WO1995004931A1

WO1995004931A1 - Diagnostic method

Info

Publication number: WO1995004931A1
Application number: PCT/GB1994/001739
Authority: WO
Inventors: Beverley Jane Randle
Original assignee: Surface Active Limited
Priority date: 1993-08-06
Filing date: 1994-08-08
Publication date: 1995-02-16
Also published as: GB9316369D0; AU7347694A

Abstract

The invention provides a diagnostic method for determining the presence, or absence of an antigen in a sample, the method comprising contacting the sample with a multispecific antibody having first and second binding sites for binding the antigen and a detectable entity respectively, binding of the detectable entity to the antibody inactivating the detectable entity in the presence of an analogue of the antigen, wherein binding of the analogue of the antigen to the antibody results in release of the detectable entity in a detectable form from the antibody but binding of the antigen to the antibody does not cause such release or causes significantly lower levels of release. Preferably, the antigen is a subunit of inhibin.

Description

DIAGNOSTIC METHOD

This invention relates to a diagnostic method and is particularly, though not exclusively, concerned with an assay for relatively small antigens such as inhibin.

Inhibin is a non-steroidal gonadai glycoprotein central in the regulation of pituitary Follicle Stimulating Hormone (FSH) secretion. Inhibin inhibits secretion of FSH by the pituitary (reviewed in Ying SY (1989), J. Steroid. 33, 705). Inhibin consequently has a key role in control of ovulation and sperm production. Inhibin and associated glycoprotein hormones have biological effects outside the reproductive system, with growth factor properties in the central nervous system and the immune response. Biotechnological applications of inhibin are widespread. In man, serum inhibin is a highly specific indicator of fertility in In Vitro Fertilisation (INF), and other clinical situations. Inhibin is also proving an excellent marker for management of certain forms of ovarian cancer. Experimental uses of inhibin biotechnology in animal husbandry have included induction of ovulation, extension of breeding season, and assessment of reproductive capacity.

The extensive commercial applications of inhibin biotechnology have yet to be realised as progress has been severely hampered by lack of an assay for bioactive inhibin and a source of the purified molecule.

Purification of inhibin in 1985 (Robertson DM et al (1985), Biochem Biophys. Res. Commun. 126, 220) led to a partial amino acid sequence and determination of the structure of the molecule (Mason AJ et al (1985), Nature 318,659). The two forms of 32 kiloDalton (kD) inhibin, A and B, consist of a common a subunit, 18 kD, and similar, but distinguishable, β subunits, β A and β B, 14kD. The α subunit is joined to either β subunit by disulphide linkage to give bioactive inhibin (Finlay JK et al (1987), J Reprod Fertil 80, 455). The biological activity of inhibin is mediated by the β subunit.

Secretion of the α subunit monomer has been detected, but the glycoprotein lacks the biological activity characteristic of inhibin (Knight PG et a\ (1989), J. Mol. Endocnnol. 2, 189). Activin is the secreted dimer of the β subunit and, in contrast to inhibin, enhances FSH secretion, despite the structural similarity to inhibin (Ling N et al 1986), Nature 321, 779). The β subunit has strong homology for Transforming Growth Factor (TGF). However, secretion of free β subunit monomers has yet to be discovered.

Production of inhibin is regulated mainly by FSH. Inhibin secretion, in response to FSH from the pituitary, originates primarily from the granulosa cells of the ovary and the Sertoli cells of the testis. Serum inhibin levels in woman rise to maximum in the mid-luteal phase of the menstrual cycle and decline prior to menstruation. In pregnancy the luteal phase does not occur, and levels increase progressively, suggesting inhibin is also produced by the corpus luteum and the placenta (Abe Y et al (1990), J. Clin. Endocnnol. Metab 71, 133). In man, inhibin is secreted in well-defined pulses from the testis, coincident with testosterone release (Winters SJ (1990), J Clin Endocnnol Metab 70, 548). Serum inhibin levels rise throughout normal male and female puberty, where gonadai inhibin production is stimulated by increasing FSH levels (Burger HG et al (1988), J Clin Endocnnol Metab

67, 689).

The biological effects of inhibin have been demonstrated by in vivo immunisation against the molecule. Antibody blocking of inhibin secretion in ewes leads to raised serum FSH and, in some studies, to an increase in ovulation (Forage G et al (1987), /. Endocnnol. 11,

PR1). In the male rat, reduction of serum inhibin by immunisation results in decreased sperm production and a significant fall in fertility (Nanage GR et al (1990), Mol. Reprod. Dev. 25, 227). With the reciprocal interaction between FSH and inhibin, inhibin has applications as a contraceptive in the male, and as an agent for treatment of infertility in the female.

Both inhibin and activin have biological effects outside the reproductive system, having functions as growth factors, and mRΝA for the glycoproteins has been detected in other tissues. Inhibin inhibits the synthesis of haemoglobin in human bone marrow whilst activin stimulates haemoglobin production (Yu J et al (1987), Nature 330, 765). Activin is also found to control oxytocin secretion in central neural pathways (Sawchenko PE et al, Nature 334, 615). In common with TGFβ, both inhibin and activin regulate T cell differentiation in vitro, suggesting a role for these glycoprotein hormones in control of the immune response (Hedger MP et al (1989), Mol Cell Endocnnol 61, 133).

Serum inhibin levels are elevated in women suffering from ovarian granulosa cell tumours.

Inhibin levels are raised months before overt tumour recurrence, at times when the clinical tumour marker of oestradiol levels is still normal (Lappohn RE et al (1989), N. Engl. J. Med. 321, 790). Inhibin may also be a useful marker for hydatidiform mole, where raised inhibin levels after surgery indicate residual trophoblastic tumour disease (Yohkaichiya T et al (1989), Br. Med. J. 298, 1684).

Inhibin biotechnology has both immediate and far reaching applications in medicine and animal husbandry. At present, medical use of inhibin biotechnology relate to measurement of serum inhibin for clinical evaluation of fertility and for tumour diagnosis.

Measurement of serum inhibin has proved useful in assessment of reproductive capacity in a variety of clinical situations. Restoration of normal sperm production in treatment of hypogonadism is indicated by increased serum inhibin levels, in excess of normal values (Tsatsoulis A et al (1990), Horm Res 33, 18). In women, decreasing serum inhibin levels are an early index of declining ovarian function with advancing age (Hughes EG et al

(1990), J. Clin. Endocnnol. Metab. 70, 358). In INF Programmes, serum inhibin is proving a valid index of follicle maturation prior to use of the ovum for in viro fertilisation (Tsuchiya K et al (1989), Fertil. Steril. 52, 88).

Measurement of serum inhibin also has major use in treatment of women suffering from granulosa cell tumours. Granulosa cell tumours account for 10% of ovarian cancer cases and arise from specialised cells in the ovarian stroma which secrete inhibin. Primary treatment is surgical, and 80% of patients die of recurrent disease. Serum inhibin has proved an excellent indicator of the size of the primary tumour and of early disease recurrence, prior to overt clinical evidence. As the recurrent disease responds well to combined chemotherapy, a serum inhibin assay has significant application in clinical management of women suffering from granulosa cell tumours.

A second generation of medical applications is indicated, related to alteration of circulating inhibin levels. Selective inhibition of FSH secretion in the male, by increasing serum inhibin levels, has potential for male contraception by reducing sperm production (Frick J and Aulizky W (1988), Hum Reprod 3, 147). In contrast, in woman, artificially-reduced inhibin levels can potentially increase FSH secretion and improve fertility. Treatment with anti-inhibin agents has great potential for INF Programmes, where conventional fertility drugs have a high risk of multiple pregnancy.

The biotechnology of inhibin is already well advanced in animal husbandry. Manipulation of serum inhibin levels by passive and active immunisation against inhibin (Mann GE et al (1990), J. Endocnnol. 125, 417 and Forage G et al (1987), J. Endocnnol. 114, PR1) has led to increased ovulation rates in sheep, and by active immunisation , in cattle (Price CA et al (1987), J. Reprod. Fertil. 81, 161). Puberty in ewe lambs can be advanced by active immunisation against inhibin (Al-Obaidi SA et al (1987), /. Reprod. Fenil. 81, 403), and the breeding season of rams can be delayed, augmented and extended by anti-inhibin immunisation (Noglmayr JK et al (1990), Biol. Reprod. 42, 81).

Full realisation of the potential applications of inhibin biotechnology is being hampered by lack of a purified inhibin source and by the inability to measure bioactive inhibin. The two problems are closely related, as lack of a purified source of inhibin has prevented development of the immunological reagents essential for inhibin assay design.

Production of a source of recombinant bioactive inhibin has proved difficult. Amino acid sequences of inhibin and activin have been deduced from cDΝA and genomic clones (Mason AJ et al (1985), Nature 318, 659 and Mason AJ et al (1989), Mol. Endocnnol. 3, 1352). Recombinant inhibin has reduced biological activity, as glycosylation of both subunits, and associated subunit assembly, is necessary for full biological activity (Bremner WJ (1989), NEngl J. Med. 121, 826). Recent studies indicate that pro-regions not present in activin or TGFβ, are essential for folding, disulphide bond formation and export of the dimeric protein (Gray AM and Mason AJ (1990), Science 247, 1328). These pro-regions must also therefore be present in isolated cDNA clones for production of recombinant inhibin. The first biologically-active recombinant inhibin A was subsequently reported in mid 1990 by Biotech Australia Ltd (Tierney ML et al (1990), Endocrinology 126, 3268).

A source of recombinant inhibin will undoubtedly aid assay development, where traditional biochemical isolation of inhibin yields only 3% recovery, despite a 2,200 fold purification (Knight PG et al (1990), Domest. Anim. Endocrinol 7, 299).

Inhibin bioactivity was first measured by a bioassay dependent on suppression of pituitary

FSH secretion in vivo in ovariectomised ewes (Findlay JK et al (1987), J. Reprod. Fertil. 80, 455) or in vitro by culture of rat pituitary cells (Lee VW et al (1987), Aust. J. Biol. Sci. 40, 105). In vivo methods proved imprecise, insensitive and necessitated use of a large number of animals. In contrast, in vitro bioassay was quantitative but time-consuming, requiring two days for completion.

Development of antisera to bovine follicular fluid extracts permitted design of the first radioimmunoassay, RIA, for inhibin (McLachlan RI et al (1987), /. Clin. Endocrinol. Metab. 65, 954). The heterologous RIA did not detect related glycoproteins of activin A or TGFβ, in comparison to a sheep pituitary cell bioassay (Robertson DM et al (1988), J.

Clin. Endocrinol. Metab. 67, 438). This lack of cross reactivity has since been found to result from the epitope specificity of the RIA being entirely localised to the α subunit of inhibin (Schneyer AL et al (1990), J. Clin. Endocrinol. Metab. 70, 1208). The validity of the existing RIA may be questioned, as a subunit monomers of inhibin are secreted into human serum (Knight PG et al (1989), J. Mol. Endocrinol. 2, 189. These monomers will be detected as bioactive dimeric α-β linked inhibin, giving a false positive result.

The first two site immunoassay for inhibin was reported at the end of 1990 by Medgenix

Diagnostics, Liege, Belgium. The assay relies on capture and detection of two epitopes on the α subunit of inhibin. Assay inaccuracy, resulting from cross reaction to free α monomer, is as yet unreported (Eliard P et al (1990), Inhibin: Clinical Investigations, Brussels, Sept 1990) but is believed to be a problem.

In our co-pending International patent application No. PCT/GB92/00769, the disclosure of which is incorporated herein by way of reference, we describe a novel assay system involving a bispecific antibody having closely adjacent binding sites for the analyte of interest and a entity such as an enzyme which is readily detectable when not bound to the bispecific antibody. For example, an enzyme active site may be blocked when the enzyme is bound to the antibody. The system is arranged such that binding of the analyte to the respective binding site of the antibody causes release of the detectable entity, as a result of steric interaction between the analyte and the entity.

Certain relatively small analytes such as inhibin may not readily be detected by such a system because their size reduces die steric interaction, even with a relatively large detectable enzyme.

It is an object of the. present invention to provide an assay system for relatively small molecules such as inhibin.

According to one aspect of the invention there is provided a diagnostic method for determining the presence, or absence of an antigen in a sample, the method comprising contacting the sample with a multispecific antibody having first and second binding sites for binding the antigen and a detectable entity respectively, binding of the detectable entity to the antibody inactivating the detectable entity in the presence of an analogue of the antigen wherein binding of the analogue of the antigen to the antibody results in release of the detectable entity in a detectable form from the antibody but binding of the antigen to the antibody does not cause such release or causes significantly lower levels of release. Thus antigen in the sample competes with the analogue for the first binding site and so reduces the amount of detectable entity released from the antibody. Thus, the present invention provides an assay for relatively small antigens, such as inhibin, in which competition for a binding site on a multispecific antibody is used to affect the level of detectable entity detected which is related to the amount of antigen in the sample. Usually, the concentration of antigen and the amount of detected detectable entity will be inversely related.

The term "analogue" as used herein embraces all antigens capable of binding to the same or closely related binding site on the multispecific antibody as the antigen of interest. The analogue may or may not include the same or similar structure as the antigen or a portion thereof. Preferably, the analogue comprises the antigen or a portion thereof linked to a larger entity so that the size of the resulting combination is sufficient to cause the release of bound detectable entity from the antibody.

In one embodiment, the analogue comprises at least a portion of inhibin or a functional equivalent thereof bound to Keyhole Limpet Haemocyanin a widely used antigen about 800 kD in size. Other large molecules may be used as larger entities to form the analogue of inhibin (or any other antigen of interest) such as Bovine Serum Albumin (BSA) although KLH is preferred because it is larger and relatively inexpensive and does not have the cross- reactivity problems of BSA. Typically, any such larger molecule will have a molecular weight exceeding 200kDaltons. Preferably the inhibin comprises at least the β subunit thereof. Functional equivalents of inhibin include recombinant polypeptides having some or all of the biological activity of natural inhibin or portions thereof.

The analogue may comprise a recombinant polypeptide including a portion capable of binding to the binding site or a closely related binding site to which the antigen of interest binds and a biologically redundant portion which is sufficiently large to cause release of the detectable entity as described above.

The antigen may be any small molecule to which a binding site on a multispecific antibody can be directed. Preferably the antigen is inhibin. Thus the invention provides a new and useful assay for inhibin.

The antigen may also be, for example, activin, oligouncleotides, saccharides, growth factors, neurotransmitters, hormones such as progesterone, oestrogen, LH, or FSH. In principle, polypeptides contaimng as few as five amino acids may be assayed using the system of the present invention.

The term "multispecific" embraces all antibodies having more than one antigen binding site such as bispecific and trispecific antibodies. Preferably the multispecific antibody is bispecific. The term "antibody" used herein embraces immunoglobulins such IgG, IgA,

IgM, IgD and IgE and other proteins or polypeptides having the antigen-binding properties of a naturally-occurring antibody, or antibodies, produced by recombinant DNA technology or any other method.

Preferably, the detectable entity is an enzyme such as glucose oxidase or more preferably β-galactosidase.

The choice of a suitable enzyme is within the scope of the skilled worker in this field.

A preferred diagnostic method in accordance with the invention comprises the steps:

1) Providing the multispecific antibody having bound detectable entity on one binding site thereof;

2) contacting the thus bound multispecific antibody with an analogue of an antigen of interest whereby binding of the analogue to a second binding site on the multispecific antibody results in release of bound detectable entity from the multispecific antibody generating a relatively high detectable signal;

3) contacting the system of step 2) with a sample of interest containing the antigen to be assayed whereby binding of the antigen to the antibody in competition with the analogue results in reduction of the signal level proportional to the amount of antigen of interest in the sample.

The diagnostic method of the invention may be operated on a convenient substrate such as a dipstick or in homogenous form as a near patient one step test.

In a further preferred embodiment, an enzyme comprising the detectable entity in a first system in accordance with the invention produces substrate for an enzyme comprising the detectable entity in a second system in accordance with the invention. Thus the two enzymes form a cascade. For example, in a diagnostic method for detecting the presence of both a and β subunits of inhibin in a sample, one system in accordance with the invention arranged to detect the β subunit is provided including the enzyme glucose oxidase comprising the detectable entity, and another system in accordance with the invention is provided arranged to detect subunit in which the enzyme horse radish peroxidase. In the presence of both inhibin subunits, released glucose oxidase produces the substrate for the horse radish peroxidase.

A diagnostic method in accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings Figures 1, 2, 3,4,5 and 6 in which:

Fig. 1 shows a multispecific antibody and associated antigen of interest and analogue;

Fig. 2 a, b and c show schematically the method of the invention;

Fig. 3 shows antigen capture of the β inhibin peptide;

Fig.4 shows antigen capture of glucose oxidase;

Fig. 5 shows partial inhibition of enzyme activity by the antibody; and

Fig. 6 shows a competitve transduction assay. The bispecific antibody 10 shown in Fig. 1 includes first and second binding sites 12 and 14. First binding site 12 is arranged to bind antigen 16 of interest. Antigen 16 is the β chain of inhibin. Alternatively binding site 12 will bind analogue 18 of the β chain of inhibin 16. Analogue 18 comprises KLH 20 bound to the β chain of inhibin 22 by conventional techniques. Specifically the KLH is succinimide linked to an amino acid inserted at position 23 of the β chain. Second binding site 14 is arranged to bind glucose oxidase 24 whereby its active site 26 is blocked when the enzyme is bound, the active site being unblocked, and therefore active, when the enzyme is released from the antibody.

Antibody 10 was produced using conventional techniques as outlined in our above mentioned co-pending International patent application.

As mentioned above KLH is a relatively large molecule, having a molecular weight of 800 kD. Binding of analogue 18 to bispecific antibody 10, when glucose oxidase 24 is already bound thereto, causes release of the bound glucose oxidase from the antibody in an active form. However, binding of the β chain of inhibin 16 to binding site 12 does not cause release of glucose oxidase bound at binding site 14, presumably because the inhibin does not sterically interact with the bound enzyme. Binding of KLH alone to the antibody will not occur.

The assay method of the present invention is illustrated in Fig. 2 a, b & c.

In the situation illustrated in Fig. 2a, binding of β subunit of inhibin 16 to bispecific antibody 10 does not cause bound glucose oxidase 24 to be released from the antibody 10. Therefore no signal (i.e a colour change) or only background levels of signal, is produced in the presence of the enzyme substrate (e.g TNBT).

In the situation illustrated in Fig. 2b, where antibody 10 having glucose oxidase 24 bound thereto is contacted with the analogue 18 of Fig. 1, glucose oxidase 24 is released on binding of the analogue 18 to binding site. This release causes a high signal. In contrast in the situation illustrated in Fig. 2c, where β subunit 16 is present in a sample is contacted with the bispecific antibody 10 having bound glucose oxidase 24, in the presence of analogue 18, competition between the β subunit of inhibin 16 and the analogue reduces the amount of bound glucose oxidase which is released leading to a signal which is intermediate in strength between the signals generated in the situations of Figs. 2a and 2b. This detectable reduction in signal is proportioned to the amount of inhibin in the sample.

A Isolation of hybridomas secreting suitable monoclonal antibodiees

Monoclonal antibody-secreting cell lines were prepared recognising a synthetic peptide fragment of the β chain of human inhibin. A polypeptide having the amino acid sequence of residues 82-114 (NPTKLRPMSMLYYDDGQNIIKKDIQNMINEECG) of the inhibin β sub unit was synthesized using, standard peptide synthesis techniques. The resulting human inhibin β chain subunit ( the β peptide ) was approximately 4kD in molecular weight. The β peptide was conjugated to bovine serum albumin in a 5:8 weight ratio using n-Maleimodobenzoyl-Ν-hydrosuccinimide ester to give a protein conjugate, β-BSA, of 7kDaltons. The β peptide was also conjugated to Keyhole Limpet Haemocyanin (KLH) in a 5:8 weight ratio using sulphsuccinimidyl 4 (Ν-maleimido methyl) cyclohexane-1-carboxylate to give a protein conjugate, β-KLH, of approximately 800kDaltons.

Hybridomas secreting antibodies reognising the inhibin β peptide were prepared by standard methods. The hybridoma cell line E26 secrets a monoclonal antibody reactive with the β peptide. The cell line was isolated from a cell fusion experiment between mouse SP2/0 myeloma cells and splenocytes from a BALB/c female mouse immunized weekly over a four week period with a 10/xg per dose of β-KLH conjugate supported with an alum adjuvant. 20μg of β-KLH was given four days prior to die cell fusion experment. Cell fusion was performed by standard techniques and cultures were screened 17 days later by indirect ELISA. 593 hybridoma cell cultures were obtained, 26 of which were found to secrete antibodies reactive with β-KLH by indirect ELISA. Four of the 26 cultures secreted antibodies which were also reactive with β-BSA and failed to recognise KLH by indirect ELISA indicating that the 4 cell lines secreted antibodies reactive with the inhibin β -peptide. One of these 4 cell lines, the hybridoma E26 was chosen for further study. The cell line secretes a mouse IgG antibody directed against an epitope on the β subunit of inhibin. The antibody does not recognise the a subunit of human inhibin, as indicated by indirect ELISA. The antibody also recognizes the β peptide and β peptide conjugates on Western protein immunoblot analyses.

10

B Selection of drug resistant clones

Hybridomas secreting suitable monoclonal antibodies were cultured in toxic media to isolate drug resistant clones for bispecific fusoma production.

15

To induce thioguanine resistance 10⁷ E26 hybridoma cells were dispensed into 2x48 well tissue culture plates containing α MEM medium (Stanners CP et al (1971) Nature, New Biol 230, 52) with 10% (v/v) heat inactivated fetal calf serum, 20%(v/v) conditioned medium from J774 macrophage cell line (Cancer Res. (1977), 37, 546) and 5μg per ml of 20 6-thioguanine (Sigma A4660).

After four weeks, 23 clonal outgrowths were visible. Clones were removed by pipette and

subcultured. All 23 cell lines were found to secrete the E26 monoclonal antibody, with no

loss of antibody secretion as a result of drug selection. Subclone 3, E26tg3, was chosen for

25 further study, this cell line having a cell doubling time of approximately 18 hours.

^• j? ^'^

C Fusoma Production

Fusomas secreting bispecific antibodies were produced by conventional techniques in cell fusion events to select those producing bispecific antibodies with an enzyme-reactive arm

and a second antibody binding site recognising the antigen of interest.

Fusoma cell line N015 secretes a bispecific immunoglobulin reactive with human inhibin

β peptide and glucose oxidase from Aspergillus niger. The cell line was isolated from a cell

fusion event between hybridoma E26tg3, a thioguanine-resistant cell line secreting

antibodies recognising β peptide, and splenocytes from a BALB/c female mouse immunised

weekly over a four week period with 10μg per dose of glucose oxidase (Sigma G7141)

supported in an alum adjuvant. 20μg of glucose oxidase was given intravenously four days

prior to the cell fusion experiment.

Cell fusion was performed by standard techniques and cultures were screened 17 days later

by indirect ELISA against glucose oxidase as me target antigen. 371 fusoma cultures were

obtained, 84 of which were found to secrete antibody reactive with glucose oxidase by

indirect ELISA. 14 cultures secreted immunoglobulin reactive with both β peptide and

glucose oxidase as determined by ELISA and Western blot analysis. One of these cell lines,

NO 15 is now described.

The cell line was routinely grown in α MEM and produces approximately 2.0 μg per ml of

immunoglobulin in unstirred monolayer culture growth conditions.

Enriched NO 15 immunoglobulin was isolated by standard affinity chromatography techniques using protein A Sepharose (Sigma P3391). 608μg of immunoglobulin was isolated from 400 ml of culture medium.

Antigen capture to demonstrate recognition of both human inhibin β peptide and

glucose oxidase.

N015 immunoglobulin can be used in an antigen capture ELISA format to detect β peptide

and glucose oxidase.

Specific β peptide antigen capture (See Fig. 3).

NO15 immunoglobulin was coated at 10 g per ml in carbonate-bicarbonate buffer, pH 9.6,

50μl per well, in a 96 well flat bottom immunoassay plate (Falcon Cat. No. 3912) overnight

incubation at 4°C. Plates were blocked with 100μl per well, 10% (V/V) fetal calf serum

in phosphate buffered saline, PBS, 2 hrs at room temperature.

Increasing concentrations of β-KLH conjugate, the specific antigen, and KLH protein, a

non-specific antigen were loaded in 50μl volumes in duplicate, from 0-20μg per ml and

incubated for 1 hour at 100m temperature. Both β-KLH and KLH antigens were substituted

in the approximate ratio of 3 biotin molecules per protein molecule. Stock β KLH biotin

was 0.33 mg per ml for dilution, and stock KLH biotin 1 mg per ml for dilution.

Wells were washed twice with 200μl PBS 0.5% (v/v) Tween 20, PBS Tween, and then

bound antigen was detected by addition of Avidin-enzyme conjugate and developing for bound enzyme. Wells were incubated with 50μl of 1:500 dilution Avidin-alkaline

phosphatase (lmg per ml stock in PBS: Sigma A2527) in PBS with 0.5% (w/v) BSA (Sigma

A7888) for 30 minutes at 4°C. Wells were washed three times in PBS Tween and then

presence of alkaline phosphatase was detected by substrate conversion of para-nitrophenyl

phosphate (Sigma 104-105E). Briefly, 50μl per well of the substrate was added at lmg per

ml in 1M diethanolamine buffer, pH9.8, comprising 97ml died anolamine, 800ml water,

lOOmg magnesium chloride hexahydrate, with 1M hydrochloric acid added until the ph is

9.8, die volume then made up to 1 litre with water. Substrate conversion by enzyme to

product was detected by measurement of optical density of 410mn.

Specific recognition of the β peptide by NO 15 bispecific antibody is illustrated in Figure

3. Significant capture of the β peptide KLH conjugate is seen from l g per ml antigen concentration. In contrast, the non-specific antigen, KLH is not recognised and captured

by the NO 15 bispecfic antibody.

Glucose Oxidase antigen capture (See Fig. 4).

Concentrations of NO 15, from 0 to 10 g per ml were coated into wells of a 96 well flat

immunoassay plate using the carbonate-bicarbonate buffer method as above. Plates were

then blocked ready for use.

Wells were incubated with 50μl per well glucose oxidase, 10μg per ml in PBS with 0.5%

(w/v) BSA, the PBS/BSA buffer. The enzyme solution was incubated for 1 hour at 4°C and me plates then washed three times with PBS Tween. Bound enzyme was dien detected by addition of the enzyme substrate and subsequent conversion to product. Substrate conversion by enzyme was measured by optical density at 630nm.

Briefly, the glucose oxidase substrate was 0.82mM 3,3'-(3,3'-dimethoxy-4,4'

biρhenylylene)-bio-(2,5,p-nitrophenyl-2H-tetrazolium chloride, TNBT, (Sigma T4000)with

6.7mg per ml β-D-glucose (Sigma G5250) and 0.0167 mg per ml phenazine methosulphate

(Sigma P9625) in 50 mM Tris buffer pH 8.4. The substrate was prepared immediately

before use and the developing microtitre plates stored in the dark.

Specific capture of the glucose oxidase enzyme by NO15 bispecific antibody is illustrated

in Figure 4. An antibody concentration of 0.625μg per ml is sufficient to capture glucose

oxidase and at increasing antibody capture concentrations, enzyme activity is detected but

at a reduced level. This suggests that NO 15 antibody partially inhibits the activity of

glucose oxidase, perhaps by recognition of an epitope near the active site of the enzyme.

NO15 antibody partially inhibits the activity of glucose oxidase (See Fig. 5).

25 μl aliquots of NO15 immunoglobulin at 10μg per ml were added to 25μl volumes of

glucose oxidase solution in PBS/BSA buffer, concentrations from 0 to 10μg per ml enzyme,

in wells of a 96 well microlitre plate. Equivalent control concentrations of glucose oxidase

were prepared in 50μl volumes, in PBS/BSA buffer. The enzyme and antibody solution and the enzyme controls were incubated for 30 minutes at room temperature. 50μl per well of

glucose oxidase substrate solution were then added to measure residual glucose oxidase

activity. Conversion to product was measured by optical density at 630nm.

Partial inhibition of glucose oxidase activity by NO15 bispecific antibody is illustrated in

Figure 5. At all concentrations of enzyme, a concentration of lOμg per ml NO15 antibody

is found to reduce substrate conversion to product.

Demonstration of competitive antibody-mediated signal transduction (see Fig. 6).

Competitive transducing antibody activity has been demonstrated with NO15 antibody.

Immobilized NO 15 immunoglobulin was loaded with glucose oxidase (the "detectable

entity") to form a transducing antibody-enzyme complex. The complex was then exposed

to a standard concentration of the high molecular weight specific β antigen conjugate β-

KLH, molecular weight 800kDaltons (the "analogue") or equivalent concentration of non-

specific high molecular weight protein KLH, molecular weight 800kDaltons, with inclusion

in both experimental sets of varying concentrations of the low molecular weight competitor,

the β inhibin peptide of 3kDaltons (the "antigen"), specifically recognised by the NO 15

bispecific antibody. Supernatant was then developed to detect for the presence of released

glucose oxidase by enzymatic conversion of substrate to product.

The two events associated witii competitive antibody-mediated transduction are illustrated

in Figure 6. First, enzyme,the detectable entity, release only occurs at significant levels in the presence of the specific antigen of sufficiently high molecular weight to cause enzyme release, namely the conjugate between β peptide and KLH, β KLH, the analogue. Although, β inhibin peptide ,the antigen, is applied in me presence of the non specific

antigen KLH, there is no significant release of enzyme, the peptide being too small to

induce antibody-mediated signal transduction by NO 15 antibody.

Second, specific competitive antibody-mediated transduction is observed in the presence of

the antigen and analogue of differing molecular weight. At low concentrations of β

peptide,the antigen, below 2.5μg per ml, transduction occurs in the presence of the standard

concentration of β KLH,the analogue, lOμg per ml. However at high concentrations of the

small antigen, competition for the antibody binding site of NO15 is seen. Enzyme release,

mediated by signal transduction wifli the high molecular weight specific β-KLH, analogue,is

reduced as the concentration of the low molecular weight specific antigen is increased.

Concentration of the small antigen β peptide, of 4kDaltons molecular weight is therefore

measured by the decrease in enzyme release generated by me competitive antibody signal

transduction event.

Briefly, NO15 immunoglobulin was coated at 5μg per well in ELISA coating buffer as

above and plates were blocked ready for use. Wells were men incubated with 50μl of lOμg

per ml of glucose oxidase in PBS/BSA buffer. The plates were incubated for 1 hour at

room temperature. Wells were then washed twice with 200μl of PBS Tween to remove

unbound enzyme from the immobilized transducing antibody complex. A series of concentrations of the specific low molecular weight β peptide were prepared

from 0-40μg per ml, all protein dilutions being performed in PBS/BSA buffer. An equal

volume of the specific high molecular weight competitor analogue, β KLH at 20μg per ml

or the non specific protein KLH at 20μg per ml was then added to the β peptide. 50μl of

the competitor mixtures were then added to the wells containing the preformed transduction

complex. For the β KLH/β peptide competition, concentrations under test were from lOμg

per ml β KLH/Oμg per ml β peptide to lOμg per ml β KLH/20μg per ml β peptide. For

the control experiment, concentrations under test were from lOμg per ml KLH/Oμg per ml

β peptide to lOμg per ml KLH/20μg per ml β peptide. Plates were incubated for 30

minutes at room temperature. The supernatants were then removed from each well and

tested for the presence of released enzyme by addition of 50μl per well glucose oxidase

substrate solution. Enzymatic conversion of substrate to product was monitored by

measuring optical density at 630nm after one hour incubation in the dark at room

temperature.

Competitive antibody transduction is demonstrated in Figure 6. In the presence of

increasing concentrations of the β peptide, specific enzyme release, mediated by recognition

of β KLH and transduction by the NO 15 bispecific antibody, is reduced. Concentration of

the molecular analyte is directly indicated by the degree of reduction in enzyme activity.

Bispecific antibody NO 15 has been deposited under the Budapest Treaty at the European

Collection of Animal Cell Cultures, Porton Down under the accesion number 94080534.

Thus the invention provides an effective assay for relatively small molecules such as inhibin.

Claims

A diagnostic method for determining the presence, or absence of an antigen in a

sample, the method comprising contacting the sample with a multispecific antibody

having first and second binding sites for binding the antigen and a detectable entity

respectively, binding of the detectable entity to the antibody inactivating the

detectable entity in the presence of an analogue of the antigen, wherein binding of

the analogue of the antigen to the antibody results in release of the detectable entity

in a detectable form from the antibody but binding of the antigen to the antibody

does not cause such release or causes significantly lower levels of release.

A diagnostic method comprising:

1) providing a multispecific antibody having bound detectable entity on one

binding site thereof;

2) contacting the thus bound multispecific antibody with an analogue of an

antigen of interest, whereby binding of the analogue to a second binding site

on the multispecific antibody results in release of bound detectable entity

from the multispecific antibody generating a relatively high detectable signal;

3) contacting the system of step 2) with a sample of interest containing the

antigen to be assayed whereby binding of the antigen to the antibody in competition with the analogue results in reduction of the signal level proportional to the amount of antigen of interest in the sample.

A diagnostic method according to claim 1 or 2 in which antigen in the sample

competes with the analogue for the first binding site and so reduces d e amount of

detectable entity released from the antibody.

A diagnostic method according to claim 1, 2 or 3 in which the antigen is inhibin, a

portion thereof or a functional equivalent thereof.

A diagnostic method according to any preceding claim in which the concentration

of antigen and the amount of detected detectable entity are inversely related.

A diagnostic method according to any preceding claim in which the analogue

comprises the antigen or a portion thereof or a functional equivalent thereof linked

to a larger entity so that the size of me resulting combination of the antigen or

portion thereof and the larger entity is sufficient to cause the release of bound

detectable entity from the antibody.

A diagnostic method according to claim 4 in which the analogue comprises at least

a portion of inhibin or a functional equivalent thereof bound to Keyhole Limpet

Haemocyanin (KLH). A diagnostic method according to claim 7 in which the inhibin comprises at least

the β subunit thereof or a functional equivalent thereof.

A diagnostic method according to any one of claims 4 to 8 in which the functional

equivalent of inhibin comprises a recombinant polypeptide having some or all of the

biological activity of natural inhibin or a portion thereof.

A diagnostic method according to any preceding claim in which, the analogue

comprises a recombinant polypeptide including a portion capable of binding to the

binding site or a closely related binding site to which the antigen of interest binds

and a biologically redundant portion which is sufficiently large to cause release of

the detectable entity as described above.

A diagnostic method according to any one of claims 1,2,3,5,6, or 10 in which the

antigen is selected from activin, oligouncleotides, saccharides, growth factors,

neurotransmitters, hormones including progesterone, oestrogen, LH, or FSH.

A diagnostic method according to any preceding claim in which the antigen

comprises a polypeptide comprising five or more amino acids.

A diagnostic method according to any preceding claim in which the multispecific

antibody is a bispecific antibody. 14 A diagnostic method according to any preceding claim in which the detectable

entity is an enzyme.

15 A diagnostic method according to any preceding claim arranged to be operated on a substrate

16 A diagnostic method according to claim 15 in which me substrate is a biosensor.

17 A diagnostic method according to claim 15 in which the substrate is a dipstick.

18 A diagnostic method according to any preceding claim arranged to be operated in homogenous system.

19. A diagnostic method according to any preceding claim in which the antibody is NO15 deposited under the Budapest Treaty on 5th August 1994 at the European Collection of Animal Cell Cultures, Porton Down under accession no. 94080534.

20. A diagnostic method according to any preceding claim in which an enzyme comprising the detectable entity in a first system arranged to operate a diagnostic method in accordance with the invention produces the substrate for an enzyme comprising the detectable entity in a second system arranged to operate a second diagnostic method in accordance with the invention . A diagnostic method according to claim 20 for detecting the presence of both α and β subunits of inhibin in a sample, in which a first system in accordance with the invention arranged to detect the β subunit is provided in which glucose oxidase comprises the detectable entity, and a second system in accordance wim the invention is provided arranged to detect a subunit in which horse radish peroxidase comprises the detectable entity, whereby, in the presence of both inhibin subunits, released glucose oxidase from me first system produces the substrate for the horse radish peroxidase of the second system.