AU599373B2 - Method of assay of inhibin - Google Patents

Description

GRIFFITH HACK CO, PATENT AN D TRADE MARK ATTORNEYS MELBOURNE SYDNEY PERTH i6 PCT WORLD EL UA BROP Oa NIaN PL W VJAU O 4 -72036/87 INTERNATIONAL APPLICATION P D DE HEATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 87/ 05702 G01N 33/541, 33/74, C07K 15/12 Al (43) International Publication Date: 24 September 1987 (24.09.87) (21) International Application Number: PCT/AU87/00070 (74) Agent: SANTER, Vivien; Clement Hack Co., 601 St.

Kilda Road, Melbourne 3004 (AU).

(22) International Filing Date: (31) Priority Application Number: 13 March 1987 (13.03.87) (81) Designated States: AT (Eui ropean patent), CH (Eu PH 5019 pean patent), DK, FR (E (32) Priority Date: 13 March 1986 (13.03.86) pean patent), IT (Europ pean patent), NL (Europ (33) Priority Country: AU pean patent), US.

(71) Applicants (for all designated States except US): BIO- Published LjKq 5 L. RoJn tu4,, VLc 3( t 0 ros i J 'c.

er S oJ oI'c- i OOC 9 4 nn-T' ns+'k?/Q on) fl 'Ic)l ne(Os aro ropean patent), AU, BE (European patent), DE (Eurouropean patent), GB (Euroean patent), JP, LU (Euroean patent), NO, SE (Euro- -eport.

NOV 1987 7 VL4 c ;af, 1r C- ,vcc 30o6s (54)Title: METHOD OF ASSAY OF INHIBIN th amendn ia:. und7r >ection a. s corect for (57) Abstract pril ing A method of immunoassay for the estimation of inhibin in an inhibin-containing sample which comprises the step of using an antibody directed against inhibin. Preferably, the antibody is contsa 4 ed in an antiserum raised by injecting an animal with an antigen selected from the group consisting of naturally-occuring or recombinant inhibin, or sub-units, fragments or derivatives thereof. The assay may suitably be a radioimmunoassay, a fluorescence-based immunoassay, or an enzyme-linked immunosorbent assay using labelled 58kD or 3 1kD inhibin as tracer. Tracrs and standards for use in the assay are described and claimed.

JOT!I',)

IOT lp 't f OL T' .V i~ ltJ i Al K.c i IU LL l ll W' 87/05702 PCT/AU87/00070 METHOD OF ASSAY OF INHIBIN This invention relates to methods for assay of inhibin, and in particular to methods for immunoassay of inhibin.

Background Art Two forms of inhibin from bovine follicular fluid have been recently purified to homogeneity, with molecular masses of 58kD and 31kD (International Patent Application PCT/AU/85/00119 and Robertson et al 1985, 1986). Under reducing conditions both forms consist of two subunits with molecular masses of 43kD and 15kD, and 20kD and respectively. Their primary amino acid structures have been 2 elucidated following cloning and analysis of cDNA species derived from bovine granulosa cell mRNA (International Patent Application PCT/AU86/00097; Forage et al 1986).

These studies indicate that 31kD inhibin is a processed form of the 58kD molecule. 31-32kD Inhibin molecules with similar sub-unit structures to bFF inhibin have been isolated from porcine follicular fluid (Miyamoto et al 1985, Ling et al 1985) and sequenced (Mason et al 1985).

Currently inhibin activity is measured by a variety of in vivo and in vitro bioassay systems (Baker et al 1981). These systems are time consuming, expensive, have limited sensitivity and precision and are of limited practicability in their application to large sample numbers (Baker et al 1981, Lee et al 1982).

Summary of the Invention 0I~0 The present invention relates to more convenient assays for the estimation of inhibin than have heretofore a been possible. The preferred assays of the invention are radioimmunoassays and the following description, whilst being directed to the preferred assays, should not be construed as limiting the invention to radioimmunoassays.

Other assays within the scope of the invention include ELISAs, immunoassays based on fluorescence detection, and related assays relying on polyclonal and monoclonal t antibodies against inhibin.

*0 Preferred Embodiments of the Invention According to one aspect of the present invention there is provided an immunoassay for the estimation of inhibin in an inhibin-containing sample which comprises the step of using an antibody directed against inhibin.

The invention therefore provides a radioimmunoassay for determining inhibin suspected to be present in a biological sample, comprising the steps of: containing the sample with an antibody directed against inhibin and incubating at 40 to 30 0 C for a period of 4 hours to 4 days, r^ 2A adding I-inhibin and incubating for a period and at temperature selected from the group consisting of at overnight room temperature, 48 to 72 hours at 4 0 C or 16 hours at 30 0

C,

adding a second antibody and incubating for minutes to 24 hours at 4 0

C,

separating precipitated material, and measuring bound 12sI-labelled inhibin in the precipitate.

Preferably the antibody is contained in an antiserum raised by injecting an animal with an antigen selected from the group consisting of naturally-occurring or recombinant inhibin, or sub-units, fragments or derivatives thereof. Particularly preferred antigens include preparations containing inhibin, purified bovine 58kD inhibin, So

*S

*S

S

S

S

SS

S**

S S

__I

i WO 87/05702 PCT/AU87/00070 3 purified bovine 31kD inhibin, human inhibin, or human or bovine inhibin or fragments thereof produced using recombinant DNA technology.

Suitable animals, include mammals such as mice, rabbits, horses, donkeys, dogs, sheep, and goats, and birds such as chickens.

Alternatively a monoclonal antibody or an IgG directed against any of the aforesaid inhibins may be used.

Most preferably the antibody is capable of neutralizing inhibin bioactivity.

Preferably the immunoassay is further characterized by the step of using labelled 58kD or 31kD inhibin as tracer.

More preferably said tracer is labelled with 125iodine (125) with an enzyme, or with a fluorescent marker.

Preferably the assay is a radioimmunoassay or an enzyme-liked immunosorbent assay (ELISA), or a fluorescencebased immunoassay.

The invention provides a method for measuring inhibin in samples such as follicular fluid or serum from various species (including humans) wherein concentrations of inhibin in standards are used to derive the concentration of inhibin in the follicular fluid or serum by competitive binding of 125I labelled inhibin and inhibin from test samples with bovine 58kD inhibin antiserum, followed by precipitation 125 and-counting of bound 125I labelled inhibin.

The preferred specific radioimmunoassay system for inhibin of the invention is applicable to bovine and human follicular fluid and serum, and can employ an antiserum against (purified bovine) 58kD inhibin with iodinated 31kD or 58kD inhibin as tracer.

According to a second aspect of the invention, there is provided a method for preparation and purification of 125I-labelled inhibin tracer which comprises the steps of iodination of inhibin using a Chloramine T procedure and purification of 125I-inhibin by an affinity fractionation step.

Preferably the affinity fractionation step uses Matrex Red A.

Preferably the purification procedure additionally comprises a gel filtration step.

WO 87/05702 PCT/AU87/00070 -4- According to a third aspect of the invention there is provided an assay standard selected from the group consisting of naturally-occurring or recombinant inhibin, or fragments or derivatives thereof. Preferably the standard displays parallelism in the assay with the samples under test.

Particularly preferred standards include bovine 31kD inhibin, and partially purified or purified human inhibin.

The conditions of the assay, in particular incubation times, may be varied in order to attain desired levels of sensitivity.

A form of the radioimmunoassay modified for increased sensitivity comprises: incubating sample and antiserum for 4 days at 4 C, followed by the addition of 2I-31kD inhibin tracer, incubating for 3 days at 4 C and then adding second antibody, 125 precipitating, and counting bound I labelled inhibin.

According to one particularly preferred embodiment, suitable for measurement of inhibin in human serum samples, the tracer is 25 -31kD inhibin, and incubation with tracer is performed at elevated temperature (30 C) in the presence of inhibin-free serum, in order to minimize non-specific effects.

Suitable sources of inhibin-free serum include steers or other castrated male animals, oophorectomized women, women with Spremature ovarian failure, and post-menopausal women.

Brief Description of the Drawings Figure 1 shows the fractionation of I-58kD and 125 1 I-31kD inhibin on analytical SDS-PAGE under reducing conditions.

Figure 2 shows the time course of immunization of a rabbit with 58kD inhibin.

Figure 3 shows the in vitro neutralization of hFF inhibin by an antiserum raised to 58kD inhibin.

Figure 4 shows the radioimmunoassay dose response curves of bFF, hFF, purified 58kD and 31kD inhibin and bovine 125

I

2 -58kD inhibin as tracers.

WO 87/05702 PCT/AU87/00070 Figure 5 shows the profile of inhibin in vitro bioactivity and immunoactivity following fractionation of bFF through the various steps of the inhibin purification procedure of Robertson et al (1986).

Figure 6 shows non-reduced SDS-PAGE profiles of 125 1-58kD and 1 25 1-31kD inhibin following incubation with bFF and serum under conditions used in the RIA of serum inhibin.

Figure 7 shows the effect of temperature on the binding of 125I-31kD inhibin to the antiserum.

Figure 8 shows logit-log dose response lines of bovine and human serum, in the plasma RIA system employing 125 12 5 I-31kD inhibin as tracer.

Figure 9 shows the ovulation induction regime and serum levels of FSH, LH, inhibin and oestradiol (E 2 in twenty-six women involved in an In Vitro Fertilisation (IVF) programme and one normal woman (FL#27).

Figure 10 is a comparison of plasma E 2 and inhibin levels plotted for some of the data in Figure 9.

Figure 11 shows the correlation between the number 2Q of ova produced and E2 or inhibin levels in serum.

Figure 12 shows the correlation between the numbers of ovarian follicles detected ultrasonically and peak inhibin levels in serum.

Figure 13 shows inhibin, FSH, progesterone and oestradiol concentrations in the sera of non-pregnant subjects (n =16) on the days following oocyte retrieval.

Figure 14 shows inhibin, FSH, progesterone and oestradiol concentrations in the sera of pregnant subjects (n 3) on the days following oocyte retrieval.

Figure 15 shows the relationship between serum inhibin and FSH during the luteal phase of non-pregnant subjects. For this analysis, non-detectable inhibin values (n 29) were assigned the limit of assay sensitivity.

Figure 16 shows inhibin, FSH, LH, oestradiol and progesterone concentrations in the sera of norm&l women during the menstrual cycle, assayed using anti-31kD inhibin.

rW 0 87/05702 PCT/AU87/00070 -6- Detailed Description Abbreviations bFF hFF oFF oRTF

HPLC

SDS-PAGE:

RIA

SS

BS

CS

PMS

HFP

of the Invention bovine follicular fluid human follicular fluid ovine follicular fluid ovine rete testis fluid high performance liquid chromatography sodium dodecyl sulphate polyacrylamide gel electrophoresis radioimmunoassay steer serum bull serum cow serum human post-menopausal serum human female plasma Preparations Purification of bFF inhibin The purification of bFF 31kD and 58kD inhibin was based on the procedures described previously (Robertson et al 1985, 1986)(Figure bFF was fractionated on a Sephacryl S200 (9 x 90 cm) gel filtration column in 0.05M ammonium acetate pH The void volume fraction from was precipitated at pH 4.75 and fractionated on Sephadex G100 (9 x cm) in 4M acetic acid.

and (d) Peak I (58kD inhibin) and Peak II (31kD inhibin) fractions fro'. were fractioned on an RPSC Ultrapore column (0.46 x 7.6 cm, Beckman) using a 0-50% acetonitrile gradient in 0.1% trifluoracitic acid. In Figure 5 the continuous line indicates optical density at 280 nm and and 254 nm (c) and Hatched area denotes inhibin bioactivity.

o RA with 125 D inhibin as tracer.

RIA with I-58kD inhibin as tracer.

1 Li WC t7/05702 PCT/A U87/00070 125 7 o o RIA with 12-3lkD inhibin as tracer.

Vo void volume.

BSA bovine serum albumin (mol. wt 67,000).

OVA ovalbumin (mol. wt 43,000).

The purified inhibin was stored in SDS electroelution buffer (approx. 3% SDS in 10 mM NH 4

HCO

3 prior I to iodination. For bioassay, samples were methanol precipitated at -20°C in order to remove SDS and solubilized by heating at 37 C for 1 hour and sonication.

Similar profiles of both bio- and immunoactive inhibin were observed at each stage of the inhibin purification procedure. The biological to immunological activity ratios for a number of purified 31kD and 58kD inhibin preparations using both tracers in the radioimmunoassay ranged from 0.30 0.43.

Sample Preparation Human follicular fluid was obtained at oocyte collection in the in vitro fertilisation programme at the Queen Victoria Medical Centre/Epworth Hospital, Melbourne. It was charcoal treated (100 mg/ml dextran-coated charcoal for 1 hour at lyophilised, stored at -20 C and resolubilized prior to assay by sonication in assay buffer or culture medium. Ovine follicular fluid (oFF) was obtained by Iaspiration of ovaries collected at a local abattoir. Ovine rete testis fluid (oRTF) is a lyophilised inhibin preparation (Baker et al 1985). Rat ovarian extract was a charcoal-treated rat ovarian cytosol preparation. Details of the biopotencies of these inhibin preparations are outlined in Table 1.

Blood was collected in lithium heparin tubes from women undergoing ovulation induction therapy (clomiphene citrate and human menopausal gonadotrophin treatment) with plasma estradiol levels at time of plasma collection ranging from 40-2,900 pg/ml. Equal aliquots of serum from each subject were combined to produce a plasma pool designated as WO 87/05702 PCT/AU87/00070 8 Human Female Plasma pool (HFP). Plasma from four post-menopausal women aged 52 and over were combined to give a pool designated Post-Menopausal Serum pool (PMS).

Bovine blood, ovaries and testes were collected on ice from a local abattoir and processed within one hour. All samples were stored at -20 C after snap freezing in solid

CO

2 /ethanol. Blood pools from adult intact (BS, n 9) and castrate (SS, n 1) male, and female (CS, n 10) cattle were allowed to clot overnight at 4 0 C prior to centrifugation and storage. Bovine ovarian follicles were hemisected and granulosa cells were collected by aspiration and cultured for 5 hours at a concentration of 10 viable cells per well (Costar 48 well plate) in 400 pl DMEM/F12 complete medium.

Media were stored frozen at -20 0 C prior to assay. Testes from four bulls were decapsulated and homogenised in equal w/v Dulbecco's phosphate buffer using an Ultra-Turrax tissue disperser (Janke and Kunkal KS, Staufen FRG) and centrifuged at 100,000g x 1 hour at 4°C and stored at -20 0 C. Prior to assay the supernatants were charcoal treated with an equal volume of 1% Norit A in Dulbecco's phosphate buffer and incubated at 4 C for 30 minutes prior to centrifugation and bioassay (Au et al 1983).

Immunization Procedure Purified 58kD inhibin (14 pg in 500 pl Dulbecco's phosphate buffer) was emulsified in an equal,volume of adjuvant (Marcol 52 [Esso, Australia]: Montanide 888 Paris] in the ratio 9:1) and injected into an intact male New Zealand white rabbit 4 intramuscular and one subcutaneous sites. Two booster injections of 14 ug under the same conditions were given at six weeks and one year. Serum was collected throughout for assessment of its in vitro neutralization activity, its ability to bind iodinated inhibin and for plasma FSH estimations.

A similar procedure was used to raise antibody directed against purified 31kD inhibin.

i I-' WO 87/05702 PCT/AU87/00070 9 Analytical SDS Polyacrylamide Gel Electrophoresis Sera and bFF.were incubated at various temperatures in an equal volume of 100 mM phosphate buffer pH 7.4 containing 0.15M NaCI, 0.1% Triton X-100 and either 0.5% BSA for studies with 125 I31kD inhibin or 0.5% Polypep for 1 I-58kD inhibin. Equal volumes (5ul) of sample and 10% SDS and Dulbecco's Phosphate buffer pH 7.4 (30 pl) were placed in a boiling water bath for 2.0 minutes then in ice. Ten microlitres of bromophenol blue (0.006%) in glycerol (62.5% in 1 10 H 2 0) was then added and the mixture centrifuged prior to electrophoresis on 12.5% slab gels (3 hours, 20-30 mA).

IPotein molecular weight markers were either co-electrophoresed with the iodinated sample in the absence of bFF and serum or on a separate track in their presence. The gels were fixed and stained overnight in ethanol: H 2 0 formaldehyde (180: 420: 100) containing 0.1% Coomassie Brilliant Blue. Each track was divided into 50 2mm slices and counted in a gamma counter.

Reversed-phase HPLC 125 I-Inhibin was applied to an Ultrapore RPSC column (0.46 x 7.5 cm, Beckman, Berkeley, Ca., USA) and fractionated using a 30 min linear gradient of 0.50% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min and 0.5 ml fractions using Waters HPLC apparatus (model 6000A pumps and a model 660 Programmer, Milford, Mass., USA).

In Vitro Bioassay Inhibin activity was determined using an in vitro bioassay based upon the dose-dependent suppression of FSH cell content in rat pituitary cell cultures utilizing a parallel line bio-ssay design (Scott et al 1980). The charcoal-treated bovine follicular fluid preparation employed a lymph reference preparation with an arbitrary unitage of 1 unit/mg (Scott et al 1980).

Hormone Assays T WO 87/05702 PCT/AU87/00070 10 Rabbit FSH Radioimmunoassay Rabbit FSH was determined using an RIA kit kindly provided by Dr. A.F. Parlow (Torrance, Ca, USA) employing 15% polyethylene glycol to separate bound and free hormone. The sensitivity of the assay was 0.9 ng/ml using rabbit FSH AFP.538.C as standard.

The within assay coefficient of variation was 8.1% and all samples were assayed in the one assay.

(ii) Rat FSH Radioimmunoassay Rat FSH generated by the pituitary cells in culture was measured by a specific radioimmunoassay using 125 reagents supplied by the NIAMDD. I-rat FSH (I 5 was used as tracer and FSH RP-2 used as standard.

The within-assay coefficient of variation was 7%.

(iii) RIA of human hormones Serum FSH was measured by RIA (Amerlex-M, Amersham, USA) using 2nd IRP FSH as standard with an interassay CV of 7.0% from 31 assays. LH was measured by RIA (LH RIA, Diagnostic Products Corp., USA) using the 2nd IRP LH as standard with an interassay CV of 10.1% from 31 assays. Both oestradiol and progesterone were measured using RIA (Coat-a-Count, Diagnostic Products Corp., with interassay CVs of 8.7% and 8.1% respectively from 150 assays. Serum beta subunit of hCG was measured by RIA (B-hCG RIA-Quant, Mallinckrodt Inc. St.

Louis, USA) using the hCG 2nd IS as standard with an interassay CV of 10.4% from 30 assays.

Calculations The RIA dose-response curves were linearised using a logit-log dose transformation. Parallelism was assessed from a comparison of slope values of dose-response curves using the multiple range test for groups of unequal size (Kramer, 1956) or by paired t-test, Potency estimates were determined using standard parallel line bioassay statistics. 'In situations where non-parallelism was observed between dose response lines WO 87/05702 PCT/AU87/00070 11 of unknown and standard preparations, potency estimates were determined from the'ratio of their ED 50 values. The sensitivity

(ED

10 was defined as the mass of hormone required to give 10% displacement in the assay whilst ED 50 corresponded to the mass required for 50% displacement.

The index of precision (Gaddum (1933); Finney (1964)) *iws used to describe assay precision. The between assay variation was calculated from the coefficient of variation of the repeated measurement of a partially purified inhibin preparation. The dissociation constant(Kdis) was 125 determined by Scatchard analysis using I-hormone and increasing amounts of unlabelled hormone. The mass of 1 25 1-hormone used in the analysis was determined from its specific activity ()iCi/pg).

The invention will now be illustrated by reference to the following non-limiting examples.

Example 1 Antiserum Characterization Antisera against 58kD and 31kD inhibin were characterized by showing that following immunization, parallel changes in plasma FSH and inhibin antibody titre were observed, indicating inhibin neutralization in vivo. The antisera neutralized bFF, hFF and purified 31kD and 58kD inhibin activity in an in vitro bioassay. The results described below refer to anti-58kD inhibin, but similar results were obtained using anti-31kD inhibin.

Response In Vitro to Immunization Following the first booster (Fig. 2a) the antibody titre was assessed by the ability of the antibody to neutralize inhibin 125 bioactivity in vitro and its capacity to bind 125I-58kD inhibin.

A sharp parallel elevation in these activities was observed between 1-8 weeks post-booster injection. During this period significant (P 0.05) elevation in serum FSH 0.95 ng/ml, n 5 vs 4.97 0.87 ng/ml, n 6 (mean 1 SD)) was noted.

Following the second booster (Fig. 2b), an immediate and 125 sustained elevation of serum 1-31kD inhibin-binding capacity and serum FSH was observed.

WO 87/05702 PCT/AU87/00070 12 Basal levels of serum FSH were assessed from the mean 2 S.D.

of 14 observations over the preceeding five months (hatched area Figure 2b).

These results indicate that purified inhibin from bovine follicular fluid can be used to immunize rabbits, producing an antiserum which has the capability of neutralizing inhibin bioactivity. The elevated levels of plasma FSH in the rabbit observed during the period of peak antiserum titre (as assessed by iodinated inhibin binding and in vitro neutralizing capacity) indicate that the antibodies produced are capable of neutralizing endogenous inhibin. The combination of the ne -ralization of inhibin both in vivo and in vitro and the close relationship of the neutralizing activity and iodinated inhibin binding capacity of the antiserum provides convincing evidence of its specificity.

Inhibin Neutralisation In Vitro Bovine follicular fluid inhibin (2 units) was quantitatively neutralised by 1 pl antiserum per culture well while 75% inhibition of bioactivity was achieved with 0.35 pU per well (Fig. 3, Table In Figure 3, the vertical dotted line indicates the volume of antiserum required to achieve neutralization of inhibin activity, an arbitrary parameter of antiserum neutralizing titre. Purified 58 and 31kD inhibin gave corresponding 75% inhibition values of 0.33 and 0.38 pl respectively. In comparison, neutralisation of hFF inhibin bioactivity required 1.32 jl antiserum (n 2, Table 1) corresponding to 27% cross-reactivity in comparison to bFF inhibin. Inhibin from ovine sources (follicular fluid, rete testis fluid) showed 8 and 6% cross-reactivity respectively.

This antiserum, at a maximum non-toxic level of 4 ul per well, did not neutralise 2 units of inhibin activity in rat ovarian cytosol extracts.

TABLE 1 Cross-reactivity of inhibin from various sources as assessed by inhibin neutralization in vitro and radioirmnunoassay IN VITRO NEUTRALIZATION RAD 101 MNOAS SAY 125 158kD Inhibin 125 I-3lkD Inhibin Preparation Inhibin Bioactivity U /ml Antiserum Titre (i-i) Cross- Reactivity ED 5 0

(U)

Cross- Reaction ED so

(U)

Cross- Reaction Follicular Fluid Bovine Human Ovine ,5200 0.35 t 0.04 (6) 1. 30 0. 25 (9) 18±32 (6) 27,000 1.32 27 5. 3 1. 6 (4) 28. 4 8. 7 (4) 0.3 0.99 (2) 2.7 (2) 320 100 320 0.1 Ovarian Extract 384 (2) 6 (2) Rete Testis Fluid Ovine .1040* 6. 0 (1) <(6.0 52 >52 *U/mg protein **No displacement with 16 U inhibin Mean SD (n) mom 7~ -i i i WO: 87/05702 PCT/AU87/00070 14 Example 2 lodination of inhibin lodination of either 58kD or 31kD inhibin has been achieved using a conventional Chloramine T iodination procedure and was associated with considerable iodination damage. Purification of the tracer was therefore necessary and it was not achieved following gel filtration chromatography on Sephadex G25. Specific binding of either iodinated hormone to Matrex Red A achieved satisfactory purification although recoveries were low. Either iodinated inhibin form thus purified had the physico-chemical properties of its non-iodinated form.

Alternative iodination procedures using lodogen, Iodobead-., lactoperoxidase, or Bolton-Hunter reagent were found to cause less damage to the inhibin molecule, but resulted in poorer incorporation of radioactivity.

Consequently iodination using Chloramine T was preferred.

Purified 58kD or 31kD inhibin (1-2 pg in 25 pl electroelution buffer was added to 25 ul 0.5 M phosphate buffer, pH 7.2. Na125I (0.5 mCi, 5 ul; Amersham, Bucks, UK) was added. Chloramine T (40 pl) was added at a ratio of 8:1 Chloramine T to hormone. The reaction proceeded for seconds at room temperature with stirring and was terminated with 20 pl sodium metabisulphite (3 mg/ml). The reaction mixture was made up to 50 pl in 20mM phosphate buffer 0.1% BSA or 0.5% Polypep (Sigma, St. Louis, Mo., USA) pH 6.0 and gel filtered on a Sephadex G25 column (PDO0, Pharmacia, Uppsala, Sweden) to remove 1I. The void volume fractions were pooled, made up to 20 ml and applied to a column of 200 pi Matrex Red A (Amicon, Danvers, Mass., USA) and then washed with phosphate buffer containing 400 mM KC1, the eluted counts being discarded. 125-inhibin was eluted with 1M KC1/4M urea in phosphate buffer. The iodinated inhibin was further gel filtered on a Sephadex G25 column (PD10) with the appropriate RIA buffer (see below) to remove the KCl/urea.

Following iodination of 58kD and 31kD inhibin, pCi and 25 pCi respectively were recovered in the void volume fractions following gel chromatography on Sephadex 1 WO 87/05702 PCT/AU87/00070 15 Approximately 30% was eluted with the 1M KC1/4M urea buffer.

125 I-inhibin, as assessed by its molecular weight on SDS-PAGE, was found in this fraction.

The specific activity of the iodinated preparations was assessed in the radioimmunoassay using a self-displacement procedure (Marana et al 1979) with the hormone used for iodination as standard. Specific activities of 50-60 pCi/pg for 58kD inhibin and 24 pCi/pg for 31kD inhibin were obtained, with recoveries ranging from 5-25%.

Example 3 Characterisation of iodinated inhibin The physico-chemical characteristics of 1I-inhibin were assessed using RP-HPLC and SDS-PAGE. A close correspondence was observed between the radioactive and bioactive profiles on RP-HPLC for both the 58kD and 31kD preparations (data not shown). The molecular mass of I-58kD inhibin following fractionation on SDS-PAGE was similar to purified non-iodinated inhibin under both non-reducing (58kD) and reducing (43kD and 15kD) conditions except that a 58kD material of unknown identity was observed in relatively low proportions under reducing conditions (Fig. The molecular weight markers employed were BSA (bovine serum albumin) 67,000; OV (ovalbumin) 43,000; CA .(carbonic anhydrase) 29,000; GL (goose egg lysozyme) 20,300; and.CL (chick egg lysozyme) 14,300. The arrow, in figure 1, refers to the point of sample application. Radioactivity found in fractions beyond fraction 47 represents free iodine in the solvent front. The purity of the inhibin used for iodination as assessed by silver staining on SDS-PAGE suggests that the 125I-58kD material is not an iodinated contaminant.

125 In support, I-58kD inhibin was fractionated by microelectrofocusing procedure on the pH range 3..5-10 and 4-8 (Foulds and Robertson 1983), and 3 peaks of radioactivity with pi values of 7.4, 6.2 and 5.2 were observed. Upon reduction on SDS-PAGE each of these peaks showed persistence of 35 1 25 58kD material. The results suggest that the presence of I-58kD material. The results suggest that the presence of 11lll i WO 87/05702 PCT/AU87100070 -16 the 125 I-58kD material is attributable to difficulties in reduction of the iodinated hormone rather than to the iodination of a contaminating protein.

Fractionation of the 125 I-31kD inhibin on SDS-PAGE revealed molecular weights of 30,200 under non-reducing conditions and 20,000 and 15,000 subunits following reduction; these values are similar to those for the non-iodinated hormone. A second antibody RIA system using either tracer yielded a parallel displacement between purified 31kD and 58kD inhibin.

Example 4 Radioimmunoassay Procedure The assay buffer used was 10 mM phosphate, 0.15 M NaCl, 0.5% BSA, pH 7.2. A delayed tracer addition, second antibody assay system was employed. The sample and antiserum were incubated in a volume of 300 pi for 16 hours at room temperature following which 125I-inhibin (10,000 cpm, 100 ul) was added and the incubation continued either overnight at room temperature or for 48 hours at 4 C. Second antibody (goat antiserum to rabbit IgG, 100 pl) was added and incubated for 1 hour at 4 C following which 1 ml 6% polyethylene glycol was added. The tubes were vortexed and incubated for a further 30 min, spun at 2000 g for 30 min at 4 C, decanted and -counted. The inclusion of Triton X-100 (final concentration 0.025%) in the assay buffer reduced non-specific binding from 4 to Radioimmunoassay procedures were established using both 31 and 58kD inhibin tracers. Following a logit-log dose transformation of the response curves, linear displacement of each tracer was observed for a range of inhibin preparations, 125 with the exception of 31kD inhibin when using I-58kD inhibin as tracer, in which a deviation from linearity below logit -0.5 (38% B/Bo) was seen (Fig. In figure 4, each value represents the mean SD of triplicates. The characteristics of each assay are outlined in Table 2.

Scatchard analysis revealed similar affinities for the antiserum of either inhibin form. Non-parallel dose response Matrex Red A.

Preferably the purification procedure additionally comprises a gel filtration step.

WO 87/05702 PCT/AU87/00070 17 lines were observed between bFF arid either 31kD inhibin with 125 I-31kD inhibin as tracer of 58kD inhibin with 125 1-58kD inhibin as inhibin tracer. The sensitivity (ED 10 and ED 50 values were comparable in each assay with either hormone.

I i i i i WO 87/05702 PCT/AU87/00070 18 TABLE 2 Characteristics of the two radioimmunoassay systems with 125 -31kD inhibin and 1 25 1-58kD inhibin as tracers 1 2 5 I-31kD inhibin 12 5 I-58kD inhibin Antibody Dilution Tracer binding (Bo) Affinity (Kdis) x 10-1 0

M

1:8000 30% 0.66 1:4000 18% 0.72

ED

1 0 (ng, fm) 31kD inhibin 0.10, 3.0 58kD inhibin 0.07, 1.2

ED

5 0 (ng) 31kD inhibin 0.30, 10.1 58kD inhibin 0.26, 4.3 Slope* bFF 1.37 0 09 a(8) 31kD inhibin 1.53 0 0 9 b(5) 58kD inhibin 1.50 t 0.07 (3) Precision** 0.036 (5) Between Assay Variation** 14 (5) Bio/Imm Ratio 31kD inhibin 0.34 0.09 (16) 58kD inhibin 0.30 0.12 (7) BGCM 0.25 (1) 0.13, 4.4 0.13, 2.2 0.51, 17.1 0.43, 1.47 0.09c(8) 1.68 0.08 (3) 1.73 t 014d(5) 0.038 8.5% 0.43 0.13 (4) 0.37 0.12 a vs b P 0.05) as assessed by paired t-test a vs c P 0.01) Mean SD Number in brackets: number of preparations Number in brackets: number of assays BGCM bovine granulosa cell culture medium For details see text.

WO 87/05702 PCT/AU87/00070 -19 Example 5 Specificity of the assay The specificity was assessed on the following grounds. First, a similar hierarchy of cross-reaction of inhibin from various species in the RIA using either tracer and in vitro neutralisation studies was observed (Table 1).

The cross-reaction in the radioimmunoassay of inhibin from different species when expressed in terms of their bioactivity was bFF 100%, hFF 30% ovine FF 1% and rat ovarian extract non-detectable. With respect to this antiserum it is apparent that both male and female bovine and human inhibin share common antigenic determinants not found in inhibin from the other two species. This implies close structural similarity between inhibin from both sexes and species. Secondly, no cross-reaction occurred for a range of purified glycoproteins and polypeptides. Rat LH and FSH, ovine LH and FSH, hCG, bovine TSH, LHRH, ovalbumin and bovine serum albumin showed less than 0.5% cross reactivity using either tracer.

SAlternatively, medium from the bovine granulosa cell culture (Fig. and bovine testis extract (data not shown), both containing inhibin bioactivity, gave parallel displacement curves to bFF inhibin in the RIA. The parallel dilution of inhibin bio- and immunoactivity of medium from bovine granulosa cell culture with the inhibin standard provides evidence for these cells being the site of inhibin production, as has been previously suggested (Erickson and Hsueh 1978; Henderson and Franchimont 1981). Thirdly, similarities were observed in the profiles of both biological and immunological activities following fractionation of bFF on gel filtration chromatography and RP-HPLC. However, in the 40-60kD molecular mass region of the Sephacryl S200 column (Figure 5a), an 8-40 fold excess of immunoactivity over bioactivity was present, accounting for 12-18% of the recovered immunoactivity.

A large variation in the ratio of biological/immunological activities with charcoal-treated bFF as standard was observed following fractionatic;n of bFF inhibin on gel filtration and RP-HPLC (Fig. 5) and between purified inhibin preparations (Table The ratios ranged WO 87/05702 PCT/AU87/00070 20 from 0.02-2.09 in fractions obtained during the purification procedure and from 0.30-0.43 with the purified inhibin preparations.

It is concluded that the RIA procedures are not detecting molecular entities devoid of biological activity and vice versa except in the lower molecular weight region (40-60kD) of the Sephacryl S200 chromatogram. Whether this lower molecular weight material represents a protein distinct from inhibin which cross-reacts in the RIA or inhibin devoid of biological activity has not been established.

The cross-reactivities of inhibin-related proteins in the RIA relative to 31 kDa bFF inhibin were as follows: porcine transforming growth factor-beta (R D Systems, Minn.

USA) 40.9%, bovine Mullerian inhibitory substance (kindly provided by Dr. J. Hutson, Royal Children's Hospital, Melbourne) purified bovine inhibin B subunit dimer 42% and the subunits of 31 kDa bFF inhibin obtained following reduction and alkylation A range of glycoproteins and growth factors have been previously tested (McLachlan et al, 1986) and showed cross reactivities against anti-58kD inhibin of less than The specificity of anti-31kD inhibin was similar, with cross-reactivities of less than SExample 6 Application of the Radioimmunoassay to Serum The RIA in its application to serum required substantial modification. Firstly 100mM phosphate buffer pH 7.4 containing 0.15M NaCI, 0.5% BSA was used, and, because of its stability in serum, 125I-31kD inhibin was preferred to 125 I-58kD inhibin as RIA tracer. Secondly, a temperature-dependent interference of steer serum with 12 5 I-31kD inhibin binding to the antiserum was observed, with an increase in binding (B/Bo) from 57% at 40C to 94% at 370C (Fig. This figure demonstrates the temperature dependence following a 16 hour incubation in the presence of various serum and inhibin preparations (bFF, 31kD inhibin, steer serum cow serum bull serum human post-menopausal serum (PMS), human female serum pool Tracer binding was maximal at 30 C in the presence of steer serum, being 87.1 j 3.4% (n Displacement of 125I-3kD inhibin L WO 87/05702 PCT/AU87/00070 21 by bFF or 31kD inhibin was largely unaffected by temperature Human PMS showed no temperature related interference upon binding although the binding was elevated (B/Bo 110-120%).

Based on these data, conditions for the assay of S 5 either bovine or human serum inhibin were established. These 125 involved using 125 -31kD inhibin as tracer in an overnight C incubation and, in order to compensate for the low level of interference by SS or PMS (presumed to contain no inhibin) standards and samples were diluted in SS or PMS accordingly.

No detectable activity was determined in steer or in human post-menopausal serum, whilst bull and human female serum showed parallel dose-response curves to their respective follicular fluid standards, with circulating levels of 0.9 and 1.1 ng respectively.

Inhibin preparations or serum samples were diluted in SS or PMS to a sample volume of 200 pl. Antiserum (100 pl, final dilution 1:8000) and samples (200 ul) were incubated for 4 hours at 30 0 C, followed by a further incubation of 16 hours at 30 C in the presence of tracer. Second antibody was added and the tubes were incubated for 24 hours at 4 C, following which 2 ml 0.15M NaCl was added and the tubes were centrifuged.

Thirdly, with respect to a choice of standard in the RIA of bovine serum inhibin, purified 31kD inhibin is favoured in view of its stability in serum. In the absence of a purified human inhibin preparation, 31kD bovine inhibin may be used as the standard in the RIA of human serum inhibin.

However, the partially purified hFF inhibin preparation described above is preferred, and purified hFF inhibin, when 30 available, would be the most preferred standard. The detectable levels of inhibin immunoactivity in serum from women under going ovarian stimulation with exogenous gonadotrophin is'analogous to the findings of Lee et al (1982), where circulating levels of inhibin activity were detected in PMSG treated immature female rats, particularly directed against 58kD and 31kD inhibin.

WO 87/05702 PCT/AU87/00070 21a Individual antisera may behave differently in the assay, and assay parameters may have to be determined for each case. Considerable variations in sensitivity between antisera have been observed, particularly between antisera directed against 31kD and 58kD inhibin. Anti-31kD inhibin appeared to give greater sensitivity than anti-58kD inhibin in the samples tasted so far.

WO 87/05702 PCT/AU87/00070 22 Example 7 Improved Sensitivity RIA for human serum In order to improve assay sensitivity, the assay procedure above was modified as follows: the total volume of the assay was reduced from 400 to 300 1l (comprising 200 pl 5 sample, 50 pi tracer and 50 pi antiserum). The assay buffer was 150 mM phosphate, 0.2% BSA pH 7.4, and the incubation of sample and antiserum was 4 days at 4 C followed by the addition of tracer and a further 3 days at 4 C prior to the addition of second antibody. Using this method, a 2.5 fold increase in sensitivity was achieved. This modified assay procedure has been applied to the measurement of human plasma inhibin. The modified assay allows the quantification of plasma inhibin in normal male plasma and in plasma throughout the normal menstrual cycle.

125 125 Example 8 Stability of 2-31kD and 125I-58kD Inhibin in Serum 125 SDS-PAGE profiles of 12 -58kD inhibin following overnight incubation with serum (SS and PMS) showed an increased formation of an 125 I-30kD component (12% of recovered activity at 4 C; 17% at 300C) in comparison with either buffer or bFF 4 C and 30 C; (Fig. The tracers were incubated overnight at either 4 or 30 C with either bFF, -steer serum (SS) or human post-menopausal serum (PMS).

Incubation of either tracer in RIA buffer alone gave similar to the bFF incubation shown. Molecular weight markers are described in Figure 1. No radioactivity was observed between the position of the marker carbonic anhydrase and the solvent front with either tracer. Results are presented as the mean I SD of three replicate experiments. In contrast, SDS-PAGE profiles of 125 I-3kD inhibin under the same incubation conditions showed no significant changes.

Recoveries of radioactivity with either tracer were not affected by either temperature or by the presence of bFF or serum.

WO 87/05702 PCT/AU87/00070 23 Example 9 Radioimmunoassay of inhibin in bovine serum The application of the inhibin RIA to serum from cattle resulted in parallel logit-log dose response lines of BS with either bFF or 31kD inhibin as standards (Fig. The response shown in figure 8 is for bovine and human serum, diluted in steer serum or post-menopausal serum respectively, in the plasma RIA system employing 125 I31kD inhibin as tracer.

Potential RIA standards (bFF, 31kD inhibin, hFF) were assayed in the presence of either phosphate buffer (200 pl, o) or steer serum 200 pl, or post-menopausal serum 200 pl, A and their logit plots were calculated using their respective non-specific binding and Bo values. Cow serum shows a minimal detectable immunological response. When the immunoactivity was expressed in terms of 31kD inhibin standard the level of inhibin in BS was 0.91 0.27 (n 3) mg/ml and CS 0.1 ng/ml.

A parallel response line of HFP was observed in the RIA with 31kD inhibin and hFF as standards corresponding to levels of 1.05 0.07 (n 3) ng/ml. The levels in normal plasma (n 8) were equal to or less than the sensitivity of the assay (0.1 ng/ml).

Example 10 Radioimmunoassay of inhibin infertile human subjects The radioimmunoassay previously described was appiied to plasma and serum from post-menopausal subjects (presumed to be inhibin free, n 8) and young women with ovarian failure (premature ovarian failure n 2, Turner's syndrome n 1, ovariectomized n There was no 125 difference in I-31kD inhibin binding in the assay between these two groups and therefore post-menopausal serum was used as a diluent in the assay.

Example 11 Radioimmunoassay of inhibin in human serum The method was applied to: Plasma from normal women during spontaneous menstrual cycles from the early follicular phase until the time of ovulation (n 2, example 'FL' #27, Fig. Inhibin WO 87/05702 PCT/AU87/00070 24 24 immunoactivity was below the limit of assay detection prior to day 13 and its increase correlated with the increase in circulating levels of oestradiol (E 2 LH and FSH on day 13 and 14.

Inhibin immunoactivity in serum from normal men (n 7) was at the lower limit of assay detection.

Plasma was obtained from 26 unselected women undergoing their fourth cycle of ovulation induction in the in vitro fertilization programme at the Epworth Hospital, Richmond, Victoria. Briefly this involved the administration of clomiphene citrate 100-150 mg daily on day 5-9 of the menstrual cycle, followed by human menopausal gonadotrophin (hMG) 75-225U daily for the next 5-7 days. Adequate follicular development was assessed by the progressive increase in plasma oestradiol and by ovarian ultrasound.

Ovulation occurred spontaneously if an endogenous LH spike was observed, or in the absence of the latter, ovulation was induced by administration of 5000 IU of human chorionic gonadotrophin (hCG). For an example of a spontaneous ovulation see 'BU' 11, Fig. 9k and for an hCG-induced ovulation sample see 'JO' 1, Fig. 9a.

Inhibin immunoactivity in the plasma samples, as defined in terms of the biological activity of a purified 31kD inhibin standard, showed a highly significant correlation with plassma oestradiol levels (Fig. 10). The correlation coefficient values have been calculated from the total data in figure 9. An example of the correspondence of plasma oestradiol and inhibin during an ovulation induction cycle is seen in example 'BE' 9, Fig. 9b. There was also significant correlation between peak plasma oestradiol concentrations and the number of oocytes recovered, and peak plasma inhibin concentrations and the number of oocytes recovered (Fig. 11), as well as a strong correlation between the peak plasma inhibin concentration and the number of ovarian follicles detected ultrasonically prior to oocyte aspiration (Fig. 12).

It is therefore apparent that both plasma inhibin and plasma c~ WO 87/05702 PCT/AU87/00070 25 oestradiol are parameters of follicular development and health, and in the majority of cases these show a close correspondence.

In certain examples 6, 'BR' 10, Fig. 9h and a dissociation between plasma inhibin and plasma oestradiol concentrations was observed, suggesting different regulation of these two parameters of follicular development.

As inhibin is a peptide produced by ovarian granulosa cells and plasma oestradiol in the human is predominantly an ovarian theca cell product, the assay of plasma inhibin is ':he first direct plasma parameter of granulosa cell/oocyte health and maturatiqn. The dissociation of plasma inhibin and E 2 may therefore be of therapeutic importance in that the plasma inhibin is a direct measure of follicular development, and its assessment may affect the timing of ovulation induction and oocyte collection.

Example 12 hFF inhibin as standard for radioimmunoassay Human follicular fluid (hFF) obtained at oocyte collection in the IVF programme was prepared for use as the radiummunoassay (RIA) standard by two gel chromatographic steps and reversed phase HPLC as described for bFF inhibin (Robertson et al, 1985). This material yielded parallel dose r-ppnse lines to human female serum inhibin obtained from women undergoing ovarian hyperstimulation for in vitro fertilisation. This partially purified human follicular fluid (hFF) inhibin standard preparation was defined in terms of its in vitro inhibin bioactivity using an inhibin bioassay based on the dose-related suppresion of FSH cell content (Scott et al, 1980). This material was used as the RIA standard and gave dose response lines parallel to serum inhibin obtained from women undergoing ovarian hyperstimulation for in vitro fertilization and also to that of pregnant serum.

The unitage of the hFF inhibin standard was calibrated in terms of an ovine testicular lymph standard preparation of defined unitage 1 U/mg using the inhibin WO 87/05702 PCT/AU87/00070 26 bioassay. The RIA has an interassay coefficient of variation (CV) of 6.4% (n 5 assay) and the sensitivity (iogit 2) was 0.37 U/ml.

The RIA was specific to bovine and human inhibin and cross-reacted less than 0.3% with a range of glycoproteins and growth factors. In addition, inhibin-related peptides cross-reacted as follows: porcine transforming growth factor bovine Mullerian Inhibitory Substance purified bovine inhibin B subunit dimer and the subunits of 31kDa bFF inhibin following reduction and alkylation No immunoactivity was detectable in the sera of castrate subjects, post-menopausal women, nor in a subject with Turner's syndrome. The RIA had an interassay coefficient of variation of 8.3% (n 5 assays), and a sensitivity of 0.37 U/ml.

Inhibin levels were determined at 1 dilution level against the partially purified hFF standard preparation using an iterative curve-fitting procedure (Burger et al., 1973).

In the calculation of results, a lognormal distribution of individual observations (Gaddum et al; 1933) was assummed, i.e. all calculations were performed using logarithmically transformed values to give geometric means and 67% confidence intervals. Statistical comparison between pregnant and non-pregnant groups was performed using the unpaired t-test.

Example 13 Inhibin levels during luteal phase and early pregnancy Nineteen women presenting consecutively for treatment in the Mondsh university IVF program were studied.

Clinically their infertility resulted from tubal disease (n endometriosis (n unkown causes (n 5) or poor semem quality (n The protocol of ovulation induction has been described elsewhere (Wood and Trounson, 1984). Briefly, all Ssubjects received clomiphene citrate (Clomid, Merell Dow, Sydney) 100-150 mg daily between days 5 and 9 of the cycle and HMG (Pergonal, Serono, Rome) 75-225 units daily from day 6.

The dosage and duration of HMG therapy were optimized WO 87/05702 PCT/AU87/00070 27 according to daily plasma oestradiol concentrations and follicular size as assessed by ovarian ultrasound. HCG (Pregnyl, Organon, Oss) 5,000 IU intramuscularly was administered to induce ovulation, and oocyte retrieval was undertaken 36 hours later. Embryo transfer was performed as described by Wood and Trounson (1984). Blood was taken on day 1 post laparoscopy and every second day from day 2 to day 14 and sera stored for measurement of FSH, LH, subunit hCG, oestradiol, progesterone and inhibin. Three of the 19 women became pregnant.

At various stages of gestation, a single serum sample was obtained from each of 24 normal pregnant women.

Samples were assayed for inhibin using the hFF inhibin standard described in Example 11. In the 16 patients who did not conceive, luteal phase inhibin levels rose to a peak level of 2.5 U/ml on day 6 and then fell to undetectable levels by day 14. These results are shown in Figure 13.

The number of subject serum samples per day was 13-16 except at day one when only eight were available.

Results are expressed as the geometric mean 67% confidence intervals. The broken line indicates the limit of sensitivity of the inhibin radioimmunoassay. The number of subjects showing non-detectable inhibin values is shown in parentheses.

Non-detectable values are not included in the mean confidence intervals.

In three subjects who conceived, inhibin levels were similar to non-conception cycles between days 2 and 8, increasing thereafter and becoming significantly higher (p 0.001) than in the non-pregnant group by day 12 post-laparoscopy. Figure 14 shows these results, expressed as the geometric mean 67% confidence intervals. The broken line indicates the sensitivity of the inhibin radioimmunoassay. *p<0.05, ***p40.001 comparing hormone values for the pregnant and non-pregnant groups on the same day. Significance values in the second panel refer to serum FSH. The late luteal phase rise in serum inhibin in the 1- 1 WO 87/05702 PCT/AU87/00070 28 pregnant patients coincided with both the rise in serumahCG and with the decline in serum FSH to values below those seen in the non-pregnant group.

Serum FSH showed a significant inverse correlation with inhibin in the luteal phase of the non-conception cycles (r 0.51, n 113, p <0.001) (Fig. 15). Similar significant inverse relationships were observed between FSH and inhibin when the data were analysed according to whether the progesterone concentrations were in the normal ovulatory luteal phase range (25-100) nm) or greater, (r 0.38, n 76, p 40.001 vs r 0.37, n 37, p <0.05, slopes not statistically different). Significant inverse correlations also existed between luteal phase serum FSH and both progesterone (r 0.64, n 113, p <0.001) and oestradiol (r 0.52, n 114, p 40.001).

Plasma inhibin and progesterone concentrations were significantly correlated in the luteal phase of cycles in which pregnancy did not occur (r 0.81, n 85, p0.001), as were plasma inhibin and oestradiol concentrations (r 0.65, n 85, p<0.001). In pregnant subjects luteal phase inhibin levels did not show significant correlations with either progesterone or oestradiol. Serum LH levels (not shown) fell sharply from day 1 (21.0 [17.0-26.1] mIU/ml) to a nadir mIU/ml) on day 8.

In a separate study of serum inhibin, levels were determined during gestation in 24 normal pregnant women. The mean level prior to 20 weeks gestation (1.31 (0.95-1.80) U/ml, n 13) was significantly lower (p <0.02) than levels after this time (2.02 U/ml (1.32-3.10) U/ml, n 11).

Thus there is a rise in circulating inhibin concentrations during the luteal phase of stimulated menstrual cycles and during pregnancy.

WO 87/05702 PCT/AU87/00070 29 Example 4 Inhibin Levels in the Normal Menstrual Cycle In a further studyserum inhibin was determined daily in 6 normal women throughout the menstrual cycle, using a radioimmunoassay employing an antiserum directed to 31kD inhibin. The normalcy of the menstrual cycle was assessed from the serum profiles of FSH, LH, progesterone, and oestradiol.

The increase in sensitivity of the assay using this antiserum permitted the detection of inhibin in over 97% of samples. The results are shown in Fig. 16.

j4*, WO 87/05702 PCT/AU87/00070 30 Advantages and applications of the assay according to the invention.

1. The assay may be used for determining inhibin concentration in a wide range of biological samples, such as serum, plasma, urine, follicular fluid, tissue homogenates, and culture fluids.

2. The assay may be used to monitor the purification of inhibin from tissue, biological fluids, or culture medium, or to monitor transfection studies.

3. Inhibin levels may be used as a marker of parameters of reproductive function, such as granulosa cell function, follicular development, number of ovarian follicles following ovarian hyperstimulation, and foetal well-being during early pregnancy, and Sertoli cell function.

It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to hereinabove.

The following terms referred to hereinbefore are trade marks: Amerlex-M, Clomid, Coat-a-Count, Marcol 5 2, Matrex Red A, Montanide 888, Norit A, Pergonal, Polypep, Pregnyl, RIA-Quant, Sephacryl, Sephadex, Triton X-100, Ultrapore, and Ultra-Turrax.

References cited herein are listed on the following pages.

I li I- WO 87/05702 PCT/AU87/00070 31

REFERENCES

Au, Robertson, D.M. and de Kretser, D.M. (1983) Endocrinology 112, 239-244.

Baker, Eddie, Higginson, Hudson, B., Keogh, E.J. and Niall, H.D. (1981) Assays of Inhibin. In P.

Franchimont and C.P. Channing (Eds), Intragonadal Regulation of Reproduction, Academic Press, pp. 193-228.

Baker, Eddie, Hudson, B. and Niall, H.D. (1985) Australian J. Biological Sciences (in Press).

Burger, Lee, and Rennie, L.C. (1983) J. Lab.

Clin. Med. 80 302-308.

Erickson, and Hsueh, A.J.W. (1978) Endocrinology 103, 1960-1963.

Finney, D.J. (1971) Statistical Method in Biological Assay, 2nd ed. C. Griffin Co. Ltd., London.

Forage Ring, Brown, McInerney, Cobon, Gregson, Robertson, Morgan, Hearn, Findlay, Wettenhall, Burger, and de Kretser, D.M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3091-3095.

Foulds, L.M. and Roberston, D.M. (1983) Mol. Cell. Endocr.

31, 117-130.

Gaddum, J.H. (1933) Medical Research Council Special Report Series, 183.

Henderson, K.M. and Francimont, P (1981) J. Reprod. Fert. 63, 431-442.

WO 87/05-702 PCT/AU87/00070 -32 Kramer, C.Y. (1956) Biometrics 12, 307-310.

Lee, McMaster, Quigg, H. and Leversha, L. (1982) Endocrinology 111, 1849-1854.

Ling, Ying, S-Y, Ueno, Esch, Denoroy, L. and Guillemin, R. (1985) Proc. Natl. Acad. Sci.. U.S.A. 82, 7217-7221.

Marana, Suginami, Robertson, D.M. and Diczfalusy, E.

4 (1979) Acta Endocrinol. (Kbh.) 92, 585-598.

Mason, Hayflick. Ling, Esch, Ueno, N., Ying, S-Y, Guillemin, Niall, and Seburg, P.H. (1985) Nature 318, 659-663.

Miyamato, Hasegawa, Fukuda, Nomura, Igarashi, Kangawa, and Matsuo, H. (1985) Biochem. Biopys Res.

Comm. 129, 396-403.

Robertson, Foulds, L.M. Leversha, Morgan, F.J., Hearn, Burger, Wettenhall, R.E.H. and de Kretser, D.M. (1985) Biochem. Biophys Res. Comm. 126, :.220-226.

Robertson, de Vos, Foulds, McLachlan, R.I.

Burger, Morgan, Hearn, M.T.W. and de Kretser, D.M.

(1986) Mol. Cell. Endocrinol. 44, 271-277 4 Scott, Burger, H.G. and Quigg, H. (1980) Endocrinology 107, 1536-1542 Wood, and Trounson, A.0. (eds). In vitro fertilization and embryo transfer (1984) Churchill Livingstone

Claims (14)

1. A radioimmunoassay for determing inhibin suspected to be present in a biological sample, comprising the steps of: contacting the sample with an antibody directed against a substantially purified 58kDa or 31kDa human or bovine follicular fluid inhibin or an inhibin at least 20% immunologically cross-reactive with said follicular fluid inhibin and incubating at 40 to 30 0 C for a period of 4 hours to 4 days; adding a substantially purified 12I- inhibin and incubating for a period and at a temperature selected from the group consisting of overnight room temperature, 48 to 72 hours at 4°C and 16 hours at 30 0 C, adding a second antibody directed against the first antibody and incubating for 30 minutes to 24 hours at 4 0 C, separating precipitated material, and 1 measuring bound 2I-labelled inhibin in the precipitate.

2. A radioimmunoassay according to Claim 1, comprising the steps of: incubating sample and said anti-inhibin antibody for 4 days at 4 0 C, adding 12 5 s-inhibin and incubating for 72 hours at 4 0 C, adding a second antibody directed against the first antibody and incubating for 30 minutes to 24 hours at 4C, see* separating precipitated material, and 125 measuring bound 2I-labelled inhibin in the precipitate.

3. A radioimmunoassay according to Claim 1 or Claim 2, in which the antibody is contained in an antiserum raised by injecting an animal with an antigen selected from the group consisting of inhibin, purified bovine 58kD inhibin, purified bovine 31kD inhibin, human inhibin, human or bovine inhibin produced using recombinant DNA l, _A 1 x~w (t 34 technology, and immunologically cross-reactive sub-units, fragments or derivatives thereof.

4. A radioimmunoassay according to any one of Claims 1, 2 or 3, in which the 1 2sI-inhibin is 58kD or 31k inhibin.

A radioimmunoassay according to any one of Claims 1 to 4, in which the sample is taken from a human and the labelled inhibin is a bovine inhibin.

6. A radioimmunoassay according to any one of Claims 1 to 4, in which the sample is taken from a human and the labelled inhibin is a human inhibin.

7. A radioimmunoassay according to any one of Claims 1 to 6, in which samples to be assayed are diluted in inhibin-free serum.

8. A radioimmunoassay according to any one of Claims S1 to 7, in which polyethylene glycol is added following i incubation with the second antibody and incubation is continued for a further 30 minutes. S. i

9. A radioimmunoassay according to any one of Claims S* 1 to 8, in which Triton X-100 is incorporated into samples S to be assayed. S.

10. A radioimmunoassay according to any one of Claims 1 to 9, in which an assay standard sample is used, said standard sample being selected from the group consisting of naturally-occurring or recombinant bovine 31kD inhibin and S. naturally-occurring or recombinant human inhibin, and immunologically cross-reactive sub-units, fragments or S* derivatives thereof.

11. A radioimmunoassay according to any one of the preceding claims, in which the 12sI-inhibin is prepared by the steps of: iodinating inhibin with Chloramine-T, S(b) isolating 12sI-inhibin by affinity chromatography on Matrex Red A, and optionally further purifying the 125I-inhibin by gel filtration.

12. A test kit for the estimation of inhibin in a /r sample, comprising at least one of: I' 1 35 12 I-labelled, substantially purified, follicular fluid-type inhibin, an antibody directed against a substantially purified, follicular fluid-type inhibin, an assay standard selected from the group consisting of naturally-occurring or recombinant bovine 31kD inhibin and naturally-occurring or recombinant human inhibin and immunologically cross-reactive sub-units, fragments or derivatives thereof.

13. A method of monitoring a parameter of I reproductive function in a mammal, selected from the group consisting of ovarian granulosa cell function, ovarian follicular development, number of ovarian follicles following ovarian hyperstimulation, foetal well-being during early pregnancy and Sertoli cell function, comprising the step of measuring inhibin content in a sample of plasma from that mammal, using a radioimmunoassay as defined in any one of Claims 1 to 11.

14. A method of assessing health and naturation of oocytes or ovarian granulosa cells of a mammal, comprising the step of measuring inhibin content in a sample of plasma from that mammal, using a radioimmunoassay as defined in any one of Claims 1 to 11. A method of selecting a preferred time for ovulation induction or oocyte aspiration for in vitro fertilization of ova from a female mammal, comprising the step of measuring inhibin content in a sample of plasma from that mammal using a radioimmunoassay as defined in any one of Claims 1 to 11. DATED this 27th day of April 1990 *o* BIOTECHNOLOGY AUSTRALIA PTY LTD; MONASH UNIVERSITY; MONASH MEDICAL CENTRE and ST VINCENT'S INSTITUTE OF MEDICAL RESEARCH By Their Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia