WO2000059945A1 - Ameliorations apportees a des dosages immunologiques d'anesthesiques - Google Patents

Ameliorations apportees a des dosages immunologiques d'anesthesiques

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Publication number
WO2000059945A1
WO2000059945A1 PCT/NZ2000/000047 NZ0000047W WO0059945A1 WO 2000059945 A1 WO2000059945 A1 WO 2000059945A1 NZ 0000047 W NZ0000047 W NZ 0000047W WO 0059945 A1 WO0059945 A1 WO 0059945A1
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WO
WIPO (PCT)
Prior art keywords
anaesthetic
antibody
antigen
propofol
anaesthetics
Prior art date
Application number
PCT/NZ2000/000047
Other languages
English (en)
Inventor
Christian John Cook
Steven Ronald Payne
Original Assignee
The Horticulture And Food Research Institute Of New Zealand 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 The Horticulture And Food Research Institute Of New Zealand Limited filed Critical The Horticulture And Food Research Institute Of New Zealand Limited
Priority to AU38468/00A priority Critical patent/AU3846800A/en
Publication of WO2000059945A1 publication Critical patent/WO2000059945A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Definitions

  • the present invention relates to novel antigenic derivatives of phenol and steroidal based anaesthetics.
  • the derivatives may be used to stimulate antibody production to the anaesthetics.
  • anaesthetic immunoassays methods for monitoring anaesthesia in a patient, and methods for determining the presence or amount of anaesthetic in a sample.
  • Biosensors and kits useful in the immunoassays and methods of the invention are also provided.
  • Anaesthesia and particularly general anaesthesia, is a high risk form of treatment for a patient.
  • the rate of metabolism of an anaesthetic in individuals varies widely, as does the level of effectiveness.
  • Patient safety requires that they be continuously observed for signs of distress and levels of consciousness. It is also desirable, for a given individual patient, to be able to establish the level of anaesthetic effectiveness, the rate at which this is achieved and the anaesthetic dosage level required to maintain an appropriate level of unconsciousness.
  • Patient care can therefore be optimised by minimising side effects and recovery time, and maximising anaesthetic effectiveness.
  • Patient monitoring techniques generally comprise physical monitoring of indicators such as heart rate, blood pressure and eye flicker. EEG monitoring is also feasible.
  • Anaesthesia monitoring generally relies on measurement of expiration gases, or more recently high performance liquid chromatography (HPLC) or ELISA for analytes in biological fluid samples.
  • HPLC high performance liquid chromatography
  • ELISA ELISA for analytes in biological fluid samples.
  • the anaesthetics described therein were generally formulated for administration other than intravenously, with assays being carried out on samples removed from the patient.
  • the anaesthetic derivatives therein comprised anilide functionality drugs, linked via an annular amino substituent to antigens to produce an antigenic conjugate.
  • the antigenic conjugate was used for the production of antibodies to the subject drug, and for use in immunoassays.
  • anaesthetics are formulated for intravenous or intramuscular administration and comprise phenol derivative anaesthetics such as propofol, and steroidal based anaesthetics such as epiallopregnanolone.
  • Propofol commonly known as Diprivan is a fast acting anaesthetic commonly intravenously administered in medical anaesthesia.
  • Epiallopregnanolone is a steroidal based progesterone analog intravenous anaesthetic. The existence of both preferred anaesthetics has been known for tens of years.
  • an antigenic compound comprising an antigenic carrier having linked thereto an anaesthetic compound, selected from steroidal based anaesthetics and phenol based anaesthetics.
  • the present invention provides an antibody capable of binding an antigenic compound of the invention.
  • the antibody is produced by immunizing an animal with an antigenic compound of the invention.
  • the invention provides an antibody capable of binding to an anaesthetic compound, selected from steroidal based anaesthetics and phenol based anaesthetics.
  • the present invention provides a method for determining the presence or concentration of a phenol or steroidal based anaesthetic in a sample, which method comprises the steps of:
  • Preferred sample materials include biological fluids such as blood, plasma and serum.
  • the present invention provides a kit comprising antibody of the invention.
  • the antibody used in the method and kit is detectably labelled.
  • the phenol or steroidal based anaesthetic is detectably labelled.
  • the present invention provides a detector means comprising:
  • a response portion comprising antibody of the invention which is responsive either directly or indirectly to an anaesthetic of interest; and (b) a sensor portion able to respond to a change in characteristic of and/or at, the response portion.
  • the response portion comprises an electrode coated with an antibody of the invention.
  • the present invention provides a method of monitoring anaesthesia in a patient, the method comprising measuring the concentration of an anaesthetic of interest in a patient sample using detection means of the invention.
  • Figure 1 is a graph illustrating relative antisera binding of propofol complex in ELISA.
  • Figure 2 is a graph of the effect of blood concentration at 0 ⁇ g/ml diprivan on relative binding in ELISA. Optical density at 450 nm is plotted against percentage of sheep blood. The error bars indicate the SEM.
  • Figure 3 is a graph showing the results of ELISA using various concentrations of diprivan in spiked 10% sheep blood. The graph shows optical density at
  • Figure 4 is a graph showing an example of ELISA assays of human blood collected from a human patient during anaesthesia (competitive format).
  • Figure 5a is a diagrammatic view of a probe useful in the present invention.
  • Figure 5b is an enlarged view of the tip of the probe electrode shown shaded in Figure 5a.
  • Figure 6 is a graph illustrating the detection of the anaesthetic epiallopregnanolone in physiological saline. DESCRIPTIO ⁇ OF THE INVENTION
  • the present invention provides an antigenic compound comprising an antigenic carrier having linked thereto an anaesthetic compound, selected from steroid based anaesthetics and phenol based anaesthetics.
  • Antigenic carriers are well known in the art. A broad selection of compounds of sufficient size and antigenicity may be employed. These include for example, proteins, polypeptides, carbohydrates, polysaccharides, lipopolysaccharides and nucleic acids. Proteins are preferred for use. It is recognised that a protein from one animal species, when introduced into another species, will be antigenic. Proteins of molecular weight between 5,000 and 10 million, preferably between 10,000 and 300,000, and more preferably between 25,000 and 250,000 may be used. Examples of these proteins include enzymes, albumins, globulins, hemocyanins, and the like. A preferred protein for use herein is bovine serum albumin (cBSA, Pierce Chemical Company, Illinois, USA).
  • cBSA bovine serum albumin
  • the anaesthetic compound is selected from the group of phenol and steroidal based anaesthetics.
  • An illustrative phenol based anaesthetic is propofol (2,6 diisopropylphenol, ICI 35,868)
  • Steroidal based anaesthetics include alphaxalone, ethanolone, saftan, predrisolone and epiallopregnanolone, but are not limited thereto.
  • An illustrative steroidal based anaesthetic is epiallopregnanolone (3 ⁇ -Hydroxy-5 ⁇ - pregnan-20-one):
  • the linking of the antigenic carrier to the anaesthetic compound may be accomplished using art established techniques. Techniques include direct bonding via single or double bonds or bonding via the plethora of linker groups known in the art. Examples of linker groups and linkage reactions are provided in Bioconjugate Techniques, ed. G.T. Hermanson (1996), Academic Press; and A Laboratory Manual (1988), E. Harlow and D. Lane, Cold Spring Harbour Laboratory, both incorporated herein by reference. All references cited hereafter are also incorporated by reference. Groups containing reactive amino, sulfhydryl, carboxyl or hydroxyl are preferred.
  • the linker group is an amine group of the carrier protein which may be linked to the anaesthetic via the Mannich reaction.
  • This reaction is detailed in Bioconjugate Techniques (above). Linking may be effected to any position on the anaesthetic compound which does not interfere with its functionality.
  • the linking will preferably be to one of the native methyl substituents on the phenyl ring, or to one of the unsubstituted positions on the propofol phenol group.
  • the linking will preferably be to methyl or hydroxyl substituents.
  • the Pierce Pharmalink immunogen kit is used to carry out the Mannich reaction link.
  • the protein conjugation may be achieved either by direct Mannich Reaction of the propofol with proteins, or by Nucleophilic Substitution of the propofol active bromide derivative with proteins (Scheme 1).
  • the present invention provides antibodies or antibody binding fragments capable of binding a novel antigenic compound of the invention.
  • Such antibodies may be polyclonal but are preferably monoclonal.
  • Monoclonal antibodies may also be produced by known art methods. These include the immunological method described by Kohler and Milstein in Nature 256: 495-497, 1975 as well as the recombination DNA method described by Huse et al.: Science 246: 1275- 1281, 1989. The use of recombinant phage antibody systems to produce single chain variable antibody fragments, and subsequent mutation (such as site specific mutagenesis) or chain shifting to produce antibodies to the anaesthetics is also contemplated.
  • the host described above may be sacrificed and its spleen removed.
  • the messenger RNA (mRNA) are then isolated and cDNA made from the mRNA using specific primers for the heavy and light chains of the variable region of the antibodies and the polymerase chain reaction (PCR) amplification.
  • the DNA sequences for the heavy and light chains are joined with a linker sequence, to ensure the correct reading frame.
  • the DNA construct will be inserted into a vector, for example, a plasmid or bacteriophage, or virus, for transformation into a host.
  • a preferred vector is a bacteriophage.
  • Suitable hosts may be selected from prokaryotic, yeast, insect or mammalian cells.
  • a prokaryotic host and most preferably Escherichia coli is used.
  • the bacteriophage produces a viral coat and the antibody fragments are expressed on the coat, a phage display library.
  • the phage display library can be screened for antibody fragments with the appropriate affinity for the specific antigens.
  • the library can be screened many times and modifications can be made to the antibody construct through protein engineering techniques, such as site directed mutagenesis and chain shuffling all of which are within the capabilities of the art skilled worker.
  • the antibodies of the invention are particularly useful in immunoassays for determining the presence and/or amount of an anaesthetic in a sample.
  • Sample materials include cells, cell membranes and biological fluids but are not limited thereto.
  • a biological fluid selected from blood, plasma or serum will be tested in vitro or in vivo.
  • the antibodies of the invention are desirably labelled.
  • Detectable labelling can be achieved using well known techniques such as use of radioisotopes, affinity labels (such as biotin, avidin and the like), or enzymatic labels (such as horse radish peroxidase, rhodamine and the like). Labelling techniques are usefully summarised in, for example, Bioconjugate Techniques, and A Laboratory Manual, referenced above.
  • the labelled antibodies can be used in vitro and in vivo to detect the presence of, or to determine the concentration of the anaesthetic of interest in a sample. If desired, the antibody may be immobilised on a solid support to facilitate media testing. Techniques for doing so are usefully described in Weir et al.: Handbook of Experimental Immunology, 4th Edition, Blackwell Scientific Publications 1986.
  • the label may be provided on the anaesthetic.
  • the techniques for labelling given above may be used to produce detectably labelled anaesthetic. Accordingly, in a further aspect, the present invention provides a method for determining the presence or concentration of an anaesthetic in a sample which method comprises the steps of:
  • Detection or determination methods include a broad range of competitive and non- competitive techniques known in the art such as agglutination, radio immunoassay, surface plasmon resonance, colourimetrical, pizoelectrical, electrochemical, acoustic, or biomolecular interaction analysis, fluorescence, or enzyme immunoassay techniques.
  • Ligands may also be required for use in these methods.
  • Ligands contemplated for use herein are molecules which bind to the antibody of the invention to inhibit or prevent it binding anaesthetic (an antagonist ligand). The production of such ligands is also well known in the art.
  • Ligands may also be detectably labelled according to the methods referenced above for use in immunoassays.
  • test kits for the qualitative or quantitative determination of anaesthetics in a sample.
  • the test kit will usually contain an antibody of the invention.
  • the antibody is conveniently disposed in a container. It may be present in solid or liquid form, and may reside in or be coated on the container.
  • kits may contain reagents capable of detecting bound anaesthetic, buffers, diluents, washing solutions, reaction containers or plates, reference standards and the like which are commonly employed in such test kits.
  • An ELISA assay test kit including an antibody of the invention is an example of such a kit.
  • the detection of the anaesthetic, or determination of concentration is carried out using detector means such as a probe.
  • a probe assembly according to the present invention will typically comprise two main portions - a response portion and a sensor portion.
  • the response and sensor portion may be integrated in the form of an electrode.
  • Many suitable sensing electrodes known in the art may be employed in the present probe assembly.
  • One useful type of electrode is an amperometric electrode.
  • the electrode may be made of appropriate materials including gold, platinum, teflon, stainless steel, carbon or alloys.
  • a preferred amperometric electrode is platinum.
  • Many amperometric electrodes are known in the art. For example, in Journal of ⁇ eurochemistry (1995) 64, 1884-1887 and Journal of ⁇ euroscience Methods (1997), 72:161-166.
  • An alternate preferred electrode is a glass insulated carbon fibre coated with porphyrin/nafionTM (Nature (1992), 358:676-678).
  • the response and sensor portions may be integrated into an immunosensor electrode.
  • immunosensor electrodes are also known in the art, for example in Biosensors, Fundamentals and Applications, Oxford Press (1987), pp 57-65; Biosensors and Bioelectronics (1987), 11:179-185; Nature Biotechnology (1997), 15:467-471.
  • Materials appropriate for amperometric electrodes may also be employed for an immunosensor electrode. Again, a preferred material is platinum.
  • the response portion whether alone or integrated will typically be an area or volume of antibodies which are capable of binding an anaesthetic of interest in the sample.
  • the antibody binds or holds the anaesthetic of interest at least temporarily. This localises the anaesthetic so quantitative determinations may be made (see later).
  • a secondary, and/or competitive, ligand may be introduced which compete for the antibody.
  • the antibody may be initially bound by secondary components which relinquish the sites to the anaesthetic. This may be dependent upon its relative concentration and/or be accumulative.
  • the antibodies will be provided on or within a suitable support. This may be a surface coating or bound layer on a two or three dimensional substrate - typically three dimensional substrates will be used to increase surface area in a particular volume. Multiple plates are another option. Dispersing the antibodies in a matrix permeable to the media is another option. In some cases this matrix may act as a filter. Such filtered probe embodiments are discussed below.
  • the antibodies may be bound to a support, including an electrode, using well known techniques.
  • One suitable absorption technique comprises cathodic copolymerisation, or the use of appropriate cross-binding agents or novel binders such as fungal origin hydrophobins.
  • an introduced standard, or ligand normally comprising a labelled substance to which the antibodies are also responsive, will be introduced to the media.
  • the introduced substance will, in these embodiments, compete with the anaesthetic occurring within the sample for receptor sites.
  • a secondary component occupying the antibody which must be displaced by the anaesthetic being determined. This may or may not be a competitive or equilibrium response.
  • a sensor portion interacting with the receptor portion may change in proportion to the sensed change, or perhaps only when a particular level of change in or at the antibody has occurred.
  • Quantitative embodiments may rely on competitive systems so that the relative proportions of anaesthetic to be determined can be gauged. This may rely on a secondary or competitive ligand in known quantities. This may be pre-bound to the antibody or otherwise introduced into the sample.
  • a sensor portion able to directly, or indirectly, determine or collect information for evaluation by either other equipment or systems of the amount or proportion of labelled standard. This may be done by evaluating a physical characteristic of the label e.g. the activity of a radioactive label may be measured locally in the region of the response portion.
  • Other possibilities are changes in colour, oxidation state, oxidation or electrochemical potentials, electrical potential, magnetism, fluorescence, affinity to a tertiary introduced component, or some other measurable quality.
  • change in electrochemical or electrical potential, particularly of redox reactive molecules and labels may be measured amperiometrically or voltametrically using known amperometers, voltmeters, or potentiostats.
  • various methods of determining the amount of labelled substance in the locality of the response portion may be adopted, and will largely depend upon the nature of the label.
  • the light emission of fluorescent dye labels may be determined, though some excitation means (e.g. light, electromagnetic radiation, or electrical stimulation) may be required and provided with the probe, or externally thereto.
  • Optical technology can be used to determine the colour (or wavelength emission) of both fluorescent, and non-fluorescent, dyes and labels which undergo changes when binding to a receptor.
  • the electromagnetic state of a receptor or label may also be monitored as a measure of amount of sample present.
  • a body may be present to house and/or support the components of the probe assembly.
  • Suitable body materials include for example plastics, stainless steel, teflon and glass. Generally, the body will be 5-100 mm in length, preferably 7-70 mm, and most preferably 10-50 mm.
  • the indicator probe may also incorporate one or more sample filters.
  • Sample filters comprise any known art filters for selectively filtering unwanted components from the response and sensor portions. Separation may be achieved based on size, molecular weight, change or other characteristic.
  • dialysis membranes are employed. Many such membranes are known including cuporphan (GFE9, Gambro Ltd) or A ⁇ 69 (Hospal Ltd) or Spectra/Por molecularporus membrane (heat sealed at one end). The membrane is passed over the body of the probe and the tip of the fibre membrane between the membrane and body appropriately sealed.
  • Preferably used is epoxy resin, although heat or glue may be used.
  • the outer surface of the dialysis membrane is electropolymerised to reduce electrointerference. Electropolymerisation may be suitably achieved using phenylenedramine or equivalents.
  • amperometric probes and/or immunosensor probes make use of amperometric probes and/or immunosensor probes.
  • the enzyme linked amperometric probe can offer real-time data together with temporal resolution, but is dependent upon the specificity of enzyme perfused through it.
  • These probes also suffer from complications in terms of the oxi dative species generated within the probe, and the effects of changing solutions of perfusing enzymes. This may well produce changes that complicate interpretation of stimulus related effects in in vivo testing.
  • Microdialysis probes have an advantage in that they can allow measurement of a large number of substances of interest simultaneously dependent on the size setting of the microdialysis membrane.
  • sophisticated analysis is needed and neither real-time nor low temporal range digrammanation are generally offered.
  • microdialysis has been coupled to amperometric enzyme detectors (J. Neuroscience Methods (1995), 60:1-9). But this method still does not overcome the sample time problem of microdialysis. There therefore remains a need for tools for real-time monitoring or determination of substances of interest in vivo.
  • the present inventors uniquely suggested an immunosensor combining an immunoassay, an electrochemical sensor and an microdialysis membrane to create a probe implantable in living tissue to monitor on-line anaesthetics of interest in vivo, as well as in vitro.
  • Figure 2 depicts an amperometric probe (10) with an electrode (12), preferably platinum, incorporating a response portion (14).
  • Reference electrode (16) is incorporated according to standard design techniques.
  • Reference electrode materials include silver, gold, platinum or stainless steel.
  • Preferred electrodes are an Ag, Ag/AgCl combination.
  • the electrodes are connected to known external points, in this case gold pins.
  • the probe assembly is fitted within a body or housing (11) to form an indicator probe.
  • the probe is provided with inlet (18) and outlet (20) tubes formed of conventional materials such as silica (outer diameter: 0.1 to 3 mm, preferably 0.15 to 2 mm, most preferably 0.19 to 1 mm; length up to 40 mm, preferably up to 30 mm) or polyethylene tubing (outer diameter: 0.3 to 30 mm, preferably 0.5 to 20 mm, most preferably 0.61 to 10 mm; length 30 to 120 mm, preferably 50 to 100 mm).
  • silica outer diameter: 0.1 to 3 mm, preferably 0.15 to 2 mm, most preferably 0.19 to 1 mm; length up to 40 mm, preferably up to 30 mm
  • polyethylene tubing outer diameter: 0.3 to 30 mm, preferably 0.5 to 20 mm, most preferably 0.61 to 10 mm; length 30 to 120 mm, preferably 50 to 100 mm.
  • Known alternatives may of course be used.
  • the stainless steel body is separated into two chambers (inlet and outlet) by a single divider (22).
  • a single divider (22).
  • Suitable divider materials include silica glass, teflon, or polyethylene epoxy amongst others.
  • the divider may be fixed in place using known techniques of heat fusion, gluing, and the like.
  • the divider is epoxy resined into place.
  • the indicator probe may also incorporate one or more sample filters (24) as discussed above.
  • the response portion may be monitored continuously, or at intervals, to provide data for subsequent evaluation.
  • a fibre optic (25) measure is depicted. This is particularly useful for use in vivo, while available miniaturisation techniques can allow the insertion and use of many probes in living biological systems.
  • the output of the sensor portion may even be coupled to a transmitter, allowing remote monitoring or checking to be performed.
  • the present invention provides a method of monitoring anaesthesia in a patient, the method comprising measuring the concentration of an anaesthetic of interest in a patient sample using detection means of the invention.
  • patients include both human and non-human patients.
  • detector means of the present invention include the use of microprobes in vivo, in situ, and in living biological systems either by internal, or surface, implantation. This can negate the need of removing sample from the biological system for measurement purposes and can allow for real-time, and continuous (over extended periods of time) measurement of anaesthesia within a patient.
  • Immunoconjugates of propofol and bovine serum albumin may be synthesised as follows:
  • This conjugation uses the Mannich reaction, which uses the active hydrogens within a compound to be condensed with formaldehyde and an amine on the BSA.
  • the conjugation was conducted using the Pierce Imject Pharmalink Immunogen kit (Pierce Chemical Company, Rockford, Illinois, USA). Briefly, 20 ⁇ l propofol was dissolved in 80 ⁇ l of dimethylsulphoxide (DMSO). 20 ⁇ l of this propofol/DMSO mix was added to 180 ⁇ l conjugation buffer. 200 ⁇ l of propofol/conjugation buffer solution was combined with 2 mg cBSA (SuperCarrier® protein) in 200 ⁇ l 0.1M MES buffer.
  • DMSO dimethylsulphoxide
  • PharmaLink Conjugation Buffer 0.1 M MES, 0.15 M NaCl, pH 4.7.
  • PharmaLink Coupling Reagent 37% formaldehyde reagent.
  • Immunoconjugates of epiallopregnanolone and bovine serum albumin may be synthesised as follows:
  • the immunisation of female BALB/c mice may be carried out according to the following protocol:
  • the antigen is an antigenic compound of the invention, and the injection is intraperitoneal.
  • Two booster injections are given at two weekly intervals.
  • the booster injections comprise 50-100 ⁇ g of immunogen (100-200 ⁇ l of antigen/adjuvant mix) per mouse with a 50:50 mix of adjuvant to antigen complex.
  • the mice are either exsanguated, their blood collected, and polyclonal antibodies isolated from serum; or the mice are killed, and their spleens removed. Spleen cells are cultured and antibodies produced are isolated according to conventional protocols.
  • Propofol was attached to either cBSA or KLH using the Pierce PharmaLinkTM immunogen kit.
  • the antigen complexes were passed through a desalting column to remove unreacted formaldehyde and the protein concentration was estimated at 280 nm.
  • the antigen complexes were mixed 1:1 with an aluminium hydroxide adjuvant and injected subcutaneously (multiple sites) into New Zealand white rabbits. Each rabbit was given either 0.4 mg cBSA complex or 2.2 mg KLH complex per set of injections. Rabbits were given booster injections at 14 d and 28 d. The rabbits were exsanguinated at 10 d following the second booster and the serum collected.
  • the immune serum from each rabbit was compared to pre-immune serum collected prior to the first injection, using ELISA based on the method described below. Serum samples were incubated in the immunoplates overnight. Also, competitive assays were performed using a range of propofol concentrations added prior to the addition of the serum samples to the immunoplates. An ELISA was developed based on a competitive assay using HRP- labelled propofol.
  • Heparinised sheep blood was collected from an abattoir and spiked with Diprivan to the appropriate concentration.
  • the spiked blood was diluted with either blood and/or PBS/T to give the appropriate blood concentrations.
  • the immunoplates were washed 3 times with 10 mM PBS/T, except after incubation with Tetramethylbenzidine (TMB)
  • Figure 3 shows the relative binding against diprivan-spiked sheep blood (at 10%> dilution) using the final anti-rabbit ELISA format.
  • concentration groupings 0 ⁇ g/ml > 0.2 ⁇ g/ml > 0.4 to 0.6 ⁇ g/ml > 0.8 to 1.2 ⁇ g/ml > 1.4 to 2.0 > ⁇ g/ml - corresponding to 0 ⁇ g/ml, 4 to 6 ⁇ g/ml, 8 to 12 ⁇ g/ml and 14 to 20 ⁇ g/ml in whole blood respectively.
  • Table 1 Example of HLPC comparison with ELISA for Human perfusion diprivan sampleess
  • heparin and EDTA tubes were plastic and the citrate tube was glass. Propofol has been shown to bind plastic and rubber.
  • Fig.5 offers a schematic representation of the probe components as detailed in the present invention.
  • These include an inlet tube (18) that allows introduction of anaesthetic into the probe which can be monitored in numerous forms, including but not exclusively by flow rates by on-line monitoring, a central body (11) of the probe (10) is included, constructed of known materials such as steels, alloys, plastics, glass in a concentric manner and including (24) a selective membrane design that separates the analysis actions within the probe from the sample and/or substrate.
  • the sensor components (12, 16, 25) Within the central body of the probe lies the sensor components (12, 16, 25) surrounded by, or in contact with, or directed towards specific antibodies (14) for the substrates to be measured.
  • the internal probe is separated by divider (22) into two chambers until a short distance prior to the actual separation membrane.
  • the probe also consists of an outlet (20) with monitoring opportunities as described for the inlet. This outlet also offers the opportunity for actual sample collection should it be desirable.
  • the sensor arrangement within the probe (12,16,25) can be connected to amplifying, displaying and quantifying devices including the provision for logging of data or radio-electric transmission to a receiver some distance away.
  • One probe of the invention depicted in Figures 5a and 5b comprises a response portion (26) comprising an area of receptors. These comprise antibody of the invention specific to the anaesthetic of interest (30), bound to a supporting substrate (32).
  • the components are housed in a body (11) allowing fluid from the sample to access the response portion (26).
  • the response portion (26) may be housed in the head of the body (11), while the bulk of equipment associated with evaluating the labeled standard can be positioned other than in the head to reduce its size.
  • the receptor may comprise antibody arranged around the base area of the probe in a number of formats. These may include conjugation onto measuring electrode (12) Fig. 5a which may be constructed of platinum, gold, stainless steel, carbon, alloys or optic fibres. Conjugation involves compounding a XH-CHO structure reacted with an ester group to introduce a formyl group which is used to bind an amino group of the antibody. This process has been documented, and is therefore known to the skilled worker. Other methods of attaching the antibodies are not excluded.
  • the antibody system may also comprise an imprinted polymer design, the preparation of which is known to skilled workers (Nature Biotechnology, 15, 354-357, 1997). The imprinted polymers are specific to the capture of anaesthetics of interest, through the imprinting process.
  • a fibre optic (25) delivers exciting electromagnetic radiation from a light source and also delivers emitted fluoresced light from the label of introduced standard at the surface of the response portion (26) to suitable electronic circuitry.
  • an anaesthetic of interest (30) in the sample may selectively travel across a membrane (34) into the measurement part of the probe. Once there (30) may bind to an antibody of the invention (28) fixed within the probe.
  • An introduced ligand (36) competitively binds to the same set of receptors (28). This introduced ligand (36) is then activated to produce energy proportional to the number of ligands (36) bound. This energy is monitored, and measured to give a relative measure of (36) bound and therefore (30) bound.
  • This relative measure is calibrated from the performance of the probe using standards of (36) and (30) in an in vitro calibration or in vivo internal standard test.
  • the probe will be calibrated, typically in a sample of pure labeled standard to obtain a 100% reading.
  • Known standards comprising known mixture of both labeled and non-labeled competitively binding substances may be used for calibration, or to obtain various data points for subsequent comparison and analysis. Calibration will normally occur in vitro, before and after use although in vivo calibration using internal standards is also possible.
  • the probe after washing, will be placed in the sample and allowed to equilibrate. A standard of labeled substance is introduced to the sample or system being monitored, allowed to distribute and competitively bind at the receptor sites. After equilibration, meaningful data from the sensor portion may be collected and analysed.
  • Probes were constructed containing antibodies to a steroidal based anaesthetic epiallopregnanolone. These were then used to measure standardised concentrations of this anaesthetic in a physiological saline solution. As can be seen in Figure 6, good prediction of concentration was obtained using probe measurement.
  • the active propofol bromide hapten (III) (18 mg, 66 ⁇ mol) was dissolved in DMF (0.2 ml). Tri-Butylamine (0.25 ml) and a trace amount of 4-(dimethylamino) pyridine were added to the above DMF solution at room temperature while stirring. Then, protein (10 mg, 0.22 ⁇ mol of OVA, or 5 mg, 0.011 ⁇ mol of KLH) dissolved in 2 ml of PBS buffer (0.2 M, pH 7.5) was added respectively and stirred at 4-6 °C (cold room) overnight. These two synthesised protein conjugates were exhaustively dialysed against phosphate buffer (0.2 M, pH 7.5) at 4-6 °C in cold room for 3 days.
  • both KLH and OVA conjugates with propofol appeared in a milky form. Then, both protein conjugates were taken to a centrifuge (14,000 RPM over 20 minutes) to remove light yellow solid, and gave each 2.3 ml of clear, colourless protein solutions.

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Abstract

L'invention concerne de nouveaux dérivés antigènes de phénol, des anesthésiques stéroïdiens et des anticorps dirigés contre ces anesthésiques. L'invention concerne également des dosages immunologiques d'anesthésiques, des méthodes destinées à surveiller l'anesthésie d'un patient, ainsi que des méthodes permettant de déterminer la présence ou la quantité d'anesthésique dans un échantillon. L'invention concerne, en outre, des biocapteurs et des kits utilisés dans le cadre des dosages immunologiques et des méthodes selon l'invention. Le propofol et l'épiallopregnanolone sont les antibiotiques préférés destinés à être utilisés dans le cadre de la présente invention.
PCT/NZ2000/000047 1999-04-06 2000-04-06 Ameliorations apportees a des dosages immunologiques d'anesthesiques WO2000059945A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU38468/00A AU3846800A (en) 1999-04-06 2000-04-06 Improvements in or relating to immunoassays for anaesthetics

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NZ33312299 1999-04-06
NZ333122 1999-04-06

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WO2000059945A1 true WO2000059945A1 (fr) 2000-10-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008453A1 (fr) * 2001-07-16 2003-01-30 Valtion Teknillinen Tutkimuskeskus Procede d'immobilisation de polypeptides
US9687496B2 (en) 2010-01-14 2017-06-27 Asarina Pharma Ab Pharmaceutical composition comprising 3-beta-hydroxy-5-alpha-pregnan-20-one with improved storage and solubility properties
CN108037283A (zh) * 2017-12-11 2018-05-15 广东海大畜牧兽医研究院有限公司 一种用于酶联免疫检测的抗体稀释液及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J.-L. GUEANT ET AL.: "Diagnosis and pathogenesis of the anaphylactic and anaphylactoid reactions to anaesthetics", CLINICAL AND EXPERIMENTAL ALLERGY, vol. 28, no. 4, 1998, pages 65 - 70 *
M.-C. LAXENAIRE ET AL.: "Life-threatening anaphylactoid reactions to propofol", ANESTHESIOLOGY, vol. 77, 1992, pages 275 - 280 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008453A1 (fr) * 2001-07-16 2003-01-30 Valtion Teknillinen Tutkimuskeskus Procede d'immobilisation de polypeptides
US7078192B2 (en) 2001-07-16 2006-07-18 Valtion Teknillinen Tutkimuskeskus Method for immobilization of polypeptides
US9687496B2 (en) 2010-01-14 2017-06-27 Asarina Pharma Ab Pharmaceutical composition comprising 3-beta-hydroxy-5-alpha-pregnan-20-one with improved storage and solubility properties
US11534446B2 (en) 2010-01-14 2022-12-27 Asarina Pharma Ab Pharmaceutical composition comprising 3-beta-hydroxy-5-alpha-pregnan-20-one with improved storage and solubility properties
CN108037283A (zh) * 2017-12-11 2018-05-15 广东海大畜牧兽医研究院有限公司 一种用于酶联免疫检测的抗体稀释液及其制备方法和应用
CN108037283B (zh) * 2017-12-11 2019-06-18 广东海大畜牧兽医研究院有限公司 一种用于酶联免疫检测的抗体稀释液及其制备方法和应用

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