72
TIBTECH - MARCH 1991 (Vol. 9]
constitutive expression of Mx proteins is deleterious to the organism or whether this would further increase the influenza virus resistance of animals with a functional endogenous Mx defense system. A third potential problem is related to the fact that Mx proteins are effective against viruses only if the proteins are abundant in the cells. Mx proteins are not secreted and must therefore be synthesized by those specialized cells of the body in which the virus is able ~o replicate. In the case of influenza virus, these cells represent the surface epithelial cell layers of the respiratory tract. Ideally, one would like to use a strong constitutive promoter that directs transgene expression specifically in these specialized cells. These promoters have yet to be characterized. Perspective Many other questions remain to be answered before Mx proteins or other IFN-induced proteins can be used for transgenic livestock production. Since Mx and other IFN-
[]
[]
[]
induced proteins are typically encoded by small families of genes, the question of which family member to choose is an important one. In the case of the Mx genes, some genes encode nuclear proteins and others encode cytoplasmic proteins. Would the nuclear murine Mxl protein, which is highly effective in mice, also be suitable for the protection of chicken from influenza virus? Would a cytoplasmic Mx protein be more effective, or would it be better to express an avian Mx protein? The transgene technology is still quite new and its application towards disease-resistant domestic animals awaits further exploration. Nevertheless, intracellular immunization, as proposed by Arnheiter and co-workers, seems to have a good chance of becoming a feasible method for introducing resistance genes into valuable livestock.
335,452-454 3 Trono, D., Feinberg, M. B. and Baltimore, D. (1989) Cell 59,113-120 4 Salter, D. W. and Crittenden, L. B. (1989) Theor. Appl. Genet. 77, 457-461 5 Abel, P. P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T. and Beachy, R. N. (1986) Science 232, 738-743 6 Arnheiter, H., Skuntz, S., Noteborn, M., Chang, S. and Meier, E. (1990) Cell 62, 51-61 7 Staeheli, P., Haller, O., Boll, W., Lindenmann, J. and Weissmann, C. (1986) Cell 44, 147-158 8 Horisberger, M. A., Staeheli, P. and Hailer, O. (1983) Prec. Natl Acad. Sci. USA 80, 1910-1914 9 Staeheli, P. and Hailer, O. (1987) Interferon 8, 1-23 10 Staeheli, P., Grob, R., Meier, E., Sutcliffe, G. and Hailer, O. (1988) Mol. Cell. Biol. 8, 4518-4523 11 Staeheli, P., Yu, Y-X., Grob, R. and Hailer, O. (1989) Mol. Cell. Biol. 9, 3117-3121
R~ferences 1 Baltimore, D. (1988) Nature 335, 395-396 2 Friedman, A. D., Triesenberg, S. J. and McKnight, S. L. (1988) Nature
PETER STAEHELI
[]
[]
[]
[]
D
D
Capturing the light and capturing the market A recent symposium on bioluminescence and chemiluminescence* concentrated on advances which are enabling the introduction of new, commercially competitive technologies based on these phenomena to a range of clinical, industrial and environmental applications. Applications are emerging in DNAand protein-blotting methodologies, diagnostics, biosensors, food hygiene and environmental monitoring, and pharmaceutical and clinical research. Chemiluminescence (CL) is the emission of radiation, usually visible or near-infrared, caused by the decay of a chemical reaction product from * Vlth International Symposium on Bioluminescence and Chemiluminescence, 10-13 September 1990, University of Cambridge, UK.
an electronic excited state to ground state. Bioluminescence (BL) is the CL produced by a wide range of organisms. Although known for several hundred years, only in the past two decades has there been analytical interest in luminescent reactions, and coupled in assays with enzymatic or immunological methods, the reactions can be adapted to detect and quantify a variety of analytes with great precision. Compared with the previous symposia in this series, presentations at this meeting reflected a shift in emphasis from theoretical and mechanistic aspects to clinical and industrial applications. Instrumentation was discussed, particularly in concurrent industrial seminars, and this area also received strong support from industrial exhibitors; great interest was shown in the new
(~) 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430191/$2.00
Department of Virology, University of Freiburg, Herman Herder Strasse 11, D-7800 Freiburg, FRG.
[]
[]
[]
detection, imaging and systems now available.
software
Molecular biology of BL The cloning and requirements for successful expression of both prokaryotic and eukaryotic luciferases were discussed and E. Meighen (McGill University, Canada), reviewed the characteristics of cloned luciferases. Addition of reduced flavin mononucleotide (FMNH2) and aldehyde to either of the two general types of bacterial luciferase identified results in light emission. Bacterial luciferase is a heteropolymeric protein with the 0b and J3 subunits of the luciferase (encoded by lux A, B genes), having arisen by gene duplication. The lux C, D and E genes encode the fatty acid reductase necessary to generate the aldehyde. G. Stewart (Nottingham University, UK) indicated that a bioluminescent phenotype could be obtained from an E. colt strain into which V. fischert lux A, B genes had been cloned, but in which lux E was absent and only derivatives of lux
TIBTECH - MARCH 1991 [Vol. 9]
C, D present. V. harveyi and V. fischeri lux genes have now been expressed in a wide range of other bacteria, including Agrobacterium,
Bacillus, Erwinia, Escherichia, Pseudomonas, Rhizobium, Anabaena and Streptomyces. In contrast with bacterial bioluminescence, light emission by crustaceans, coelenterates and fireflies is dependent on ATP rather than pyridine nucleotides. Firefly lucfferase uses benzothiazole as luciferin, while crustacean (Vargula), and coelenterate (Aequorea), luciferases both use imidazolopyrazine. Expression of luciferases in eukaryotic systems has, until recently, been more or less limited to firefly luciferase, but there is reasonable promise for bacterial luciferases provided an appropriate promoter is introduced and the lux A and B genes fused (E. Meighen; A. A. Szalay, University of Alberta, Canada). Bacterial iV. harvey~] luciferase has been expressed in plant, mammalian, yeast and also in insect cells (baculovirus system), where very high levels of expression (up to 10% total protein) have been achieved. Interesting evolutionary implications were thrown up by K. Wood (Promega Corp., WI, USA), reporting significant protein sequencc homology between firefly luciferase and a CoA ligase from plants. The luciferase had previously been shown to possess CoA ligase activity and it appears that oxidase activity of luciferase arose by development of a new catalytic mechanism within the existing protein structure of an ancestral CoA ligase. Cloned firefly luciferases are being used extensively in assays as genetic reporters, and modifications to enzyme structures which cause changes in colour of light emitted may lead to applications using multiple luciferases simultaneously (K. Wood). A reminder that BL was not created for the benefit of biotechnologists was provided by J. Morin (UCLA, USA), who discussed the natural role of BL in communication in marine organisms. Many speakers emphasized the importance of considering not only the replication and transcription of cloned lux genes, but also transcript stability and processing, translation and protein stability and folding. A. A. Szalay indicated that the
73
thermostability of cloned proteins in mammalian cells should be borne in mind - incorporation of bacterial beat-shock chaperonin GroELS could facilitate correct protein folding. A. K. Campbell (University of Wales College of Medicine, Cardiff, UK), reported cloning both luciferase genomic and cDNA, coupled to promoters which allow tissue-specific, inducible expression in mammalian cells and whole organisms.
Luminescent labels and substrates The trend away from isotopic labelling of proteins and other molecules, particularly for diagnostic kits, is increasing. In addition to the health risks, staff training and radioactive waste-disposal problems, isotope half-life limits the useful shelf life of diagnostic kits. Luminol was the first luminescent isotopesubstitute, and is still widely used in commercial immunoassay kits. Most CL reactions for immunoassay are based on luminol, lucigenin or other cyclic hydrazides, acridinium esters or oxalates. Dioxetanes and acridinium esters appear to be the compounds targeted for most attention and development. Horseradish peroxidase (HRP), with luminol as indicator, is probably the commonest enzyme label, though glucose and xanthine oxidases, alkaline phosphatase, and J3-galactosidase are also used. The majority of molecular manipulations are aimed at improving compound stability, light yield, and reaction times compatible with signal reading range of instrumentation. Dioxetanes, perhaps the most efficient organic compound emitters, are unstable - stability has been achieved by the addition of an adamantyl group. P. Schaap (Lumigen Inc., USA), has further developed this approach to produce a highly efficient substrate for alkaline phosphatase ( A P ) , commercial applications for which have already appeared. The synthesis and commercialization of enzyme-activated, stabilized chemiluminescent dioxetanes is also being hotly pursued by Tropix Inc. I. Bronstein (Tropix, Inc.), presented the uses of dioxetane labels and substrates in sensitive immunoassays and blotting protocols. P. Schaap presented a simple, rapid DNA fingerprinting protocol using AP coupled to a synthetic oligo derived from the Jeffreys mini-
satellite sequences. Sensitivity is reported to be as good as that using conventional probes and 32p, and has the advantage over other noncolorimetric signals that filter reprobing is easy. The main challenge in nonisotopic systems for the detection of DNA and protein has been to achieve sensitivity comparable with that of radioactive labels (D. Pollard-Knight, Amersham, UK). The coupling of enzymes or chemical labels to nucleic acid probes (e.g. HRP/luminol enhanced chemiluminescence, chemically triggered acridinium esters and AP cleavage of substituted dioxetanes), combined with probe and target amplification strategies, are now approaching the requisite sensitivity, with detection limits claimed to be 0.2-5.0 attomoles. Enhanced chemiluminescence (ECL), a well-established method for the detection of HRP labels has recently been applied to filter-based detection of DNA and protein (A. C. Simmonds, Amersham, UK), and can be used to detect single-copy genes in Southern blots. Appropriately derivatized bioluminescent proteins can replace enzyme labels in solid-phase immunoassays. Aequorin (derived from recombinant apoaequorin expressed in E. cohl, is highly stable, and may be derivatized with biotin and then coupled to antibodies to produce stable, specific bioluminescent detection reagents (M. T. Cormier, University of Georgia, USA and ELA Technologies, GA, USA).
Applications Commercial growth of BL and CL technologies is undoubtedly following the progress in developing new luminescent substrates and cloning the genes for bioluminescent, proteins. To date, most applications have been based on the fireflysystem detection of ATP. E. Schram (Free University, Brussels, Belgium), indicated that, despite the introduction of recombinant luciferase and the use of lipids as protective agents, methodology development (separation methods and automation), has lagged behind that of reagents and instrumentation. Despite its lack of specificity in biomass assays, its rapidity and sensitivity ensures the firefly §ystem remains a popular method in the dairy and food indus-
74
TIBTECH- MARCH 1991 [Vol. 9]
--Fig. 1 (a) Image of microtitre tray containing varying levels of A TP and firefly luciferase/luciferin. Emitted light was measured using a sensitive intensified CCD imaging system, the Biomedical Image Quantifier (BIQ BioView) Image Research Ltd, Cambridge, IlK. Light levels (photon counts) in 96 wells may be quantified rapidly and simultaneously. The kinetic profile of light emission can be followed by measuring successive images of the tray. ApplicPtion of BL.imaging techniques in "rapid microbiology ~permits identification of discrete colonies without long incubation periods: (b, c) images of light emission from genetically engineered E. coli expressing luciferase (i.e. the lux gene), inoculated onto agar in a 90 mm petri dish• (b) Light emission after 10 h incubation at 30°C; and (©) light emission after overnight incubation (large and sometimes confluent colonies) with computer superposition of colonies seen in (b) as red spots. Photographs b and c are reproduced, with permission,
from Trends Anal Chem. (1990) 9, pp. 269-277. C
/ J
/ _J
~
~
.
J
•
tries, for clinical chemistry and hygiene and environmental monitoring.
'Rapid microbiology' Problems with applications in sensitive microbial detection in the food and beverage industry were highlighted by D. A. Stafford (Dynatech, USA). Suitable extractants, to overcome variations in the efficiency of ATP extraction from microbial cells in food contamination assays, have been developed. [The use of detergents to liberate ATP, thought to be bound to proteins within cells, was also reported for use with erythrocytes by M. Kellerman (University Medical School, Pecs, Hungary).] Methods proposed for removing somatic ATP and cells involved filtration of milk to leave bacteria on a filter which can then be assayed (E. Schi'am), or destruction of nonbacterial ATP prior to bacterial lysis by treatment with surfactant-sodium iodate mixtures (N. N. Ugarova, Lomonosow University, Moscow,
USSR). Improvements in signal and enzyme stability by the use of phospholipids which protect luciferase enzyme (D. Stafford) were reported, and detection of 10-20 microorganisms m1-1 is now claimed to be routinely possible. However, for acceptance of these techniques on a large scale, both Stafford and Schram pointed out that costs of assays need to be reduced, and that, whereas large labs need automated systems, on-site testing necessitates simple-to-use, portable 'dip-stick' technology. The lack of appropriate methodology also probably accounts for the failure of luminescence-based technology to make a major impact on clinical microbiology. J. G. M. Hastings (Queen Elizabeth Hospital, Birmingham, UK), indicated that assays which are easy to use for no great increase in cost, giving quicker results and tangible advantages in patient management, are required. Development of luminometers, for
~
microtitre plates in particular, will push bioluminescent assays into the lab. Use of CL assays in estimating bacterial ATP in screening for bacteriuria, microbial antibiotic-susceptibility/sensitivity testing and hygiene testing has encountered little commercial success: again, the recent development of dipstick luciferinluciferase assays should help (see Fig. 1).
Clinical research CL is finding applications in clinical chemistry and medical physiology. Many assays (mostly immunoassays), developed for analytes of medical, veterinary and forensic importance (hormones, drugs), were reported. Altered protein glycosylation, characteristic of certain disease states, may be monitored using bind= ing to plant lectins coupled with use of a recombinant aequorin tag (M. J. Cormier, University of Georgia, USA). It has been known for some
TIBTECH- MARCH 1991 [Vol. 9]
years that chloroplasts exhibit endogenous luminescence. More recently it has been reported that phagocytic cells such as alveolar macrophages luminesce and, because of its high sensitivity, cellular luminescence may be used as a clinical test for activation and function of PMNLs arid other phagocytic cells. An introduction to this phenomenon and its applications was provided by P. de Sole (Catholic University, Rome, Italy). I. Cree (University of Dundee, UK), discussed the use of microtitre assays in clinical investigation of opsonization and cellular defects (acquired or inherited) and the study of the effects of cytokines and drugs on phagocyte function in vitro. Data on inherited disorders of phagocyte metabolism (B. Descamps-Latscha, H6pital Necker, Paris, France), treatment of asthma (Ph. Godard, Montpellier, France; I. Cree), and effectiveness of UV treatment of psoriasis by measurement of phagocyte activity of psoriatic PMNLs (I. Cree), were presented. The sensitivity of luminescence assays means that very little tissue is required: A. Lundin (Karolinska Inst., Sweden), reported measurement of mitochondrial ATP production rates from individual muscle fibres in detection of various muscle orders, with comparable results to those obtained using HPLC and spectrophotometry; and in studies on lipid and glucose metabolism, glycerol-utilizing bacterial luciferases have been used to measure lipolysis rates in fat cells collected by needle biopsy (A. Lundin). P. E. Andreotti (MCL Technologies, FL, USA), reported using microtitre-plate ATP-luciferase assays in selecting chemotherapeutics for cancer patients; to maximize tumour remission, an assay must be capable of predicting both resistance and sensitivity of tumour to agent. Such assays have indicated that dividing and non-dividing cells have different spectra of sensitivity and have even detected some drugs with tumour-stimulating effects. Hardware and software Parameters to consider in the selection of appropriate labels and instrumentation for particular applications were discussed in the main symposia and received still greater emphasis in separate, industrially sponsored seminars. Luminescent
75
reactions can occur very rapidly (
TIBTECH - MARCH 1991 (Vol. 9]
constitutive expression of Mx proteins is deleterious to the organism or whether this would further increase the influenza virus resistance of animals with a functional endogenous Mx defense system. A third potential problem is related to the fact that Mx proteins are effective against viruses only if the proteins are abundant in the cells. Mx proteins are not secreted and must therefore be synthesized by those specialized cells of the body in which the virus is able ~o replicate. In the case of influenza virus, these cells represent the surface epithelial cell layers of the respiratory tract. Ideally, one would like to use a strong constitutive promoter that directs transgene expression specifically in these specialized cells. These promoters have yet to be characterized. Perspective Many other questions remain to be answered before Mx proteins or other IFN-induced proteins can be used for transgenic livestock production. Since Mx and other IFN-
[]
[]
[]
induced proteins are typically encoded by small families of genes, the question of which family member to choose is an important one. In the case of the Mx genes, some genes encode nuclear proteins and others encode cytoplasmic proteins. Would the nuclear murine Mxl protein, which is highly effective in mice, also be suitable for the protection of chicken from influenza virus? Would a cytoplasmic Mx protein be more effective, or would it be better to express an avian Mx protein? The transgene technology is still quite new and its application towards disease-resistant domestic animals awaits further exploration. Nevertheless, intracellular immunization, as proposed by Arnheiter and co-workers, seems to have a good chance of becoming a feasible method for introducing resistance genes into valuable livestock.
335,452-454 3 Trono, D., Feinberg, M. B. and Baltimore, D. (1989) Cell 59,113-120 4 Salter, D. W. and Crittenden, L. B. (1989) Theor. Appl. Genet. 77, 457-461 5 Abel, P. P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T. and Beachy, R. N. (1986) Science 232, 738-743 6 Arnheiter, H., Skuntz, S., Noteborn, M., Chang, S. and Meier, E. (1990) Cell 62, 51-61 7 Staeheli, P., Haller, O., Boll, W., Lindenmann, J. and Weissmann, C. (1986) Cell 44, 147-158 8 Horisberger, M. A., Staeheli, P. and Hailer, O. (1983) Prec. Natl Acad. Sci. USA 80, 1910-1914 9 Staeheli, P. and Hailer, O. (1987) Interferon 8, 1-23 10 Staeheli, P., Grob, R., Meier, E., Sutcliffe, G. and Hailer, O. (1988) Mol. Cell. Biol. 8, 4518-4523 11 Staeheli, P., Yu, Y-X., Grob, R. and Hailer, O. (1989) Mol. Cell. Biol. 9, 3117-3121
R~ferences 1 Baltimore, D. (1988) Nature 335, 395-396 2 Friedman, A. D., Triesenberg, S. J. and McKnight, S. L. (1988) Nature
PETER STAEHELI
[]
[]
[]
[]
D
D
Capturing the light and capturing the market A recent symposium on bioluminescence and chemiluminescence* concentrated on advances which are enabling the introduction of new, commercially competitive technologies based on these phenomena to a range of clinical, industrial and environmental applications. Applications are emerging in DNAand protein-blotting methodologies, diagnostics, biosensors, food hygiene and environmental monitoring, and pharmaceutical and clinical research. Chemiluminescence (CL) is the emission of radiation, usually visible or near-infrared, caused by the decay of a chemical reaction product from * Vlth International Symposium on Bioluminescence and Chemiluminescence, 10-13 September 1990, University of Cambridge, UK.
an electronic excited state to ground state. Bioluminescence (BL) is the CL produced by a wide range of organisms. Although known for several hundred years, only in the past two decades has there been analytical interest in luminescent reactions, and coupled in assays with enzymatic or immunological methods, the reactions can be adapted to detect and quantify a variety of analytes with great precision. Compared with the previous symposia in this series, presentations at this meeting reflected a shift in emphasis from theoretical and mechanistic aspects to clinical and industrial applications. Instrumentation was discussed, particularly in concurrent industrial seminars, and this area also received strong support from industrial exhibitors; great interest was shown in the new
(~) 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430191/$2.00
Department of Virology, University of Freiburg, Herman Herder Strasse 11, D-7800 Freiburg, FRG.
[]
[]
[]
detection, imaging and systems now available.
software
Molecular biology of BL The cloning and requirements for successful expression of both prokaryotic and eukaryotic luciferases were discussed and E. Meighen (McGill University, Canada), reviewed the characteristics of cloned luciferases. Addition of reduced flavin mononucleotide (FMNH2) and aldehyde to either of the two general types of bacterial luciferase identified results in light emission. Bacterial luciferase is a heteropolymeric protein with the 0b and J3 subunits of the luciferase (encoded by lux A, B genes), having arisen by gene duplication. The lux C, D and E genes encode the fatty acid reductase necessary to generate the aldehyde. G. Stewart (Nottingham University, UK) indicated that a bioluminescent phenotype could be obtained from an E. colt strain into which V. fischert lux A, B genes had been cloned, but in which lux E was absent and only derivatives of lux
TIBTECH - MARCH 1991 [Vol. 9]
C, D present. V. harveyi and V. fischeri lux genes have now been expressed in a wide range of other bacteria, including Agrobacterium,
Bacillus, Erwinia, Escherichia, Pseudomonas, Rhizobium, Anabaena and Streptomyces. In contrast with bacterial bioluminescence, light emission by crustaceans, coelenterates and fireflies is dependent on ATP rather than pyridine nucleotides. Firefly lucfferase uses benzothiazole as luciferin, while crustacean (Vargula), and coelenterate (Aequorea), luciferases both use imidazolopyrazine. Expression of luciferases in eukaryotic systems has, until recently, been more or less limited to firefly luciferase, but there is reasonable promise for bacterial luciferases provided an appropriate promoter is introduced and the lux A and B genes fused (E. Meighen; A. A. Szalay, University of Alberta, Canada). Bacterial iV. harvey~] luciferase has been expressed in plant, mammalian, yeast and also in insect cells (baculovirus system), where very high levels of expression (up to 10% total protein) have been achieved. Interesting evolutionary implications were thrown up by K. Wood (Promega Corp., WI, USA), reporting significant protein sequencc homology between firefly luciferase and a CoA ligase from plants. The luciferase had previously been shown to possess CoA ligase activity and it appears that oxidase activity of luciferase arose by development of a new catalytic mechanism within the existing protein structure of an ancestral CoA ligase. Cloned firefly luciferases are being used extensively in assays as genetic reporters, and modifications to enzyme structures which cause changes in colour of light emitted may lead to applications using multiple luciferases simultaneously (K. Wood). A reminder that BL was not created for the benefit of biotechnologists was provided by J. Morin (UCLA, USA), who discussed the natural role of BL in communication in marine organisms. Many speakers emphasized the importance of considering not only the replication and transcription of cloned lux genes, but also transcript stability and processing, translation and protein stability and folding. A. A. Szalay indicated that the
73
thermostability of cloned proteins in mammalian cells should be borne in mind - incorporation of bacterial beat-shock chaperonin GroELS could facilitate correct protein folding. A. K. Campbell (University of Wales College of Medicine, Cardiff, UK), reported cloning both luciferase genomic and cDNA, coupled to promoters which allow tissue-specific, inducible expression in mammalian cells and whole organisms.
Luminescent labels and substrates The trend away from isotopic labelling of proteins and other molecules, particularly for diagnostic kits, is increasing. In addition to the health risks, staff training and radioactive waste-disposal problems, isotope half-life limits the useful shelf life of diagnostic kits. Luminol was the first luminescent isotopesubstitute, and is still widely used in commercial immunoassay kits. Most CL reactions for immunoassay are based on luminol, lucigenin or other cyclic hydrazides, acridinium esters or oxalates. Dioxetanes and acridinium esters appear to be the compounds targeted for most attention and development. Horseradish peroxidase (HRP), with luminol as indicator, is probably the commonest enzyme label, though glucose and xanthine oxidases, alkaline phosphatase, and J3-galactosidase are also used. The majority of molecular manipulations are aimed at improving compound stability, light yield, and reaction times compatible with signal reading range of instrumentation. Dioxetanes, perhaps the most efficient organic compound emitters, are unstable - stability has been achieved by the addition of an adamantyl group. P. Schaap (Lumigen Inc., USA), has further developed this approach to produce a highly efficient substrate for alkaline phosphatase ( A P ) , commercial applications for which have already appeared. The synthesis and commercialization of enzyme-activated, stabilized chemiluminescent dioxetanes is also being hotly pursued by Tropix Inc. I. Bronstein (Tropix, Inc.), presented the uses of dioxetane labels and substrates in sensitive immunoassays and blotting protocols. P. Schaap presented a simple, rapid DNA fingerprinting protocol using AP coupled to a synthetic oligo derived from the Jeffreys mini-
satellite sequences. Sensitivity is reported to be as good as that using conventional probes and 32p, and has the advantage over other noncolorimetric signals that filter reprobing is easy. The main challenge in nonisotopic systems for the detection of DNA and protein has been to achieve sensitivity comparable with that of radioactive labels (D. Pollard-Knight, Amersham, UK). The coupling of enzymes or chemical labels to nucleic acid probes (e.g. HRP/luminol enhanced chemiluminescence, chemically triggered acridinium esters and AP cleavage of substituted dioxetanes), combined with probe and target amplification strategies, are now approaching the requisite sensitivity, with detection limits claimed to be 0.2-5.0 attomoles. Enhanced chemiluminescence (ECL), a well-established method for the detection of HRP labels has recently been applied to filter-based detection of DNA and protein (A. C. Simmonds, Amersham, UK), and can be used to detect single-copy genes in Southern blots. Appropriately derivatized bioluminescent proteins can replace enzyme labels in solid-phase immunoassays. Aequorin (derived from recombinant apoaequorin expressed in E. cohl, is highly stable, and may be derivatized with biotin and then coupled to antibodies to produce stable, specific bioluminescent detection reagents (M. T. Cormier, University of Georgia, USA and ELA Technologies, GA, USA).
Applications Commercial growth of BL and CL technologies is undoubtedly following the progress in developing new luminescent substrates and cloning the genes for bioluminescent, proteins. To date, most applications have been based on the fireflysystem detection of ATP. E. Schram (Free University, Brussels, Belgium), indicated that, despite the introduction of recombinant luciferase and the use of lipids as protective agents, methodology development (separation methods and automation), has lagged behind that of reagents and instrumentation. Despite its lack of specificity in biomass assays, its rapidity and sensitivity ensures the firefly §ystem remains a popular method in the dairy and food indus-
74
TIBTECH- MARCH 1991 [Vol. 9]
--Fig. 1 (a) Image of microtitre tray containing varying levels of A TP and firefly luciferase/luciferin. Emitted light was measured using a sensitive intensified CCD imaging system, the Biomedical Image Quantifier (BIQ BioView) Image Research Ltd, Cambridge, IlK. Light levels (photon counts) in 96 wells may be quantified rapidly and simultaneously. The kinetic profile of light emission can be followed by measuring successive images of the tray. ApplicPtion of BL.imaging techniques in "rapid microbiology ~permits identification of discrete colonies without long incubation periods: (b, c) images of light emission from genetically engineered E. coli expressing luciferase (i.e. the lux gene), inoculated onto agar in a 90 mm petri dish• (b) Light emission after 10 h incubation at 30°C; and (©) light emission after overnight incubation (large and sometimes confluent colonies) with computer superposition of colonies seen in (b) as red spots. Photographs b and c are reproduced, with permission,
from Trends Anal Chem. (1990) 9, pp. 269-277. C
/ J
/ _J
~
~
.
J
•
tries, for clinical chemistry and hygiene and environmental monitoring.
'Rapid microbiology' Problems with applications in sensitive microbial detection in the food and beverage industry were highlighted by D. A. Stafford (Dynatech, USA). Suitable extractants, to overcome variations in the efficiency of ATP extraction from microbial cells in food contamination assays, have been developed. [The use of detergents to liberate ATP, thought to be bound to proteins within cells, was also reported for use with erythrocytes by M. Kellerman (University Medical School, Pecs, Hungary).] Methods proposed for removing somatic ATP and cells involved filtration of milk to leave bacteria on a filter which can then be assayed (E. Schi'am), or destruction of nonbacterial ATP prior to bacterial lysis by treatment with surfactant-sodium iodate mixtures (N. N. Ugarova, Lomonosow University, Moscow,
USSR). Improvements in signal and enzyme stability by the use of phospholipids which protect luciferase enzyme (D. Stafford) were reported, and detection of 10-20 microorganisms m1-1 is now claimed to be routinely possible. However, for acceptance of these techniques on a large scale, both Stafford and Schram pointed out that costs of assays need to be reduced, and that, whereas large labs need automated systems, on-site testing necessitates simple-to-use, portable 'dip-stick' technology. The lack of appropriate methodology also probably accounts for the failure of luminescence-based technology to make a major impact on clinical microbiology. J. G. M. Hastings (Queen Elizabeth Hospital, Birmingham, UK), indicated that assays which are easy to use for no great increase in cost, giving quicker results and tangible advantages in patient management, are required. Development of luminometers, for
~
microtitre plates in particular, will push bioluminescent assays into the lab. Use of CL assays in estimating bacterial ATP in screening for bacteriuria, microbial antibiotic-susceptibility/sensitivity testing and hygiene testing has encountered little commercial success: again, the recent development of dipstick luciferinluciferase assays should help (see Fig. 1).
Clinical research CL is finding applications in clinical chemistry and medical physiology. Many assays (mostly immunoassays), developed for analytes of medical, veterinary and forensic importance (hormones, drugs), were reported. Altered protein glycosylation, characteristic of certain disease states, may be monitored using bind= ing to plant lectins coupled with use of a recombinant aequorin tag (M. J. Cormier, University of Georgia, USA). It has been known for some
TIBTECH- MARCH 1991 [Vol. 9]
years that chloroplasts exhibit endogenous luminescence. More recently it has been reported that phagocytic cells such as alveolar macrophages luminesce and, because of its high sensitivity, cellular luminescence may be used as a clinical test for activation and function of PMNLs arid other phagocytic cells. An introduction to this phenomenon and its applications was provided by P. de Sole (Catholic University, Rome, Italy). I. Cree (University of Dundee, UK), discussed the use of microtitre assays in clinical investigation of opsonization and cellular defects (acquired or inherited) and the study of the effects of cytokines and drugs on phagocyte function in vitro. Data on inherited disorders of phagocyte metabolism (B. Descamps-Latscha, H6pital Necker, Paris, France), treatment of asthma (Ph. Godard, Montpellier, France; I. Cree), and effectiveness of UV treatment of psoriasis by measurement of phagocyte activity of psoriatic PMNLs (I. Cree), were presented. The sensitivity of luminescence assays means that very little tissue is required: A. Lundin (Karolinska Inst., Sweden), reported measurement of mitochondrial ATP production rates from individual muscle fibres in detection of various muscle orders, with comparable results to those obtained using HPLC and spectrophotometry; and in studies on lipid and glucose metabolism, glycerol-utilizing bacterial luciferases have been used to measure lipolysis rates in fat cells collected by needle biopsy (A. Lundin). P. E. Andreotti (MCL Technologies, FL, USA), reported using microtitre-plate ATP-luciferase assays in selecting chemotherapeutics for cancer patients; to maximize tumour remission, an assay must be capable of predicting both resistance and sensitivity of tumour to agent. Such assays have indicated that dividing and non-dividing cells have different spectra of sensitivity and have even detected some drugs with tumour-stimulating effects. Hardware and software Parameters to consider in the selection of appropriate labels and instrumentation for particular applications were discussed in the main symposia and received still greater emphasis in separate, industrially sponsored seminars. Luminescent
75
reactions can occur very rapidly (
