Background of the Invention Various lanthanide complexes have been developed for use in time-resolved fluoroimmunoassays and other immunoassay techniques, as they are well suited to act as probes for biological molecules such as proteins. They may for example be used to attach to protein molecules which are antibodies, in order to form a probe for the corresponding antigen.

Suitable compounds for forming such lanthanide complexes contain a beta-diketone structure, which is well suited to complexing with lanthanide ions. In order to be suitable for use in immunoassays, the complex must be attachable to a target molecule, which may be a protein, in such a manner that the structure of the target molecule is not unduly disrupted.

Matsumoto et al (US 5,859, 297 and US 6,166251) have published the structures of several compounds that can be complexed with a lanthanide ion to form fluorescent probes. One of the structures described by Matsumoto is a o-terphenyl structure containing two beta-diketone groups connected to perfluoroalkyl groups, and with a tether group for attaching the molecule to proteins. Of the tether groups described by Matsumoto, the preferred group is the chlorosulfonyl group. Although this group has a high reactivity towards amino groups in proteins, it has several disadvantages. It is a highly reactive and non-selective group, and can potentially react with many other molecules in the system, for example alcohols. Further, it is a small group, so that the aromatic nucleus of the fluorescent probe will be located very close to the protein molecule, potentially leading to significant disruption of the protein structure. The terphenyl compound containing the chlorosulfonyl group described by Matsumoto is susceptible to hydrolysis, requiring storage in strictly anhydrous conditions. Also, it is difficult to purify and decomposes under the conditions of many common purification methods such as hplc. Consequently a greater amount of the compound is required in order to get a desired level of activity relative to a more readily purified compound. Since some of the impurities may be capable of binding to a protein, but be incapable of complexing with a lanthanide, more attachments to the protein molecule will likely be formed, leading to greater potential for disrupting the structure of a protein to which it is

attached. Additionally, the compound is quite hydrophobic, making its reaction with proteins in aqueous solution problematic. Finally, the adduct of the terphenyl compound containing the chlorosulfonyl group described by Matsumoto with a protein is commonly quite unstable, particularly at high fluorophore to protein (F/P) ratios, and requires storage at low temperature and prompt use following its manufacture.

Matsumoto also describes other tether groups, however no methods are described for attaching those tether groups to an aromatic nucleus. Many of the tether groups described are not easily attached to the o-terphenyl nucleus.

There is therefore a need for a purifiable compound with improved hydrophilicity, capable of complexing with a lanthanide ion, and which contains a tether group capable of binding to proteins, wherein the compound itself and its adduct to a protein are relatively stable. Furthermore, there is a need for such a compound in which the tether group allows the bulky portion of the molecule to reside at a distance from the protein molecule to which it may be attached such that there is minimal disruption to the structure of the protein molecule.

Object of the Invention It is the object of the present invention to overcome or substantially ameliorate at least one of the above disadvantages.

Summary of the Invention In one form, the invention provides a chelating compound comprising a metal complexing moiety, a linker group joined to the metal complexing moiety through a -SO2-group, and a coupling moiety joined to the linker group and capable of reacting with an amine containing species to couple the metal complexing moiety to the amine containing species through the linker group.

The metal complexing moiety may comprise a terphenyl nucleus, and may comprise two beta-diketone groups (or keto-enol forms thereof) joined thereto such that they are capable of complexing a metal species (for example a metal atom or a metal ion). The two beta-diketone groups may be capable of complexing the same metal species. They may have electron withdrawing groups such as perfluoroalkyl groups (e. g. perfluoropropyl groups) bonded thereto. Thus the metal complexing moiety may comprise a terphenyl nucleus and two electron withdrawing groups, each of which is joined to the terphenyl nucleus through a beta-diketone group. The linker group may be a hydrophilic linker group, and may have at least 4 atoms in its main chain. The coupling moiety may comprise a carbon atom having three chalcogens attached thereto, and may comprise for

example a carbonate, thiocarbonate, dithiocarbonate or trithiocarbonate group. It may comprise an activated leaving group, i. e. a leaving group capable of being displaced by an amine group (for example the amine group on a protein) in order to couple the metal complexing species to an amine containing species of which the amine group is a portion.

The activated leaving group may comprise a nitrogen heterocycle, for example an N- succinimido group.

In an embodiment, there is provided a chelating compound of formula I

wherein: - T is a terphenyl nucleus; - EWG1 and EWG2 are electron withdrawing groups; - L is a linker group; and - C is a coupling moiety; whereby the two beta-diketone groups are capable of complexing a metal species.

The two beta-diketone groups may be oriented so as to be capable of complexing a metal species. They may be capable of complexing the same metal species. The metal species may be for example a metal atom or a metal ion.

In a first aspect of the invention there is provided a chelating compound of formula 1 wherein: EWG1 and EWG2 are electron withdrawing groups, - X, Y and Z are chalcogens, - R is selected from the group consisting of hydrogen, an alkyl group, an alkenyl group, an alkynyl group and an aryl group,

-A is a group such that-N (R)-A-X- is a hydrophilic linker group with at least 4 atoms in its main chain, and - Grp-Y is an activated leaving group.

EWG1 and EWG2 may the same or they may be different. They may independently be, for example, straight chain or branched chain perfluoroalkyl groups of formula- CnF2n+). Integer n may be between 1 and 15 or between 1 and 10 or between 1 and 5, and may be 1,2, 3,4, 5,6, 7,8, 9,10, 11,12, 13,14, or 15 or may be greater than 15.

X, Y and Z may be the same or they may be all different or two may be the same and one different. They may be independently selected from the group consisting of oxygen, sulfur, selenium and tellurium.

- N (R)-A-X- may have at least 4 atoms in its main chain, or at least 5,6, 7,8, 9,10, 15 or 20 atoms in its main chain, or it may have between 4 and 20 atoms or between 5 and 18 or between 6 and 16 or between 7 and 14 or between 8 and 12 atoms, and may have 4, 5, 6,7, 8,9, 10,11, 12,13, 14,15, 16,17, 18,19 or 20 atoms in its main chain.

A may for example be (CH2) p, where p is a non-negative integer, and may be for example 1, 2,3, 4 or 5. One or more of the hydrogen atoms of A may be substituted by a group B, which may independently be a halogen, an alkyl, aryl, alkenyl or alkynyl group, an alcohol group, an amine group, an ether group or some other group. A may contain one or more ether groups, and may contain for example 1,2, 3,4 or 5 ether groups. For example A may be (CH2CH20) qCH2CH2, where q is a non-negative integer and may be for example 0, 1, 2,3, 4 or 5.

R may be hydrogen, or it may be an alkyl group or an alkenyl group or an alkynyl group or an aryl group. The alkyl group may be straight chain or branched and may have between 1 and 10 carbon atoms, or between 1 and 8 atoms or between 1 and 6 carbon atoms, and may have 1,2, 3,4, 5,6, 7,8, 9 or 10 carbon atoms. The alkenyl group may have between 2 and 10 carbon atoms, or between 2 and 8 carbon atoms or between 2 and 6 carbon atoms and may have 2,3, 4,5, 6,7, 8,9 or 10 carbon atoms. The alkynyl group may have between 2 and 10 carbon atoms, or between 2 and 8 carbon atoms or between 2 and 6 carbon atoms and may have 2,3, 4,5, 6,7, 8,9 or 10 carbon atoms. The aryl group may be aromatic or it may be heteroaromatic. The alkyl, alkenyl, alkynyl or aryl group may be substituted and may be multiply substituted. There may be between 0 and 5 substituents, or between 0 and 4 or between 0 and 3 or between 0 and 2 substituents.

There may be 0,1, 2,3, 4 or 5 substituents. The substituents may be the same or they may be different.

Grp may be non-aromatic or it may be aromatic. The atom of Grp that is attached to Y may be a heteroatom, for example N, P, O or S, or may be carbon. Grp may be for example a heterocyclic group, an aromatic group, a heteroaromatic group or an aliphatic group.

In an embodiment, EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is (CH2) 3 and Grp is N-succinimido.

In a second aspect of the invention there is provided a process for making a chelating compound comprising the steps of : - reacting a sulfonyl chloride of formula 2, with a compound of formula HN (R) -A-XH to form an intermediate, and - reacting the intermediate with a compound of formula 3, wherein EWG1, EWG2, X, Y, Z, A, R and Grp are defined as in the first aspect of the invention.

X'is selected from the group consisting of oxygen, sulfur, selenium and tellurium.

Grp'-X'is an activated leaving group. Grp'may be non-aromatic or it may be aromatic. The atom of Grp'that is attached to X'may be a heteroatom, for example N, P, O or S, or may be carbon. Grp'may be for example a heterocyclic group, an aromatic group, a heteroaromatic group or an aliphatic group. X'may be the same as or different to any one or more of X, Y and Z, and Grp'may be the same as or different to Grp.

The process may additionally comprise one or more of the steps of purifying the intermediate and purifying the chelating compound.

In an embodiment, EWG1 and EWG2 are both C3F7, X, X', Y and Z are all oxygen, R is hydrogen, A is (CH2) 3 and Grp and Grp'are both N-succinimido.

In a third aspect of the invention there is provided a chelate comprising: - a chelating compound according to the invention, and - a metal ion,

wherein the metal ion is chelated by the chelating compound. The metal ion may be a lanthanide ion or an actinide ion. The metal ion may be selected from the group consisting of Sm3+, Eu3+, Tb3+ and Dy3+ for example. The metal ion may be chelated by the beta-diketone groups of the chelating compound.

In an embodiment, the chelating compound has structure (1), the metal ion is Eu3+, EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is (CH2) 3 and Grp is N-succinimido.

In a fourth aspect of the invention there is provided a process for making a chelate comprising treating a chelating compound according to the invention with a metal ion.

The metal ion may be a lanthanide ion or an actinide ion. The metal ion may be selected from the group consisting of Sm3+, Eu3+, Tb3+ and Dy3+ for example. The treating may comprise treating the chelating compound with a solution of the metal ion, and the solution may be an aqueous solution.

In an embodiment, the chelating compound has structure (1), the metal ion is Eu3+, EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is (CH2) 3 and Grp is N-succinimido.

In a fifth aspect of the invention there is provided a tagged species comprising: - a species, and - one or more chelating groups chemically bonded to the species, wherein the chelating group has the structure shown in formula 4 and EWG1, EWG2, X, Z, R and A are as described in the first aspect of the invention.

The chelating group (s) may be attached to the species through nitrogen atom (s) of said species. The nitrogen atom (s) may be part of amine group (s) and the amine group (s) may be primary or secondary amine group (s). The species may be for example a protein, an antibody, a portion of a protein, a portion of an antibody, a peptide, an aminosaccharide, a synthetic aminopolymer or an aminofunctional surface.

In an embodiment the species is a protein or a portion of a protein or a peptide and the tagged species is a tagged protein or a tagged peptide. There may be on average

between about 1 and 20 chelating groups per molecule of tagged protein or tagged peptide, and may between about 1 and 10 or between about 1 and 8 or between about 1 and 6 or between about 1 and 4, and there may be about 1,2, 3,4, 5,6, 7,8, 9,10, 11,12, 13,14, 15,16, 17,18, 19 or 20 chelating groups per molecule of tagged protein or tagged peptide. The chelating group (s) may be attached to the protein or portion of a protein or peptide through for example lysine residue (s) therein.

In another embodiment, EWG1 and EWG2 are both C3F7, X, and Z are both oxygen, R is hydrogen and A is (CH2) 3.

In a sixth aspect of the invention there is provided a process for producing a tagged species comprising reacting a species with a chelating compound according to the invention. The process may comprise reacting the species with a solution, suspension or emulsion of the chelating compound and the solution, suspension or emulsion may be an aqueous solution, suspension or emulsion, and may also comprise one or more other components. The one or more other components may be for example surfactants, emulsifiers, buffers or other additives. Alternatively the solution, suspension or emulsion may comprise a non-aqueous solvent, and said solvent may be miscible with water. The solvent may be for example DMF, THF, acetone or 1,4-dioxane. The process may comprise reacting amine group (s) on the species with the chelating compound, and the amine groups may be primary or secondary amine group (s).

In an embodiment the species is a protein or a portion of a protein or a peptide and the tagged species is a tagged protein or a tagged peptide. The process may comprise reacting amine group (s) of, for example, lysine residue (s), with the chelating compound.

In another embodiment, the chelating compound has structure (1), EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is (CH2) 3 and Grp is N-succinimido.

In a seventh aspect of the invention there is provided a labelled species comprising: - a tagged species according to the fifth aspect of the invention, and - at least one metal ion chelated by the chelating groups of the tagged species.

The metal ion may be a lanthanide ion or an actinide ion. The metal ion may be selected from the group consisting of Sm3+, Eu3+, Tb3+ and Dy3+ for example. There may be one metal ion per chelating group, or there may be on average less than one metal ion per chelating group. There may be between about 0.5 and 1 metal ion per chelating group, or between about 0.6 and 1 or between about 0.7 and 1 or between about 0.8 and 1 or between about 0.9 and I metal ion per chelating group. There may be about 0.5, 0.6, 0.7,

0.8, 0.9 or 1 metal ion per chelating group. The chelating group (s) may be attached to the species through nitrogen atom (s) of said species. The at least one metal ion may be chelated by beta-diketone group (s) of the chelating group (s).

In an embodiment the tagged species is a tagged protein.

In another embodiment, the metal ion is Eu3+, EWG1 and EWG2 are both C3F7, X and Z are both oxygen, R is hydrogen and A is (CH2) 3.

In an eighth aspect of the invention there is provided a process for making a labelled species, said process comprising reacting a tagged species according to the fifth aspect of the invention with a metal ion. The metal ion may be a lanthanide ion or an actinide ion. The metal ion may be selected from the group consisting of Sm3+, Eu3+, Tb3+ and Dy3+ for example. The reacting may comprise reacting the tagged species with a solution of the metal ion, and the solution may be an aqueous solution.

In an embodiment the tagged species is a tagged protein.

In another embodiment, the metal ion is Eu3+, EWG1 and EWG2 are both C3F7, X and Z are both oxygen, R is hydrogen and A is (CH2) 3.

In a ninth aspect of the invention there is provided a process for making a labelled species, said process comprising reacting a species with a chelate according to the third aspect of the invention. The process may comprise reacting the species with a solution, suspension or emulsion of the chelate and the solution may be an aqueous solution, suspension or emulsion, and the solution, suspension or emulsion may also comprise one or more other components. The one or more other components may be for example surfactants, emulsifiers, buffers or other additives. Alternatively the solution, suspension or emulsion may comprise a non-aqueous solvent, and said solvent may be miscible with water. The solvent may be for example DMF, THF, acetone or 1,4-dioxane. The process may comprise reacting amine group (s) on the species with the chelate, and the amine groups may be primary or secondary amine group (s).

In an embodiment the species is a protein or a portion of a protein. The process may comprise reacting amine group (s) of, for example, lysine residue (s), with the chelate.

In another embodiment, the metal ion is Eu3+, EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is (CH2) 3 and Grp is N-succinimido.

In a tenth aspect of the invention there is provided a reagent comprising a chelating compound according to the invention or a chelate according to the invention. There is also provided the use of a chelating compound according to the invention or a chelate according to the invention for labelling a species. The species may be for example a protein or a peptide.

In an eleventh aspect of the invention there is provided the use of a chelating compound according to the invention, or a chelate according to the invention, or a tagged species according to the invention, a labelled species according to the invention or a reagent according to the invention, in an immunoassay.

The present invention also provides a kit for use in an immunoassay comprising: - a first part comprising a tagged species according to the invention; and - a second part comprising a metal ion capable of being chelated by the chelating groups of the tagged species.

Detailed Description of the Invention In the present specification the following definitions are used: complex: a chemical species in which at least one metal ion is associated with pi- electrons of a complexing compound; chelate : a chemical species in which at least one metal ion is associated with pi- electrons of a chelating compound in at least two distinct regions of the chelating compound, so that a metal ion and the chelating compound form a cyclic structure; complexing compound: a chemical compound capable of forming a complex with a metal ion; chelating compound: a chemical compound capable of forming a chelate with a metal ion; chelating group: a portion of a chelating compound, said portion having pi- electrons in at least two distinct regions, so that a metal ion and the chelating group are capable of forming a cyclic structure activated leaving group : a leaving group capable of being displaced by an amine group, for example a primary or secondary amine group.

In the above definitions, it will be understood that it is not necessary that all metal ions are capable of forming a complex or a chelate, only that there exists at least one metal ion so capable.

Throughout this specification, EWG1, EWG2, X, Y, Z, R, A and Grp are defined as in the first aspect of the invention.

The present invention relates to a chelating compound comprising a metal complexing moiety, a linker moiety joined to the metal complexing moiety through a -SO2-group, and a coupling moiety joined to the linker group and capable of reacting with an amine containing species to couple the metal complexing moiety to the amine containing species through the linker moiety. An example of such a chelating compound

is provided in formula (1). Conveniently, the metal complexing moiety may comprise a terphenyl nucleus and two electron withdrawing groups, each of which is coupled to the terphenyl nucleus by a beta-diketone group. This structure has previously been shown to complex metal ions, for example lanthanides and actinides to provide a fluorescent product. When complexed with a lanthanide or an actinide, for example Sm3+, Eu3+, Tb3+ or Dy3+, the resulting chelate may be fluorescent, and may exhibit high fluorescence intensity. A labelled species comprising the chelate may also exhibit high fluorescence.

A feature that may be exhibited by the chelating compound of the present invention is relative ease of synthesis. Features of the chelating species that provide ease of synthesis include the-S02-group and, if present the carbonate group of the coupling moiety and the amine function of the linker moiety. Thus the-S02-group may readily be introduced, using known methods, into the aromatic nucleus in the form of an-S02CI group, which may be reacted with an aminoalcohol to introduce an aminofunctional linker group. This may be reacted with a suitable carbonate species to introduce a coupling moiety into the chelating compound in the form of a carbonate group comprising an activated leaving group. As is described herein, other variations of the groups described above are envisaged within the scope of the present invention.

The site of chelation of a metal ion to chelating compounds of formula 1 is thought to be at the beta-diketone groups. It will be readily understood by one skilled in the art that the structure shown in formula 1 (a conjugated keto-enol) is a tautomeric structure to the beta-diketone structure and may be considered equivalent thereto. The postulated site of chelation is embedded in a hydrophobic region of the molecule, having a large aromatic nucleus attached to it. In an example the chelation site is also near a perfluoroalkyl group which enhances the hydrophobicity of the region. The hydrophobicity of this region is thought to enhance the fluorescence of a chelate which comprises a chelating compound of formula 1 and a metal ion by reducing the access of water molecules to the chelated metal ion. Water molecules can reduce the fluorescence of the chelate by providing a radiationless decay pathway for an excited metal ion. The electron withdrawing groups of formula 1 may be perfluoroalkyl groups, which may be linear or they may be branched, or one may be linear and one may be branched. They may be the same or they may be different. The numbers of carbon atoms in the perfluoroalkyl groups may be selected to provide a suitable level of hydrophobicity. They may independently be between 1 and 15 or between 1 and 10 or between 1 and 5, and may be 1,2, 3,4, 5,6, 7, 8, 9,10, 11, 12,13, 14, or 15 or one or both may be greater than 15.

It is postulated that chelation of the beta-diketone with a metal ion is enhanced if that group is in the keto-enol form C (O)-C=C (OH). The keto-enol form is thought to be favoured when the group is attached to one electron-donating group and one electron- withdrawing group. The terphenyl nucleus is a suitable electron-donating group, and perfluoroalkyl groups as described above are suitable electron-withdrawing groups (EWG1 and EWG2). Other electron-withdrawing groups that may be suitable for use in the present invention include nitrated, polynitrated, halogenated (fluorinated, chlorinated, brominated or iodinated) and polyhalogenated alkyl, alkenyl and aryl groups, for example trinitromethyl, trihalomethyl, perhaloethyl, perhaloethenyl, trihalophenyl, tetrahalophenyl, pentahalophenyl or trinitrophenyl, nitrile groups or other electron- withdrawing groups known to those skilled in the art. The alkyl group may be straight chain or branched and may have between 1 and 10 carbon atoms, or between 1 and 8 atoms or between 1 and 6 carbon atoms, and may have 1,2, 3,4, 5,6, 7,8, 9 or 10 carbon atoms. The alkenyl group may have between 2 and 10 carbon atoms, or between 2 and 8 carbon atoms or between 2 and 6 carbon atoms and may have 2,3, 4,5, 6,7, 8,9 or 10 carbon atoms and may have one or more double bonds.

The tether group Grp-YC (Z) X-A-N (R)-S (O) 2- of the chelating compound of formula 1 should be hydrophilic, so that said chelating compound is sufficiently hydrophilic to be conveniently reacted with a protein in aqueous solution. A sufficient degree of hydrophilicity may be achieved when the compound HX-A-N (R) H is water soluble.

A may for example be (CH2) p, where p is a non-negative integer, and may be for example 1,2, 3,4, 5 or more than 5. One or more of the hydrogen atoms of A may be substituted by a group B, which may independently be a halogen, an alkyl, aryl, alkenyl or alkynyl group, an alcohol group, an amine group an ether group or some other group.

A may contain one or more ether groups, and may contain for example 1,2, 3,4 or 5, or more than 5 ether groups. For example A may be (CH2CH20) qCH2CH2, where q is a non- negative integer and may be for example 0,1, 2,3, 4 or 5 or more than 5. Alternatively A may be of the form Li, LjOLz, LiOL20L3 or LlOL2O. Lr lOLrX where r is a non- negative integer and may be for example 4 or 5 or more than 5, and each of Li, L2,..., and Lr is an alkylene group with between 1 and 4 carbon atoms, and may be straight chain or branched chain and may be substituted or unsubstituted. LI, L2,..., Lr may be the same or they may be different or some may be the same and some may be different.

R may be hydrogen, OR', SR', OH, SH, alkyl, alkenyl, alkynyl or aryl, where R' may be alkyl, alkenyl, alkynyl or aryl. The alkyl group may be straight chain or branched

and may have between 1 and 10 carbon atoms, or between 1 and 8 atoms or between 1 and 6 carbon atoms, and may have 1,2, 3,4, 5,6, 7,8, 9 or 10 carbon atoms. The alkyl group may be for example methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-1- propyl, 2-methyl-2-propyl or some other group. The alkenyl group may have between 2 and 10 carbon atoms, or between 2 and 8 carbon atoms or between 2 and 6 carbon atoms and may have 2,3, 4,5, 6,7, 8,9 or 10 carbon atoms and may have one or more double bonds. The alkenyl group may be for example vinyl, allyl, 2-propenyl, but-1-en-1-yl, but- 1-en-2-yl, but-1-en-3-yl, but-1-en-4-yl or some other alkenyl group. The alkynyl group may have between 2 and 10 carbon atoms, or between 2 and 8 carbon atoms or between 2 and 6 carbon atoms and may have 2,3, 4,5, 6,7, 8,9 or 10 carbon atoms. The alkynyl group may be for example ethynyl, 1-propynyl, 2-propynyl, but-1-yn-1-yl, but-1-yn-3-yl, but-1-yn-4-yl or some other alkynyl group. The aryl group may be aromatic or it may be heteroaromatic, and may be for example phenyl, naphthyl, anthracyl, pyridyl, furyl, thiophenyl, pyrazinyl or some other aromatic or heteroaromatic group. The alkyl, alkenyl, alkynyl or aryl group may be substituted and may be multiply substituted, and the substituent may be for example alkyl, alkoxycarbonyl, nitro, hydroxyl, a halogen or some other group. There may be between 0 and 5 substituents, or between 0 and 4 or between 0 and 3 or between 0 and 2 substituents. There may be 0, 1, 2,3, 4 or 5 substituents or there may be more than 5 substituents. The substituents may be the same or they may be different.

X, Y and Z may be the same or they may be all different or two may be the same and one different. They may be independently selected from the group consisting of oxygen, sulfur, selenium and tellurium.-N (R)-A-X- may have at least 4 atoms in its main chain, or at least 5,6, 7,8, 9 or 10 atoms in its main chain, or it may have between 4 and 20 atoms or between 5 and 18 or between 6 and 16 or between 7 and 14 or between 8 and 12 atoms, and may have 4,5, 6,7, 8,9, 10,11, 12,13, 14,15, 16,17, 18,19 or 20 atoms in its main chain or it may have more than 20 atoms in its main chain.

The atom of Grp that is attached to Y may be a heteroatom, for example N, P, O or S, or may be carbon. Grp may be for example a heterocyclic group, an aromatic group, or a heteroaromatic group. Grp may have between 4 and 12 atoms in one or more rings, each of said atoms being either carbon or a heteroatom. Grp may have between 4 and 8 atoms or between 4 and 6 atoms, and between 1 and 3 rings, and may have 4,5, 6,7, 8,9, 10,11 or 12 atoms and may have 1,2 or 3 rings. For example Grp may be N-succinimido, N- maleimido, N-pyrrolidinyl, N-imidazolyl, N-pyridyl, N-pyrrolyl, N-indolyl, N-pyrazolyl, N-quinolinyl, N-morpholinyl, N-oxazolidinyl,, N-hydroxy-sulfo-succinimido, phenyl, p-

nitrophenyl, pentafluorophenyl, phenylsulfonyl, a substituted form of any of these, or some other group such that Grp-Y is a good leaving group. Grp may have one or more substituents, and the substituents may be for example halo (fluoro, chloro, bromo or iodo), nitrilo, nitro, Cl to C6 straight chain or branched alkyl, alkoxycarbonyl, aryloxycarbonyl, alkoxysulfonyl or aryloxysulfonyl, aldehyde, arylcarbonyl or alkylcarbonyl. The leaving group may be such that the compound Grp-YH is water soluble. Grp-YH may for example be N-hydroxy-sulfo-succinimide.

Representative tether groups therefore may be Succ-OC (O) 0- (CH2) 3-N (H)-S (0) 2-, or Succ-OC (O) 0- (CH2) 2-0- (CH2) 2-N (H)-S (O) 2-, or Succ-OC (O) 0- (CH2) 2-0- (CH2) 2-0- (CH2) 2-N (H)-S (0) 2-, where Succ represents N-succinimido or N-hydroxy-sulfo- succinimido.

Chelating compounds of formula 1 may be conveniently made from the corresponding sulfonyl chlorides of formula 2. In a first step, a sulfonyl chloride of formula 2 is reacted with an amine of formula HX-A-N (R) H. A catalyst may be employed in the reaction. The catalyst may be a tertiary amine, and may be for example dimethylaminopyridine (DMAP). The reaction may be conducted in a solvent, said solvent being a solvent for the sulfonyl chloride of formula 2, the amine of formula HX- A-N (R) H and DMAP. The solvent may be for example acetonitrile, 1,4-dioxane or tetrahydrofuran. The reaction may be conducted at room temperature or above room temperature or between room temperature and the boiling point of the solvent, and may be conducted at the reflux temperature of the solvent. The first step produces a sulfonamide intermediate which contains an-XH group.

In a second step, the-XH group of the sulfonamide intermediate is reacted with a compound of formula 3. The compound of formula 3 may be a carbonate, or it may be a thiocarbonate, a selenocarbonate, a dithiocarbonate, or any other compound of formula 3 wherein X', Y and Z are chalcogens which may be the same or they may be different or two may be the same and the other may be different. Grp is as defined earlier. Grp'may be a heterocyclic group, an aromatic group, or a heteroaromatic group. For example Grp' may be N-succinimido, N-maleimido, N-pyrrolidinyl, N-imidazolyl, N-pyridyl, N- pyrrolyl, N-indolyl, N-pyrazolyl, N-quinolinyl, N-morpholinyl, N-oxazolidinyl,, N- hydroxy-sulfo-succinimido, phenyl, p-nitrophenyl, pentafluorophenyl, phenylsulfonyl, a substituted form of any of these, or some other group such that Grp'-X'is a good leaving group. Grp'may have one or more substituents, and the substituents may be for example halo (fluoro, chloro, bromo or iodo), nitrilo, nitro, CI to C6 straight chain or branched alkyl, alkoxycarbonyl, aryloxycarbonyl, alkoxysulfonyl or aryloxysulfonyl, aldehyde,

arylcarbonyl or alkylcarbonyl. Grp and Grp'may be the same or they may be different. A catalyst may be employed in the reaction. The catalyst may be a tertiary amine, and may be for example DMAP. The reaction may be conducted in a solvent, said solvent being a solvent for the sulfonamide intermediate, the compound of formula 3 and DMAP. The solvent may be for example acetonitrile, THF, acetone or 1, 4-dioxane. The reaction may be conducted at room temperature or above room temperature or between room temperature and the boiling point of the solvent, and may be conducted at the reflux temperature of the solvent. Both the sulfonamide intermediate and the chelating compound of formula I may be purified by common purification methods. For example the chelating compound of formula 1 may be purified by precipitation from a solvent, for example from hexane, and may also be purified by HPLC, preferably reverse phase HPLC.

A chelate may be formed from a chelating compound of formula 1 and a suitable metal ion, for example a lanthanide ion or an actinide ion. Suitable ions include Sm3+, Eu3+, Tb3+ and Dy3+. In a chelate of a metal ion and a chelating compound of formula 1, the metal ion is thought to be associated with the two beta-diketone groups. The chelate may be formed by exposing a chelating compound of formula 1 to a solution of the metal ion. The chelating compound of formula 1 may be in solution. The solvent may be for example an alcohol or an ether or a cyclic ether or a mixture of two or more of these, and may be for example methanol, 1,4-dioxane or a mixture thereof. The solvent preferably does not contain amine or amide groups since these may react with the tether group of the chelating compound of formula 1 or its metal chelate. The metal ion may have a counterion and the counterion may be any counterion that enables the metal ion to be present in solution in sufficient concentration to form a suitable chelate with the chelating compound of formula 1. The counterion may be for example a halide, such as chloride, bromide or iodide or it may be some other suitable counterion. The solution of the metal ion may be in water.

The tether group of a chelating compound of formula 1, or of a chelate comprising a chelating compound of formula 1, may be used to attach a said chelating compound or chelate to a species to form a tagged species or a labelled species respectively. The species may have one or more amine groups, which are capable of reacting with the Grp- YC (Z) X-A- group of the chelating compound or chelate, according to equation 1 : Grp-YC (Z) X-A-+ spec-NH2 o spec-N (H) -C (Z) X-A- (1) where spec-NH2 is a species with one or more amine groups. The species may be a protein, and the protein may be for example an antibody. However other species may be

used, providing the species is capable of reacting with the group Grp-YC (Z) X-A-. The species may for example be an aminosaccharide or a natural or synthetic aminopolymer or an aminofunctional surface.

The species may be reacted in solution or it may be reacted while it is not in solution, for example using a two-phase process. During the reaction the chelating compound or chelate may be in solution or suspension or emulsion, and said solution suspension or emulsion may be aqueous or it may be non-aqueous. The reaction may be conducted at a convenient temperature at which both the chelating compound or chelate and the species are stable. The reaction temperature may be between about 15 and 35°C, or between about 20 and 30°C, and may be about 15,20, 25,30 or 35°C. The reaction time may be between 1 minute and 24 hours or between about 5 minutes and 20 hours or between about 10 minutes and 16 hours or between about 15 minutes and 12 hours or between about 20 minutes and 8 hours or between about 25 minutes and 4 hours or between about 30 minutes and 2 hours or between about 45 minutes and 1.5 hours, and may be about 1,2, 3,4, 5,10, 15,20, 25,30, 40 or 50 minutes or about 1,1. 5,2, 3,4, 5, 6,8, 12,16, 20 or 24 hours. Following the reaction, the tagged species or labelled species may be separated from unreacted chelating compound or chelate by for example a Sephadex column or a centrifugal filter, for example a 50KDa centrifugal filter.

If the reaction shown in equation 1 is conducted using a compound of formula 1, the product of that reaction is a tagged species, having structure spec-N (H) -Ter, where Ter is a chelating group of formula 4.

A chelate may be formed from a tagged species and a suitable metal ion, for example a lanthanide ion or an actinide ion. Such a chelate is a labelled species according to the seventh aspect of the invention. Suitable ions include Sm3+, Eu3+, Tb3+ and Dy3+. In the chelate of a metal ion and the tagged species, the metal ion is thought to be associated with the two beta-diketone groups of the Ter group of the tagged species. The chelate may be formed by exposing the tagged species to a solution of the metal ion. The tagged species may be in solution, or may be in suspension or emulsion. The solvent may be aqueous, and will depend on the nature of the species spec. If the species is a protein, a solvent should be chosen that will not disrupt the structure of said protein. The solvent preferably does not contain amine groups since these may react with the tether group of the tagged species or its metal chelate. The metal ion may have a counterion and the counterion may be any counterion that enables the metal ion to be present in solution in sufficient concentration to form a suitable complex with the compound of formula 1. The

counterion may be for example a halide, such as chloride, bromide or iodide or it may be some other suitable counterion. The solution of the metal ion may be an aqueous solution.

A chelating compound, or a chelate, a tagged species, a labelled species or a reagent according to the present invention may be used in an immunoassay. Thus a metal-bound conjugate may be made which incorporates the antigen to be assayed together with an antibody bearing one or more groups derived from a chelating compound complexed with a metal. Antibodies are commonly proteins or protein fragments which may be bound to a chelating compound of the present invention as earlier described. Complexation of the beta-diketone groups of the Ter group of formula 4 may be performed at any of the following stages: after formation of a conjugate by reaction of a tagged species bearing one or more Ter groups with an antigen; after formation of a tagged species bearing one or more Ter groups, to form a labelled species which can be reacted with an antigen; after formation of a chelating compound having a Ter group.

Thus a metal-bound conjugate may be made by exposing an antigen to a labelled species, or by exposing an antigen to a tagged species followed by exposing the resulting conjugate to a metal containing solution. The labelled species may be a labelled antibody and the tagged species may be a tagged antigen.

The present invention also provides a kit for use in an immunoassay comprising: - a first part comprising a tagged species according to the invention; and - a second part comprising a metal ion capable of being chelated by the chelating groups of the tagged species.

The metal ion may be selected from the group consisting of Sm, Eu, Tb3+ and Dy+ for example. The metal ion may be in solution. The solution may comprise other components, for example a buffer. The tagged species may be a tagged protein, a tagged antibody, a tagged portion of a protein, a tagged portion of an antibody or a tagged peptide.

In using the kit in an immunoassay, the first part, or a portion thereof, may be combined with a sample which may or may not comprise a species of interest, said species of interest being capable of combining with the tagged species. The resulting mixture may then be treated with the second part, or a part thereof, in order render the species of interest, if present, fluorescent. The species of interest may then, if present, be detected by fluorescence microscopy or some other suitable technique.

Advantages provided by the present invention compared to the prior art include:

labelled species derived from proteins and chelating compounds of formula 1 are relatively stable: they may be stored for up to about 6 months while retaining sufficient activity (potentially longer with sodium azide, BSA and other additives). By comparison, labelled species derived from proteins and compounds of formula 2 may be stored for only a few days before decomposing to an unacceptable degree.

- chelating compounds of formula 1 are relatively stable to hydrolysis, but are capable of binding readily to amine groups such as those commonly present in proteins.

- chelating compounds of formula 1 are relatively hydrophilic, due to the presence of a hydrophilic linker group, and are consequently easier to react with species such as proteins which are conveniently present in aqueous solution, suspension or emulsion form ; - chelating compounds of formula 1 may be reacted with a protein to provide a tagged protein wherein the hydrophobic region of the chelating group of the chelating compound is kept at a distance from the protein backbone such that there is minimal interaction between said region and said backbone, so that there is reduced risk of disruption of the protein structure; - chelating compounds of formula 1 may be readily synthesised from available starting materials, and do not require use of dicyclohexylcarbodiimide (DCC) as a coupling reagent. DCC produces a by-product, dicyclohexylurea, which is often difficult to separate from the desired product of a reaction.

- chelating compounds of formula 1 are sufficiently stable that they may be purified using common techniques such as HPLC without substantial decomposition, and may thus be obtained in a substantially pure state. As a consequence, accurate control of the conjugation process can be achieved, so that conjugates may be prepared with prescribed labelling ratios.

Brief Description of the Drawings A preferred form of the present invention will now be described by way of example with reference to the accompanying drawings wherein: Figure 1 is a synthetic scheme for making a chelating compound of formula 1; Figure 2 is a synthetic scheme for making a labelled protein according to the present invention; Figure 2a is another synthetic scheme for making a labelled protein according to the present invention;

Figure 3 is a synthetic scheme showing two routes for making a metal-bound immunoconjugate; Figure 4 is a graph of density of precipitate formed on addition of 1,4 dioxane solutions of four different chelating compounds to 0. 1 M NaHC03 solution at pH 8.2, measured using optical density at 600 nm on an Eppendorf BioPhotometer instrument; Figure 5 a) is a plot of the UV absorbance at 280nm of conjugate fractions collected as single (1 ml) fractions after passage through a Sephadex size exclusion chromatography column; Figure 5 b) is a plot of F/P ratio against BHHST concentration for Sephadex purified conjugates, showing that, with the exception of P4, F/P increased linearly with BHHST concentration; Figure 6 is an image of Giardia cysts labelled with conjugates PI to P6 captured with gate-delays of 60 us at gains varying from 55 to 60; Figure 7 shows two graphs of absorbance at three different stages in the centrifugal filter operation centrifugal wash operation: a) for BHHST conjugate O, ; and b) for BHHCT ands BHHST; Figure 8 shows two graphs of absorbance at 260,280 and 320 nm over a period of 92 days for the four immunoconjugates: a) absorbance at 280 nm, which is an indication of protein concentration, and b) showing that, with the exception of conjugate 04, the F/P ratio was observed to drop with the passage of time; and Figure 9 shows post-labeled (antimouse-FITC) fluorescence intensity line profiles comparing 0 and CRY104 labeled oocysts (filter &num 5 and #6).

Detailed Description of the Preferred Embodiments With reference to Figure 1, compound 10 is o-terphenyl, which is a freely available chemical. Reagent 15 is acetyl chloride, compound 20 is terphenyl acetate, reagent 25 is an ester of a perfluoroalkanoic acid. Reagent 25 as shown in Figure 1 is ethyl heptafluorobutanoate, however other esters of heptafluorobutanoic acid may also be used.

Compound 30 is a bis- (perfluoropropyl-beta-diketone). Reagent 35 is chlorosulfonic acid and compound 40 is a sulfonyl chloride derived from compound 30. Reagent 45 is a water-soluble aminoalcohol, which in Fig. 1 is shown as 3-aminopropanol, however other aminoalcohols such as 3-aminopropyl-2-hydroxyethyl ether or 3-aminopropyl-3- hydroxypropyl ether could also be used. Intermediate 50 is a hydroxysulfonamide derived from compound 40 and reagent 45. Reagent 55 is a bis (N-heterocyclo) carbonate, for example bis (N-succinimido) carbonate or bis (N-maleimido) carbonate and chelating

compound 60 is a carbonate derived from compound 50 and reagent 55. In this specification, if R is hydrogen, X, Y and Z are all oxygen and Grp is N-succinimido, then compound 30 is referred to as BHHT, compound 40 as BHHCT, intermediate 50 as BHHPA and chelating compound 60 and BHHST.

The synthesis of chelating compound 60, BHHST, which has the structure of formula 1 wherein EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is CH2CH2 and Grp is N-succinimido, is described below. Acetylation of o- terphenyl with reagent 15, using an aluminium chloride catalyst, generates compound 20.

Reaction of this with reagent 25 in ether in the presence of sodium ethoxide produces compound 30, BHHT. The terphenyl nucleus of BHHT may be then activated by reaction with reagent 35 to produce compound 40, BHHCT. The reactions described above are known and are described in the open literature.

A convenient process for making chelating compound 60 comprises the steps of reacting compound 40 with reagent 45, 3-aminopropanol, to form intermediate 50, and reacting intermediate 50 with reagent 55. The process additionally comprises the step of purifying chelating compound 60. Reaction of compound 40 with reagent 45 may be conducted in acetonitrile, using a tertiary amine catalyst, for example dimethylaminopyridine (DMAP). The reaction may be conveniently conducted at reflux and is substantially complete after a reaction time of about 3 hours. The reaction may be conducted at a lower temperature, and may be conducted for a longer reaction time.

Reaction of intermediate 50 with regent 55 may be conducted in acetonitrile, using a tertiary amine catalyst, for example dimethylaminopyridine (DMAP). The reaction may be conveniently conducted at reflux and is substantially complete after a reaction time of about 48 hours, producing chelating compound 60, BHHST. The reaction may be conducted at a lower temperature, and may be conducted for a longer reaction time.

Chelating compound 60 may be purified by precipitation from a solvent, for example from hexane, and in purified form is an amorphous white powder.

Figure 2 is a synthetic scheme for making a labelled protein according to the present invention. In Figure 2, species 110 is a protein, Prot-NH2, with at least one primary amine group and reagent 115 is the chelating compound of formula 1 wherein EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is CH2CH2 and Grp is N-succinimido. Species 110 may be for example an antibody, such as antimouse antibody. Tagged protein 120 has a protein attached to at least one group, Gr, of formula 4 through nitrogen atom (s), and conjugate 130 contains tagged protein 120 and a lanthanide ion Lan3+ chelated by the group of formula 4 of tagged protein 120.

A convenient process for producing labelled protein 130 comprises initially reacting a protein with reagent 115 to produce tagged protein 120. The process may comprise reacting protein 130 with a solution, suspension or emulsion of reagent 115 and the solution, suspension or emulsion may be an aqueous solution, suspension or emulsion, and may also comprise one or more other components. The one or more other components may be for example surfactants, emulsifiers, buffers or other additives.

Tagged protein 120 may then be reacted with a metal ion. The ion may be selected from the group consisting of Sm3+, Eu3+, Tb3+ and Dy3+. The reacting may comprise reacting the tagged protein with a solution of the ion, and the solution may be an aqueous solution.

The reaction time may be between 1 minute and 1 hour, or between 15 minutes and 1 hour, at room temperature, or may be between 1 hour and 1 day, or overnight, at 4°C.

Figure 2a is another synthetic scheme for making a labelled protein according to the present invention. In Figure 2a, compound 210 is the chelating compound of formula 1 wherein EWG1 and EWG2 are both C3F7, X, Y and Z are all oxygen, R is hydrogen, A is CH2CH2 and Grp is N-succinimido. Chelate 220 is a chelate formed from chelating compound 210 and a lanthanide ion Lan3+, protein 225 is a protein with at least one primary amine group, and labelled protein 230 comprises protein 225 bound to a chelating group of structure 4 and a lanthanide ion chelated by the chelating group of formula 4. Lanthanide Lan3+ may be Eu3+, and protein 325 may be an antibody, for example antimouse antibody.

A convenient process for producing labelled protein 230 comprises initially reacting chelating compound 210 with a lanthanide ion to produce chelate 220. The lanthanide ion may be selected from the group consisting of Sm3+, Eu3+, Tb3+ and Dy3+. The lanthanide ion may be in the form of a solution of a halide of said lanthanide ion, and the halide may be chloride. A preferred lanthanide ion is Eu3+. The reaction time may be between about 1 minute and about 1 hour, or between about 20 minutes and about 1 hour. Chelate 220 may then be reacted with a protein to produce labelled protein 230. Preferably the protein is in aqueous solution, suspension or emulsion, and said solution, suspension or emulsion may also comprise one or more other components. The one or more other components may be for example surfactants, emulsifiers, buffers or other additives.

Figure 3 is a synthetic scheme showing two routes for making a metal-bound immunoconjugate which may be used in an immunoassay. In Figure 3, labelled protein 310 contains a protein Prot that is an antibody to antigen 320. Antigen 320 may be for example a surface protein on a microorganism. Labelled protein 310 may be for example made as described in Figure 2 or Figure 2a. Antigen 320 is the antigen to be determined

in the immunoassay. Metal-bound conjugate 330 is formed from labelled protein 310 and antigen 320. Tagged protein 340 may be made as described in Figure 2. Conjugate 350 is formed from tagged protein 340 and antigen 320. Reaction of conjugate 350 with a lanthanide ion Lan3+ provides an alternate route to metal-bound conjugate 330. Thus in a first route to metal-bound conjugate 330, antigen 320 is isolated on a filter, and allowed to bind with labelled protein 310 on the filter. The filter is then washed to remove excess reagent, leaving metal-bound conjugate 330 on the filter. In a second route to metal- bound conjugate 330, antigen 320 is isolated on a filter, and allowed to bind with tagged protein 340 to form conjugate 350. The reaction time may be between about 15 and about 30 minutes. The filter contents are then transferred to a microreaction vial and there treated with Lan3+, TOPO and Triton X-100 to form metal-bound conjugate 330.

Example 1: BHHST was synthesised as follows.

4,4'-bis (1", 1", 1", 2", 2", 3", 3"-heptafluoro-4", 6"-hexanedion-6"-yl)- (3-hydroxy- propyl)-sulfonamide)-o-terphenyl.

A solution of hexane precipitated BHHCT (1.21 g; 142 mM) in dry acetonitrile (25 ml) was transferred to a 100 ml round bottom flask containing a magnetic stirrer bar. To this was added, alternately in 500 ul aliquots, solutions of dimethylaminopyridine in acetonitrile (4.1 mM; 500 mg in 5 ml) and 3-aminopropanol in acetonitrile (2.9 mM; 220 mg in 5ml). On addition of the two solutions, the BHHCT solution became momentarily cloudy but rapidly cleared to form an orange-yellow solution that was stirred for a further 3 hrs before terminating the reaction. The reaction proceeded rapidly as followed using reverse-phase TLC (3: 2 MeOH: H20) and was essentially complete after 1 hour.

The appearance of byproducts occurred simultaneously with formation of BHHPA and these impurities were removed by acid-wash in the workup. Acetonitrile was removed at 45°C on the rotary evaporator and the residue dissolved in ethyl acetate/KHS04 (200 ml) solution and transferred to a separatory funnel. The organic material was insoluble in the ethyl acetate until acidified. The organic phase was washed (3 x 20 ml) with aqueous KHS04 (15 g/100 ml) followed by 2 x 50 ml brine washes and then dried over drierite overnight. Ethyl acetate was removed on the rotary evaporator to give 1 g of crude BHHPA (79% yield) that was refluxed for 2 hrs with a litre of water saturated hexane (I ml water in 1 L hexane). The hexane solution was filtered and reduced on the rotary evaporator to give 120 mg of purified BHHPA and the process was repeated several times until all the BHHPA has been extracted. BHHPA has low solubility in wet hexane (about 120 ng. mol'') and very low solubility in dry hexane,

however the impurities are far less soluble in this solvent, wet or dry. IH NMR 8 (400 MHz, CDC13) 1.80 (2H quintet), 3.25 (2H quartet), 3.81 (2H t), 5.22 (1H, t), 6.58 (2H, s), 7.28 (4H, split doublets), 7.61 (1H, d), 7.86 (4H, split doublets), 7.97 (2H, t), 7.99 (1H, d); Electrospray-MS, m/e 843.

(4, 4'-bis- (l", 1", 1", 2", 2", 3", 3"-heptafluoro-4", 6"-hexanedion-6"-yl) sulfonylamino- propyl ester N-succinimide ester-o-terphenyl) Purified BHHPA (270 mg; 320 uM) was dissolved in acetonitrile (15 ml) in a 50 ml round bottom flask equipped with a stirrer bar. Acetonitrile solutions of di- (N- succinimidyl) carbonate (DSC: 600 uM ; 165 mg in 1 ml) and dimethylaminopyridine (DMAP: 800 uM ; 100 mg in 1 ml) were prepared. The BHHPA solutions were added in alternate aliquots. After addition of the DSC/DMAP, a TLC sample was taken and the flask filled with argon, stoppered and stirred magnetically. A second TLC sample was taken 3 hrs later and the contents stirred for a further 45 hrs before solvent was removed on the rotary evaporator at 45°C and the residue dissolved in ethyl acetate (200 ml). The organic phase was washed (3x 20 ml) with aqueous KHS04 (15 g/100ml) followed by 2 x 50 ml brine washes and then dried over drierite overnight. The ethyl acetate was removed on the rotary evaporater to give 200 mg of crude BHHST (63% yield) that was further purified by solution in hexane approximate solubility 1 mg. mol''). Four fractions were obtained in each (100 ml) extraction weighing successively 100 mg, 65 mg, 17 mg and 3 mg; the remaining red coloured hexane insolulble residue, weighed 20 mg. Sample from the 65 mg hexane fraction was reserved for NMR analysis.'H NMR 5 (400 MHz, CDC13) 2.18 (2H quintet), 3.10 (2H t), 4.04 (2H t), 4.35 (2H t), 6.57 (2H s), 7.29 (4H, t), 7.61 (1H, s), 7.63 (1H, s), 7. 86 (4H, t), 8.13 (iH, s), 8.15 (2H, d).

Solubility index Since improved hydrophilicity was one of the motives for synthesizing BHHST, a method of comparing the relative solubilities of each of the chelating compounds was devised. Precipitation of chelating compounds was observed during protein conjugations with both BHHCT and BPPCT. The optical density of the precipitate was measured using excitation at 600 nm (OD600) since this wavelength does not evoke fluorescence from the chelating compounds. Aliquots of BHHST, BHHPA, BHHCT and BPPCT (BPPCT is the homologue of BHHCT with a pentafluoro group substituted for the heptafluoro group on the beta-diketone moiety) were prepared in 1,4 dioxane and concentration was determined using the appropriate extinction coefficient (3. 32*104, 3. 34* 104, 2. 80*104 and 3. 39*104 cm''. M. L-1 respectively). The OD600 of a sodium bicarbonate solution (95 AL, 0. 1 M, pH 8. 2) in a UVette (Eppendorf GmBh, Germany) was measured after incremental addition

of the chelating compound-dioxane solution (1 RL increments) and plotted against concentration.

Conjugate preparation requires the addition of chelating compound solvated in an organic solution to aqueous bicarbonate, thus it is a good practical means of evaluating the hydrophilicity of the chelating compound. At sufficiently high concentrations, the chelating compounds precipitate from solution as a suspension that scatters light and increases optical density. At lower concentrations, precipitates that formed initially would dissolve as the concentration of the organic solvent (1,4 dioxane) was increased, leading to a humped curve when OD600 was plotted against concentration of chelating compound. Concentrations of BHHST, BHHPA, BHHCT and BPPCT stock solutions were estimated using the extinction coefficients detailed above and from absorbance measurements made on serially diluted stock solution (33: 1 in 1,4 dioxane then 33: 1, O. IM, NaHCO3).

The stock solutions of chelating compounds were adjusted to approximately 26 mmolar for solubility tests. Molar concentration of chelating compounds in bicarbonate solution were plotted against optical density to produce the curves shown in Fig. 4.

Conjugate preparation Conjugates were prepared using the concentrations and reagent volumes shown in Table 1.

Table 1 Volume and concentration of reagents used for conjugate preparation. Code Antibody Ab. conc. B#HST vol. BHHST conc. P, 100 µL of Antimouse 3. 3 mg.ml-1 2.5 µL 242 ptg. mi" P2 100 µL of Antimouse 3.3 mg.ml-1 5.0 µL 484 µg.ml-1 P3 100 µL of Antimouse 3.3 mg.ml-1 7.5 µL 727 µg.ml-1 P4 100 µL of Antimouse 3. 3 mg.ml-1 10.0 µL 969 µg.ml-1 Ps 100 µL of Antimouse 3. 3 mg.ml-1 5.0 µL 484 µg.ml-1 100 µL of Antimouse 3. 3 mg.ml-1 10.0 µL 969 µg.ml-1

An aliquot of BHHST was dissolved in 66 uL of DMF and concentration was estimated at 9.9 mg. mol'' using the extinction coefficient (s320) 3.32 x 104. The conjugations were performed at 24°C for one hour. Conjugates P I to P4 were isolated from unbound BHHST using Sephadex columns whereas conjugates Ps and P6 were purified using 50 KDa centrifugal filters. Protein content of purified conjugates was determined using both the BCATM assay and spectrophotometric methods. BCATM is a commercial protein assay from Pierce of Rockford, Illinois, USA.

Conjugate activity determination Giardia cysts were pre-labelled with G203 anti-Giardia Mab and post-labelled with the anti-mouse conjugates P, to P6 using the concentrations recorded in table Table 2.

A fluorescence-enhancing buffer FEB was used with PVA-Eu3+solution for mounting slides (4 pL cyst suspension, 3 RL FEB and 2pL PVA-Eu3+). Fluorescence intensity of labelled cysts prepared with each conjugate was noted and the result recorded in Table 2.

Table 2 Fluorescence intensity observed for Giardia cysts indirectly labelled with conjugate (observation using an epifluorescence microscope using the longpass DAPI filter) Code FlP Protein Ab Conc. Label fluorescence intensity mg. ml', µg.ml-1 P 1 16.9 0.298 30 good fluorescence intensity P2 7.6 0.955 190 good fluorescence intensity P, 6. 1 1. 568 157 faint blue fluorescing cysts Pa 4.5 1. 324 132 few faint labeled cysts, most labeled Pa 9. 0 1.905 190 good fluorescence intensity Po 21. 3 1. 579 158 few well labeled cysts, most labeled Conjugation of BHHST with antimouse Anti-mouse was supplied as a lyophylized powder that was accurately weighed and prepared at a concentration of 10 mg. ml-1. Absorbance measurements were performed in triplicate for the immunoglobulin and the DMF-BHHST solution; the averaged absorbance values are shown in Table 3. The factor used to convert absorbance at 280 nm to protein weight in mg. ml-1 was confirmed to be 1.38 using equation 2: protein weight (mg. ml-1) = A2go/1. 38 * dilution (2) Table 3 Absorbance measurements for antibody and chelate used to prepare conjugates Pi to P6 Compound DiR A260 A280 A320 A280/A320 mg.ml- Antimouse 33 : 1 0. 255 0. 412 0. 037 10. 0 BHHST 667 : 1 0. 364 0.357 0.502 0. 712 9. 9

Conjugates P1 to P4 Following reaction with BHHST, conjugates P to P4 were separated from unbound chelate on Sephadex columns. Fractions collected were monitored by absorbance at 280 nm and typically the conjugate was contained in a single fraction. F/P ratios increased linearly with BHHST concentration for conjugates P to P3 and then sharply leveled off for conjugates P4, as shown in Fig. 5.

Protein content of the principal conjugate containing fraction is reported in Table 4 and excludes protein carried in fractions preceding and following the main fraction.

Table 4 The protein content of conjugates determined with the BCA assay and by absorbance at 280nm to determine F/P. BCA determined F/P BioPhotometer FlP Jter 30 days Code Protein Std. Dev. Abs540 F/P ratio Protein Std. Dev. F/P ratio Protein F/P ratio µg.ml-1 µg.ml-1 Abspooled µg.ml-1 P1 910 0.01672 1.5 786 0.00127 1.8 299 16.9 P2 1050 0. 01106 2. 0 944 0. 00121 2. 2 955 7. 6 F, 879 0.01334 4.8 1169 0.00201 3.6 1568 6.1 P4 773 0.01614 6.1 1133 0.0019 4.2 1325 4.5 P5 1942 0.00662 8.4 2131 0.0089 7.7 1906 9.0 P6 1893 0.10379 18.0 1924 0.00457 17.8 1580 21.3 Although 80% or more of the conjugate was collected in a single (1 ml) fraction, up to 20% was lost in the fractions preceding or following since they were not processed. Thus protein was under-reported compared to levels observed for conjugates Ps and P6 isolated with centrifugal filters. The Sephadex fractions were concentrated to 100 uL volumes using 50 KDa centrifugal filters in preparation for BCA analysis. Protein recovery from the Sephadex columns averaged 72% when the unprocessed fractions were included.

Conjugates P5 and P6 Although conjugates P2 and P4 were prepared in a similar manner to Ps and P6. the F/P ratios of the latter were respectively more than 2-fold and 4-fold greater (Table 4).

Removal of unbound BHHST on Sephadex columns has previously been observed to

result in decreased F/P ratios compared to centrifugal filtration methods and is believed to be due to loss of more heavily labelled conjugate on the column. Conjugate was analysed using the BCATM assay and absorbance values converted to protein estimates using the BSA standard curve modelled as a quadratic (Eq. 3).

Abss4o= 0. 15454 + (0. 01077 [conc. ] )- (2. 08551E-5 [conc. ] 2) (3) The R2 coefficient for the curve was 0.9976 and conjugate samples were analysed in triplicate with a pooled standard deviation of 0.0279 for absorbance (540 nm) readings.

Analysis of conjugate absorbance data indicated that protein contributed slightly to absorbance at 320nm and thus was factored into the method employed to calculate F/P.

Conjugate activity Giardia cysts were pre-labelled with G203 anti-Giardia Mab (135. ug. ml~1) and post-labelled with conjugate at the concentration shown in Table 2. Absorbance of the conjugates was measured at a dilution of 10: 1 a month after the conjugates were prepared and some significant changes were noted (Table 4). Conjugate P, protein level was 30% of the initial post-conjugation value and F/P had climbed 8-fold. Although protein content was relatively unchanged for conjugate P2, the F/P ratio was double that initially observed. With the exception of conjugate P4, the F/P for all conjugates was higher than the initial determination. Giardia cysts were labelled with the 10: 1 dilutions, P2 concentration was doubled by the addition of a second lOuL aliquot of conjugate. Pl was anticipated to provide little or no labelled cysts since concentration was low, however the cysts were surprisingly well labelled and only conjugate Ps provided superior fluorescence. Fluorescence intensity of labelled cysts was ranked in order of decreasing intensity: P5>P1>P2>P4>P6>P3 (Fig. 6). No fluorescence cysts were obtained with conjugate P3.

Example 2 : Preparation of immunoconjugate CRY104 antibody (500uL ; 3.3mg. mol-1) was desalted and resuspended in 200pal of filtered (0. 2um) conjugation buffer at pH 9.04 at a measured concentration of 5. 9mg. ml-1.

Lyophilized goat-antimouse was dissolved in conjugation buffer (2.3mg ; 760µL) to produce a 3mg. ml-1 solution from which 100tL aliquots were taken for each conjugation.

Details of antibodies used are shown below. Antibody Subclass Target Supplier CRY 104 IgG X Cryptosporidium parvum *AusFlow G203 IgG, Giardia lamblia *AusFlow Goat Anti-mouse polyvalent Mouse IgG, IgA and IgM Sigma (#M8019) Goat Anti-mouse-polyvalent Mouse IgG, IgA and IgM Silenus FITC Antibody was diluted (33: 1; 3uL in 97gL of PBS) and absorbance recorded in Table 5 to determine protein concentration using Equation 2.

Table 5 Absorbance reading for de-salted antibody diluted 33: 1 in PBS Sample I Sample 2 Protein estimate Antibody 260 A280 A320 A260 A280 A320 Diln. # mg.ml-1 CRY104 0. 148 0. 248 0 0. 144 0.250 0 33 : 1 1. 38 5. 95 Anti mouse 0. 067 0.109 0 0. 058 0.094 0 33 : 1 1. 38 2. 42

Reagents Fluorescence enhancing buffer (FEB) was prepared by adding Triton X-100 at 3 mg. mi-1, TOPO at 0.6 mg. ml-1 and Tween 80 at 2.6 mg. ml-1 to 0. 1M bicarbonate buffer at pH 8.5. FEB was combined with PVA-Eu3+ for mounting (oo) cysts on slides.

Preparation of BHHST solution A 1 mg aliquot of BHHST (purified by precipitation from hexane) was used for this conjugation. The concentration of BHHST was determined using the molar absorbance coefficient (a26o) of 3.32 x 104. BHHST was dissolved in 80 uL of DMF and a 3 gL sample serially diluted (33: 1 then 20: 1 in Conj. Buf. ) to give a final dilution of 667: 1.

Duplicate samples were analyzed and averaged to give the values shown in Table 6 from which BHHST concentration was estimated at 14.4 mg. ml-1.

Table 6 Absorbance reading (averaged) for BHHST/DMF solution diluted 667: 1 Compound DZ s A280 A320 A280/A320 mg.ml-1 Final Vol. BHHST 667 : l 0. 624 0. 567 0. 908 0.624 14.4 80 µL Immunoconjugate preparation Four immunoconjugates were prepared using the concentrations and reagent volumes shown in Table 7.

Table 7 Volume and concentration of reagents used in conjugation Code Antibody Ab. conc. BHHST vo BHHSTconc. O1 100 µL of CRY104 5.9 mg.ml-1 11 µL 1.42 mg.ml-1 0z 100 µL of CRY104 5.9 mg.ml-1 15 µL 1.88 mg.ml-1 O3 100 µL of Antimouse 3.3 mg.ml-1 11 µL 1.42 mg.ml-1 O4 100 µL of Antimouse 3.3 mg.ml-1 15 µL 1.88 mg.ml-1

Unbound BHHST was removed using Ultrafree Biomax 50K centrifugal filters and centrifuging at 13000rpm at 4°C. Eluate from filtration cycles was collected for absorbance measurements results of which are shown in Table 8. Duplicate samples of purified immunoconjugate were diluted (33: 1; 3 uL in 97 L PBS) and absorbance readings recorded in Table 9.

Table 8 Centrifugal wash absorbance values-note that values recorded for Filtration 1 should be multiplied by a factor of 8 to correct for dilution.

. X X X X E Em m w 6'N CRY104 O, 1. 358 0. 557 0. 051 0. 702 0. 290 0. 049 0. 231 0. 105 0. 034 CRY 104 02 1. 824 0. 769 0. 074 1. 120 0. 498 0. 141 0. 323 0. 156 0. 071 Antimouse o3 1. 279 0. 517 0. 033 0. 749 0. 319 0. 070 0. 202 0. 095 0. 038 Antimouse 104 11. 996 10. 853 ! 0. 151 Table 9 Immunoconjugate absorbance readings Sample I Sample 2 Conjugate Code Diln. A260 A280 A320 A260 A280 A320 CRY104 Ol 33 : 1 0. 114 0.150 0. 100 0. 115 0.147 0. 098 CRY 104 O2 33 : 1 0. 090 0. 115 0. 078 0. 092 0. 118 0.077 Antimouse O3 33:1 0. 064 0.069 0. 069 0. 059 0. 065 0. 068 Antimouse O4 33:1 0.051 0. 054 0. 053 0. 050 0. 050 0. 050

In-situ labeling of (oo) cysts with immunoconjugate Labeling of (oo) cysts was performed 36 days after preparation of the immunoconjugates, and protein concentration and F/P ratios were determined.

Cryptosporidium oocysts were diluted (10 µl @ 7.5 x 107 oocysts.ml-1; 90 1L PBS) and mixed with 200 µL of water concentrate in a micro-reaction vial before applying to

membrane filters #1 and #2. Membrane filter #3 was used to collect a Giardia spiked water concentrate (10 uL @ 5 x 106 cysts. ml-1 in 100 uL water concentrate) whereas filter #4 carried Giardia cysts alone (10 µL @ 5 x 106 cysts. Mi-I in PBS). (Oo) cysts were labelled using the concentrations shown in Table 10. Post-labelling of membrane filters #5 and #6 was performed with 100tl aliquots of FITC antimouse (Silenus) at a dilution of 20: 1. For membrane preparations &num 1 to #4, a 5 uL aliquot of the (oo) cyst suspension was transferred to a 0.5 ml micro-reaction vial containing 4 uL of FEB and 3 PtL of the PVA/Eu3+ solution, incubated for 30 minutes and transferred (5 uL) to a slide for TRFM analysis using a DAPI filter box. FITC labeled oocysts were suspended and analysed in PBS.

Table 10 Pathogen, labeling method and reagents # Description Pre-label post-label 1 Cryptosporidium + water cone. direct label 0, (208 µg. ml-1) none 2 Cryptosporidiur + water conc. indirect label CRY104 (165 µg.ml-1) O3 (66 µg.ml-1) 3 Giardia + water conc. indirect label G203 (135 Ag. ml*') 03 (66 pg. ml") 4 Giardia + indirect label (stability test) G203 (135 µg. ml-1) O3 (132 µg.ml-1) 5 Cryptosporidium affinity test (O1) O1 (100 µg.ml-1) FITC antimouse 6 Cryptosporidium affinity control (CRY104) CRY104 (100 µg.ml-1) FITC antimouse Results Absorbance readings for protein, BHHST and filtration eluate Absorbance measurements recorded for the de-salted antibody prior to conjugation with BHHST (Table 5) show zero absorbance at 320 nm. Protein content was estimated using the p value shown in the table and Equation 3.

The BHHST A2so/A320 ratio of 0.624 shown in Table 6 is within expected limits.

Absorbance readings shown in Table 8 for the eluate collected after the first filtration cycle are 1/8 their actual value since the initial volume (200 uL) was twice that used for the other filtrates and measurements were made using a 1: 4 dilution. Corrected values were used for the plot shown in Fig. 7; maximum absorbance of the wash eluate occurs at 260 nm corresponding to a peak in N-hydroxysuccinimide absorbance.

Immunoconjugate characterization and F/P determination Duplicate samples were taken from each immunoconjugate (diluted 33: 1; 3 1 in 97 p1 PBS) to obtain the absorbance values recorded in Table 9. For all immunoconjugates, the average variation in absorbance measurements between duplicates was 1. 7%.

Averaged absorbance values were recorded in Table 11 together with calculated protein and chelate concentrations. The parameter in column AdjA280 was the corrected value for absorbance at 280 nm due to protein.

Table 11 Immunoconjugate F/P ratio

Conjugate Code Diln. A280 A320 Adj. Protein Chelate F/P ratio A280 µg.ml-1 µg.ml-1 CRY104 O, 33 : 1 0. 149 0.099 0.087 2142 82. 8 5. 9 CRY104 °2 33 : 1 0. 117 0.078 0.068 1678 65. 3 5. 9 Antimouse O3 33:1 0.067 0. 069 0. 024 612 63. 5 15. 8 Antimouse O4 33:1 0.052 0. 052 0. 019 497 47. 6 14. 6 Immunoconjugate stability Stock solutions of the immunoconjugates were diluted with PBS and were free of sodium azide, BSA or stabilizing agents. Absorbance was measured after 36,45, 64 and 92 days to monitor protein loss and to provide an estimate of the immunoconjugate half-life (Fig.

8, Table 12).

Table 12 Estimated half-lives for each of the immunoconjugates based on extrapolation of the last 4 points for the curve shown in Fig. 8a. O1 O2 O3 O4 186 days 176 days 81 days* 64 days As a general observation, CRY104 conjugates (O1 and 02) were significantly more stable than anti-mouse conjugates (03 and 04). With the exception of conjugate 04, the F/P ratio of the immunoconjugates decreased steadily over the 92 day observation period.

Conjugate 04 rapidly degraded from day-36 onwards and the deterioration was characterized by loss of protein (day 92: 100 llg. ml~l) and an unexpected rise in the F/P ratio (day 92: F/P = 42).

The low absorbance values recorded for conjugate 04 may have exposed a non- linearity in the method used to calculate F/P. There were two instances (at different times) when protein concentration for both CRY104 conjugates was recorded at levels higher than immediately after the conjugation. All measurements were performed in triplicate, however worst-case standard deviation for A28o readings reached 0.02 absorbance units and this translated to a difference of 300tig protein between measurements. Thus protein levels for conjugates O, and 02 are believed to have remained relatively unchanged, although F/P ratio decreased by an average of 27% over 92 days. The period immediately

following the conjugation was notable for the rapid decrease in F/P experienced by all immunoconjugates. CRY104 conjugates again proved more stable with an average drop of 15% by day-36 whereas anti-mouse F/P ratio declined by an average of 19%.

Conjugate 03 was expended before the completion of the observation period, however F/P ratio was observed to drop sharply when last measured. Observations that the rate-of- decline of F/P nearly halved for the remaining 56 days provided further evidence of the superior stability of CRY104. The half-life of conjugate 04 occurred within the observation period and half-life of the remaining conjugates was estimated based on the rate of decline of the F/P ratio. Due to the more relaxed rate-of-decline observed for F/P from day 36 onwards, only the last 4 observations were used to estimate decay rate and the process was assumed to follow a linear trajectory.

The overall success of (oo) cyst immunolabeling performed with 36-day old immunoconjugate (Ol) proved that a small drop in F/P ratio was not indicative of a significant loss in immunolabel activity.

Immunoconjugate activity Fluorescence intensity of FITC labeled oocysts (filters &num 5 and #6) was measured using the line profile tool to generate the profiles shown in Fig. 9. Fluorescence intensity was averaged across the cupped peaks and recorded above the line profile peaks for each cyst. Fluorescein is reported to suffer concentration quenching, however concentrations of the anti-mouse-FITC label were maintained at low levels to ensure quenching was not a problem. The FITC labeled oocysts were representative of their respective populations and indicate qualitatively, there was no distinction between the activity of native CRY104 and that of immunoconjugate O1.