The present invention relates to the use of known materials as selective binding agents, to novel forms of these materials, particularly immobilised forms, and to test kits, filter apparatus and assay methods incorporating or using these. Particularly provided are materials comprising indole ring compounds, particularly polymeric materials of repeat unit including an indole ring, preferably in particulate or immobilised forms, eg. on a solid phase support.

In the food industry there is appreciable concern about the potential consumer health hazards associated with illegal use of growth promoting agents on animals destined for food use. Of particular concern are, for example, the °> ₂ -adrenoceptor agonists such as clenbuterol, salmeterol and salbuterol, and certain steroids. The use of clenbuterol as a drug for treating bovine and equine respiratory diseases or as a tocolytic is legal in many countries. In order to minimise consumer exposure in the UK there is a twelve day withdrawal period specified for this drug aimed at limiting residue concentration in edible tissue to below the maximum residue limit (MRL) .

When used at therapeutic levels (eg. at about 0.8μg/kg body weight) clenbuterol shows high specificity for β ₂ -adrenoreceptors with minimal f^-adrenoceptor side effects such as tachycardia. However, it may also be used illegally at ten times the therapeutic dose for the purpose of producing leaner carcases by mobilising fat deposits and by protein accretion (repartitioning) ; this leading to increased profitability for the producers. There is an appreciable health risk with such illegal use as the drug is administered at high doses for prolonged periods close to slaughter, without official monitoring or control. This illegal, uncontrolled use of high doses of drugs provides a health risk to the person providing the treatment as well as to the consumer, and is particularly hazardous to those with heart disease. Indeed, there have been cases of food poisoning in Spain and

France in which the consumption of liver containing clenbuterol was indicated (C.Pulce et al. Vet. Human Toxicol 33 (1991) 480 and J.F. Martinez-Navarro, Lancet, 336 (1990) 1311)- Thus detection of the use of clenbuterol is required both on animals recently slaughtered and those destined for slaughter.

Detection of the presence of such drugs at low levels is also required when monitoring illegal taking at sporting events. Many drugs are banned or controlled and there is therefore a need to develop sensitive tests for these. A further need to determine the presence of drugs and their residues arises when treating a person that has taken an overdose. In order to prescribe the correct antidote, and the amount of that to be administered, an accurate estimate of the drug taken needs to be determined.

One of the difficulties in detecting clenbuterol in animals intended to be slaughtered arises due to the rapid rate at which it is eliminated from the body. Selected methods must be capable of detection at very low levels because the safe maximum residue limit is low. Drugs may be extracted, concentrated and estimated on the basis of differences in their physiological properties, however many of the known physico-chemical techniques such as HPLC are unable to achieve limits of detection down to the safe maximum residue level.

Physico-chemical (liquid-liquid or liquid-solid) methods of extracting and isolating drugs and residues, both from body fluids and tissue preparations, often require appreciable empirical development for each residue. Methods may thus lack general applicability even within drug classes due to the wide variations in chemical characteristics such as partition coefficients and pKa's. Thus they are often limited in their ability to bind whole or different classes of drugs as is required where general screening is necessary.

Immunoaffinity methods overcome some of the problems facing

physiochemical methods, and are able to detect target molecules with related structure and/or function at low levels. However, to produce antibodies with the required specificity may require an intensive immunisation programme and good fortune. The fact that antigens and antibodies are very sensitive to external conditions also provides difficulties in the use of such techniques.

Solid-phase extraction procedures may also be used to detect target molecules with related structure and/or function. Although common adsorbents are produced on a large scale, they must be selected to suit particular analytes and various chemical groups may be attached to aid selectivity. Extensive method development specific to the analyte may be required to enable the bonded phases to detect drugs effectively and often product is too impure for analytical purposes.

A particular problem associated with screening for β-adrenergics is their variation in ionising characteristics. Whereas salbutamol may be ionised at high pH, clenbuterol is not, thus use of ion exchange resins to screen a sample will not bind both these drugs. Similarly, other classes of drugs include members of differing binding characteristics, and thus which will not necessarily be bound by the same capture agent, eg. ion exchange resin.

It is known that melanins have some ion exchange properties, and further that they do bind certain molecules in vivo and in vitro (see N G Linquist, Acta Radiol. (Stockh) , Suppl 325 (1973) 1) and these properties have been used in binding studies for toxicological and pharmacological studies.

The present inventor has now determined that indole ring containing materials such as melanin are not only capable of specifically binding a number of drugs, particularly fj-adrenoreceptor agonists such as clenbuterol, salmeterol and salbuterol, but that they are capable of doing this with high capacity. Furthermore, that when these are

immobilised as solid bodies or on a solid phase support they are well suited as adsorbents which can greatly facilitate the extraction of a variety of drugs, including those such as β-adrenergic agonists or antagonists and their residues. Not only can such compounds do this at physiological pH, unlike many commercially available extractants, but preferred forms can adsorb and desorb the drugs relatively rapidly.

This not only provides broad inter-class extraction capability, but also concentration of drugs from a variety of biological sources. Using optional washing steps together with desorbent/displacement reagents, the drug or drugs can be recovered in purified form.

A first aspect of this invention provides the use of an indole ring containing compound, particularly polymeric compounds having a repeating unit having an indole ring, as a specific binding agent for the selective capture and/or retention and/or release of one or more members of one or more classes of chemical compound. Preferably the selective capture and/or retention and/or release is carried out for the purpose of screening fluids for presence and/or amount of one or more target compounds; preferably the binding agent is used to concentrate the target compound or class of compounds.

Particularly suitable compounds targeted to be screened for are drugs, eg. β-adrenergic agonists and/or antagonists and steroids. Preferred polymeric materials are melanins or analogues thereof.

Melanins are polymeric pigments which can be found in pigmented tissues such as the choroid tissue and the retinal epithelium of the eye. There are several sources suitable for the use of the invention; it may be extracted from pigmented tissues (JP04175377) , produced by fermentation processes and/or genetic methods (W09200373, W08802372, SU1063834) or produced synthetically. The tyrosinase mediated pathway for melanogenesis from tyrosine or dopa is shown in Figure 10, as discussed in Lindquist N G, Acta Radiol (Stockh) Suppl. 325 (1973) 1.

Compounds for use in the present invention may suitably be in a particulate form, an extruded form, in the form of granules, pellets or spheres or may be immobilised on a solid phase support. The support may be selected from polymers, aluminas, silicas or derivatives or mixtures of any of these, eg. glasses, but other suitable materials will be apparent to those skilled in the art. Suitable examples of polymer supports include polypeptides, proteins, polysaccharides, cellulose, starch, agarose, polyacrylamide and copolymers or mixtures of these. Examples of suitable glass beads are available as preactivated beads from Dynal, Wirral, UK or as HIPAC (RTM) beads from Chromachem, Missoula, Montana USA.

The compound may be attached to a solid phase support using multivalent coupling agents, or directly, under conditions which do not alter its activity to below useful levels. Suitable coupling agents include but are not limited to dialdehydes such as glutaraldehyde, maleic acid and its derivatives, hydrazides and diazo compounds. Suitable glass or other silica based beads may be commercially obtained in preactivated form with tresyl, tosyl, aldehyde, hydrazine or tosyl activating groups.

A preferred form of polymeric material having a repeating unit comprising an indole ring is that provided by polymerising particulate bound tyrosine or dopa, or one of their analogues, conveniently by use of tyrosinase or an oxidising reagent. Suitable oxidising reagents for converting these compounds to indole form include ferricyanide (see Analytical Biochemistry (1988) 170, p335~34θ) and silver oxide while tyrosinase may be used to completely convert them to melanin like polymer (see J Pharm Sci (1976) 65, pl057"lθ6θ) . It will be realised that strong oxidising reagents may be used to induce a cerain degree of polymerisation without the presence of tyrosinase.

A second aspect of the present invention provides specific binding materials comprising indole ring compounds, particularly polymeric materials having a repeating unit having an indole ring per se. Forms

of melanin like material suitable for such materials and examples of target compounds which may be selectively bound by the materials are those discussed for the first aspect of the invention. Preferred materials are those comprising immobilised tyrosine, dopa or analogues thereof that have been oxidised to provide indole ring compounds, more preferred materials being melanin or polymerised oxidised tyrosine or dopa or analogues thereof. Preferably the melanin is in particulate form such as granules.

A third aspect of the present invention provides an assay for a target substance, including a target substance capture step, and a target substance identification and/or quantification step, characterised in that a specific binding material comprising an indole ring compound, preferably a polymeric material having a repeating unit having an indole ring, is used as capture agent in the capture step. Particularly such capture agent concentrates the target such that when desorbed from the binding material using a displacing agent such as a buffer or an alcohol, a more concentrated solution can be produced for assay purposes. Examples of the forms of melanin like materials suitable as a capture agent and examples of target compounds which may be selectively captured will be as discussed for the first and second aspects of the invention.

A further aspect of the present invention provides selective retention apparatus comprising a specific binding material comprising an indole ring compound, particularly a polymeric material having a repeating unit having an indole ring, as a specific binding agent for capturing and/or retaining target materials, eg such as β-adrenergic agonists and/or antagonists or steroids, particularly at physiological pH. The form of melanin like substances suitable as a specific binding agent and examples of target compounds which may be selectively bound will be as discussed for the first, second and third aspects of the invention. Particularly such apparatus is capable of concentrating adrenergic materials, such as β ₂ adrenergics, and steroids, from

physiological solutions.

A first embodiment of this further aspect of the invention provides a specific binding material comprising an indole ring compound, preferably a polymeric material having a repeating unit having an indole ring, retained immobilised within a chamber or a conduit having an inlet means and an outlet means. A suitable chamber or conduit may be a column containing melanin through which a test material or material to be purified may be passed. A second embodiment provides a filter, particularly of filter bed type, including the polymeric material as provided by the second aspect. The specific binding material in both these embodiments may be any of the forms of the second aspect of the invention.

A still further aspect of the present invention provides test kits for performing an assay for a target drug comprising a specific binding agent as described in the use or material of the invention together with one or more further reagents selected for their requirement in performing the assay.

A first embodiment of this aspect of the present invention provides a test kit wherein the indole ring compound, eg. as immobilised or as polymeric material, is retained in a chamber or conduit having an inlet means and an outlet means as described above, together with reagents for displacing target drugs so bound and reagents for specific binding assay of the displaced drug.

The reagents for displacing target drug may include, for example, solvents such as alcohols, eg methanol and ethanol, or THF or DMF, that cause their liberation, ie. solvents selected for their ability to dissolve the particular drug of interest with high partition coefficient between it and melanin, or may include, eg, buffered solutions of pH suitable for changing the ionisation state of the melanin and/or bound drug such that it is freed. The reagents for

identifying displaced drugs for assay purposes may include labelled binding agents capable of competing for binding sites with the drug displaced from the specific binding agent. Such assay may be carried out in the known manner to determine an unknown amount of compound displaced from the agent. For use without laboratory equipment such label will preferably be one that evokes a colour forming reaction, thus allowing visualisation of the amount of labelled agent on a binding surface; that surface being provided in the kit. Radiolabels will of course be usable where laboratory equipment is available. Immunoassay formats may also be used with such embodiments optionally being of dipstick format if required. \

A still further aspect of the present invention provide the use of a specific binding material, particularly one of the polymeric materials, as provided by the second aspect of the invention, as a binding agent for the retention of drugs in slow release formulations. Such formulations may combine, for example, melanin as provided by the third aspect, onto which has been bound a suitable quantity of drug, eg. clenbuterol, such that implantation or ingestion of the melanin results in the slow dissociation of the drug into the body of a patient or user. The present inventor has determined that the rate of desorption of adrenergics and steroids from such materials makes hem suitable, under physiological conditions, for such use.

The method and materials of the present invention will now be described by way of illustration only with reference to the following non-limiting Examples and Figures. Further embodiments of the invention will occur to those skilled in the art in the light of these.

FIGURES

Figure 1: Shows a comparison of the capacity for melanin granules to bind β-agonists and salicylic acid in microtitre plate wells.

Figure 2: Shows the binding of 3H-clenbuterol to melanin granules in the absence and presence of unlabelled clenbuterol at increasing concentration.

Figure 3∑ Shows comparative % binding of chlorpromazine, salicylic acid, clenbutarol, salbutamol, nortestosterone, alpha-trenbolene and DES to Sepia melanin, a particulate form of melanin available from Sigma Chemical Co as a suspension.

Figure 4: Shows comparative % binding for the drugs bound in Fig 3 using commercially available cyano-silicate binding agent.

Figure 5* Shows the kinetics of clenbutarol binding to Sepia melanin.

Figure 6: Shows the comparative % binding with time of clenbutarol and 19-nortestosterone to oxidised non-polymerised L-dopa coated Clifmar glass beads and similar oxidised dopa beads that have been further exposed to L-dopa and tyrosinase and oxidised to form an indole ring containing polymer similar to melanin.

Figure 7' Graphs Ja to 71; show the comparative % binding for a number of drugs with varying amount of bovine melanin derived by the method described in Example 1. The drugs so bound are indicated at the top of each graph.

Figure 8: Graphs 8a to 8c show the comparative % binding of Azaperone, Xylazine and Carazol to uveal melanin granules provided by the method of Example 1.

Figure 9- Graphs 9& to 9c show the binding of clenbutarol and nortestosterone to tyrosinase polymerised oxidised L-dopa coated glass beads and the % release therefrom on exposure to 1ml of ethanol demonstrating varying selectivity of retention.

Figure 10: Shows the Raper-Mason scheme of melanogenesis from tyrosine and dopa.

Example 1. Preparation of melanocyte granules

Stock solutions of phosphate buffer were prepared by dissolving 0.8l7g of sodium dihydrogen phosphate dihydrate and l6.856g of disodium orthophosphate dihydrate in 98θml water. The pH was adjusted to 8.0 with concentrated hydrochloric acid and the solution made up to one litre.

Choroid tissue and pigmented retinal epithelium was obtained from bovine eyes by dissection and stored at -20°C until required. Frozen protease (Subtilisin A - Sigma) was removed from the freezer and left to reach room temperature. 1ml of phosphate buffer, 0.1M, pH 8.0 was added to 0.02g of protease in a 1.5 ml polypropylene Starstedt screwtop tube and the protease dissolved by gentle mixing.

2g of each type of tissue was weighed out and transferred to separate labelled 50ml polypropylene screwtop tubes. 8ml of 0.1M, pH 8.0 phosphate was added to each tube followed by 200μl of 20ng/ml protease (Subtilisin A) . The contents of the tubes were then vortexed ensuring all the tissue was in suspension before adding a magnetic stirrer. A stainless steel water bath was filled with enough water to cover the tubes and placed on a hot plate. A temperature probe was set at 57°C and placed in the water bath. The water temperature was allowed to reach 57°C before the tubes containing tissue were placed in the waterbath and incubated for an hour with stirring.

After an hour the tubes were removed and the temperature probe set at 99°C. Once the water temperature had reached 99°C the tubes were returned to the waterbath and incubated for a further 15 minutes to denature the protease. The magnetic stirrer was then removed and the tubes centrifuged at 13000g for ten minutes in order to precipitate pellets.

The supernatants were discarded and δmls of fresh phosphate buffer transferred to each tube. The pellets were resuspended by vortex mixing and centrifuged at 13000g for ten minutes before the supernatants were again discarded. This procedure of the pellets being resuspended and centrifuged was then repeated twice.

After the third wash 8ml phosphate buffer was added to the tubes and the pellets were resuspended by vortex mixing. Eight 1ml aliquots of the resulting solution from each tube was then transferred into preweighed and labelled 1.5ml polypropylene screwtop tubes for storage purposes. These were centrifuged at 130000g for ten minutes and the supernatant discarded. The tubes were then reweighed and the net weight of the melanin granules calculated by subtracting the final tube weight from the original tube weight. The pellets were then stored at -20°C. For use these were formed into desired bodies or coated onto wells or other substrates, chemically or by mere association. The dimensions of the ovoid granules in the pellet product are 770 ± 150nm long by 50 ± 120nm wide.

Example 2: Preparation of modified L-dopa coated beads. L-dopa may be attached to solid phase supports by a number of standard chemistries. For this example commercially available derivatised and/or preactivated glass beads may be used to enable attachment of ligand, either via the primary amino-group of L-dopa or via its carboxylic acid moiety. In this instance derivatised controlled pore glass beads (Clifmar Associates Ltd; Guildford, Surrey) were used with coupling being achieved by Schiff's base formation via the amino group of L-dopa. Beads were subsequently oxidised using an oxidising solution to convert the dopa to an indole form (see Figure 10) and these beads were used in binding capacity studies and further converted to melanin like material as set out below.

A melanin like indole ring containing repeat unit polymer is produced on the surface of the oxidation polymerised L-dopa bead by adding

additional L-dopa and the enzyme tyrosinase. Thus L-dopa (300mg) is dissolved in phosphate buffer (120ml, pH7.5« 0.1M) in a 250ml graduated cylinder. The beads (3«5g) are kept in suspension by continuous bubbling of air through the solution. Tyrosinase (lOmg) was added and the mixture left bubbling overnight at room temperature before washing repeatedly with water and straining over a nylon mesh.

The comparative binding of a variety of drugs, including β-adrenergics and steroids, is shown in the graphs in Figure 6. It should be noted that comparing Figures 5 and 6, the polymerised L-dopa coated beads equilibrate much faster than melanin itself; Sepia melanin equilibrates within about 10 minutes while the L-dopa tyrosinase induced polymer equilibrates within 1 to 2 minutes. This equilibration was reflected in similarly rapid desorption times using alocoholic desorbants.

The binding of clenbutarol and 19 nortestosterone to oxidised glass immobilised L-dopa (i) that has been oxidised and (ii) that has then been exposed to a solution of L-dopa and tyrosinase as described above is shown in Figure 6 and the adsorption values derived from the graphs set out there are given below.

Glass beads (i) Capacity Affinity Dissociation (moles/mg) constant(Ka) constant(Kd)

Clenbutarol 4.6xl0 ^"10 l.OlxlO ⁵ M- ¹ 9.9xlO ^_6 M

19 Nortestosterone 4.6xlO- ¹⁰ 1.27xl0 ⁵ M- ¹ 7.9xl0 ^"6 M

Glass beads (ii)

Clenbuterol 7.δxlO ^-10 β.βxlO^M- ¹ ) 1.5xlO ^_6 M

19 Nortestosterone 6.7xl0 ^"10 17-Oxl0 ⁵ (M- ¹ ) 0.6xl0- ⁶ M

Example ^ . Test kit for assay of clenbuterol in body fluids. A test kit according to the invention is provided comprising essentially, a melanin granule filled column, a displacement reagent

consisting of methanol, anti-clenbuterol antibody coated microtitre, alkaline phosphatase-clenbuterol conjugate,

Microtitre well plates were coated with antibodies raised to clenbuterol diazotised to carrier protein (see Yamamoto & Iwata. J. Immunoassay, 3 (1982) 155); ovalbumin being used as carrier protein and 40:1 molar ratio diazotised clenbuterol:ovalbumin was used in the reaction mixture (pH 9-5) at coupling.

Antibodies were produced using an inoculant in an emulsion of sterile saline (0.9 w/v) with Freund's complete adjuvant for primary inoculation and Morris' incomplete adjuvant for boosters. Sheep of about 2 years of age were inoculated with 3mg (in 1.3ml emulsion, primary) or lmg (in 2.3ml, boost) of immunogen per animal. Boost injections were given at intervals of three months after primary inoculation and blood samples taken by jugular venepuncture nine days later. The gamma-globulin fraction was prepared using ammonium sulphate (33% saturated) precipitation by the method of Garvey et al (Methods in Immunology, Benjamin, Massachusetts, 3rd Edit, 1977.P 218) and stored in aliquots at -20°C until required.

Plates were produced by reconstituting the globulin fraction to the original serum volume, diluting 1:1 in glycine buffer (0.3M, pH 2.2-Ishikawa et al. J. Immunoassay, 1 (1980) 385) and incubated at ambient temperature 22-25°C for 35 minutes. The preparation was then diluted as appropriate (1/16000) in tris(hydroxymethyl)methylamine (Tris) buffer (0.05M, pH 8.5) and incubated at ambient temperature for 1 hour before addition of 0.2ml to each plate well. Plates were sealed with adhesive film and incubated for 3 hours (22-25°C) before emptying and washing with PAS-gelatin buffer (0.3ml), further emptying and PAS-gelatin buffer addition (0.3ml) before final sealing with adhesive film and storing at 4°C until required. Binding activity is retained for several months under such conditions.

The conjugate was prepared using the procedure of Yamamoto and Iwata (as reference above) but alkaline phosphatase was used instead of β-galactosidase as the enzyme label. This was exhaustively dialysed against PS buffer prior to conjugation using 20:1 molar ratio of diazotised hydroxyclenbuterol: enzyme. Purification was by the method of Sauer et al (J. Reprod. Fert. 76 (1986) 375)- Other conjugates suitable for performing colour forming reactions can be prepared by analogous methods known to those skilled in the art.

Example 4. Assay of β ₂ -adrenergics. clenbuterol. in urine . Urine samples obtained from cows were applied to a melanin column were washed with water, or pH 7 PBS buffer, then eluted with methanol into test tubes. Eluate was concentrated to dryness, under nitrogen where possible, and redissolved in PAS-gelatin buffer (100-500μg), preferably with the aid of sonication (10 minutes) and in a water bath at ambient temperature (l8-23°C).

Enzyme immunoassay may be performed as described by Sauer et al (as ref above) using appropriate clenbuterol standards instead of progesterone (lOμl, range 0.1~50ng) and hydroxyclenbuterol-alkaline phosphatase conjugate (8θng/ml). Adhesive film is used to seal the plates during incubations and absorbance is determined using a MR710 automatic plate reader (Dynatech, Billinghurst, UK). This method was also used with antibodies raised to other adrenergic drugs immobilised in respective wells.

Example : Assay using melanin as binding agent.

Melanin itself can be used as the binding substrate on which labelled and eluted β-adrenergics compete for binding sites. A number of granules of melanin as prepared above were placed in microtitre plate wells and ^l4 C salmeterol or ³ H clenbuterol were added in 1:1 ratio with redissolved sample as described in Example 3- The concentration of β-blocker in the sample can be determined by comparing bound label, ie. counts, in the known manner.