WO2000059945A1 - Improvements in or relating to immunoassays for anaesthetics - Google Patents

Abstract

The invention relates to novel antigenic derivatives of phenol and steroidal based anaesthetics and antibodies to the anaesthetics. Also provided are 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. Propofol and epiallopregnanolone are preferred antibiotics for use in the invention.

Description

IMPROVEMENTS IN OR RELATING TO IMMUNOASSAYS FOR

ANAESTHETICS

FIELD OF THE INVENTION

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. Also provided are 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.

BACKGROUND

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.

For optimised patient care, a rapid and specific analytical method is needed for measuring concentrations of anaesthetics in biological fluids, as distinct from closely related compounds. This need has led to the development of a variety of procedures for monitoring both levels of consciousness in patients and levels of anaesthetic in blood or plasma.

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. Some twenty years ago US Patent No. 4,069,105 disclosed enzyme immunoassays for measuring levels of anaesthetics involving anilides, lidocaine being illustrative. 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. In turn, the antigenic conjugate was used for the production of antibodies to the subject drug, and for use in immunoassays.

In US 4,650,771 in 1983, the art was further developed by providing anilide derivative anaesthetics conjugated to antigens via one of the aromatic methyl substituents. Immunoassays on fluid samples were again proposed.

In the 15 to 20 years which followed the publication of these US patents, there have been many developments in the fields of anaesthesia. Currently preferred 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.

While these anaesthetics can be measured using HPLC methods, there are currently no methods to measure the concentration of these intravenous/intramuscular anaesthetics in patients in real-time, on-line or with easy convenience in a clinical setting. Moreover, no known antibody detection methods to these types of anaesthetics have been described. As the mechanism of action of these anaesthetics are also poorly understood, the development of antibodies to these anaesthetics to enable rapid and clinically relevant detection methods using kits or off-line or in real-time biosensors would therefore fulfill a long felt want.

It is an object of this invention to provide antigenic compounds and antibodies for use in anaesthetic detection, and biosensors which go some way to addressing the foregoing or at least provide the public with a useful choice. SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided an antigenic compound comprising an antigenic carrier having linked thereto an anaesthetic compound, selected from steroidal based anaesthetics and phenol based anaesthetics.

In a further aspect, the present invention provides an antibody capable of binding an antigenic compound of the invention.

Conveniently, the antibody is produced by immunizing an animal with an antigenic compound of the invention.

In a further aspect, the invention provides an antibody capable of binding to an anaesthetic compound, selected from steroidal based anaesthetics and phenol based anaesthetics.

In a further aspect, 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:

(a) contacting the sample with an antibody of the invention; and

(b) detecting if, or determining the amount of, anaesthetic that becomes bound.

Preferred sample materials include biological fluids such as blood, plasma and serum.

In a still further aspect, the present invention provides a kit comprising antibody of the invention.

Preferably, the antibody used in the method and kit is detectably labelled.

In an alternative embodiment, the phenol or steroidal based anaesthetic is detectably labelled.

In a still further aspect, the present invention provides a detector means comprising:

(a) 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.

Preferably, the response portion comprises an electrode coated with an antibody of the invention.

In a still further aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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 μg/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

450 nm plotted against concentration of diprivan (μg/ml) in 10% sheep blood. The error bars indicate the SEM. The letters a-f show significant differences where the letters are different.

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. DESCRIPTIOΝ OF THE INVENTION

As discussed above, there has long been a need for rapid and clinically relevant methods for monitoring intravenously or intramuscularly administered anaesthetics and or monitoring or detecting steroid and phenol based anaesthetics in common use today. An antibody detection method would fill this need. It is recognised in the art that low molecular weight compounds are generally not antigenic. Anaesthetic compounds fall into this category. As a pre-step to producing an antibody to an anaesthetic it is therefore necessary to link it to an antigenic carrier.

Accordingly, in a first aspect, 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).

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)

commonly known as Diprivan or Disoprivan.

Steroidal based anaesthetics include alphaxalone, ethanolone, saftan, predrisolone and epiallopregnanolone, but are not limited thereto. An illustrative steroidal based anaesthetic is epiallopregnanolone (3α-Hydroxy-5α- pregnan-20-one):

Epiallopregnanolone

Derivatives or analogs of propofol and epiallopregnanolone which are functionally equivalent are also known and contemplated for use herein.

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.

Preferably, 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. In the case of propofol and its derivatives, 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.

In the case of epiallopregnanolone and its analogs and derivatives, the linking will preferably be to methyl or hydroxyl substituents.

Conveniently, 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).

Scheme 1. Synthesis of Propofol-Protein Conjugates

In a further aspect, 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.

Conventional procedures for generating polyclonal antibodies are detailed in Koelle et al.; Cell 67: 59-77, 1991 incorporated herein by reference. Briefly, the protocol requires immunisation of a selected animal host such as a rabbit, goat, rat or mouse (usually a mouse), with an antigenic compound of the invention on a number of spaced occasions, with one or more test bleeds preceding exsanguination and blood collection. Serum may be separated from clotted blood by centrifugation. Serum may be tested for the presence of polyclonal antibodies using ELISA competitive assays or art equivalent methods.

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.

For example, 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. Then 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. Preferably, 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. In terms of the present invention, usually a biological fluid selected from blood, plasma or serum will be tested in vitro or in vivo.

For use in the immunoassays, 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.

In an alternate embodiment, 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:

(a) contacting the sample with an antibody of the invention; and

(b) detecting if, or determining the amount of, anaesthetic that becomes bound.

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.

These techniques are usefully summarised in Handbook of Biosensors and Electronic Noses., Ed. E.K. Rogers CRC Press 1997, incorporated herein by reference.

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.

The test methods form the basis of kits for the qualitative or quantitative determination of anaesthetics in a sample. As a minimum, 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.

In addition to the antibodies, the 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.

In a preferred embodiment, 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. In one embodiment, 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 Νeurochemistry (1995) 64, 1884-1887 and Journal of Νeuroscience Methods (1997), 72:161-166.

An alternate preferred electrode is a glass insulated carbon fibre coated with porphyrin/nafion™ (Nature (1992), 358:676-678).

In a preferred embodiment, the response and sensor portions may be integrated into an immunosensor electrode. Such 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.

In most embodiments the antibody binds or holds the anaesthetic of interest at least temporarily. This localises the anaesthetic so quantitative determinations may be made (see later). In competitive systems a secondary, and/or competitive, ligand may be introduced which compete for the antibody. In non-competitive systems, 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.

Typically 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.

In most methods of use, 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. By determining the balance of "component to be determined" vs. "competitive component" occupying antibody, detective and/or quantitative determinations may be made. The balance is generally arrived at by ascertaining a value for the antibody occupied by labelled standard.

This may be compared with a value for the probe subjected purely to a standard of introduced substance, i.e. all antibody occupied by labelled standard according to standard probe principles.

In non-quantitative systems, there may be present 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.

For instance, to determine the amount of labelled standard occupying the antibody, use can be made of 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. Conveniently, 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.

As can be appreciated, 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. For instance, 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. Conveniently, dialysis membranes are employed. Many such membranes are known including cuporphan (GFE9, Gambro Ltd) or AΝ69 (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. Preferably, the outer surface of the dialysis membrane is electropolymerised to reduce electrointerference. Electropolymerisation may be suitably achieved using phenylenedramine or equivalents.

The reader will appreciate that the proposed embodiments make use of amperometric probes and/or immunosensor probes. As discussed above, 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. However, sophisticated analysis is needed and neither real-time nor low temporal range digrammanation are generally offered. In a recent development 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.

To permit on-line monitoring, 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). Known alternatives may of course be used.

The stainless steel body is separated into two chambers (inlet and outlet) by a single divider (22). The advantage of using such a divider are that dividers serve to separate inlet and outlet perfusion flows. 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. Preferably, 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. In Figure 2 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 ability for real-time continuous monitoring of anaesthetic in a patient in vivo during surgery is therefore provided.

Accordingly, in a further aspect, 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.

The term "patients" referred to herein include both human and non-human patients.

Specific uses of the 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.

All references cited herein are specifically incorporated by reference.

Certain preferred aspects and applications of the present invention will now be described in relation to the following non-limiting examples.

EXAMPLE 1

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 μl propofol was dissolved in 80 μl of dimethylsulphoxide (DMSO). 20 μl of this propofol/DMSO mix was added to 180 μl conjugation buffer. 200 μl of propofol/conjugation buffer solution was combined with 2 mg cBSA (SuperCarrier® protein) in 200 μl 0.1M MES buffer. 50 μl of coupling reagent was added to the cBSA/propofol solution, and Incubate the reaction at 42 °C for 17 hours. Following incubation, the conjugation mix was passed through a desalting column to separate any unbound propofol from the antigen complex. Fractions were checked by measuring their absorbance at 280 nm and fractions containing the conjugate were pooled, ready for injection.

SuperCarrier® Immune Modulator 2 mg of cationized BSA, lyophilized from 200 μl of a 10 mg/ml solution containing 0.1 M MES [2-(N-Morpholino)-ethanesulfonic acid], 0.15 M NaCl including stabilisers, pH 4.7.

PharmaLink Conjugation Buffer, 0.1 M MES, 0.15 M NaCl, pH 4.7. PharmaLink Coupling Reagent, 37% formaldehyde reagent.

EXAMPLE 2

Immunoconjugates of epiallopregnanolone and bovine serum albumin may be synthesised as follows:

As per Example 1, using the Pierce PharmaLink Immunogen kit but dissolving 5 μg epiallopregnanolone in 100 μl methanol and adding this to 100 μl conjugation buffer before adding this to the reaction mixture and incubating.

EXAMPLE 3

The immunisation of female BALB/c mice may be carried out according to the following protocol:

In all cases, 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 μg of immunogen (100-200 μl of antigen/adjuvant mix) per mouse with a 50:50 mix of adjuvant to antigen complex. 4-5 days following the last injection, 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.

EXAMPLE 4

Materials Propofol was obtained from Research Biochemicals International, USA. Diprivan™ was obtained from ICI New Zealand. Keyhole limpet hemocyanin (KLH), bovine serum, albumin (cBSA), and the PharmaLink™ Immunogen and Immobilisation kits were obtained from Pierce Chemical Company, USA. Ovalbumin (OVA) and phosphate buffered saline + 0.05% Tween 20 pH 7.4 (PBS/T) were obtained from Sigma Chemical Company, Australia.

Propofol was attached to either cBSA or KLH using the Pierce PharmaLink™ immunogen kit. The antigen complexes were passed through a desalting column to remove unreacted formaldehyde and the protein concentration was estimated at 280 nm.

Immunisation Protocol

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.

Antibody ELISA Assays

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.

Final ELISA Format Used with Purified Antibody and Spiked Blood

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.

Between each step of the process, the immunoplates were washed 3 times with 10 mM PBS/T, except after incubation with Tetramethylbenzidine (TMB)

1. Coat immunoplates with 10 μg/ml ovalbumin-propofol complex (ONA-propofol) in 0.05M bicarbonate buffer pH 9.6 for at least 1 h at room temperature.

2. Block immunoplates with 1% OVA in PBS/T pH 7.4 for at least 1 h at room temperature or overnight at 4°C. 3. Add 50 μl diprivan sample then add 50 μl anti-propofol antibody solution (10 μg/ml) and incubate at room temperature for 30 mins.

4. Add 100 μl horseradish peroxidase labelled anti-rabbit IgG at 0.25 μg/ml for 30 mins at room temperature.

5. Add 100 μl TMB reaction solution and incubate at room temperature for 5 mins. 6. Stop reaction with 2M H₂S0₄ and read optical density at 450 nm on a plate reader.

Statistical Analysis

ELISA data were analysed by one way repeat measures AΝOVA using Jandel Sigmastat for Windows version 2.0 statistical package.

Results

The results from ELISA (Figure 1) and competitive assays (data not shown) indicate that antibodies have been produced against the propofol-protein complex, which can recognise free propofol.

Sheep blood reduced the amount of binding of anti-propofol antibodies. Binding increased with higher dilution of the blood at 0 μg/ml diprivan as shown by lower optical density (Figure 2).

Figure 3 shows the relative binding against diprivan-spiked sheep blood (at 10%> dilution) using the final anti-rabbit ELISA format. We were able to detect and measure the difference between several concentration groupings; 0 μg/ml > 0.2 μg/ml > 0.4 to 0.6 μg/ml > 0.8 to 1.2 μg/ml > 1.4 to 2.0 > μg/ml - corresponding to 0 μg/ml, 4 to 6 μg/ml, 8 to 12 μg/ml and 14 to 20 μg/ml in whole blood respectively.

The immune antisera results indicated that we were successful in producing polyclonal antibodies that recognised the protein-propofol complex. They could also bind free propofol in the commercial preparation Diprivan. We were able to purify antibodies from the crude Νa₂S0₄ precipitated antisera using an affinity column, which were used in later experiments.

The final format ELISA that we have developed gave consistent results but was affected by the concentration of blood. Dilution of the blood gave better results (Figure 2), indicating that blood components were non-specifically binding the anti-propofol antibodies, preventing some of these from binding to the OVA-propofol complex coating the immunoplates. These antibodies were removed from the wells during the washing step and were not available to be bound by the HRP-labelled anti-rabbit antibodies, therefore lowering the observed binding.

Using a 10- fold dilution of spiked sheep blood, we were able to detect and measure concentration differences that correspond to the typical clinical concentrations in whole blood. We were able to obtain results that could be used in categories; low (< 4 μg/ml), medium (4 to 12 μg/ml) and high (> 14 μg/ml) of propofol.

Human Blood Trials

Current trials were conducted with human blood collected before and after the administration of diprivan. Blood was collected before injection, at 2, 5 and 15 minutes post injection of diprivan. Samples are collected into blood tubes containing anticoagulant (heparin, EDTA, or citrate). The samples were analysed by ELISA and HPLC. We have detected false positives in the pre-injection samples, in both ELISA and HLPC, when the blood samples were collected in the heparin tubes, but the samples collected in citrate tubes have not shown this problem. Citrate and heparin are different anti- coagulants and the citrate tube are glass whereas the heparin tubes are plastic. The time/concentration profile shown in the patient samples have been consistent with those expected from HLPC experiment from other researchers (Figure 4 & Table 1). 15 patients have been assessed for the propofol profile during anaesthesia.

Table 1: Example of HLPC comparison with ELISA for Human perfusion diprivan sampleess

We tested 3 types of collection tubes, heparin, EDTA and citrate. The heparin and EDTA tubes were plastic and the citrate tube was glass. Propofol has been shown to bind plastic and rubber. We assessed the effect of position of the tube, upright or lying down. We collected spiked blood samples (10 μg/ml) from the tubes at 0, 3, 6 and 72 hours, extracted and analysed on the HLPC. We found that the heparin tubes were had the largest decrease in concentration of propofol over time, with the tube lying down being the highest drop by a considerable margin. We recommended to change the collection procedure to using citrate tubes and to store them upright.

We have tested various dilution factors on spiked human blood samples from 100%, 50%, 25% and 10%> whole blood, with the samples being diluted in phosphate buffered saline with tween 20. The lower dilutions (10% and 25% blood) seem to give more consistent results.

We have also tested the effects of filtering the blood through a 0.2μm syringe filter. Removal of red blood cells and large components from the blood sample reduces variability between replicate samples.

EXAMPLE 5

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. 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. EXAMPLE 6

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.

In Fig 5b it can be seen that in use 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.

According to one method of use, 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.

By using multiple labels to the single bound antibody it is possible to amplify the measurement potential of the probe to measure very small concentrations of anaesthetic.

EXAMPLE 7

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.

EXAMPLE 8

4-Hydroxymethyl-2, 6-diisopropylphenol (II) — To an ice-cold aqueous solution (0.4 ml) of NaOH (0.5 g) was added the solution of propofol (I) (0.46 g) in MeOH (3 ml). Formaldehyde solution (35%, 5 ml) was further added to the solution while stirring. The temperature was raised slowly to room temperature and the reaction mixture was kept for 3 hours. After cooling in an ice-cooled bath and careful neutralisation to pH 7.25 using cold, dilute hydrochloric acid (0.1 M), the aqueous solution was extracted with chloroform (3 x 20 ml). The organic phase was then washed with water and dried over sodium sulphate. After removing solvent the residues was purified by column chromatography (4% MeOH in CHC1₃) and it gave the pure product (160 mg, 30% yield, R^ = 0.48) and the propofol starting material (165 mg, R_F = 0.69).

4-Bromethyl-2, 6-diisopropylphenol (III) — Glacial acetic acid (3 ml) was added to a dry flask containing 4-hydroxymethyl-2, 6-diisopropylphenol (II) (0.16 g, 0.77 mmol) and stirred on an ice-cold bath. Hydrobromic acid (33 % in glacial acetic acid, 1 ml) was added to the above solution while stirring and the whole reaction mixture was kept stirring for another 0.5 hour at 4-6 °C. After addition of cold chloroform (30 ml) and ice-cold water (10 ml) the organic phase was immediately separated out by separation funnel and dried over anhydrous calcium sulphate. After thin-layer chromatography (TLC) check (4% MeOH in CHC1₃, single spot at TLC plat R_F = 0.74 for active propofol bromide) and filtration, the organic solvent was removed by rotary evaporation and high vacuum system at room temperature. The pink crystals (188 mg, 90% yield) as the product was obtained. The unstable product could not be further purified and were used immediately for the next protein conjugations.

• Synthesis of Propofol-Protein Conjugates by Nucleophilic Substitution:

The active propofol bromide hapten (III) (18 mg, 66 μmol) 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 μmol of OVA, or 5 mg, 0.011 μmol 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.

After dialysis, 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.

A simple spectrophotometric method (2,4,6-trinitrobenzene 1-sulfonic acid TNBS titration) was used for the estimation of hapten (propofol) to carrier protein (KLH or OVA) ratio. The percent conjugation of hapten to protein was calculated using the following equation:

(concentration of ε-amino group in carrier protein - concentration of ε-amino group in hapten protein conjugate)

% Conjugation X 100% concentration of ε-amino group in carrier protein

For KLH-propofol conjugate: % Conjugation = [( 19%-7%)/l 9%] X 100% - 63%

For OVA-propofol conjugate:

% Conjugation < 5% (very low conjugation) The results from ELISA test (previously prepared anti-propofol antibody from KLH or cBSA conjugate and the above KLH-propofol conjugate or OVA-propofol conjugate as coating antigen) also showed that KLH-propofol conjugate had much higher propofol attachment to the KLH-proteins than that of the OVA-proteins.

• Immunisation of Mice:

The above KLH-propofol conjugate (0.75 ml) in phosphate buffer (0.2 M, pH 7.5) was mixed well with Imject Alum (0.75 ml, Pierce) as adjuvant to make 1.5 ml of mixture. Eleven mice were injected with the resulted mixture (0.1 ml) each for raising anti-propofol antibodies.

It will be further appreciated by those persons skilled in the art that the present description is provided by way of example only and that the scope of the invention is not limited thereto.

Claims

CLAIMS:

1. An antibody capable of binding an anaesthetic compound selected from steroidal based anaesthetics and phenol based anaesthetics.

2. The antibody of claim 1 wherein the anaesthetic is a phenol based anaesthetic.

3. The antibody of claim 2 wherein the anaesthetic is propofol.

4. The antibody of claim 1 wherein the antibody is a steroidal based anaesthetic.

5. The antibody as claimed in claim 4 wherein the anaesthetic is selected from alphaxalone, ethanolone, saftan, predrisolone and epiallopregnanolone.

6. The antibody as claimed in claim 5 wherein the anaesthetic is epiallopregnanolone.

7. An antigen comprising an antigenic carrier linked to an anaesthetic compound selected from steroid based anaesthetics and phenol based anaesthetics.

8. An antigen as claimed in claim 7 wherein the antigenic carrier is a protein.

9. An antigen as claimed in claim 8 wherein the protein is selected from enzymes, albumins, globulins and hemocyanins.

10. An antigen as claimed in claim 8 wherein the anaesthetic compound is a phenol based anaesthetic.

11. An antigen as claimed in claim 10 wherein the anaesthetic is propofol.

12. An antigen as claimed in claim 8 wherein the anaesthetic is a steroidal based anaesthetic.

13. An antigen as claimed in claim 12 wherein the anaesthetic is selected from alphaxalone, ethanolone, saftan, predrisolone and epiallopregnanolone.

14. A method of preparing an antibody to a steroidal based anaesthetic or a phenol based anaesthetic comprising immunising an animal with an antigen as claimed in any one of claims 7 to 13.

15. An antibody produced by the method of claim 14.

16. An antibody according to any one of claims 1-6 and 15 which is a polyclonal antibody.

17. An antibody according to any one of claims 1-6 and 16 which is a monoclonal antibody.

18. An antibody as claimed in any one of claims 1-6 which is a recombinant antibody.

19. A method for determining the presence or concentration of a phenol or steroidal based anaesthetic in a sample comprising the steps of:

(a) contacting the sample with an antibody as claimed in any one of claims 1-6 or 15-18; and

(b) detecting if, or determining the amount of, anaesthetic that becomes bound.

20. A method as claimed in claim 19 wherein the anaesthetic is a phenol based anaesthetic.

21. A method as claimed in claim 20 wherein the anaesthetic is propofol.

22. A method as claimed in claim 19 wherein the anaesthetic is a steroidal based anaesthetic.

23. A method as claimed in claim 22 wherein the anaesthetic is selected from alphaxalone, ethanolone, saftan, predrisolone and epiallopregnanolone.

24. A method as claimed in claim 23 wherein the anaesthetic is epiallopregnanolone.

25. A method as claimed in claim 24 wherein the antibody used in the method is detectably labelled.

26. A method as claimed in claim 19 wherein detectably labelled anaesthetic is detected.

27. A method as claimed in claim 19 which is an ELISA method.

28. A method of claim 27 wherein a protein-anaesthetic complex competes with anaesthetic for binding to an anti-anaesthetic antibody which is subsequently detected by an enzyme linked anti-antibody.

29. A kit for determining the presence or concentration of a phenyl or steroid- based anaesthetic sample, the kit comprising an antibody of any one of claims 1-6 and 16- 19 together with an antigen capable of binding to it.

30. A kit as claimed in claim 29 wherein one of the components is linked to an enzyme.

31. An antigen as claimed in any one of claims 7-13 wherein the antigen is linked to an amine group in the carrier.

32. An antigen as claimed in claim 31 wherein the carrier is a protein.

33. An antigen as claimed in claim 32 wherein the link between the amine group of the carrier protein and the anaesthetic is obtainable via the Mannich reaction or nucleophilic substitution.

34. An antigen as claimed in claim 33 wherein the anaesthetic is propofol.

35. An antigen as claimed in claim 34 wherein the anaesthetic is epiallopregnanolone.

36. A detector means comprising:

(a) a response portion comprising antibody as claimed in any one of claims 1-6 and 15-18 which is responsive either directly or indirectly to an anaesthetic of interest; and (b) a sensor portion able to respond to a change of characteristic and/or at the response portion.

37. A detector means as claimed in claim 36 wherein the response portion comprises an electrode coated with an antibody as claimed in any one of claims 1-6 or

15-18.

38. A method of monitoring anaesthesia in a patient comprising measuring the concentration of an anaesthetic in a patient sample using a detection means of claim 36 or claim 37.