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WO1996012188A1 - Procede de diagnostic et dispositif de realisation de celui-ci - Google Patents

Procede de diagnostic et dispositif de realisation de celui-ci Download PDF

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Publication number
WO1996012188A1
WO1996012188A1 PCT/GB1995/002342 GB9502342W WO9612188A1 WO 1996012188 A1 WO1996012188 A1 WO 1996012188A1 GB 9502342 W GB9502342 W GB 9502342W WO 9612188 A1 WO9612188 A1 WO 9612188A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
repoπer
diagnostic method
binding
Prior art date
Application number
PCT/GB1995/002342
Other languages
English (en)
Inventor
Beverley Jane Randle
Deborah Karen Scott
Joanne Elizabeth Harris
Original Assignee
Surface Active Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Surface Active Limited filed Critical Surface Active Limited
Priority to AU35742/95A priority Critical patent/AU3574295A/en
Publication of WO1996012188A1 publication Critical patent/WO1996012188A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • This invention relates to a diagnostic method and also to apparatus for performing the diagnostic method.
  • a "reporter” generally a readily detectable entity, such as an enzyme, to produce a detectable signal event in a single step.
  • WO95/04931 we disclose a diagnostic method for determining the presence, or absence of an antigen in a sample, the method comprising contacting the sample with a multispecific antibody having first and second binding sites for binding the antigen and a detectable entity respectively, binding of the detectable entity to the antibody inactivating the detectable entity in the presence of an analogue of the antigen, wherein binding of the analogue of the antigen to the antibody results in release of the detectable entity in a detectable form from the antibody but binding of the antigen to the antibody does not cause such release or causes significantly lower levels of release.
  • a convenient assay format for a number of applications and which is suitable for use by less skilled personnel or for over the counter sale for such as for home use. It is an object of the present invention to provide such convenient assay formats and these are provided based on the invention described in the above European patent application.
  • a diagnostic method in which binding of a first antigen to a first antibody antigen binding site causes release of a second, reporter, antigen from an adjacent second antibody antigen binding site characterized in that the released reporter causes a detectable signal at a separate second site.
  • the present invention is advantageous in that the transduction and signalling events are physically separated to provide a convenient assay apparatus
  • the sample may be contacted with the antibody in the presence of an analogue of the first antigen, wherein binding of the analogue to the antibody results in release of the reporter from the antibody but binding of the first antigen to the antibody does not cause such release of the reporter or causes detectably lower levels of release characterised in that the released reporter is moved, away from the antibody and causes the production of detectable signal at a second site in accordance with the method of WO95/04931.
  • the first and second antibody antigen binding sites may be provided by the same multispecific antibody or by different antibodies which are in very close proximity to each other.
  • multispecific antibody embraces all antibodies having more than one antigen binding site such as bispecific and trispecific antibodies.
  • antibody-mediated signal transduction The release of a reporter from the second site through binding of another molecule to a first site, may be termed antibody-mediated signal transduction.
  • antibody used herein embraces immunoglobulins such as IgG, IgA, IgM, IgD and IgE and other proteins having the antigen-binding properties of a naturally-occurring antibody or antibodies produced by recombinant DNA technology or any other such methods.
  • analogue as used herein embraces all antigens capable of binding to the same or closely related binding site on the multispecific antibody as the antigen of interest.
  • the analogue may or may not include the same or similar structure as the antigen or a portion thereof.
  • the analogue comprises the antigen or a portion thereof linked to a larger entity so that the size of the resulting combination is sufficient to cause the release of bound detectable entity from the antibody.
  • the sample is preferably fluid and may be, for example, a bodily fluid, drinking water, food, etc.
  • the reporter can be for example an enzyme such as ⁇ -galactosidase, or urease, glucose oxidase, carbonic anhydrase, or horseradish peroxidase, all of which are well characterised and easily assayable enzymes, charged ion group, photoactivatable substrate, radioactive tracer, dye, lectin, fluorescent ligand che ⁇ uluminescent cell,
  • enzyme such as ⁇ -galactosidase, or urease, glucose oxidase, carbonic anhydrase, or horseradish peroxidase, all of which are well characterised and easily assayable enzymes, charged ion group, photoactivatable substrate, radioactive tracer, dye, lectin, fluorescent ligand che ⁇ uluminescent cell,
  • the reporter or one of its reaction products if it is, for example, an enzyme or other catalytic molecule, or a reaction product of a reaction catalysed by it, at a third site on an adjacent or the same antibody causing release of a bound third antigen from an adjacent fourth antigen binding site.
  • the antibody can be
  • an IgM antibody may be used which features ten different reaction steps.
  • a method of indicating risk of respiratory distress syndrome in which the antigen detected by a method of the first aspect of the invention is SP-A.
  • a method of detecting pregnancy in a mammal in which the antigen detected is chorionic gonadotrophin.
  • the mammal is human, the detected antigen being human chorionic gonadotrophin.
  • CKMB creatine kinase MB
  • Myocardial infarction can be indicated by CK-MB mass assay or be deterrnining the ratio between tissue and serum iso forms of the enzyme.
  • an apparatus for performing the method of the invention in which the binding of the antigen or analogue to be detected, the transduction event, is separated from the signalling event.
  • the reporter is carried/moved away from the first antigen binding site.
  • the reporter may be carried by the fluid of the sample under test or a flow of medium, such as a buffer, between the site of binding of the first antigen to a signal position, preferably under an imposed hydrostatic pressure, or, where the reporter is charged, by movement due to a potential difference between the site of binding of the first antigen and a signal position.
  • a biosensor is an analytical device which exploits the unique specificity conferred by a biological sensing element for an analyte of interest in association with suitable physicochemical transducers which detect a biological change mediated by the biological sensing element to detect or measure the analyte.
  • suitable physicochemical transducers which detect a biological change mediated by the biological sensing element to detect or measure the analyte.
  • concentration of an analyte is measured and expressed by the transducer as a digital electronic signal.
  • the biosensor electrode is a transducer for biochemical change in which the biological sensing element may measure an electrochemical,, optical, calorimetric, or acoustic change.
  • the change is electrochemical, amperometric or potentiometric electrodes.
  • Amperometric electrodes including the Clarke oxygen electrode, measure current flow as a linear function.
  • potentiometric electrodes use ionic species and measure potential difference as a logarithmic function.
  • An example of the use of a potentiometric electrode is ammonium ion measurement by the urea electrode.
  • Biosensing electrodes are being paralleled in development by MOSFETS, metal oxide semi-conductor field effect transistors. These transducers can be ion selective ISFETS for example for measurement of oxygen by incorporation of glucose oxidase, or chemically sensitive, CHEMFETS, where for example urease can be used to measure blood urea nitrogen levels.
  • Biosensor applications have so far been limited particularly to blood gas, metabolite, and electrolyte measurements, including sodium, potassium, chloride, urea, glucose and bicarbonate.
  • the width of biosensor application would be greatly improved by provision of improved methods for including antibodies as the biological sensing element.
  • Such antibodies may be polyclonal or monoclonal in origin but have the advantage of sustainable reproducible specificity.
  • Markers that can be detected by immunoassay include proteins and polypeptides such as tumour-associated antigens, hormones, neuropeptides, viral and bacterial particles; lipid and carbohydrate antigen species.
  • antibodies as the biological sensing element in a biosensor. These include biosensors to detect human gonadotropin (Robinson GA et al 1987
  • biosensors there is a general need to incorporate antibodies into biosensors to increase the range of analytes that can be measured by biosensors.
  • the need can be met by incorporation of transducing antibodies into such devices.
  • the biosensor can provide near instant diagnostic information. This is in marked contrast to the biosensor and immunoassay disclosed
  • the method of the invention can be arranged so that release of bound reporter causes or results in the production of a detectable electrical signal.
  • a secondary electrical signal is generated from the primary antibody transduction event, providing a means to incorporate the benefits of the use of these generic antibodies into electronic biosensor devices.
  • a biosensor comprising an immunoassay system according to either of the two previous aspects of the invention adjacent to an appropriate electrical transducer.
  • the release of the repo ⁇ er may generate charged ions or particle groups to produce a current or generate a potential difference in the cases of amperometric and potentiometric devices respectively.
  • the antibody is held covalently immobilized in the biolayer.
  • the antibody remains bound covalently, whilst interaction with the diagnostic signal molecule is ionic - giving a dynamic phase for physiochemical signalling which can be arranged adjacent to an electronic component for detecting the release of the reporter.
  • This biolayer produces an enzymatically-de ⁇ ved product or releases an electrically charged molecule. The resulting charge flow can then be detected electronically.
  • the required biolayer can be a cross-linked protein structure which can be spun into
  • the pattern of the applied biolayer can be established using negative photoresist technology.
  • a solution of antibody protein, and gelatin can be crosslinked to form a gel by chemical means, including acrylamide-bisacrylamide cross-linking, by haemagglutination, or by aggregation of beads, made of dextran, agarose or latex, carrying covalently bound transducing antibodies.
  • the protein solution can include negative photoresist components and suitable crosslinkers, such as chromium dioxide.
  • the biolayer immobilises the transducing antibody in a covalently bound matrix.
  • the biolayer can then be activated for use by brief incubation with the
  • biosensor biolayer is then ready for use.
  • GAL30 recognises human surfactant apoprotein A and NO15 type specimen antibody recognises human ⁇ inhibin. Release of the reporter occurs as a result of specific antibody-mediated signal transduction and leads to a physicochemical change which can be measured as an electrical signal.
  • RDS respiratory distress syndrome
  • Fig. 1 shows a diagnostic device 10 comprising an upper portion 12, an intermediate gasket 14, and a lower portion 16.
  • the upper and lower portions of the device are moulded from a suitable plastics material; the gasket is formed in a similar way from another suitable plastics material, preferably santoprene.
  • the upper portion 12 is a snap fit on to the lower portion 16 to form a substantially liquid-tight unit (a bleed hole 17 for air displaced by the incoming sample fluid is provided).
  • the upper portion 12 comprises a central sample supply aperture 18 which is adapted to receive a suitable portion of sample collection devices for example the tip of a syringe.
  • the aperture 18 may be arranged to securely hold the sample collection device.
  • the aperture may be provided with locking means to lock the sample collection device.
  • the upper portion 12 of the device has three windows 20, 22, and 24 respectively.
  • the windows may be formed from a translucent or transparent material whereby colour changes in materials within the device 10 can be viewed by a user from outside the device through the windows to indicate a best result.
  • the gasket 14 which retains an applied liquid sample within the lower portion 16, defines apertures 26, 28, 30, which are concentric with windows 20, 22, and 24 respectively; further apertures 32, 34, 36; and a central sample receiving area 38.
  • An incoming sample is passed through a glass fibre filter (not shown) (GF51 , Schleicher & Schuell, Dassel, Germany) 40 to remove macro particles such as red blood cells and to ensure an even flow rate amongst subsequent flows.
  • the filtered sample is divided into three flows of equal volume by means of baffles in the central sample
  • the lower portion 16 comprises a substantially coplanar surface 42 below which extend a central sample receiving recess 44, which is in flow communication with test
  • Test area recesses 46, 48, and 50 through gaps 47, 49, 51, which in turn lead to through further gaps 53, 55, 57, signal recesses 52, 54, and 56.
  • Test area recess 50 and signal recess 56 provide a main sample test track; test area recess 48 and signal recess 54 provide a positive control track; and test area recess 46 and signal recess 52 provide a negative control track. The three tracks keep the main test flow and positive and negative control flows separate thus avoiding cross-contamination.
  • Each test area recess 46, 48 and 50 contains a nitrocellulose paper disc (not shown) (0.45 ⁇ Schleicher & Schuell) on which is immobilized a bispecific antibody GAL30 described in our copending European patent application described above to which is, or is not, bound to the second antigen binding site a reporter, ⁇ galactosidase, according to the following scheme:
  • Main test area 50 bispecific antibody bound to the nitrocellulose specific for
  • Negative control test area 46 multispecific antibody GAL30, as in main test area, but which has been depleted for its ability to bind SP-A (such a defective monospecific antibody may be selected from a mixed population of the basic bispecific antibody by ion exchange chromatography for example using a Mono Q HR5/5 column (Pharmacia) in lOmM Tris HC1 pH 8.5 and altering NaCl concentrations); ⁇ -galactosidase is bound to its second antigen binding site;
  • Positive control test area 48 bispecific antibody GAL30 specific for SP-A and ⁇ -galactosidase; control amounts of SP-A (0.1-1 ⁇ g in 10 ⁇ l.) and ⁇ - galactosidase.
  • the multispecific antibody can be immobilized in the device in a variety of other ways for example on inert materials, such as beads - agarose, etc; in sol-gel solutions, such as photoresists.
  • the signal recesses 52, 54, and 56 each contain a further nitrocellulose disc (0.8 ⁇ ) which is impregnated with lO ⁇ l dots of substrate of the reporter enzyme, ⁇ - galactosidase, VBzTMgal (lOO ⁇ g to 2 mg ml 1 in PBS (Melford Labs, England) which is yellow in colour but which changes colour to red by substrate conversion of ⁇ -galactosidase.
  • a sample of amniotic fluid is collected during delivery of a premature baby, for example, using a syringe.
  • An aliquot (1ml) of the fluid sample is then introduced into the device from the syringe through the aperture 18.
  • the sample is filtered by the filter 40 and divided into three equal flows which pass along the respective main, positive and negative control, test tracks under the pressure from the syringe.
  • ⁇ -galactosidase is released from the bispecific antibody in the test area recess 50 and is passed along by positive hydrostatic pressure due to the syringe into the signal recess 56 where it reacts with the enzyme substrate VBz TMgal resulting in generation of a red colour which can be viewed through the main test track window 24. If the sample does not contain SP-A, then no ⁇ -galactosidase is released from the bispecific antibody to which it is bound in the test area recess 50 and therefore the substrate in the signal recess 56 remains yellow.
  • the positive control signal recess 54 will show in a yellow to red colour change which can be viewed through the positive control window 22 due to the release of ⁇ - galactosidase by the control of SP-A and/or SP-A from the test sample in either case; whereas the negative control signal recess 52 as viewed through negative control window 20 will remain yellow.
  • control windows may be surrounded by annular colour indications which correspond to expected colour changes, i.e the positive control window 20 will be surrounded by a red annular marking; and the negative control window 22 will be surrounded by a yellow annular marking; and the test window 24 is surrounded by a red half annulus (indicating a positive result) and a yellow half annulus (indicating a negative result).
  • the method of assessing the risk of RDS, using the device described above, is advantageous in that it can be performed by non technical staff on the labour ward; it is rapid due to the flow of liquid under pressure caused by the syringe taking a maximum of four minutes, and uses amniotic fluid, often contaminated with blood, furthermore the result is unequivocal in interpretation.
  • the device is easy and economical to produce.
  • the device described above may be simply revised to provide a diagnostic tool for other indications.
  • the bispecific antibody GAL30 may be replaced by another multispecific antibody which is specific for human chorionic gonadotrophin and a suitable reporter whereby the device becomes useful for diagnosing pregnancy in humans.
  • the antibody may be specific for CK MB and a suitable reporter whereby the device is useful for diagnosing the occurrence of myocardial infarction.
  • the method of the invention may be arranged to operate in a biosensor as exemplified below in the following description of various biosensor formats:
  • a transducing antibody recognising a glucose oxidase reporter molecule, such as NO 15. ⁇ inhibin and glucose oxidase.
  • the non- electroactive substrate glucose is present in the transducing biolayer.
  • Antibody-mediated release of glucose oxidase diagnostic signal molecule from the transducing antibody matrix in the specific presence of ⁇ - inhibin permits reduction of glucose, consuming molecular oxygen, a physico-chemical event that can be measured by a Clarke electrode or ISFET device.
  • Antibody-mediated signal transduction is achieved in one step by using a ⁇ inhibin-keyhole limpet hemocyanin, KLH conjugate of molecular weight 803 kD, which displaces bound glucose oxidase to provide the mobile reporter for subsequent physico- chemical transduction.
  • a transducing antibody recognising a ⁇ -galactosidase reporter, such as GAL30, bispecific immunoglobulin recognising human surfactant apoprotein A, SP-A, and ⁇ -galactosidase.
  • the non-electroactive substrate lactose is present in the transducing biolayer. Release of ⁇ - galactosidase from the transducing antibody immobile matrix in the presence of SP-A leads to the hydration of lactose to produce ⁇ -D galactose and D-glucose. The liberated non-electroactive glucose then provides a substrate for glucose oxidase, consuming molecular oxygen, a physico-chemical event that can be measured by a Clarke electrode or ISFET device.
  • a transducing antibody recognising the antigen under test and the diagnostic signal molecule, urease. Release of urease results in the hydrolysis of urea to ammonium ions, which can then be measured by a suitable ISFET.
  • a transducing antibody recognising the antigen under test and a large ionic group, such as an ionic repo ⁇ er such as a calcium-carrying molecule. Release of this repo ⁇ er molecule can be measured by interaction with a suitable ionophore, such as A23187 .
  • the diagnostic signal molecule is a metalloprotein where release of the mettaloprotein from the transducing antibody generates a small change in electrical
  • the transducing antibody biolayer generates a detectable electrical signal by release of ion groups and/or generation of small molecular products by enzymatic action, signals.
  • the signal can be enhanced by imposing a direction of flow on the charged panicles, and molecular species.
  • Directional flow can be assisted by use of an ionophore layer to polarize released ionic groups.
  • the biolayer can overlay the electrode surface by interfacing with a selective silane layer.
  • the production of molecular species, including bicarbonate, ammonium ions, molecular oxygen and ionic calcium, is caused by direct action of the repo ⁇ er and is measured by an electrical signal.
  • the release of the repo ⁇ er is caused by antibody- dependent molecular signal transduction in the presence of the diagnostic marker under test. There is a consequent relation, both qualitative and quantitative, between the presence of the diagnostic marker under test and the production of an electrical signal by the biolayer of the transducing immunosensor.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de diagnostic permettant de détecter la présence d'un premier antigène dans un échantillon dans lequel la fixation du premier antigène à un premier site de fixation anticorps-antigène provoque la libération d'un second antigène rapporteur du second site adjacent de fixation anticorps-antigène, ce procédé étant caractérisé en ce que le rapporteur libéré est éloigné de l'anticorps et entraîne la production d'un signal détectable au niveau d'un second site éloigné. L'invention concerne également un dispositif destiné à la réalisation de ce procédé de diagnostic.
PCT/GB1995/002342 1994-10-18 1995-10-03 Procede de diagnostic et dispositif de realisation de celui-ci WO1996012188A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35742/95A AU3574295A (en) 1994-10-18 1995-10-03 Diagnostic method and apparatus for performing the method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9421468.1 1994-10-18
GB9421468A GB9421468D0 (en) 1994-10-18 1994-10-18 Biosensor

Publications (1)

Publication Number Publication Date
WO1996012188A1 true WO1996012188A1 (fr) 1996-04-25

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PCT/GB1995/002342 WO1996012188A1 (fr) 1994-10-18 1995-10-03 Procede de diagnostic et dispositif de realisation de celui-ci

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AU (1) AU3574295A (fr)
GB (1) GB9421468D0 (fr)
WO (1) WO1996012188A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2345133A (en) * 1998-12-24 2000-06-28 Hypoguard Limited Polyspecific antibody immunodiagnostic device
CN100415896C (zh) * 2004-08-16 2008-09-03 中国科学院电子学研究所 肌酸激酶生物传感器及其试剂的制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003679A1 (fr) * 1982-04-12 1983-10-27 Hybritech Inc Anticorps ayant des doubles specificites, leur preparation et utilisation
EP0150999A2 (fr) * 1984-01-26 1985-08-07 Serono Diagnostics Limited Méthodes d'examen
EP0303110A2 (fr) * 1987-08-14 1989-02-15 Boehringer Mannheim Italia S.P.A. Dispositif immunodiagnostique et méthode
EP0511011A1 (fr) * 1991-04-26 1992-10-28 Surface Active Limited Anticorps nouveaux et méthodes pour leur utilisation
WO1994017409A1 (fr) * 1993-01-22 1994-08-04 Northeastern University Quantification d'analytes a l'etat de trace par electrophorese capillaire d'affinity
WO1995004931A1 (fr) * 1993-08-06 1995-02-16 Surface Active Limited Procede diagnostique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003679A1 (fr) * 1982-04-12 1983-10-27 Hybritech Inc Anticorps ayant des doubles specificites, leur preparation et utilisation
EP0150999A2 (fr) * 1984-01-26 1985-08-07 Serono Diagnostics Limited Méthodes d'examen
EP0303110A2 (fr) * 1987-08-14 1989-02-15 Boehringer Mannheim Italia S.P.A. Dispositif immunodiagnostique et méthode
EP0511011A1 (fr) * 1991-04-26 1992-10-28 Surface Active Limited Anticorps nouveaux et méthodes pour leur utilisation
WO1994017409A1 (fr) * 1993-01-22 1994-08-04 Northeastern University Quantification d'analytes a l'etat de trace par electrophorese capillaire d'affinity
WO1995004931A1 (fr) * 1993-08-06 1995-02-16 Surface Active Limited Procede diagnostique

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2345133A (en) * 1998-12-24 2000-06-28 Hypoguard Limited Polyspecific antibody immunodiagnostic device
CN100415896C (zh) * 2004-08-16 2008-09-03 中国科学院电子学研究所 肌酸激酶生物传感器及其试剂的制备方法

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Publication number Publication date
AU3574295A (en) 1996-05-06
GB9421468D0 (en) 1994-12-07

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