88702-02pct.632

PROCESS FOR CROSS-LINKING MOIETIES

The invention relates to a linkers for linking moieties such as nucleic acids, proteins, antibodies and polypeptides with other moieties such as enzymes, antibodies and solid supports, and processes using such linkers.

Crosslinking reagents are often used in the diagnostic, pharmaceutical, chemical, electronic and biotechnology industries to produce conjugates such as DNA and antibodies with enzymes, and proteins with biotin. These conjugates ^' find applications as nucleic acid probes and in immunoassays, amongst others. Crosslinking reactions are also used in the immobilisation of macromolecules on solid supports such as glass, microprocessors, membranes, dipsticks and magnetic beads.

A number of crosslinking reagents and methods are known in the prior art. For example, Pierce market a kit for controlled protein-protein crosslinking which depends on the reaction of a thiol with a maleiimide group (Perbio Science UK 05/06, Pierce 23456) and disuccinimidyl suberate, a homobifunctional agent (Pierce 21655). Other reagents include the photoactivatable sulfo-SANPAH (sulfosuccinimidyl 6-(4'-aizido-2'-nitro- phenylamino)hexanoate) .

A key disadvantage of the methods mentioned above is that the thiol/maleiimide approach is an all-day procedure requiring several complicated steps using unstable reagents. In particular, the thiols readily oxidise and dimerise, and the maleiimides can hydrolyse. Furthermore, the homobifunctional reagents suffer from self-conjugation and polymerisation. Additionally, rapid hydrolysis leading to poor yields and the required use of high concentrations of such toxic reagents are also disadvantages, Photoactivatable reagents also show poor discrimination and are often low yielding.

There exists a need therefore for processes for linking moieties such as macromolecules which do not suffer from the above disadvantages.

The Applicant has now developed an improved method for the conjugation of moieties such as biological macromolecules. This method takes less time than prior art methods; and all reactions steps can be carried out in a single reaction vessel using a single buffer without the need to purify intermediates. High concentrations of toxic reagents are avoided using the method of the invention. Furthermore, the final conjugate may be easily purified using standard means.

The invention therefore provides linkers which are suitable for linking a first moiety with a second moiety and processes using such linkers. The invention also provides "activated" first moieties which comprise first moieties conjugated to linkers of the invention in a form in which they can be used for conjugation to a second moiety, and processes for the production of such "activated" moieties.

The invention therefore provides a process for linking a first moiety with a linker, wherein the first moiety comprises one or more free amino groups and wherein the first moiety is not a cell membrane or cell membrane fraction, the process comprising the steps: ,

(i) conjugating the first moiety with a linker of formula I:

or a dimerised linker of formula II:

wherein

R ¹ to R ⁴ , R ⁷ to R ¹⁰ _' R ¹¹ to R ¹⁴ and R ¹⁷ to R ²⁰ are independently selected from H ₃ halogen, Ci-io-alkyϊ, Cs-s-cycloalkyl, OH, O-Ci- ₃ -alkyl, SH, NH ₂ , NO ₂ , CO ₂ H, SO ₃ H and SO ₃ X ₃ wherein X denotes a counter ion, for example, NH ₄ ⁺ , Na ⁺ or K ⁺ ;

R ⁵ , R ⁶ , R ¹⁵ and R ¹⁶ are independently selected from H ₃ halogen, Ci-β-alkyl, OH ₃ O-Ci-3-alkyl, SH ₃ NH ₂₃ CO ₂ H ₃ and CO ₂ -C i- ₃ -alkyl ₃

m and n are independently I ₃ 2 or 3, and

Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone.

The invention further relates to a process for conjugating a first moiety with a second moiety, wherein the first and second moieties independently comprise one or more free amino groups, the process comprising the steps:

(i) conjugating the first moiety with a linker of formula I:

or a dimerised linker of formula II:

wherein

R ¹ to R ⁴ , R ⁷ to R ¹⁰ , R ¹¹ to R ¹⁴ and R ¹⁷ to R ²⁰ are independently selected from

H ₃ halogen, Ci-ip-alkyl, C ₃ -8-cycloalkyl, OH ₃ O-Ci-3-alkyl ₃ SH ₃ NH ₂ , NO ₂ , CO ₂ H ₃ CO ₂ -C i-3-alkyl, SO ₃ H and SO ₃ X ₃ wherein X denotes a counter ion, for example ₃ NH ₄ ⁺ , Na ⁺ or K ⁺ ;

R ⁵ ₃ R ⁶ ₃ R ¹⁵ and R ¹⁶ are independently selected from H ₃ halogen, Ct-e-alkyl,

OH ₃ O-Ci-3-alkyl, SH ₃ NH ₂₃ CO ₂ H ₃ and CCVC.-s-alkyl;

m and n are independently I ₃ 2 or 3, and

Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone,

and then

(ii) reacting the first moiety-linker conjugate formed in (i) with the second moiety.

Preferably, step (i) is carried out under the conditions:

molar ratio of first moiety: linker 1:45 to 1:140; and/or time 15 to 120 minutes.

Preferably, step (ii) is carried out under the conditions:

molar ratio of first moiety used in step (i):second moiety 1: 1 to 1:8; and/or time 15 to 120 minutes.

In the context of the present invention, the following definitions are used both hereinbefore and hereinafter:

By the following

is meant one double bond or a plurality of up to n conjugated double bonds wherein each pair of R ⁵ and R ⁶ groups may, independently of any other R ⁵ and R ⁶ pairs, be either in the cis or in the trans geometrical isomeric configuration; and wherein each R ⁵ may be the same or different, and wherein each R ⁶ may be the same or different. The same applies, mutatis mutandis, to groups comprising R ¹⁵ and R ¹⁶ and m.

The term Ci-io-alkyl denotes a branched or unbranched alkyl group having between 1 and 10 carbon atoms. The group includes methyl, ethyl, n-propyl, wσ-propyl, w-butyl, x ^' so-butyl, sec-butyl, tert-butyl etc.

The term C3-s-cycloalkyl denotes a cyclic alkyl group having between 3 and 8 carbon atoms in the ring. The group includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term O-Ch-3-alkyl denotes an alkoxy group having between 1 and 3 carbon atoms in the alkyl portion. The group includes methoxy, ethoxy, n- propoxy and wσ-propoxy.

The term CO2-Ci-3-alkyl denotes an ester group having between 1 and 3 carbqn atoms in the alkyl portion. The group includes methyl carboxylate, ethyl carboxylate, rø-propyl carboxylate and wσ-propyl carboxylate.

The term halogen denotes an atom of a group VII element selected from among fluorine, chlorine, bromine and iodine.

The term "free amino group (s)" refers to amino groups which are capable of reacting with the isothiocyanate groups of the linker. Examples of such free amino groups include those present in the amino acids lysine, arginine, asparagine and glutamine of polypeptides or at the N terminus of polypeptides.

Preferably, R ¹ to R ⁴ , R ⁷ to R ¹⁰ , R ¹¹ to R ¹⁴ and R ¹⁷ to R ²⁰ are each independently selected from H, methyl, ethyl and SO3X.

Preferably, R ⁵ , R ⁶ , R ¹⁵ and R ¹⁶ are each independently selected from H and Ci-3 alkyl.

In some embodiments of the invention, only one or two of R ¹ to R ⁴ and R ⁷ to R ¹⁰ are SO3X. Particularly preferably, only one of R ¹ to R ⁴ and/or only one of R ⁷ to R ¹⁰ are SO3X. Most preferably, only one of R ¹ to R ⁴ and/or only one of R ⁷ to R ¹⁰ are SO ₃ X wherein R ⁴ is SO ₃ X and/or R ⁷ is SO ₃ X.

In other embodiments of the invention, only one or two of R ¹¹ to R ¹⁴ and R ¹⁷ to R ²⁰ are SO ₃ X. Particularly preferably, only one of R ¹¹ to R ¹⁴ and/or only one of R ¹⁷ to R ²⁰ are SO ₃ X. Most preferably, only one of R ¹¹ to R ¹⁴ and/or only one of R ¹⁷ to R ²⁰ are SO ₃ X wherein R ¹⁴ is SO ₃ X and/or R ¹⁷ is SO ₃ X.

m and n are preferably 1. If n = 1, then R ⁵ and R ⁶ are preferably arranged in the trans configuration. If m=l, then R ¹⁵ and R ¹⁶ are preferably arranged in the trans configuration.

X is preferably Na ⁺ .

In some embodiments of the invention, the linker is a compound of formula III:

or a dimerised linker of formula IV

or a compound of formula V

or a dimerised linker of formula VI, VII or VIII:

wherein the R groups, X, m and n are as defined above.

In the compounds of formulae III-VIII, R ¹ to R ²⁰ , where present, are preferably independently selected from H, methyl and ethyl. Most preferably, R ¹ to R ²⁰ , when present, are all H.

A particularly preferred linker is 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS, Aldrich 28,561-7). The corresponding dimerised linker (i.e. the compound of formula IV, wherein R ¹ to R ³ , R ⁵ , R ⁶ , R ⁸ to R ¹⁰ , R ⁿ to R ¹³ , R ¹⁵ , R ¹⁶ and R ¹⁸ to R ²⁰ are all H, and n and m are both 1) is also particularly preferred.

The organic linker Y is preferably a linear C2-6-alkyl, most preferably C2-4- alkyl group, optionally substituted at one or more of the backbone carbon atoms by one or more groups selected from halogen, Ci-3-alkyl, O-Ci-3-alkyl, CO ₂ H and CO ₂ -Ci- ₃ -alkyl.

In one particularly preferred embodiment, Y is -CH2CH2-.

In another particularly preferred embodiment, Y is -(CHa) ₄ CH(COOH)-.

In particularly preferred embodiments, the dimerised linkers are selected from the compounds of general formulae IX and X:

X

wherein X is as defined above.

In the present invention, the first and second moieties, which may be the same or different, are preferably a chemical entity and/or a solid support.

Preferably, the chemical entity is an isolated or purified Chemical entity. Examples of chemical entities include macromolecules, most preferably biological macromolecules. Examples of biological macromolecules include nucleic acid molecules, proteins, polypeptides and peptides. Preferably, the biological macromolecule is in isolated or purified form, e.g. an isolated nucleic acid molecule or a purified protein, polypeptide or peptide. Examples of nucleic acid molecules include DNA and RNA. The nucleic acid molecule may be single-stranded or double-stranded or part single- and part double- stranded. In some preferred embodiments of the invention, the macromolecule is a single-stranded oligomer, preferably a DNA oligomer. Examples of proteins and polypeptides include enzymes and antibodies. Preferably the enzyme is an enzyme which is capable of -producing a detectable reaction, such as horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose oxidase or a restriction enzyme.

In some embodiments of the invention, the first and/or second moiety is not a cell membrane or a cell membrane fraction. By the term "cell membrane or cell membrane fraction" is meant that the first and/or second moiety is not a biological cell membrane (e.g. a membrane from a red blood cell) or a fraction therefore, for example a fraction that has been obtained by centrifugation of the cell membrane in a solvent. In other embodiments, the first and/or second moiety is not a cell membrane protein, in particular not an anion channel protein.

The first and/or second moieties may also be a structural binding peptide such as streptavidin or avidin, or a label, such as biotin.

In some embodiments of the invention, one or both of the moieties may be an antibody. The antibodies may be of any suitable source, e.g. monoclonal, polyclonal, chimeric, bispecific, single-chain or fragments thereof. Antibody fragments include, but are not limited to. Fab, Fab' and F(ab')2, Fd, single- chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region (s) alone or in combination with the entirety or a portion of the following: hinge region, CHl, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHl, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals, or derived from phage or ribosome display libraries. Preferably, the antibodies are human, murine (e.g. mouse or rat), donkey, rabbit, goat, guinea pig, camel, horse, or chicken. The antibodies of the invention may be monospecific, bispecific, trispecific or of greater multispecificity.

The antibodies may be labelled in any suitable manner, thus allowing for , detection in a suitable assay.

In other embodiments of the invention, one or both of the moieties is a solid support, such as a particle or bead, or magnetic bead, or matrix such as a sheet, gel, filter, membrane, fibre, tube, microtitre plate or column. Other examples of solid supports according to the invention are dipsticks, polymers, fibre optics and glass and electronic devices (e.g. diodes and transistors). Such solid supports may either already comprise one or more free amino groups or they may be amino-functionalised.

In some embodiments of the invention, one or both moieties is a medicament, for example an antibiotic, antifungal or anticancer agent.

In other embodiments of the invention, one or both moieties is a dye, for example, fluorescein, rhodamine or Texas Red. Preferably the dye is fluorescein.

In yet other embodiments of the invention, one or both moieties is a moiety as

- ii - defined above which incorporates a radiolabel. The radiolabel may, for example, be ¹²⁵ I or Europium.

The molar ratio of the first moiety to the linker in step (i) is preferably in the range 1:45 to 1: 140. The molar ratio is chosen to maximise the efficiency of the conjugation without unduly lengthening the overall process time. Preferably, the molar ratio is 1:60 to 1: 120, more preferably 1:80 to 1: 100, and most preferably about 1:90.

The molar ratio of the second moiety to the first moiety in step (ii) is preferably in the range 1:1 to 8:1. The molar ratio is chosen to maximise the efficiency of the conjugation without unduly lengthening the overall process time. Preferably, the molar ratio is 2: 1 to 6: 1, more preferably 3: 1 to 6: 1, and most preferably about 4:1.

The buffer is chosen as one that is suitable for conjugating the chosen first and second moieties with the chosen linker. Suitable buffers are well known in the art. Preferably the buffer is an aqueous buffer. If the first and/or second moiety is a biological molecule, the buffer should ideally be a physiologically-acceptable buffer. Examples of suitable aqueous buffers include PBS, borate-based buffers, Tris-HCl, etc. Most preferably, the buffer is a sodium borate buffer.

One or more co-solvents may also be used, e.g. DMF, DMSO and/or an alcohol, for example ethanol, propanol or butanol.

The pH will be chosen to maximise the efficiency of the conjugation. Examples of suitable pHs include pH 5.5 to 9.5. If the first and/or second moiety is a biological molecule, the pH should ideally be a physiologically- acceptable pH, for example pH 6 to 9, more preferably pH 6 to 8.5, most preferably about pH 8.0.

The reaction times for steps (i) and (ii) are chosen to maximise the efficiency of the conjugation without unduly lengthening the overall process time. The reaction times for steps (i) and (ii) may the same or different. If times of less than 15 minutes are used, an undesirable amount of linker and first/second moieties may be unreacted. If times of more than 120 minutes are used.

polymer formation may occur. Examples of suitable reaction times are about 15, about 30, about 45, about 60, about 75, about 90, about 105 or about 120 minutes. Preferred reaction times are 15 to 45 minutes, 30 to 90 minutes and 45 to 75 minutes. Most preferably, the reaction time is about 30 minutes or about 60 minutes.

The temperature of the reaction is chosen to maximise the conjugation efficiency. The reaction temperatures for steps (i) and (ii) may the same or different. Examples of suitable temperatures are 4 to 50°C.

If the first and/or second moiety is a biological molecule, reaction temperatures should preferably be kept within physiological ranges, for example between 4 and 37°C. Preferably the reaction temperature is 4-37°C, most preferably about room temperature, e.g. about 21 ⁰ C.

In one preferred embodiment of the invention, step (ii) is carried out directly after step (i). By the term "directly after", it is intended to mean that step (ii) is carried out without a delay or intermediate step after step (i) .

In other preferred embodiments of the invention, when carrying out step (ii), the second moiety is added to the reaction mixture obtained from step (i), i.e. the first moiety-linker conjugate obtained from step (i) is not purified or is not treated in any other way before the second moiety is added to the reaction mixture.

Preferably, both step (i) and step (ii) are carried out in a single reaction vessel using the same buffer.

At the end of the conjugation process, the conjugate may be purified by any suitable process. Examples of purification processes include chromatographic means, for example ion exchange, anion/cation exchange chromatography, FPLC or reverse phase, hydrophobic interaction, gel filtration, membrane separation, extraction, gels, capillary electrophoresis, affinity chromatography, precipitation, centrifugation and osmosis. Preferred purification processes are anion exchange, gel filtration and membrane separation (e.g. dialysis).

For example, the conjugate may be purified by FPLC using a Tris/salt

gradient.

After purification, the conjugate may be stored, for example at 4° C in 2OmM Tris pH 7.5, 0.1-0.5 M sodium chloride etc. Under such conditions, the conjugate should remain stable for at least 5 months.

Alternatively, the conjugate may be freeze-dried or lyophilised. Stabilising agents (such as BSA, antibacteriocides, detergents, surfactants and sugars) may also be added to the conjugates (either in solution, freeze-dried or lyophilised).

Preferred conjugates from step (i) are antibody- or enzyme-linker conjugates, particularly those wherein the enzyme is horseradish peroxidase or glucose oxidase, and particularly those wherein the linker is DIDS. Further preferred conjugates from step (i) include solid support-linker, for example "activated" beads, dip-sticks, micro-titreplates, polymers, fibre optics, glass and electronic devices.

Preferred conjugates from step (ii) include enzyme-antibody, enzyme-enzyme, enzyme-oligonucleotide, enzyme-DNA conjugates, enzyme-solid support and antibody-solid support. Particularly preferred conjugates include horse-radish peroxidase-antibody and horse-radish peroxidase-oligonucleotide conjugates; glucose oxidase-antibody and glucose oxidase-oligonucleotide conjugates. Specific examples of particularly preferred conjugates are horse radish peroxidase-DIDS-antibody and horse-radish peroxidase-DIDS- oligonucleotide conjugates; glucose oxidase-DIDS-antibody and glucose oxidase-DIDS-oligonucleotide conjugates.

In one particularly preferred embodiment of the invention, an enzyme is reacted with about a 90 fold molar excess of crosslinker for about 30 minutes.

The linker-enzyme conjugate is then reacted in about 2.4 fold molar excess (enzyme:antibody) with an antibody for 30 minutes. Preferably, the enzyme is horseradish peroxidase, the linker is DIDS, and the antibody is an anti- HGG antibody.

The invention also relates to a process for conjugating a first moiety with a second moiety, as herein defined, which additionally comprises the step of

reacting one or more compounds of formula I with a diamine of formula XI:

XI

H ₂ N NH ₂

wherein Y is as defined above, in order to produce a dimerised linker of formula II.

^• The linkers of the invention can be used, for example, to attach DNA or polypeptides to solid supports such as polystyrene beads, plastic wells, magnetic particles, electronics or optical fibres.

The invention also provides a kit comprising:

(a) a linker of any one of formulae I, III, and V as herein defined; and

(b) a diamine of formula XI as herein defined.

The invention also provides a kit comprising:

(i) a first moiety-linker conjugate, wherein the linker is of any one of formulae I, II, III, IV, V, VI, VII, VIII, IX or X; and (ii) a coupling buffer.

The first-moiety-linker conjugate is preferably in lyophilised or freeze-dried form.

The coupling buffer is preferably one which is suitable for coupling the first- moiety-linker conjugate to a second moiety, wherein the second moiety comprises one or more free amino groups. Most preferably, the coupling buffer is 0. IM sodium borate, pH 8. The coupling buffer may be provided in dry or aqueous form.

The kit optionally includes instructions for coupling the first moiety-linker conjugate to a second moiety.

The invention also extends to conjugates which are produced using the processes of the current invention.

The invention further provides a compound of formula II:

wherein

R ¹ to R ⁴ , R ⁷ to R ¹⁰ > R ¹¹ to R ¹⁴ and R ¹⁷ to R ²⁰ are independently selected from H, halogen, Ci- ₁₀ -alkyl, C ₃ - ₈ ~cycloalkyl, OH ₅ O-d-s-alkyl, SH ₃ NH ₂ , NO2, CO ₂ H, CO ₂ -C i-3-alkyl, SO ₃ H and SO3X, wherein X denotes a counter ion, for example, ISTHU ⁺ , Na ⁺ or K ⁺ ;

R ⁵ , R ⁶ , R ¹⁵ and R ¹⁶ are independently selected from H, halogen, Ci-6-alkyl, OH, O-Ci-3-alkyl, SH, NH ₂ , CO ₂ H, and CO ₂ -Ci- ₃ -alkyl;

m and n are each independently 1, 2 or 3, and

Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone.

Preferably, R ¹ to R ⁴ , R ⁷ to R ¹⁰ , R ¹¹ to R ¹⁴ and R ¹⁷ to R ²⁰ are each independently selected from H ₃ methyl, ethyl and SO3X.

The invention further provides a compound .of formula IX or X:

X

wherein X is NH ₄ ⁺ , Na ⁺ or K ⁺ .

The invention also provides a moiety-linker conjugate, wherein the linker is one of formulae I ₅ II, III, IV, V, VI, VII, VIII, IX or X as defined herein. Preferably, the moiety is as defined herein.

The invention also provides a first moiety-linker-second moiety conjugate wherein the linker is one of formulae I, II, III, IV, V, VI, VϊI, VIII, IX or X as defined herein; and wherein the first and/or second moiety is as defined herein.

The invention also provides a conjugate as defined herein for use as a medicament; and a conjugate as defined herein for use in a diagnostic method practised on the human or animal body.

The invention also provides the use of a conjugate as defined herein in an ex vivo diagnostic method; and the use of a conjugate as defined herein in the manufacture of a diagnostic tool for use in a diagnostic method practised on the human or animal body.

The invention further provides the use of a conjugate as defined herein in the manufacture of a medicament for use in a method of treatment of the human or animal body.

The invention also provides a method of treating a patient comprising administering to that patient a conjugate as defined herein; and a method of diagnosing a condition in a patient comprising administering to that patient a conjugate as defined herein.

The following abbreviations are used throughout this specification:

BSA Bovine serum albumin

DIDS 4,4'-diisothiocyanato-2j2'-stilbenedisulfonic acid

5 DMF Dimethylformamide

DMSO Dimethyl sulfoxide

ELISA Enzyme-linked immuno-sorbent assay

ELOSA Enzyme-linked oligo-sorbent assay

FPLC fast protein liquid chromatography

10 HCG human chorionic gonadotropin

HPLC High performance liquid chromatography

HRP horseradish peroxidase

PBS phosphate-buffered saline

Tris tris (hydroxymethyl) aminomethane ^• 15

BRIEF DESCRIPTION OF FIGURES

Figure 1 illustrates the purification of a HRP-DIDS-anti-HCG antibody conjugate on an ion exchange column on an Akta Prime liquid

20 chromatographic system.

Figure 2 illustrates the purification of a HRP-DIDS-anti-Inhibin A antibody conjugate on an ion exchange column on an Akta Prime liquid chromatographic system. 25

Figure 3 illustrates an ELISA of a HRP-DIDS-anti-Inhibin A conjugate of Example 3 in comparison with a commercial standard (control) assay for Inhibin A.

30 Figure 4 shows the purification of a HRP-DIDS-oligonucleotide by anion exchange chromatography.

Figure 5 illustrates an ELOSA of two HRP-DNA conjugates of the invention.

EXAMPLES

Example 1

Preparation of HRP-DIDS-anti-HCG antibody conjugate

Materials

Monoclonal anti-HCG antibody, British Biocell International, batch 7416, 5.3 mgs/ml.

Crosslinking agent DIDS, Aldrich 285617. Horse radish peroxidase, Bio2yme Labs, code HRP 4B, Batch 935 C Sodium Borate buffer pH 8, 0.1 M

Reagent concentrations

Reagent m.wt mass(mg) moles in reaction

HRP 40000 0.27 6.8 nmol

DIDS 498 0.3 0.6 μmol

ANTIBODY 150000 0.4 2.7 nmol

Protocol

To the HRP dissolved in borate buffer (45μl) was added DIDS in borate buffer (5μl). The reaction mixture was shaken for 0.5 hours and then the antibody (75.6μl of supplied solution + 124μl borate buffer) was added and the shaking continued for a further 0.5 hours. ^■

The product was purified by anion exchange chromatography using a Q- Sepharose column and AKTA prime chromatography unit. The product was eluted in a gradient of 0 to IM sodium chloride in 2OmM Tris pH 7.5. (Fig

1).

Example 2

Preparation of bead-DIDS-anti-HCG antibody conjugate

Antibodies can be linked to amino functionalised beads (for example, polystyrene) using.a similar procedure to that described in Example 1.

Materials

Polyscience amino-functionalised polystyrene beads 2μm.

Lot GK002050 IT, 50mg/ml, 4nmol of amine/mg.

DIDS and antibody as Example 1.

Protocol

1 mg of beads contained in a ImI Eppendorf tube(with the cap removed and placed in a FALCON tube) were washed with borate buffer (2x300μl) and concentrated by centrifugation (2000rpm, 4 minutes per buffer wash). To the concentrated beads was added 200μg DIDS dissolved in 50μl borate buffer. The mixture was shaken at 50 revolutions per minute for 1 hour and then washed with borate buffer (2x300μl ) and concentrated. 20μl (O.lmg) of antibody in 80μl borate buffer was added to the beads and the mixture shaken for a further 1 hour. The conjugated beads were washed with borate buffer (2x300μl) and then stored at 4°C in 2OmM Tris pH 7.5.

Enzyme antibody conjugates are quality controlled by immunoassay. A capture antibody is immobilised on polystyrene beads' and the beads dried onto a membrane (dipstick). The immunogen+conjugate dissolved in an elution buffer is then applied to the dipstick and the mixture allowed to run up the stick. Enzyme substrate is added and any signal observed at the application point of the beads is noted.

Example 3

Preparation of HRP-DIDS-anti-Inhibin A conjugate

An HRP-DIDS-anti-Inhibin A (a fertility marker) conjugate was prepared using a similar procedure to that described in Example 1. Figure 2 shows the chromatographic (AKTA prime) purification profile obtained.

This conjugate was quality controlled using the DSL Ltd Inhibin A (DSL-10- 28100) ELISA kit. Figure 3 demonstrates the superior performance of the prepared conjugate versus a commercial control in an ELISA for Inhibin A.

The level of detection of target Inhibin A by the present conjugate was lOpg/ml. In comparison, the commercial product was at least 10 times less sensitive (detection level of 250pg/ml).

Example 4

Preparation of HRP-DIDS-oligonucleotide

Materials

Amino-link oligonucleotide to DNA target:

Amine-30mer oligonucleotide in EbO, lOOμM, HPLC purified (Sigma

Genosys);

Cross linking reagent: DIDS;

HRP: HRP 4B - Batch 947C (50mgs); and

Sodium Borate buffer pHδ.O, 0.1M.

Reagent concentrations

Reagent m.wt. mass(mgs) mols in PW Sodium borate buffer vol

HRP 40000 0.675 17nmols 45μl

DIDS 498 _. 0.75 1.5μmol 5μl

Amino-linked oligonucleotide 9395 0.063 6.7nmols 67μl (or 134μl for 2x)

Protocol

To the HRP dissolved in sodium borate (45μl) was added the coupling agent DIDS in borate buffer (5μl). The reaction mixture was shaken for 0.5 hours and the oligonucleotide (67μl or 134μl for 2x) was added and the shaking continued for a further 0.5 hours (total volume =117μl or 184μl). 2OmM Tris pH 7.5 (250μl) was added to stop the reaction.

The product was purified by anion exchange chromatography using a Q- Sepharose column and AKTA Prime chromatography unit. The product was eluted in a gradient of 0 to IM sodium chloride in 2OmM Tris pH7.5 (Figure 4).

Figure 5 demonstrates the comparative performance in an ELOSA of two HRP-DNA conjugates differing only in the structure of the linker connecting the DNA strand to the enzyme (linker 1 is DIDS ₃ linker 2 is DIDS-lysine- DIDS).

Application of linker 1 led to a level of detection of 400pg/ml (lfmol of target).

Example 5

Kit comprising a pre-activated moiety

A kit is produced comprising (i) a lyophilised or freeze-dried HRP-DIDS conjugate as described in Example 1, and (ii) an aqueous coupling buffer (0. IM sodium borate, pH 8).

At the time of use, the coupling buffer is added to the lyophilised or freeze- dried HRP-DIDS conjugate. An antibody is then added and conjugation is allowed to proceed for 0.5 hours. The reaction is stopped by the addition of 20 mM Tris/HCl, pH 7.5.