration, the enzymic activity of an enzymeimmunosensor can be detected by measurement of its heat of reaction 6'7, but sensitivities are low due to the small heat output and the need to shield from the relatively large temperature fluctuations of a laboratory environment (unshielded signal:noise ratio of 1:1000). A related system making use of a concept developed for pH and glucose sensors 8'9, would involve an optical fibre, near the tip of which is attached the antibody (Fig. 2). Light of the appropriate excitation wavelength would be passed down the fibre, which would also be used for inspection of the emitted light. Devices could be designed in 'two site' or 'competitive' configurations, requiring reagent additions (Fig. 2 i) or reagent-independent (Fig. 2 ii). Such arrangements are essentially' alternative forms of established heterogeneous immunoassay principles, adapted to a probe format. They are unlikely to be the direct forbearers of reagent independent and reversible immunosensors. Direct-acting sensors Very much more effort has been committed to developing transducers which rely upon direct direction of antigen becoming attached to an antibodycoated surface (or vice versa). These transducers have in common their fundamental non-specificity- any molecule bound to the surface affects the measured parameter to some extent. The specificity of analyte detection therefore relies entirely on achieving high ratios of specific to nonspecific binding; in the context of low concentrations of an analyte in blood, this can represent a formidable problem. Measurable surface parameters which are modified by the presence of bound molecules include local changes in density and dielectric constant. These effects can be monitored using electromechanical or optical sensing techniques and have formed the basis for experimental immunosensors. Piezoelectric systems Electromechanical sensing has been carried out using piezoelectric transducers and surface acoustic wave
Trends in Biotechnology, VoL 3, No. 7, 1985
devices. The piezoelectric effect describes the ability of quartz crystals and certain plastics to generate a small electrical charge in response to mechanical stress. Alternatively, a voltage
propagation of acoustic waves across the surface of a crystal. Surface acoustic wave (SAW) devices are widely used as radiofrequency filters in consumer electronics and are already manufactured in high volume at low unit cost and, / t3~XTEELECTRODE therefore, attractive as potential transducers for low-cost immunoprobes. Surface deposits on the crystal modify the acoustic wave velocity and attenuation, thereby altering the frequency of an oscillator circuit controlled by the device. By covalently attaching antibodies to SAW devices, Roederer and Bastiaans 12 constructed immunosensors for human IgG and for influenza type A virus. The C~IN ~ T ~ crystals respond quickly (within 20 s) Fig. 3. Insulated gate field effect transistor. The and detect nanogram quantities of antisource-to-drain current (Io) is determined by gen ~3, but it is claimed that their full the gate potential and the source--drain voltage potential has yet to be realized. These difference. devices function in liquid - the resonances and wave velocities being applied to such a material results in a damped in a characteristic manner small mechanical deformation. By determined largely by the viscosity of choice of device structure, an electrical the medium. In an early report 12, signal can be used to induce a mechan- specificity was obtained by comparison ical resonance in the device. Such a of SAW crystals with and without transducer can also be used to launch a antibody pre-coating. Later studies surface acoustic wave (SAW) along the involved comparing devices coated surface of the crystal, which can be with antibodies of different specificity. detected by a second transducer a short Given these observations and the low distance away. cost of piezoelectric devices, the The interaction between the attached prospects for this approach to immunomass and these piezoelectric systems sensing are exciting. can be detected in two disntict ways, which may be conveniently categorized as 'intrinsic' and 'extrinsic'. In the ~,ATEVOLTAG~ ~ OATFELECTROD~ 'intrinsic', bulk crystal mode, the change in characteristic resonant frequency of a crystal in an oscillator cricuit is a function of the attached mass Si O-z INSULATOR (in much the same way as a weight on a spring). It is usual to compare matched pairs of crystals to avoid spurious signals arising from temperature and electronic fluctuations. Piezoelectric crystals operated in this mode are very sensitive; a crystal with a characteristic frequency of 15 MHz will display a shift of approximately 2 600 Hz per microgram of attached material - Fig. 4. Antibody-coated field effect transistor (immunoFET). Analogous devices have been giving a theoretical detection limit of constructed with antigen-coated membranes. about 10 12 g (Ref. 10). A number of highly sensitive gas, metal and vapour sensors have been developed using this Potentiometric systems principle 1°'12. Similarly, Karube et al. Since biological macromolecules measured cell concentrations in a carry a number of both positive and fermentation broth using paired negative charges, a layer of such molepiezoelectric crystals H. cules attached to an electrode surface is The 'extrinsic' mode of operation of expected to be detectable as an altered piezoelectric mass sensors involves potential. Using both glass electrodes detection of changes in the velocity of enveloped in antibody-coated mere-
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Trends in Biotechnology, Vol. 3, No. 7, 1985
branes and antibody-coated amino silanized titanium elec- i trodes, a number of authors reported between 1977 and 1980 that such direct potentiometric measurement of antigen is feasible ~4-17.Thus, for example, hCG could be detected in the urine of pregnant women 15. It is likely that the detected potential is a mixed potential resulting from the double layer of buffer ions and ionized groups of the analyte and antibody molecules at the electrode surface. Owing to differential ion permeation across the thin metal oxide insulating surfaces of these electrodes, the detected potential is subject to change with alterations in buffer strength, pH and ionic composition. The consequent dependence of potential readings on factors unrelated to analyte concentration may have some bearing on ii the lack of reported progress in studies of this type over the intervening years 16.
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Another reason for the shortage of data on direct potentiometric immunoelectrodes is that an equivalent, and far more elegant, measurement can be made using an antibody-coated field effect transistor - immunoFET. Field effect transistors (FETs) are devices in which the conductivity of the 'channel' region (Fig. 3) - measured by application of a voltage between 'source' and 'drain' electrodes - is controlled by the strength of the electrical field generated by the 'gate', thus providing a miniaturizable Fig. 5. Evanescent light. This occurs when light is totally reflected at a reamplifier. Conventional FET devices have an insulating fractive index interface, such as that between a waveguide and the surlayer - usually of SiO2 - over the doped silicon channel rounding medium. (i) A light wave is a rapidly oscillating electromagnetic between source and drain, which is overlaid with a metal field (like a radio wave). When totally reflected, the wave induces a correselectrode (gate electrode) for applying the controlling ponding field which decreases exponentially at the other side of the intervoltage. By replacing the gate electrode with a layer of face. This short-range field is called the evanescent wave. (ii) When large antigen and exposing this surface to a buffer or antibody molecules adhere to the interface, the evanescent field is disturbed and solution, immunoFET devices have been made (Fig. 4) total reflection breaks down allowing light to escape from the waveguide. which appear to be sensitive to anti-albumin or anti-syphilis Detectors based on this system are consequently sensitive to the number and size of surface-adherent particles. antibodies 1s,19. While showing enormous promise in their early stages of development, effective, reliable and analyte-selective immunoFET sensors are yet reason to believe that the theoretical to be constructed. A number of their problems are shared with other FET-based predictions of sensitivity for immunobiosensors such as ion-sensitive FETs and enzyme-FETs; in general these shared FET sensors (10 mV signal for 10 -7problems can now be overcome. For example, the SiO2 insulating layer can be 10 -11 M analyte)22 are misleading; protected from pinhole generation upon exposure to aqueous buffer using a layer immunoFET probes may yet become of Si3N4 (Ref. 18), and silicon hydration can be prevented by encapsulating the commercially successful. whole device in multiple layers of photosetting polymers 2° or using sapphire substrates (silicon on sapphire)2L A hurdle peculiar to the immunoFET concept is the need to provide a very thin Optical s y s t e m s The changes in local dielectric probut fully insulating layer (membrane) between the antigen or antibody coating and the semiconductor surface. Such a membrane must be thin enough to allow a small perties associated with the binding of charge redistribution - occurring as a result of analyte (antibody or antigen) antigens to a surface can also be binding - to exert a detectable change in electrical field. However, it must also detected by a variety of optical measureprovide adequate insulation to prevent dissipation of the field by leakage of ions or ments; refractive index and dielectric by electron tunnelling effects. After some years of study, Janata recently constant being closely related indices of concluded that manufacture of satisfactory membranes will require the use of new the interaction between materials and technologies, as yet undeveloped 22. Pending this breakthrough, the development electromagnetic energy. Fourier transform infra-red spectroscopy (FTIR) has ofimmunoFET sensors appears to be stagnant. Even assuming that the ideal insulating membrane can be developed, a further been applied to inspect thin aqueous hurdle may need to be overcome. Surface charges and hydrogen-bonding sites of layers (a few micrometers) near the surproteins cause a counter-ion shell (double-layer) and structured water shells to face of IR-transparent crystals. The use surround the molecules; these regions of structured charge will inevitably of F T I R for measuring antibodycontribute to the electrical field affecting the FET gate. It is likely that the size of, antigen reactions has been extremely and charge density within, such regions of structured charge will be affected by the limited and requires sophisticated and species and concentration of buffer ions. Nevertheless, there is no persuasive expensive instruments.
Trends in Biotechnology, FoL 3, No. 7~ 1985
184 Ellipsometric measurements also reveal dielectric changes - seen as alterations in optical polarization phenomena - but their use for monitoring antibody- antigen reactions at a useful range of sensitivity also demands large, complex and expensive intruments. Neither FTIR nor ellipsometry appear to be amenable to the miniaturization and low cost implied in the concept of immunosensor probes.
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Compared with the evanescent wave approach, surface plasmon systems ii At the resonance may provide immunoprobe devices which are more readily manufactured at low Evanescent wave unit cost4'24. While making In contrast, both evanuse of a similar electroescent wave and plasmon magnetic field configuraresonance systems have tion to probe the molecular been conceived in probe layer, plasmon resonance format. An immunosensor devices do not involve evansystem has been investigated in which molecules escent light waves. Both gas sensors and dye detection bound to the external Hi surface of a waveguide (e.g. systems have been conoptical fibre) interact with Fig. 6. Surface plasmon structed using this Finresonance. A surface light propagating through plasmon is a collective ciple2~-2!. the waveguide2L Such light motion of electrons in the The particular optical is carried by multiple total surface of a metal conducconditions required for internal reflections and, at tor, excited by the impact generation and use of each reflection, generates an of light of appropriate surface plasmons for evanescent light wave wavelength at a particular immunosensing applicawhich exponentially decays angle, Op. The exciting tions have been generated into the external medium light could be evanescent using metallized diffraction Angle of incidence gratings 24 and using the (Fig. 5 i). Since the dis- light from a waveguide or if the metal is flat, or tance of penetration of the prism metallized surface of glass a collimated beam iraNo bound material evanescent wave is of the pinging on a metallized difprisms 4. For chemical ..... Bound protein same order of magnitude as stability in aqueous buffer fraction grating. For a given wavelength of light a surface plasmon effect is the molecular dimensions of observed as a sharp minimum in light reflectance when the angle of inci- environment, gold is the antibody-antigen com- dence is varied (compare i with ii). This is known as a "Woods anomaly" preferred metal. Model plexes, it is only surface- when observed on metallized diffraction gratings (iii). The critical angle is immunoprobes have been bound molecules which in- very sensitive to the dielectric constant of the medium adjacent to the metal constructed using antiteract with the light. surface and is therefore affected by analytes binding to that surface. human IgG, anti-human Probes embodying this albumin and other macroprinciple can therefore dismolecular systems, curtinguish between bound and free com- must have a characteristic A ~ (unique rently showing sensitivities in the low ponents in the medium surrounding within the sample mixture) and (b) in its nanogram range and with indications the waveguide. Reaction kinetics have sensitivity - the absorbed light is only a that these can be improved markedly. been studied and devices have been very small % of the total transmitted In general, surface plasmon systems constructed for both light absorption, light. respond to the average optical thickness scattering and fluorescence systems, These problems can be overcome by of the layer of bound molecules, this For absorption, a narrow frequency adding antibody-linked fluorophores to being related to molecular volume as band of incident light corresponding to the bound antibody-antigen complex well as concentration. the absorption max (A~,,) of the analyte as in conventional fluorescent immunoComparison of diffraction grating of interest is used. Binding of the assays. A requirement for external and prism systems indicates that the analyte by the antibodies on the surface reagents is, however, an obvious dis- performance of the latter is heavily is detected as a change in transmitted advantage in designing biosensor con- dependent upon the deposition of very light intensity. The absorption configu- figurations. thin (~30 nm) and highly reproducible ration is limited (a) in the range of Claimed sensitivities of evanescent metal layers. Volume manufacture to analytes that can be measured - each wave systems are at present in the nano- these tolerances is a significant hurdle.
185
Trends in Biotechnology, VoL 3, No. 7, 1985
On the other hand, the reproducibility of grating systems depends upon the quality of the diffraction grating - a hurdle similar to that already overcome during the manufacture of compact audio discs. As the optical requirements for inspection of surface plasmort resonance effects can be met using inexpensive instrumentation, and volume manufacture of the appropriate antibody-sensitized metallized surfaces appears feasible, the prospects for commercially viable plasmon immunoprobes are exciting.
Conformation-sensitive transducers The approaches to direct acting immunosensors described above have in common their use of antibodies merely to provide selective binding of analyte to a surface; some property of that surface being inspected by the transducing element. A limitation to this approach is that no fundamental distinction can be made by the transducer between specifically and nonspecifically bound molecules. Such a distinction could be made if a transducer were sensitive to changes in the antibody binding site conformation or changes in the electron probability distribution around the binding site, which occur when antigen binds. All available immunochemical evidence indicates that conformational transitions in individual binding sites are not transmitted past the hinge region of antibody molecules to the Fc region; interaction between transducer and antibody is, therefore, required in the Fab or, preferably, Fv regions (for recent review of antibody structure, see Ref. 28). A number of speculative concepts, which provide for transduction of local events in the binding site, involve the insertion of reporter groups close to the binding site. For example, the activity of an ionophore attached to antibody might be modulated by antigen binding 29,just as the activity of antigenconjugated ionophore has been shown to be modified by antibody binding 3°. Quantifying the ionophore function would require the incorporation of the complex into a stable plastics 3° or lipid bilayer 3~membranes and the provision of buffers and circuitry to measure ion flux. Construction of stable and reproducible devices of this type will depend on advances in membrane biochemistry and in conjugation procedures.
Akernative reporter groups, particularly sensitive to subtle changes in their electronic environment, are fluorochromes and luminophores. Ideally, antigen binding would lead to reversal of the quenching of a binding-site associated fluorochrome or luminophore. An immunoprobe incorporating such a fluorescent conjugate might well embody evanescent wave or surface plasmon optics for transmission of excited and emitted light, while a luminescent conjugate could be excited by exposure to a pulsed electrical field. The routine construction of such devices, specific for a wide range of antigens, will depend upon advances in our detailed understanding of the various alterations in electron distribution which can follow antibody-antigen combination. Furthermore, directed covalent attachment of reporter groups to well-defined sidechains close to the antigen-binding site will become essential. There are exciting challenges here.
repulsions, etc., will prevent incorrect analyte engaging sufficiently closely with the binding site and will thereby provide specificity. Naturally, screening for highly selective but low affinity monoclonal antibodies which perform in this way will be carried out on a different basis from screening for high affinity antibodies. Further specificity in such systems might be achieved by the use of panels of immunosensors, each with different selectivity in low affinity antigen binding, where the 'fingerprint' of the binding characteristics of the desired analyte would be known. Whichever routes to the ideal immunosensor turn out to be the most technologically elegant and commercially viable, their development will necessarily involve close cooperation between scientists and engineers from different parts of the technological spectrum; a truly multidisciplinary opportunity,
Reversibility
I wish to thank my colleagues Nick Higgins and Craig Sawyers for numerous invaluable discussions during the preparation of this manuscript, Sheila Patterson for patient retyping, and PA Technology for making time and resources available to me.
Once binding-site reporter systems become available for commercially important antibody-antigen systems, there will be an opportunity for creation of truly reversible, or continuously reading, immunosensors. In most systems of analysis, the functional specificity of an antibody results from its higher affinity for one antigen over the others present in the sample. When the antigen of relevance is present at low concentration relative to other components in the sample, as is commonly the case, good functional specificity relies upon high affinity. Since the affinity of antibody-antigen interaction is largely controlled by the dissociation rate rather than the association rate, high affinity binding of antigen is essentially irreversible without alterations in pH and/or ionic strength of the buffer. Consequently, most immunosensors of high specificity will be irreversible unless pulsed with protein denaturing conditions. Where a close interaction between antigen and the combining-site of the antibody can be monitored using reporter-groups, the necessity for very long antigen residence times (high affinity) diminishes. Antibody specificity can be determined by whether the analyte sufficiently modifies electron distributions to affect reporter group function. Steric hindrance, charge
Acknowledgements
References 1 Lowe,C. R. (1984) Trends Biotechnol. 2, 59-65 2 Kfhler, G. and Milstein, C. (1975) Nature 256, 495-497 3 Gronow, M. (1984) Trends Biochem. Sci. 9, 336-340 4 Liedberg, B., Nylander, C. and LundstrSm, I. (1983) Sensors and Actuators 4, 299-304 5 Matsuoka,H., Karube, I. and Suzuki, I. (1983) International Symposium on Electroanalysis, Proc. Roy. Soc. Chem. 6 Aizawa,M. (1983) in Proceedings of the International Meeting on Chemical Sensors., pp. 683-692, Kodansha
7 Mattiasson, B., Danielsson, B. and Mosbach, K. (1980)Food Process Eng. 2, 59-68 8 Peterson, J. I., Goldstein, S. R. and Fitzgerald, R.V. (1980)Anal Chem. 52, 864-869 9 Schultz,J., Mansouri, S. and Goldstein, I. J. (1982)Diabetes Care 5, 245-253 10 Guilbault, G. G. (1980) Ion-selective Electrode Rev. 2, 17-71 11 Ishimori, Y., Karube, I. and Suzuki, S. (1981) Appl. Environ. Microbiol. 42, 632-636 12 Roederer, J. E. and Bastiaans, G. J. (1983) Anal Chem. 55, 2333-2336 13 Chem. Eng. News (1984)April, pp. 18 19 14 Aizawa, M., Kato, S. and Suzuki, S. (1977)J. Membr. Sci. 2, 125-132
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69, 4001-4011 21 Kuriyama,T., Kimura, J. and Kawana, Y. (1984) Announcement by NEC 26 Weber, W. H. and Eagen, C. F. (1979) Optics Lett. 4, 236-238 Corporation at International Conference on Solid State Devices and Materials, 27 Nylander, C., Liedberg, B. and Lind, T. of the International Meeting on Chemical (1982/83) Sensors and Actuators 3, 79-88 Kobe City, Japan Sensors, pp. 699-704, Kodansha 22 Janata, J. and Blackburn, G. F. (1984) 28 Burton, D. R. (1985) Mol. Immunol. 22, See bibliography in Ref. 22 Ann. N Y Acad. Sci. 286-292 161-206 Janata,J. and Huber, R. J. (1980) in: Ionsensitive Electrodes in Analytical Chemis- 23 Carter, T., Dahne, C. and Place, J. F. 29 Petty-Saphon,S. (1983) UK patent appli(1983) Patent number WO83/01112, cation 8325267, assigned to PA try (Freiser, H., ed.), 2, 107-174, Plenum assigned to the Battelle Memorial Consulting Services,Ltd Press 30 Keating, M. Y. and Rechnitz, G. A. Institute Collins, S. and Janata, J. (1982) Anal, (1984) Anal Chem. 56, 801-806 24 Pettigrew, R. M. et al. (1984) Patent Chim. Aaa 136, 93-99 number WO84/02578, assigned to Com- 31 Thompson, M., Krull, U. J. and Sibbald, A., Whalley, P. D. and Worsfold, P. J. (1980)Anal. Chim. Acta. tech Research Unit Ltd Covington, A. K. (1984) Anal Chim. 117, 121-132 25 Pockrand, I. et al. (1978)J. Chem. Phys, Acta 159, 47-62
15 Yamarnoto,N. et al. (1980) Clin. Chem. 26, 1569-1572 16 Yamamoto,N. et aL (1983) in Proceedings 17 18
19 20
There are no standard procedures RECOMBINANT DNA METHODOLOGY b y J. R. Dillon, A. Nasim and E. R. Nestmann, John Wiley & Sons, 1985. £22.95 (xx + 219 pages) 1 S B N 0 471 89851 1 Recombinant D N A Methodology is an
introductory laboratory manual for students and research workers venturing into the field for the first time, and not an encyclopaedic compilation of methods. In this respect, its scope is more limited than that of the indispensable Molecular Cloning by Maniatis et al. How well does the book by DiUon et al. fulfil its aims? Books giving practical details of techniques pose several problems for reviewers. How should such books be criticized? Can it be done by simply examining the procedures and comparing them with the methods used in one's own work? This is clearly unacceptable. There are no 'standard' procedures, for these are always modified, and the number of variations in use must equal the number of laboratories using the procedure! There can be very few methods that are beyond improvement, but procedures enshrined in manuals may become fossilized and used uncritically. The habit of referring to such manuals as 'bibles', with the implication that they contain commandments to be followed unquestioningly, is a reflection of this attitude. Dillon et al. have made a valiant attempt to minimize this danger by adding a commentary to the practical procedures. The pages are divided vertically into halves, the left column
containing the steps of the method while the right column contains comments on the individual steps. These comments may give the rationale for the step, or give hints as to how to perform it, or suggest alternatives that have been used by others. This gives the reader a grasp of what is going on and will perhaps encourage a more critical approach. One consequence of this split page format is that the layout looks rather confused. I frequently found that my eyes were drawn to the comments rather than the method and it would have been better to have made them more distinct by using different typefaces. Major sections and subsections should begin on a new page, although this would make a longer book. The size of the book makes it easy to handle; at 219 pages it is about 215 the size of Maniatis. I hope that the binding is sufficiently strong to withstand rough treatment at the bench.
There are some unfortunate omissions for a book intended to be a reference manual for recombinant DNA methodology. Detailed maps and restriction enzyme sites are not provided for the common vectors, nor is a table giving restriction enzyme recognition sequences. These are admittedly readily available elsewhere but they should be included in a text of this sort. They could have been put in the space given to a list of suppliers, surely unnecessary when companies go to great lengths to make sure their wares are known. Overall, I think that Dillon et al. have achieved their aims. The book cannot replace that by Maniatis et al. as the reference work for a research laboratory, but it should find wide acceptance as a laboratory manual for undergraduates and postgraduates new to the field. I recommend it:
JAN WITKOWSKI
Imperial Cancer Research Fund Laborawries, St Bartholomew's Hospital, Dominion House, Bartholomew Close, London E C I A 7BE, UK.
Introducing enzymes ENZYMES IN ORGANIC SYNTHESIS. (C1BAFOUNDATION SYMPOSIUMIII) edited by Ruth Porter and Sarah Clark, Pitman, 1985. £2Z95 (viii + 248 pages) 1 S B N 0 272 79785 5
When a 'real' organic chemist reads such a title, he or she will very likely ask: 'Why should I want to use enzymes as catalysts in my syntheses?' This is a highly pertinent point given the stateof-the-art in the field. Factors such as limited availability, instability, single
batch-use and high costs continue to restrict the use of enzymes and the fact is that most organic chemists are unfamiliar with the application of enzymes. In the last two decades, however, several new techniques, especially genetic engineering, immobilization of biocatalysts, and affinity chromatography have strongly stimulated the application of enzymes, not only in organic chemistry. Assessment, discussion and criticism of the topic