NZ236879A - Lolitrem type compounds; lolitrem antibodies; antigens; hybridomas; vaccines; affinity chromatography columns and immunoassays - Google Patents

Description

? Z 8 7 PATENTS FORM NO. 5 Fee No. 4: $260.00 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional No; 236§79 Dated: 23rd January 1991 LOLTTREMS - ANTIBODIES AND ASSAY TECHNIQUES Her Majesty the Queen in right of New Zealand acting by and through the Minister of Agriculture, c/- Ministry of Agriculture and Fisheries, East Street, Hamilton, New Zealand hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: f /, <r 'T 9 r k 7 C' This invention relates to lolitrem, lolitrem antibodies, lolitrem assay techniques, and methods and compositions pertaining to same.

The lolitrems are a group of compounds having the general structure shown below: Lolitrems Several Lolitrems are known, these being Lolitrem A (as above wherein X = CsHgO), Lolitrem B (as above wherein X = CsHg), Lolitrem C (as above wherein X = C5H10) and Lolitrem D (as above wherein X = C^H^O). These Lolitrems 10 are known tremorgenic mycotoxins, are naturally occuring and are produced by at least the endophytic fungus Acremonium Lolii. Lolitrem B may be extracted from infected perennial rye grass (Lolium Perenne L) and infected rye grass seed. The known naturally occuring Lolitrems, as Mycotoxins, exhibit insecticidal and growth inhibitory activity against insects.

However, the known Lolitrems have exhibited some adverse and even toxic effects on larger animals, depending upon the quantity of Lolitrems ingested. This may be of commercial point importance for domestic animals such as cattle. While it is rare and unusual for death of a beast to occur directly as a result of Lolitrem poisoning, it is more common for death or injury to result 20 from drowning or a fall when a disorientated beast suffering from the symptoms of Lolitrem poisoning encounters what is normally a minor hazard such as a bank, stream or swamp. 2 o ■? q Because of the potential importance of Lolitrems, albeit in a negative manner, it is also desirable to develop a sensitive assay technique for determining quantitatively and/or qualitatively the presence of various Lolitrems. Such assay techniques may be useful in the monitoring of Lolitrem levels in affected or slaughtered beasts, or even monitoring levels in animal feed stocks and new plant varieties.

It is an object of the present invention to address the above problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

According to one aspect of the present invention there is provided a A compound, useful for the preparation of haptens and conjugates, of the formula According to an another aspect of the invention there is provided a method for the synthesis of a compound substantially as described above comprising the hydrolysis of a lolitrem.

According to an another aspect of the invention there is provided a method for the synthesis of a hapten or lolitrem derivative which includes a fragment of the formula 15 comprising the reaction of the compound of claim 1 to add a conjugate bridge to the position indicated A, said conjugate bridge comprising a member of the group: n-alkyl, iso-alkyl, alkenyl, aryl, alkynyl, aromatic, amide, oxime, epoxide, carboxylic acid, ester, activated ester, aldehyde, acetyl and ketal groups.

According to an another aspect of the invention there is provided a hapten or compound including a fragment of the formula prepared via a method substantially as described above.

According to an another aspect of the invention there is provided a method for the preparation of an antigen or immunogen comprising the reaction of a hapten substantially as described above or as prepared by a method substantially as described above, with a earner.

According to an another aspect of the invention there is provided an antigen or immunogen having the lolitrem skeleton as an antigenic determinant.

According to an another aspect of the invention there is provided a monoclonal antibody, or fragment thereof, able to recognise and react with a substance whose antigenic determinant comprises: 4 According to an another aspect of the invention there is provided a hybridoma capable of producing a monoclonal antibody substantially as described above.

According to an another aspect of the invention there is provided a method of generating a hybridoma capable of producing a monoclonal antibody substantially as described above, comprising antibody secreting cells or lymphocytes obtained by immunising an animal with a substance having an antigenic determinant comprising: According to an another aspect of the invention there is provided a polyclonal antibody able to recognise and react with a substance whose antigenic determinant comprises: According to an another aspect of the invention there is provided a method of preparing a polyclonal antibody substantially as described above by the administration of an antigen or immunogen, substantially as described above, to an animal.

According to an another aspect of the invention there is provided a method for the purification of antibodies, substantially as described above, comprising the use of an affinity chromatography column having an immobilised compound or derivative including a fragment of the formula r!?' ; • r, If r" v According to an another aspect of the invention there is provided an affinity chromatography column useful for the separation or purification of antibodies substantially as described above having a compound or derivative including a fragment of the formula 0- bound to a column support.

According to an another aspect of the invention there is provided a passive vaccine comprising an antibody and/or an antibody fragment, substantially as 10 described above, together with a diluent.

According to an another aspect of the invention there is provided a method of addressing lolitrem toxicity comprising the administration of a passive vaccine substantially as described above.

According to an another aspect of the invention there is provided an in vitro 15 immunoassay method of determining lolitrems or substances incorporating the lolitrem skeleton, using an antibody substantially as described above, according to standard ELISA or immunoassay techniques.

According to an another aspect of the invention there is provided an immunoassay kit comprising an antibody substantially as described above. 6 It is perhaps useful to define several of the terms used within the specification: Lolitrems 45 The above molecule represents the Lolitrem molecule and illustrates the numbering sequence for the carbon skeleton. A Lolitrem is any compound having the above carbon/nitrogen skeleton. The nature of X is unimportant for the purpose of the definition.

Lolitriol is a Lolitrem having the above specific structure.

Analyte Is the substance in a test sample whose presence or quantity is to be determined. For the current invention, the analyte in question will typically be a lolitrem.

Antibody A synthesized binding protein produced by the immune system of an animal in response to the presence of a foreign organism or immunogen.

Antigen A substance which will react with its specific antibody.

Antigenic Determinant A feature of characteristic of an antigen defining the recognition pattern of an antibody.

Antiserum A preparation containing polyclonal antibodies to an antigen.

Carrier A large molecular weight compound to which haptens are linked and which typically comprises an enzyme, protein, protein or DNA fragment, polysaccharide or other macromolecule.

Conjugate A carrier to which a hapten has been linked.

Conjugate Bridge An intermediary group attached to the analyte or antigen and enabling same to be bound to a carrier.

Enzyme Immunoassay An assay procedure based on the reversible and non-covalent binding of an antigen by a specific antibody, in which one of the reactants is labelled with an enzyme.

ELISA Enzyme ■ linked immunosorbent assay An enzyme immunoassay suitable for use in conjunction with the present invention, in which one of the reactants is absorbed on the surface of the well of a microtitration plate. 8 Emit Enzyme mediated immunoassay technique A homogeneous enzyme immunoassay suitable for use in conjunction with the present invention, for haptens in which the enzymes link to the hapten in such a way that the enzyme activity is altered when the hapten combines with the 5 antibody.

Fab Fragments A fragment including the antigen combining site after digestion of an immunoglobulin molecule with an enzyme such as papain.

Hapten An antigen, normally not immunogenic until coupled to a larger molecule, such as a carrier.

Immunoassay An assay technique which relies on the reversible non-covalent binding of an antigen by antibody. Typically either the antigen or antibody is labelled. The 15 technique may therefor be used to analyse for antigens or antibodies.

Immunogen A substance stimulating the production of antibody or antibodies which may combine with similar substances as an antigen.

Monoclonal Antibodies, Hybridoma -20 Antibodies derived from a single clone of lymphocytes. Typically these are produced by a hybridoma cell line prepared by the fusion of a single J3- lymphocyte clone and a plasmisoma cell line.

Polyclonal Antibodies Antibodies to a particular antigen which are derived from multiple lymphocyte 25 populations.

The antigenic determinant of interest is the lolitrem skeleton as apparent in the following diagram: 9 lolitrem skeleton H H This skeleton is common to all the lolitrems, including those of specific interest such as the identified Lolitrems A through D. It is therefore desirable that haptens and immunogens associated with lolitrem assay techniques, and 5 more importantly with vaccine/remedy preparations, recognise the lolitrem skeleton.

Consequently, this specification is directed primarily towards techniques relating to the preparation and use of antigens possessing the lolitrem skeleton; typically this involves the preparation and use of haptens and 10 immunogens wherein any conjugate bridge connects to the 10 and/or 14 carbon atoms, preferably via the 11 and 13 (respectively) oxygen atoms - see labelling scheme below: 45 Further aspects of the present invention will now be discussed with reference 15 to the accompanying diagrams in which: Figure 1 is an ELISA derived competition curve for lolitrem B. 7 9 The compound lolitriol (iride supra) is useful for the preparation of haptens and immunogens of this nature i.e. where the conjugate bridge is connected to carbon 10 and/or 14. The hydroxy groups are reactive and may be quite readily replaced with a variety of conjugate bridging groups. Alternatively, conjugate 5 bridging groups may directly substitute X (refer lolitrem structure) on any known lolitrem, including derivatives A through D. Typically however, many of these lolitrems are readily converted to lolitriol and preparation of haptens, immunogens etc. will proceed via this compound.

Preparation of Lolitriol Lolitriol may be readily prepared via the hydrolysis of a lolitrem. Taking lolitrem B as a specific example, Y mg is dissolved in Y ml of ethanol. To this is added Y ml of 0.1M hydrochloric acid. The solution is stirred at 45°C for two hours and the reaction mixture made weakly alkaline through the addition of Y ml saturated sodium hydrogen carbonate solution. 2Y ml of 15 dichloromethane is used to extract the lolitriol. Drying may be performed over sodium sulphate, anhydrous. The product may be filtered and isolated. A virtually quantitative conversion is normally achieved.

It is envisaged that variation of the above parameters and reactants may also be made and may be necessary for other lolitrems.

Conjugate bridges A wide variety of conjugate bridges may be used to produce haptens and immunogens. In a broadest sense, a conjugate bridge may comprise any group containing one or more of the following fragments: n-alkyl, iso-alkyl, alkenyl, aryl, alkynyl, aromatic. More specifically, it is likely that the bridge 25 group will comprise one or more reactive fragments or functionalities enabling it to bind or join to a suitable carrier. Thus in many, but not all, instances the conjugate bridge may contain or be - a(n) amide, oxime, epoxide, carboxylic acid, ester or activated ester, aldehyde, acetal, ketal. 11 Other functional groups which may enable the hapten to bind or conjugate to a carrier of interest, especially those described within this specification (vide infra), are also envisaged. The epoxide is one such group, reacting with the £-amino groups of lysine residues in polypeptides: The size of the conjugate bridge may also be a consideration. Normally the portion comprising the bridge will not exceed two times the size or mass of the lolitrem skeleton. If the bridge is too large or introduces its own character, then the lolitrem skeleton may comprise only part of the antigenic determinant for any hapten or immunogen derived and used for the production of 10 antibodies/antisera. It may be desirable if a generic antibody is to be created, that the conjugate bridge in any immunogen is restricted in size to substantially the same or less than that of the lolitrem skeleton, though it is recognised that size may not be the only feature introducing such effects and that any such other factor should also be considered.

+ H-,Nrv_/\/ polypeptide r- epoxide derivative OH ~ NH polypeptide ' ' The nature of the compound chosen to form a conjugate bridge with a lolitrem will reflect these factors. Reactions will follow known organic syntheses though several examples will be given later within this specification.

Carriers Numerous macromolecules may be used as a carrier for the preparation of an immunogen. This area of technology is well established and known carrier molecules and binding techniques may be employed. 12 It is envisaged that often the carrier will be an enzyme, protein, polysaccharide or other high weight molecular compounds or fragments. Specific examples include ovalbumin, BSA, thymoglobulin, haemocyanin, polystyrene, DNA fragments. Enzymes may be used, though preferably these 5 should be fairly active with a high turnover number e.g. horse radish peroxidase, alkaline phosphatase etc. The use of enzymes will commonly be directed towards haptens and immunogens for use with assay techniques - in these instances enzymes which have a substrate producing a detectable reaction (typically colorimetrically, though also including fluorescent, 10 radioactive tracer and other techniques) may be preferred. Such enzyme/substrate combinations are well documented with respect to assay techniques.

A chosen carrier may also be a column support, such as Sepharose or the equivalent, enabling the hapten to be immobilised and used for 15 chromatographic techniques. This may be useful for purification and assaying, such as will become apparent later in the specification.

Preparation of haptens and immunogens Several representative reactions will be given as specific examples of the addition of a conjugate bridge to a lolitrem. In these examples, the analyte 20 reactant is lolitriol due to the relative ease by which reaction at the 10, 14 carbon atom hydroxy sites occurs. via acetal or ketal formation Reaction 1 / OMc Lolitriol R" R 13 Reaction 2 Oxidation of the 14 carbon atom hydroxy group followed by formation of a carboxymethyl) oxime.

OCT oxidise Lolitriol OH OH O oxime formation, e.g.

H2N0CH2C02H COOH / + pyridine, R.T. J / overnight -o 0 /X via acylation Reaction 3 RCH ,COCI OH pyridine Lolitriol Reaction 4 minor V R OH 14 236879 In each case the mixture is normally stirred. Reaction may be at room temperature or gently warmed to hasten reaction. Reaction times will vary on the nature of R. R is typically an alkyl (n- or iso-) fragment comprising 1 to 25 carbon atoms. In many instances, R will comprise between 3 to 10 carbon atoms in its skeleton.

Other Specific Examples Some further examples illustrating the chemistry of lolitriol are given - these are indicative of the types of reactions which may be used to introduce conjugate bridges to the lolitrem skeleton. It is noted however that the specific compounds which appear in the examples are not necessarily the most suitable for forming a conjugate bridge - in practice it is likely that a functional group such as a carboxylic acid, amide etc. (see above) will be present. Alternatively compounds of the types described below may be further reacted to modify the conjugate bridge portion to a form which more readily links to a chosen carrier.

Example Esterification of the 10, 14 carbon hydroxy groups using succinic anhydride.

O succinic anhydride derivative Example R O ^ The above compound where R = Methyl may be prepared via the following reaction: 9"„ H v\OH J?—OMe ppts or pts Lolitriol is dissolved in a suitable non-hydroxylic solvent, such as 5 dichloromethane or chloroform. To this is added an excess of 2-methoxypropene (assuming a 1:1 stoichiometric ratio). A trace of a catalyst such as pyridinium p-toluenesulphonic Acid (PPTS) or p-toluenesulphonic Acid (PTS) may be used. There is also a possibility that silica gel may also act as a catalyst for reaction, and the reaction mixture could be allowed to pass 10 through a silica column. The reaction is generally completed within approximately 30 seconds when PPTS or PTS is used as a catalyst. This reaction is fairly straightforward and the reaction with 2-methoxypropene yields a product in which the 1, 3 hydroxyls are protected. The product is also readily converted to Lolitriol via a hydrolysis reaction such as described earlier 15 within this specification.

Example Lolitrem B and iso-lolitrem B may be prepared by either of the following two reactions: >= 0", H >„OH stererisomer mixture H + MeOH 16 The same conditions and procedures as described for the 2-methoxypropene reactions may be followed. The rate of the reaction is generally in the order of days (approximately 1-2 days at room temperature). The Lolitrem B product of both reactions are actually a mixture of stereoisomers. For the latter reaction, methanol is a bi-product of the reaction. It is suspected that an equilibrium may be achieved for the latter reaction, and the use of a 4A Molecular sieve to absorb the methanol may influence the reaction towards the Lolitrem B product.

Immunogens, antibodies, antisera Methods have been described for producing haptens and immunogens which include the desired lolitrem skeleton determinant. These compounds may often be produced in high degrees of purity, which may be advantageous. The use of such high purity immunogens, especially at low doses, increase the chances of stimulating clonal cells to produce high affinity antibodies.

The methods also allow a wide range of haptens and immunogens with differing conjugate bridges to be produced. Use of such immunogens with structurally dissimilar bridges may reduce antibody recognition of the immunogen bridge.

Production of polyclonal antibodies Immunogens produced according to the present invention may be useful for the production of polyclonal antisera as well as for monoclonal antibodies. These procedures, based on the administration of an immunogen to an animal to elicit the formation of antibodies, are well known and documented and may be employed.

In trials, anti-lolitrem antibodies have been raised in sheep and mice. To confirm the binding of free lolitrem, competition ELISAs have been performed. 17 A typical competition curve in cELISA, performed substantially as described later in the specification1" , is shown in Figure 1.

Example— Method of Preparing Anti-Lolitrem Antibodies For each antigen/antibody combination, up to twenty (depending on antigen 5 availability) sheep are immunised according to the schedule below. The antibody titres and the affinity and competitive characteristics of the antisera are monitored so that those animals (typically 3-10) producing the most suitable antibodies are retained for antibody production.

The wethers retained for antibody production will be re-immunised as 10 required to elevate serum antibody levels prior to blood collection for antibody isolation. Animals rejected may be immunised with a different antigen or culled from the flock.

Antigens may incorporate variations in the protein carrier linkage group and linkage site as is appropriate for each mycotoxin. Initial trials were 15 performed with BSA-lolitrem, ovalbumen-lolitrem and thyroglobulin-lolitrem derivatives.

Each ewe received sets of 0.5 ml of antigen/adjuvent as two 0.25ml injections containing 0.3mg of antigen into the rump/hindleg area on three separate occasions each four weeks apart. This site is chosen to avoid any site reaction 20 interfering with blood collection from the jugular. Tests to date indicate that site reactions are minimal and not a problem.

If antibody titre measurements indicat a necessity, the ewes will receive further injections of anitgen as required to elevate antibody concentrations to suitable levels. Freund's complete adjuvent (FCA) is used for the first 25 injection, and Freund's incomplete adjuvent (FIA) for all booster injections. t The procedure was modified through the use of a colorimetric disclosure employing an alternative substrate; rather than using ABTS, plates were disclosed by incubation with 3,3'5,5'tetramethylbenzidine (TMB) for 15 minutes followed by the addition of 2M II2SO4 and read spectrophotometrically. 18 ^ ~y - ■ —i r :> 6o/9 If alternative adjuven'ts causing a lesser site reaction than Freund's complete adjuvent become available while still stimulating the immune system to a comparable degree then they may be used preferentially.

Blood samples for antibody titre determinations are taken approximately to the 5 following schedule (actual bleeding dates will depend on the antibody titres). DayO Bleed Inject (FCA) Day 28 Inject (FIA) Day 56 Inject (FIA) Day 70 Bleed Day 74 Inject (FIA) Blood is collected from selected sheep with high titres using blood donor packs (approx. 500ml) on several occasions over several weeks. No more than three 500ml collections are normally taken in any one week period and no more than six collections during any four week period. The sheep may be retained and 15 reimmunised as required to produce antibodies according to need and following the same protocols.

Example— For Mice The above procedures are followed with the following exceptions: O.lmg (100[ig) antigen (e.g. protein-lolitrem conjugate) is administered in 20 lOOjil total volume lots, which are injected intraperitoneally. Further information on these procedures is contained in Marlow, E., Lane & D., (editors) Rider, C.C.: "Antibodies. A Laboratory Manual" Publ. Cold Spring Harbor Labs (1988) pp 92-113.

Production of hybridoma cell lines for monoclonal antibody 25 secretion Due to the possibility of obtaining relatively high purity immunogens according to the previously described syntheses and an increasing trend towards monoclonal antibody propagation techniques, it is envisaged that 19 monoclonal techniques will more frequently be used in conjunction with the present invention.

Hybridoma cell lines were produced by the fusion of J3-lymphocytes from an immunised animal with cells of a myeloma cell line, in an adaptation of the method of Kohler and Milstein (1974). For example: Balb/c derived myeloma cells (of line NS1 or P3X63-Ag8.653, Kearney et al, 1975) were maintained in log-phase growth at 37°C in a humidified atmosphere of 8% C02 , for at least 14 days prior to fusion. The immunised animal whose antiserum contained a high titre of specific antibodies was given a final intravenous boost of antigen (0.1 to 0.2 ml) three days prior to the cell fusion. The animal was exsanguinated and the spleen aseptically removed. Splenocytes were harvested by mechanically rupturing the organ and a cell suspension produced. The cells were pelleted (200g, 5 min at RT), and resuspended in 10ml 0.17mol/l NH4 CI buffer (pH 7.2) to lyse red blood cells. The splenocytes were again pelleted, and resuspended in 20ml serum-free medium, together with approximately twice their number of myeloma cells. The cell pellet formed after centrifugation was equilibrated at 37°C.

Sterile PEG-4000 (Merck Ltd) (lg) was melted and mixed with 1ml of warmed serum-free medium. This 50% PEG solution was maintained at 37°C and slowly added to the cell pellet, in the ratio of 100|il per 2xl07 total cells, over a period of 1 min, and stirred gently with a warmed pipette; the cells were left in this PEG solution for a further 1 min. Serum free medium was added to achieve a doubling of the volume per min, to a final volume of 20-25 ml. The cells were allowed to settle under gravity for 5 min, before being centrifuged at very low speed (200g, 3 min), and the PEG-containing medium aspirated. The cells were gently resuspended in medium containing HAT selection components and 20% FCS, allowing 1ml medium for every 1-I.5xl06 spleen cells, and this suspension distributed into microtitre wells containing peritoneal cell feeders at 100f.il per well.

Both hybridoma cells and their parent myelomas were cultured in medium consisting of Dulbeco's modified Eagle's medium (DMEM) (Gibco Ltd) supplemented with foetal calf serum (FCS, Gibco Ltd) at between 5 and 20%, penicillin / streptomycin, glutamine and 2 mercapto-ethanol. This medium 5 was supplemented with hypoxanthine, aminopterin and thymidine (HAT) for the selection of hybridomas.

Purification of Antibodies Affinity chromatography may be used to purify or separate antigens and immunogens. A lolitrem derivative, such as one of the haptens or conjugates 10 described earlier, may be bound to a suitable column support in place of another carrier. Sepharose, which is widely used in the field, is one such support though others may also be used.

Techniques for the preparation and use of an affinity column are as outlined in Horstmann, B.J., Chase H.A. & Kenney, C.N.: "Purification of Anti-Paraquat 15 Monoclonal Antibodies bv Affinity Chromatography on Immobilised Hapten". Journal of Chromatography 516 (1990)433-441. Use of a hapten incorporating the lolitrem skeleton, such as whose preparation has been described, bound to a Sepharose 4B has been found to be useful in practice. Details of different Sepharose supports suitable for different binding sites are disclosed in 20 literature and manufacturer's data e.g. "Affinity Chromatography Principles". Publ. Pharmacia LKB Biotechnology, Sweden (1991).

Characterisation data of Antisera & Antibodies Lolitrem antisera have been raise according to the procedures described 25 herein. One has been fully characterised and under standard ELISA conditions this antiserum was found to cross-react only with lolitriol. Monoclonal antibodies have also been raised and a lolitriol specific antibody has thus far been identified. 21 ?7,6 Table 1 Cross-reactivity data for a Lolitrcm-B specific antiserum raised in sheep is as follows: Analogue % Cross Reactivity Lolitrem-B 100 Lolitriol 1400 Paxilline <0.25 a-paxitriol <0.25 fl-paxitriol <0.25 fl-paxitrem <0.25 Aflatrem <0.25 The detection limit of this assay is 700pg Passive Vaccines - Compositions and Tx*eatments Preparations of antibodies and/or antisera thus produced may find several applications. Perhaps most important is as a passive vaccination for lolitrem 10 neurotoxins. A preparation comprising generic antibodies recognising antigens processing the lolitrem skeleton is one envisaged preparation. These antibodies may be polyclonal or monoclonal, such as have been described earlier. Another composition comprises antibodies specific to particular individual lolitrems or groups thereof. Yet a further composition may 15 comprise antibody fragments (Fab) typically immunoglobulin M and/or G classes. Various combinations of the foregoing are also envisaged and in most cases the antibodies and/or fragments will be added to a pharmacologically acceptable carrier or diluent. Methods of preparing such preparations are in accordance with standard techniques of vaccine preparation.

Methods of administration are typically through intravenous injection. Oral administration is not normally possible due to lysis and denaturation of protein matter by the digestive system. It is envisaged that the greatest 22 potential use of such a passive vaccine will be for domestic grazing animals, especially those likely to ingest rye grasses or other food-stuffs containing lolitrem compounds. The application to other animals is also envisaged.

An example of the preparation and administration of compositions suitable as 5 passive vaccines or remedial treatments, the procedures as outlined in Wenger, T.L., Butler, V.P., Haber E., & Smith, T.W.: "Treatment of 63 Severely Digitalis-Toxic Patients With Digoxin-Specific Antibody Fragments". JACC (Journal of the American College of Cardiology) Vol 5 No. 5 (May 1985) ppll8A-123A may be followed. In lieu of digoxin-specific antibodies/fragments 10 may be substituted antibodies and fragments reactive to the lolitrem skeleton. Some modification of these procedures may be necessary according to the nature of the animal patient, though the nature of these changes would be apparent to a skilled addressee of the art and/or after typical preliminary investigative trials to fine-tune the procedures.

The aforementioned preparations may therefore be useful as a remedial treatment for lolitrem intoxication or poisoning. Known procedures and knowledge relating to such remedial treatments may be employed in the preparation and administration of any such treatment or remedy. The nature of vaccine and remedial preparations and dosage will vary according to 20 animal type, severity of complaint etc.

A further application of haptens, immunogens and antibodies/antisera produced herein is in association of lolitrem assay techniques. A wide variety of standard immunoassay techniques are known and are reviewed in publications such as "Immunoassays for Veterinary and Food Analysis -1" 25 edited by B.A. Morris, M.N. Clifford & R. Jackman, Publ. Elsevier 1988. Typical techniques which are envisaged include LISA and ELISA though any other appropriate or comparable technique may also be used.

Immunoassay employing anti-lolitrem-B antibodies The standard competition ELISA was employed for initial assay (e.g. 30 Garthwaite et al, 1990). Briefly, 50f.il BSA-lolitrem-B conjugate at l-10|ig/ml 23 o ^ 7 c was incubated in each well of a 9G-\vell microtitre plate at 4°C overnight. Wells were washed once with PBS, and 200|il blocking agent (e.g. 1% ovalbumin [w/v] in PBS) added and incubated for 1 hour at room temperature. These were aspirated and washed once in PBS before addition of sample antibody (total 5 100(il). This solution was incubated for 1-2 hour at room temperature. Wells were washed twice with PBS-NP40 (0.05% v/v), twice with PBS and aspirated.

HRP-labelled goat anti-mouse Ig was diluted 1/1500 in blocking solution, and distributed to the plate (100j.il/well). This was incubated for 2 hours, before a final wash step (twice in PBS-NP40 and twice in PBS). Bound antibody was 10 disclosed by the addition of substrate solution, e.g. ABTS (Sigma 1888); the substrate (ABTS) was dissolved at 0.5mg/ml in 0.05M citrate buffer pH 4.0 immediately before use 0.25(.il of 30% hydrogen peroxide was added per ml buffer. After the substrate solution had been added (200|il per well), the plates were incubated in the dark at RT for 15 min and the absorbence at 414 nm 15 determined. Wells lacking in analyte showed an intense green colour, whilst those containing high concentrations remained colourless.

The ability to readily add various groups to the lolitrem skeleton, especially through the use of lolitriol, is advantageous in immunoassay techniques for 20 lolitrems. Conjugate bridges containing readily traceable functions or features are readily incorporated to produce a range of suitable antigens or immunogens. Thus radioactive, fluorescent or otherwise detectable moieties may be readily incorporated. A common method would revolve around the use of a tritium containing conjugate bridge obtained by reacting a tritium labelled 25 compound with lolitriol or a lolitrem. Other labelled atoms and isotopes may also be employed.

That a wide range of conjugate bridges are possible, also enhances the ability to bind the lolitrem skeleton to a wide range of known enzymes. Some enzymes are more suitable for immunoassay use than others (see previous comments 30 on colorimetric detection); the selection of enzymes having a high activity or specific quality is made easier. 24 An assay or diagnostic test kit, incorporating an antibody recognising or reactive to the lolitrem skeleton, or fragment thereof, may be provided. The ability of incorporate a wide range of variously detectable moieties will broaden the range of conditions (be it field or labj under which the kit may be used. An 5 embodiment of a kit may be based upon any of the assay techniques described or referenced herein and may be used (or be specifically adapted) for a wide range of applications including detecting the presence/and or quantity of lolitrems, lolitrem antigens, antibodies etc.

The invention described herein relates to methods of producing various 10 lolitrem derivatives including haptens, immunogens and antigens as a whole, and antibodies and antisera specific to lolitrem type compounds. While several different methods and syntheses have been described, it is noted that these are representative only and do not cover all variations which are envisaged to be within the scope of the invention. Uses of these compounds and groups have 15 also been described, including preparations for use as a passive vaccination and remedial treatment for the neurotoxic effects of lolitrems. Use in immunoassay techniques have also been described. It is once again noted that only several representative methods and techniques have been described. Known scientific methods and procedures which would be readily known to a 20 skilled laboratory worker or addressee of the art have not been described in detail - to do so would be laborious and time consuming. Insofar as various assay techniques are concerned, these are commonly known and performed - a reference text has been previously mentioned and is included in this specification by way of example. The neurotoxic effects of some lolitrems and 25 their structural data and characterisation may be found in the article "Tremoreenic Neurotoxins from Perennial Ryegrass causing Ryegrass Staggers Disorder of Livestock: Structure Elucidation of Lolitrem B" -Gallagher, Hawkes, Steyn and Vleggar, Journal of the Chemical Society, Chemical Communications (J. Chem. Soc., Chem. Commun.,) (1984) 614-6, 30 which is also included by way of example.

Other references (included by way of example) to which the reader may refer relating to the production of hybridoma cell lines and immunoassay techniques, are: Garthwaite, I., Stead, A.D., & Rider, C.C.: "A monoclonal antibodv-based 5 immunoassay for the polvmines spermine and spermidine." Biochem, Soc. Trans. 17(1989) 1056-1057.

Kearney, J. F., Radbruch, A., Liesegang, B., & Rajewsky, K.,: "A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines." J. Immunol. 124 (1979) 10 1548-1551.

Kohler, G. & Milstcin, C.: "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature 256 (1975)495-97.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made 15 thereto without departing from the scope thereof as defined in the appended claims. 26

Claims (47)

WHAT WE CLAIM IS: 23 9

1. A compound, useful for the preparation of haptens and conjugates, of the formula: O- H,OH OH

2. A method for the synthesis of the compound of claim 1 comprising the hydrolysis of a lolitrem.

3. A method for the synthesis of a lolitrem derivative which includes a fragment of the formula O- 1 I - comprising the reaction of the compound of claim 1 to add a conjugate bridge to the position indicated A, said conjugate bridge comprising a member of the group: n-alkyl, iso-alkyl, alkenyl, aryl, alkynyl, aromatic, amide, oxime, epoxide, carboxylic acid, ester, activated ester, aldehyde, acetyl and ketal groups.

4. A method as claimed in claim 3 wherein the compound of claim 1 is acylated.

5. A method as claimed in claim 3 wherein the compound of claim 1 is reacted to form an acetal or ketal. 27 gz\ *"7;p i I %;) \J;79;

6. A method as claimed in claim 3 wherein the compound of claim 1 is esterified.;

7. A method as claimed in claim 3 wherein the compound of claim 1 is reacted with an alkoxypropene.;

8. A compound including a fragment of the formula;O;prepared via a method according to any one of claims 3 through 7.

9. A compound as claimed in claim 8 of the general formula:;OH;hnr;

10. A compound as claimed in claim 8 of the general formula:;0-;• \;V \;C';i3si;28;

11. A compound as claimed in claim 8 of the general formuTa:;O;36879;COOH;

12. A compound as claimed in claim 8 of the general formula:;0-;

13. A compound as claimed in claim 8 of the general formula:;O-;OH COOH;HnV;

14. A method for the preparation of an antigen or immunogen comprising the reaction of a hapten as claimed in any one of claims 8 through 13 or as prepared by a method as claimed in any one of claims 3 through 7, with a carrier.;29;235379;

15. A method as claimed in claim 14 wherein a carrier comprises a member of the group comprising:;a polypeptide, a protein, an enzyme, ovalbumin, BSA, thyroglobulin, haemocyanin, polystyrene or a DNA fragment, horseradish peroxidase, alkaline phosphatase, an enzyme having a detectable reaction with a substrate, and an agarose.;

16. An antigen or immunogen having the lolitrem skeleton as an antigenic determinant.;

17. An antigen or immunogen as claimed in claim 16, prepared by a method according to either claim 14 or claim 15.;

18. A monoclonal antibody, or fragment thereof, able to recognise and react with a substance whose antigenic determinant comprises:;

19. The antibody of claim 18, immunoreactive to a lolitrem.;

20. The antibody of claim 18, immunoreactive to a compound as claimed in any one of claims 8 through 13.;

21. The antibody of claim 18, immunoreactive to an antigen or immunogen as claimed in either claim 16 or claim 17.;

22. A hybridoma capable of producing a monoclonal antibody as claimed in any one of claims 18 through 21.;30;23687;

23. A method of generating a hybridoma capable of producing a monoclonal antibody as claimed in any one of claims 18 through 21, the method comprising the derivation of the hybridoma from antibody secreting cells or lymphocytes obtained by immunising an animal with a substance having an antigenic determinant comprising:;

24. A polyclonal antibody able to recognise and react with a substance whose antigenic determinant comprises:;

25. A method of preparing a polyclonal antibody as claimed in claim 24 by the administration of an antigen or immunogen as claimed in any one of claims 14 through 17 to an animal.;31;OJT7;// H;e.*J ^ /

26. A method for the purification of antibodies as claimed in any one of claims 18 through 21 or in claim 24, comprising the use of an affinity chromatography column having an immobilised compound or derivative including a fragment of the formula: -tC\

27. An affinity chromatography column useful for the separation or purification of antibodies as claimed in any one of claims 18 through 21 or in claim 24 having a compound or derivative including a fragment of the formula 0- bound to a column support.

28. An affinity chromatography column as claimed in claim 27 wherein the column support is an agarose.

29. An affinity chromatography column as claimed in either claim 27 or claim 28 wherein a compound as claimed in any one of claims 8 through 13 is reacted to bind to the column support.

30. A passive vaccine comprising an antibody as claimed in any one of claims 18 through 21 or claim 24, and/or an antibody fragment, together with a diluent. 32 n

31. A passive vaccine according to claim 30 in a form suitable for intravenous injection.

32. A composition addressing lolitrem poisoning comprising an antibody or fragment thereof, reactive to an antigenic determinant comprising: -tC\

33. A method addressing lolitrem toxicity, other than in humans, comprising the administration of a composition as claimed in claim 32.

34. An in vitro immunoassay method of determining lolitrems or substances incorporating the lolitrem skeleton, using an antibody as claimed in any one of claims 18 through 21 or claim 24, according to standard ELISA or immunoassay techniques.

35. An immunoassay kit comprising an antibody as claimed in any one of claims 18 through 21 or claim 24.

36. A hapten including a fragment of the formula: O— ~?C\ HQ-] substantially as described herein with reference to the contained examples. 33 C-) \ ■ £ O L 0 ^

37. An antigen or immunogen including a fragment of the formula: O- 79 substantially as described herein with reference to the contained examples.

38. A monoclonal antibody substantially as described herein with reference to the contained examples.

39. A method of producing a hapten, substantially as described herein with reference to the contained examples.

40. A method of producing an antigen or immunogen, substantially as described herein with reference to the contained examples.

41. A method of producing a monoclonal antibody, substantially as described herein with reference to the contained examples.

42. A hybridoma substantially as described herein with reference to the contained examples.

43. A passive vaccine substantially as described herein with reference to the contained examples. 34 r-J

44. A composition addressing lolitrem poisoning, said composition including an antibody or fragment thereof, reactive to an antigenic determinant comprising: said composition being substantially as described herein with reference to the contained examples.

45. A method of immunoassay, substantially as described herein with reference to the contained examples.

46. An immunoassay test kit, substantially as described herein with reference to the contained examples.

47. A Lolitrem-B specific antiserum substantially as described herein with reference to Table 1. Her Majesty the Queen in right of New Zealand acting by and through the Minister of Agriculture hv Her Attorneys 35