International Collaborative Study byin vitroBioassays and Immunoassays of the First International Standard for Inhibin, Human Recombinant

Biologicals (1996) 24, 1–18

International Collaborative Study by in vitro Bioassays and Immunoassays of the First International Standard for Inhibin, Human Recombinant 1

M. P. Rose1 and R. E. Gaines Das2 Division of Endocrinology and Informatics Laboratory, WHO International Laboratory for Biological Standards, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, U.K. 2

Abstract. The First International Standard for Inhibin, Human Recombinant, (ISI), a lyophilized preparation of rDNA-derived human 32 kDa Inhibin A in ampoules coded 91/624, was evaluated by international collaborative study for its suitability to serve as an International Standard. This study, which involved 15 laboratories in nine countries, included a variety of in vitro bioassays and immunoassays. The ISI was compared with two other lyophilized preparations of human recombinant inhibin, the International Standard for Porcine Inhibin (ISP) and preparations of human follicular fluid inhibin. Predicted loss of activity based on estimates of potency of contents of ampoules which had been stored under conditions of accelerated thermal degradation indicated that the ISI has satisfactory stability. On the basis of the results of this study, the ISI was deemed suitable to serve as a standard for in vitro bioassays and immunoassays and was established by the Expert Committee on Biological Standardization of the World Health Organization as the First International Standard for Inhibin, recombinant human, with an assigned unitage of 150 000 International Units per ampoule. This unitage maintains an approximate continuity of units with the ISP. © 1996` The International Association of Biological Standardization

Introduction Inhibin is a gonadal dimeric glycoprotein which has an inhibitory effect on the secretion of follicle stimulating hormone (FSH) by the pituitary gland. The concept of a non-steroidal gonadal hormone with this biological activity was postulated some 70 years ago.1 Following isolation of the protein2 and cloning of the complementary DNA and determination of the full structure of bovine3 and human4,5 inhibin, it was found that inhibin shared structural homology with a family of glycoproteins which includes Mullerian inhibiting substance, transforming growth factor β, activin and bone morphogenic proteins. This structural homology suggests that inhibin may exert paracrine or autocrine actions in a wide range of biological systems. Such actions have been observed experimentally, for example in erythrodifferentiation.6 Clinically, inhibin determinations may have uses in the diagnosis of some forms of cancer including * To whom correspondence should be addressed. 1045–1056/96/010001118 $18.00/0

granulosa cell tumours, cystadenocarinoma of the ovary and hydatidiform mole and in physiology and pathology of pregnancy including placental function.7,8 In 1984, the World Health Organization (WHO) Special Program of Research, Development and Research Training in Human Reproduction identified the need for an ampouled stable reference preparation of inhibin to be made widely available to serve as a research standard for in vitro bioassays of inhibin. An extract of porcine follicular fluid, which was estimated to be about 1–3% pure (w/w), was provided by the National Institutes of Health (NIH) Contraceptive Development Branch (Bethesda, U.S.A.). This material was ampouled by the National Institute for Biological Standards and Control (NIBSC) in 1986 and was assessed by an international collaborative study initiated in 1988.9 Research on inhibin depended, until recently, upon in vitro biological assays in which classically defined inhibin bioactivity could be detected and measured. However, immunoassays and novel in vitro bioassays for inhibin have now been developed which may © 1996 The International Association of Biological Standardization

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M. P. Rose and R. E. Gaines Das

require a highly purified preparation of inhibin to serve as a reference preparation. The only source of highly purified human inhibin in sufficient quantity to serve as a standard for such assays is through rDNA technology. Two preparations of highly purified recombinant DNA-derived human inhibin were ampouled in 1990–91 by NIBSC following recommended procedures for international standards.10 These preparations have now been evaluated as candidate International Standards for both in vitro bioassay and immunologically based assays by international collaborative study. The aims of this study were: (1) To compare the biological potency and immunoreactivity of the candidate materials and assess their suitability to serve as an International Standard (IS) for human inhibin. (2) To calibrate the proposed IS for recombinant DNA-derived human inhibin by both bioassay and immunoassay. (3) To compare the recombinant DNA-derived human inhibins with the International Standard for Porcine Inhibin (ISP) and with natural human inhibin. (4) To assess the specificity and reproducibility of the different assay systems currently in use for estimating highly purified inhibin and inhibin in biological fluids or extracts. (5) To assess, in different assay systems, the stability of recombinant DNA-derived inhibin lyophilised in ampoules by assay of the contents of ampoules exposed to accelerated thermal degradation. Materials and methods

Ampouled preparations included in the study Participants were also asked to include their inhouse standard where possible. Materials included in the study and their abbreviations throughout this report are summarized in Table 1. 1. First International Standard for Porcine Inhibin (ISP). This consists of a batch of ampoules (code 86/690) containing approximately 20 µg of inhibin, of about 1–3% purity (w/w) from porcine follicular fluid. Further details of this preparation can be found in Gaines-Das et al.9 2. Recombinant DNA-derived 32 kDa Human Inhibition A 90/522 (hI90). This consists of a batch of ampoules coded 90/522 containing recombinant 32

kDa human inhibin A. The bulk material was shipped to NIBSC as 2·5 mg of purified inhibin lyophilized in the presence of 5·0 mg bovine serum albumin (repurified by reverse phase HPLC). This material was reconstituted in 10 ml 4·0 M acetic acid containing 1 mg/ml repurified human serum albumin, filtered (nominal pore diameter 0·22 µm) and made up to a final volume of 2000 ml with a solution containing 1 M acetic acid, 5 mg/ml mannitol and 1 mg/ml albumin. This solution was distributed into ampoules in approximately 1 ml aliquots. The mean weight of solution of 43 weighed ampoules was 1·0061 g with a range of 0·16%. The contents of the ampoules were freeze dried and secondarily desiccated. The nominal content of these ampoules is 1·25 µg human inhibin A. Preliminary characterisation suggested that the content is 1·78 µg/ampoule by immunoassay (RIA performed by manufacturer against bulk material from which this preparation was derived) of the contents of three ampoules and 2080 u/ampoule in terms of a bovine follicular fluid standard or 53 800 6 5830 IU/ampoule in terms of the ISP by bioassay of the contents of six ampoules.

3. First International Standard for Inhibin, Human Recombinant (ISI). This consists of a batch of ampoules coded 91/624 containing recombinant 32 kDa human inhibin A. The bulk material arrived at NIBSC as a solution (22 ml) of sodium chloride (0·15 M) tris (0·05 M, pH 7·5) containing human recombinant inhibin (0·5 mg/ml). Twenty millilitres of this solution was diluted to 200 ml with a solution containing trehalose (0·2% w/v), repurified human albumin (AK12, Lister Institute, Elstree, U.K. (0·5% w/v), sodium chloride (0·15 M) and tris (0·05 M, pH 7·52), filtered (nominal pore diameter 0·22 µm) and diluted to a final volume of 2000 ml with a solution containing trehalose (0·2% w/v), repurified human albumin (0·5% w/v), sodium chloride (0·15 M) and tris (0·05 M, pH 7·52). This solution was then distributed in glass ampoules in approximately 1 ml aliquots. The mean weight of solution in 36 weighed ampoules was 1·0073 g with a range of 61·44%. The contents of the ampoules were freeze-dried and secondarily desiccated. The nominal content of these ampoules is 5 µg recombinant human inhibin A. Preliminary characterisation indicated that the content was 4925 ng inhibin A per ampoule by immunoassay against preparation hI90, and recovery against the parent lot was 100% by ELISA and 84% by rat pituitary bioassay. 4. Human follicular fluid 92/640 (hffQ). This consists of a batch of ampoules coded 92/640 containing

in vitro bioassays and immunoassays

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Table 1. Summary of materials used Ampoule code

Material (study abbreviation)

Nominal content

Study code

90/522

32 kDa rDNA human inhibition (hI90)

1·25 µg (5 mg Mannitol. 1 mg albumin)

E, J or M

91/624

32 kDNA human inhibin (ISI)

5 µg 32 kDa inhibin (2 mg trehalose, 5 mg albumin, 1 3 1024 moles NaCl, 5 3 1025 moles tris)

K or A

91/628

32 kDNA human inhibin (hIL)

5 µg (5 mg albumin, 2 mg trehalose)

L

92/640

Human follicular fluid (hffQ)

1–2 ng 32 kDa inhibin (5 3 1025 moles tris, 1·5 3 1024 moles NaCl, 5 mg albumin)

Q

92/696

Human follicular fluid (post FSH) (hffP)

5 ng 32 kDa inhibin

P

92/650

Immunoaffinity purified human inhibin (ipIR)

26·2 ng 32 kDa inhibin (5 3 1025 moles tris, 1·5 3 1024 moles NaCl, 5 mg albumin, 2 mg trehalose)

R

86/690

IS porcine inhibin (ISP)

(See Ref 1)

N

Accelerated thermal degradation samples

hI90 after storage for 981 days at: 20°C 37°C 45°C

As hI90

ISI after storage for 400 days at: 4°C 20°C 37°C 45°C

As ISI

lyophilized, charcoal treated human follicular fluid (hff) diluted with a solution containing tris (0·05 M, pH 7·5), NaCl (0·15 M), human serum albumin (0·5% w/v). The bulk material arrived at NIBSC as a frozen solution of 15 3 0·5 ml aliquots. This material was diluted to 50 ml with the above solution and was distributed in ampoules as approximately 1 ml aliquots. The contents of the ampoules were freeze-dried and secondarily desiccated. The nominal content is approximately 1–2 ng 32 kDa inhibin per ampoule.

5. Human follicular fluid 92/696 (hffP) . This consists of a batch of ampoules coded 92/696 which contain lyophilised, charcoal treated hff collected from patients undergoing oocyte retrieval after FSH stimulation. The follicular fluid (ff) was distributed into ampoules in approximately 1 ml aliquots, freezedried and secondarily desiccated. The nominal con-

I F B G C D H

tent is approximately 5 ng per ampoule 32 kDa inhibin as estimated by immunoassay of the ff.

6. Immunoaffinity purified natural human inhibin (ipIR). This consists of a batch of ampoules coded 92/650 containing immunoaffinity purified natural human inhibin. The bulk material arrived at NIBSC as a frozen solution consisting of 13 3 200 µl aliquots. The inhibin was purified on an immunoaffinity column containing an antibody directed against the α chain of inhibin. The bulk solution was diluted to a final volume of 50 ml with a solution containing tris (0·05 M, pH 7·5), sodium chloride (0·15 M), human serum albumin (0·5% w/v) and trehalose (0·2% w/v). This solution was distributed into ampoules in approximately 1 ml aliquots and the contents of the ampoules were freeze-dried and secondarily desiccated. The nominal content as estimated by prelimi-

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M. P. Rose and R. E. Gaines Das

nary immunoassay is 26·2 ng per ampoule of 32 kDa inhibin.

7. Recombinant DNA-derived human inhibin A 32 kDa 91/628 (hIL). This consists of a batch of ampoules coded 91/628 containing recombinant 32 kDa human inhibin A. The bulk material arrived at NIBSC as a preparation containing 0·25 mg recombinant inhibin lyophilized in the presence of 1·25 mg bovine serum albumin. This material was reconstituted with 1 ml 4 M acetic acid and diluted to a final volume of 50 ml with a solution containing 4 M acetic acid, human serum albumin (AK12, Lister Institute, Elstree, U.K.) (0·5% w/v) and trehalose (0.2% w/v). The solution was distributed into ampoules in approximately 1 ml aliquots and was freeze-dried and secondarily desiccated. The nominal content is 5 µg 32 kDa inhibin per ampoule. 8. Coded samples. Ampoules coded A-I comprised samples of hI90 and the ISI which had been stored at elevated temperatures (220°C, 14°C, 120°C, 137°C, 145°C) for estimation of accelerated thermal degradation.

Participants and design of the study Fifteen participants in nine countries took part in the study. These are listed in Table 2. Throughout this report each laboratory is identified by a number which is not related to the order of listing. Participants were asked to carry out their usual assay procedure, to include as far as possible each of the ampouled materials described above, to use freshly reconstituted ampoules and to use sufficient replicates at multiple doses to provide data for analysis of variance and assessment of linearity and parallelism. Participants were asked to provide the raw data for centralised analysis at NIBSC and their own calculated or computed results for comparison of potency estimates. To provide an independent assessment of intraassay variation, each participant received a coded duplicate, which was an ampoule of hI90 relabelled M. This also provided a ‘base line’ sample for analysis of degradation data as handling was expected to be the same as that for the ‘degraded’ samples. Restrictions on the availability of some preparations together with limitations on the capacity of some assay systems meant that all preparations were not assayed by all participants. Assays contributed Three of the 15 laboratories provided data for in vitro cell based assays only. Five laboratories used

Table 2. List of participants Professor N. Groome, School of Biological and Molecular Sciences, Oxford Polytechnic, Gipsy Lane, Headington, Oxford OX3 0BP, U.K. Professor R B Billiar and Ms Patricia Smith, Division of Reproductive Biology, McGill University, Royal Victoria Hospital, Women’s Pavilion, 687 Pine Avenue West, Montreal, PQ, Canada H3A 1A1. Dr F H de Jong, Department of Endocrinologie and Reproduction, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands. Professor J. France and Mr J. Keelan, Department of Obstetrics and Gynaecology, University of Auckland School of Medicine, National Women’s Hospital, Claude Road, Auckland 3, New Zealand. Professor P. Franchimont and Dr E. Poncelet, Département d’Endocrinologie, Laboratoire de Radioimmunologie, Universite de Liege, C.H.U. Sart Tilman, 4000 Liege, Belgium. Professor S. G. Hillier and Dr C. D. Smyth, Reproductive Endocrinology Laboratory, University of Edinburgh, 37 Chalmers Street, Edinburgh EH3 9EW, U.K. Dr H. S. Juneja and Ms P. Parte, Institute for Research in Reproduction, Jehangir Merwanji Street, Parel, Bombay 400 012, India. Dr P. G. Knight and Dr S. Muttukrishna, Department of Biochemistry and Physiology, University of Reading, Reading RG6 2AJ, U.K. Professor Yoshihisa Hasegawa, Department of Laboratory Animal Science, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aamori 034, Japan. Dr J. P. Mather*, Dr L. Krummen†, Dr T. Woodruff* and‡, Miss E. Mann‡, *Cell Biology Department, †Cell Culture CCFR&D Department and ‡MAC Department, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, California CA 94080, U.S.A. Dr D. Robertson and Mr N. Cahir, Prince Henry’s Institute of Medical Research, PO Box 152, Clayton, Victoria 3168, Australia. Dr W. R. Robertson, Department of Medicine, Section of Clinical Biochemistry, University of Manchester, Hope Hospital, Eccles Old Road, Salford M6 8HD, U.K. Dr S. Sasamoto and Dr G. Watanabe, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan. Dr A. Balen and Miss J. Er, Cobbold Laboratories, The Middlesex Hospital, Mortimer Street, London W1N 8AA U.K. Dr M. Wheeler and Miss L. Simmons, Department of Chemical Pathology, St. Thomas Hospital, London SE1 7EH U.K.

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Table 3. Summary of assay methods and house standards used in study Laboratory code

House standard code

House standard

Units of house standard

Bovine follicular fluid

µg (1 µg BFF ; 2·162 IU IS)

BFF

‘Units’ (ø 400 pg rDNA inhibin) ‘Units’ (ø 60 pg rDNA inhibin)

16H4

ELISA αBA (ELB)

Immunoaffinity purified human inhibin Immunoaffinity purified human inhibin

02

Female rat thecal cells (ROVA)

rDNA-derived human inhibin

ng/ml

13140–8I

03

ELISA A (EIA)

rDNA-human inhibin in post menopausal human serum Follicular phase human serum

µl/ml

PMS

µl/ml

PMS

32 kDa bovine inhibin

ng/ml

32 kDa bovine inhibin

ng/ml

32 kDa bovine inhibin

ng/ml

Bovine follicular fluid

µl/ml

01

Assay type

Male rat pituitary cells (RPT) ELISA αα (ELA)

ELISA B (EIB) RIA (RIA) 04

Oxidised dimeric inhibin IRMA (IRMA_O)

16H4

Normal dimeric inhibin IRMA (IRMA_N)

α subunit RIA (RIA)

bff

Adult female sheep pituitary cells (OPT) 05

12d male rat pituitary cells (RPTM_BF)

None

06

Adult female rat pituitary cells (RPTF) RIA (RIA)

32 kDa bovine inhibin

‘Units’/well

32 kDA bovine inhibin (also porcine follicular fluid bovine inhibin for bioassay)

ng/ml (1 ng 32 kDa ;155·3 units)

Adult male rat pituitary cells (RPT) RIA (RIA)

Bovine follicular fluid (Charcoal treated rDNA-derived human inhibin

IU

bff 2/4

ng

HIS B

RIA (RIA)

Charcoal stripped bovine fluid

µl/0·5 ml

bff

09

10

Adult male rat pituitary cells (RPT_F) 11

Two-site IFMA (IFMA)

None

12

Adult female rat pituitary cells (RPT) Radioimmunoassay (RIA)

32 kDa bovine inhibin

ng/ml

86/690 Is Rat inhibin A Rat inhibin B 34 kDa human inhibin in follicular fluid 30 kDA human inhibin in follicular fluid

IU/ml ng/ml

rDNA-derived human inhibin

ng/l

13

Two-site IRMA (IRMA)

ng/ml ng/ml R-9011

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M. P. Rose and R. E. Gaines Das

Table 3 (continued) Laboratory code

Assay type

Units of house standard

House standard

House standard code

14

Adult male rat pituitary rDNA-derived human cells (RPTM) inhibin K562 cells (K562) RIA (RIA) Two-site ELISAs (11B5:ck:9A9:CK:CK:CK)

µg/ml

13559–83

15

Adult female rat pituitary cells (RPTF_FN)

1st IS porcine inhibin

IU/ml

86/690

16

Two-site assay (ELISA)

rDNA-derived human inhibin

17

RIA (RIA)

rDNA-derived human inhibin rDNA-derived human

ng/ml

R-90/1 13140–40/R

Two-site ICMA (ICMA)

inhibin immunometric assays only and the remainder used both immunometric and cell based assays. These are summarized in Table 3.

In vitro cell based assays. Eight laboratories used dispersed rat pituitary cells; four used cells derived from adult male rats;11–14 three used cells derived from adult female rats15,16 and one used cells derived from 12-day-old male rats.17 One laboratory used pituitary cells derived from adult female sheep.18 In these assays, participants assessed the inhibitory effects of inhibins on the basal or LHRH-stimulated (two laboratories) release of FSH from cells into the culture medium. In addition to rat pituitary cell systems one laboratory measured androgen production by rat ovarian thecal cells from 21-day-old rats19 and one laboratory measured haemoglobin synthesis in K562 cells.20 Eight laboratories included their own laboratory standard(s) (Table 3). These local standards varyingly included rDNA-derived human inhibin, 32 kDa bovine inhibin and bovine ff. For purified preparations the amount added was given as mass units and for unpurified preparations the amount added was given as ‘units’. One laboratory included purified rat inhibin and one laboratory included human inhibin. Immunometric assays. Seven laboratories used radioimmunoassays (RIA),21–23 four laboratories used enzyme linked immunosorbent assays (ELISA,24–26 two laboratories used immunoradiometric assays (IRMA),27,28 one laboratory used an immunofluor-

escent assay (IFMA) and one laboratory used an immunochemiluminescent assay (ICMA).29 Seven laboratories used rDNA-derived human inhibin as standard and other laboratories used, varyingly, immunoaffinity purified human inhibin, 32 kDa bovine inhibin or charcoal stripped bovine ff (Table 3).

Statistical analysis Data for each assay were assessed both graphically and statistically for any deviations from a consistently increasing (or decreasing, according to the nature of the responses) relation between doses and responses and for the occurrence of outlying observations. Responses were deleted from analysis if they occurred at doses for which the relation between doses and responses was not consistently increasing (or decreasing), i.e. for doses so small that there was no detectable response or for doses larger than that giving the maximal response. Outlying responses, identified as statistical outliers within the group of responses (at a particular dose) which increased the variance of that group of responses so that it was significantly larger than the pooled variance of all other groups of responses, were also deleted. If the group giving a significantly increased variance comprised only two observations, both were deleted. Fewer than 0·2% of the total of over 10 000 response values were deleted as outliers. For many assays, the relation between doses and responses was sigmoidal and could be satisfactorily described using a four-parameter logistic function. For each of these assays the fitted asymptotic values

in vitro bioassays and immunoassays

were used to transform the responses to logits and the resulting log dose—logit response data were analyzed using weighted linear regression analysis. Linearity and parallelism were assessed, and the logs of the relative potencies were determined as the displacement of fitted parallel log dose—logit response lines. For assays for which there was insufficient data for fitting four-parameter logistic functions or for which this function was not a satisfactory description, the most nearly linear part of the log dose– response relation was analysed using the methods for parallel line bioassays. Estimates of relative activity were combined as geometric means, and fiducial intervals have been determined using the variance of the logs of the estimates combined. Groups of estimates have been compared using unweighted analysis of variance of the logs of the estimates. Results

Dose response relation The relation between doses and responses was satisfactorily described by an appropriate four-parameter logistic function for most assays. The exceptions were the bioassays of laboratories 2 and 5, in each of which there were differences in the dose– response relations of the various preparations, and for which data analyzed were restricted to a common response range for all preparations. The data from the immunoassays in laboratory 11 indicated that the ff preparations (hffP and hffQ) had dose–response relations different to the other preparations. In some assay systems some preparations, generally the ISP, hffP, hffQ or ipIR (human inhibin) did not give a detectable response at the dose levels tested; such preparations were deleted from analysis. Comparison of the slopes of the transformed dose– response lines showed significant consistent differences between some groups of preparations in about half of these assay systems. In most cases the preparations showing differences were the ISP and hffP, hffQ and/or ipIR. In a few systems differences were detected between the two rDNA derived preparations hI90 and the ISI.

Variability of estimates The potencies of the coded duplicate preparations relative to one another showed good agreement with the expected value of 1 in most cases, (Fig. 1). Relative potencies differing from the expected value of 1 by a factor of more than 2·5 were obtained in four

7

assays. These values were omitted from calculations since, although means were essentially unchanged if they were not omitted, the variances of groups including them were unduly inflated. This observation is in contrast to that made in the collaborative study of the ISP, where it was noted that estimates for activity of coded duplicates varied by up to fourfold.1 Within assay variability as determined by the deviation of the relative potency of the coded duplicates from the expected relative potency of 1·0 was about three times larger for bioassays than for immunoassays. For each type, the between assay, within laboratory variability was no larger than the within assay variability, but the between laboratory variability was slightly larger than the within laboratory variability (1·8 times larger for bioassay and 1·3 times for immunoassay). The overall geometric mean (gm) of all laboratory gm estimates of relative activity of coded duplicate preparations was 0·99 (95% limits 0·93–1·05). Means obtained separately for bioassays or immunoassays or for individual pairs of coded duplicates were also in good agreement with the expected value of 1·0.

Comparisons of the rDNA-derived preparations Estimates of the potency of the ISI relative to hI90 (ampoules of hI90 equivalent in activity to one ampoule of the ISI) and of the potency of hIL relative to each of hI90 and the ISI (ampoules of hI90 or ISI equivalent in activity to one ampoule of hIL) are given in Table 4 and Fig. 2a–c. For each of these comparisons, the within laboratory, between assay variability was similar to that obtained for comparisons of the coded duplicate preparations. Comparison by bioassay of hI90 with the ISI gave estimates which were, overall, no more variable between laboratories than estimates of the coded duplicates if estimates from laboratory 4 were excluded. The mean of laboratory means (excluding 4) was 6·32 ampoules of hI90 equivalent in activity to 1 ampoule of ISI (95% limits 5·29–7·53). Comparison by immunoassay of these preparations gave estimates which were some three times more variable between laboratories than estimates of the coded duplicates if the immunoassays from laboratory 10 were excluded. The overall mean (excluding laboratory 10) was lower than that for the bioassays, being 5·42 ampoules of hI90 equivalent in activity to 1 ampoule of ISI (95% limits 4·75–6·18). The immunoassay estimates tend to fall into two groups, one group composed largely of ELISAs with estimates

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M. P. Rose and R. E. Gaines Das

Figure 1. Histogram of estimates of the relative activity (expected to be 1) of ampoules identical except for code compared in the same assay. The horizontal axis gives relative activity on a log scale. Each square denotes an individual assay estimate with the number in the square denoting the laboratory code and assay type. Using ampoule codes as given in Table 1, estimates are for ampoules coded M having equivalent activity to one ampoule coded J, ampoules of M/ampoule of E, ampoules of J/ampoule of E or ampoules of K/ampoule of A. ‘Double’ or ‘triple’ squares denote two of three of these duplicate comparisons made in the same assay.

tending to be less than 5 ampoules of hI90 equivalent to 1 ampoule of ISI, and the other group of, largely, RIAs with estimates in excess of 6 ampoules of hI90 equivalent to 1 ampoule of ISI. Comparison by either bioassay or immunoassay of hIL with ISI gave estimates which were twice as variable between laboratories as comparisons of hIL with hI90. For bioassays, the between laboratory variability, for either comparison, was not substantially different from the between laboratory variability for the coded duplicates. For immunoassays, the between laboratory variability was double for comparisons of hIL with hI90 and three times larger for comparisons of hIL with ISI than that for the coded duplicates. Estimates by bioassay and immunoassay were similar for comparison of hIL with hI90 (overall mean of laboratory means, 95% limits, 4·24, 3·79–4·74 ampoules of hI90 equivalent in activity to 1 ampoule of hIL; mean for bioassays 4·16, for immunoassays 4·28) and for comparison of hIL with ISI (0·71, 0·61–0·82 ampoules of ISI equivalent in activity to 1 ampoule of hIL, mean for bioassays 0·67, for immunoassays 0·73).

Comparisons of the ISI and hI90 with the ISP and with the various in house standards of inhibin Estimates of the activity of the candidate standards were determined as IU of the ISP (Fig. 3) and as µg, ml or ‘units’ of the various in house standards. The within laboratory, between assay variability for comparison, by bioassay, of either of the candidate standards with the ISP was similar to that obtained for comparison of the coded duplicate preparations. However, that was double for immunoassays. In many immunoassay systems, the dose–response line for the ISP was near the end of the response range and/or the slope for the dose–response line for the ISP was different from that for the candidate standards. In such cases the estimated relative potencies are imprecise and likely to vary greatly between assays, even within the same laboratory. For comparisons, both by bioassay and by immunoassay, of each of ISI and hI90 with the ISP, the variability between laboratories was significantly larger than that within laboratories. However, for either rDNA-derived preparation, the variability

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Table 4. Potency of the candidate preparations relative to one another expressed as ampoules of hI90 (90/552) equivalent in activity to one ampoule of ISI and of hIL (91/628) relative to each of the candidate standards and the IS of porcine inhibin (expressed as ampoules of hI90, ISI or ISP equivalent in activity to one ampoule of hIL (91/628) hIL vs Assay type

Laboratory code

ISI vs hI90

hI90

ISI

86/690

01 02 04 05 06 09 10 12 14 14 15

6·38 4·31 1·28 5·92 9·47 6·75 8·31 5·30 4·66 7·55 6·26

2·86 5·50 — — 3·98 3·03 6·64 4·02 3·01 5·92 —

0·45 1·28 — — 0·42 0·45 0·80 0·76 0·65 0·95 —

16·25 9·57 — — 74·06 36·05 69·78 54·77 114·5 — —

01 01 03 03 03 04 04 04 06 09 10 11 12 13 14 14 14 14 16 17 17

3·69 3·60 3·88 7·33 6·60 5·48 7·40 3·61 6·93 6·68 14·33 4·00 7·81 4·79 6·31 5·01 5·07 6·61 6·75 3·66 7·08

4·71 2·81 — — — 4·33 5·02 4·02 4·61 2·71 5·82 3·72 5·87 — 4·57 4·43 3·88 5·08 3·99 — —

1·26 0·74 — — — 0·79 0·68 1·11 0·67 0·41 0·41 0·93 0·75 — 0·72 0·88 0·77 0·77 0·59 — —

328·1 471·7 — — — 216·3 218·2 26·74 137·5 30·73 28·28 459·0 858·1 — — — — 43·31 — — —

Bioassays RPT ROVA OPT RPTM_BF RPTF RPT RPTM_F RPT K562 RPTM RPTF_FN Immunoassays ELA ELB EIA EIB RIA IRMA_N IRMA_O RIA RIA RIA RIA IFMA RIA IRMA 11B5:CK 9A9:CK CK:CK RIA ELISA ICMA RIA

between laboratories was three times larger for immunoassays than for bioassays. Valid calibration by all bioassays in this study of either ISI or hI90 in terms of the ISP is not possible; that is, the unitage assigned to either candidate standard is dependent on the particular bioassay system in which the preparations are compared. The overall geometric mean of laboratory mean estimates by bioassay suggests that the activity of hI90 is 20 000 IU per ampoule, and that of ISI is 75 000 IU per

ampoule. Restriction to only those bioassays using adult rat pituitary cells substantially reduced the between laboratory variability and gave geometric means of laboratory means of 25 000 IU of the ISP per ampoule of hI90 and 150 000 IU of the ISP per ampoule of ISI. These assays tend to reflect the FSH inhibiting activity of inhibin, originally used to define this material. Overall estimates by immunoassay are larger than those by bioassay, probably reflecting the different

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M. P. Rose and R. E. Gaines Das

Figure 2a. Histogram of estimates of the relative activity of the rDNA-derived human inhibins ISI and hI90 compared in the same assay. The horizontal axis gives relative activity on a log scale. Each square denotes an individual assay estimate with the number in the square denoting the laboratory code and assay type. The relative potency is expressed as ampoules of hI90 having equivalent activity to 1 ampoule of ISI.

Figure 2b. Histogram of estimates of the relative activity of hIL (91/628) and hI90 (90/522) compared in the same assay. The horizontal axis gives relative activity on a log scale. Each square denotes an individual assay estimate with the number in the square denoting the laboratory code and assay type. The relative potency is expressed as ampoules of hI90 having equivalent activity to 1 ampoule of hIL.

in vitro bioassays and immunoassays

11

Figure 2c. Histogram of estimates of the relative activity of hIL (91/628) and the ISI (91/624) compared in the same assay. The horizontal axis gives relative activity on a log scale. Each square denotes an individual assay estimate with the number in the square denoting the laboratory code and assay type. The relative potency is expressed as ampoules of ISI having equivalent activity to 1 ampoule of hIL.

cross-reactivities of the human and porcine inhibins in the assays included in this study, and also the relative impurity of the ISP compared with rDNAderived human inhibin preparations. Even in biological assays where both porcine and human inhibin exert a similar effect it is not necessarily expected that these materials will behave in similar ways. Comparisons of ISI or hI90 with the various in house standards differed, not surprisingly in view of the range of preparations and units used. However, comparisons of these preparations with stated mass of house standard where this was available showed broad agreement with the nominal ampoule contents based on the reported nature of the bulk preparations and preliminary characterization of the ampouled preparations.

Comparison of the preparations of follicular fluid with the ISI, hI90 and the ISP (hffP 55 92/696, hffQ 5 92/640, ipIR 592/650) Comparisons of each of hffP, hffQ and ipIR with the ISI, with hI90, with the ISP and with one another are given in Table 5a–d. Estimates for hffQ and ipIR in terms of either ISI or hI90 or the ISP extend over a

10-fold or greater range and no attempt has been made to combine them. Estimates for hffP by bioassay in terms of either ISI or hI90 are as consistent, both within and between laboratories, as the coded duplicates (if estimates from laboratories 1 and 10 are omitted). Estimates for hffP by immunoassay in terms of either ISI or hI90 are as consistent within laboratories as the coded duplicates (omitting estimates from laboratories 1, 4, 14 RIA and 17 ICMA) but differ significantly between laboratories, with the between laboratory variability for estimates in terms of hI90 being three times larger than that for estimates in terms of ISI. Estimates for hffQ in terms of hffP (omitting laboratory 1 ELB and 10 IMM) are remarkably consistent between laboratories and between bioassays and immunoassays. However, estimates for ipIR in terms of hffP are not consistent between laboratories.

Stability It was reported that some of the thermally accelerated degradation samples were difficult to dissolve and the possibility of incomplete reconstitution of samples should be borne in mind when considering these data.

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M. P. Rose and R. E. Gaines Das

Figure 3a. Histogram of estimates of the relative activity of rDNA-derived inhibin hI90 (90/522) and ISP (86/690) compared in same assay. The horizontal axis gives relative activity on a log scale. Each square denotes an individual assay estimate with number in the square denoting the laboratory code and assay type.

Figure 3b. Histogram of estimates of the relative activity of rDNA derived inhibin ISI (91/624) and ISP (86/690) compared in same assay. The horizontal axis gives relative activity on a log scale. Each square denotes an individual assay estimate with number in the square denoting the laboratory code and assay type.

in vitro bioassays and immunoassays

In the majority of assay systems there was no consistent significant difference between the slopes of the log dose—logit response lines for the thermally accelerated degradation samples and the samples of the same material stored continuously at 220°C. The potency for each of the thermally accelerated degradation samples was obtained relative to the mean potency of samples of the same material stored continuously at 220°C. For each sample, the within laboratory, between assay variability was similar to that obtained for the coded duplicates except for the samples of hI90 which had been stored at 145°C. The samples of hI90 stored at 145°C showed substantial loss of activity and, in consequence, the dilutions based on an assumed relative potency of 1 gave responses which tended to be near the end of the response range with resulting imprecision in estimates of potency.

13

Relative potency of thermally accelerated degradation samples of hI90. Estimates of the relative potency of the samples stored for 981 days at 120°C did not differ significantly from estimates of potency for the coded duplicates, in terms of mean estimate or within, or between, laboratory variability of estimates. Estimates of the relative potency of the samples stored at 137°C showed a significant loss of activity which was marginally greater by bioassays than by immunoassays. For each type of assay there was a significant difference in estimates between different laboratories; for example, by immunoassay, laboratories 1ELA and 4-RIA detect 35% of activity remaining while the other laboratories detect only 20% remaining activity. Estimates of the relative potency of the samples stored at 145°C showed an almost complete loss of bioactivity and substantial losses of immunoreactivity. Predicted losses of activity assuming monomolecular degradation according to an Arrhe-

Table 5a. Potency of the human follicular fluid preparation 92/696 (hffP) relative to the preparations of rDNA derived inhibin and the IS for porcine inhibin expressed as ampoules of standard equivalent in activity to 100 ampoules of hffP Standard preparation Assay type

Laboratory code

hI90

ISI

ISP

01 02 05 06 09 10 12 14

2·17 1·19 1·23 1·11 2·03 14·47 0·72 0·62

78·72 0·28 0·21 0·12 0·31 1·74 0·14 0·15

16·25 2·07 2·10 21·17 17·57 152·1 9·88 —

01 01 03 04 04 04 06 09 10 12 14 14 16 17 17

161·7 — 3·88 0·13 0·83 97·67 13·05 9·73 31·57 2·23 39·32 7·37 0·22 457·4 8·54

— — — 0·02 0·11 27·04 1·88 1·45 2·20 0·29 9·15 1·11 — 88·84 1·21

11846 20·65 — 6·47 36·14 650·4 389·4 110·3 153·5 326·2 — 62·77 155·2 2083 102·7

Bioassays RPT ROVA RPTM_BF RPTF RPT RPTM_F RPT RPTM Immunoassays ELA ELB EIA IRMA_N IRMA_O RIA RIA RIA RIA RIA CK:CK RIA ELISA ICMA RIA

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M. P. Rose and R. E. Gaines Das

Table 5b. Potency of the human follicular fluid preparation 92/640 (hffQ) relative to the rDNA-derived inhibin and the IS for porcine inhibin expressed as ampoules of standard equivalent in activity to 100 ampoules of hffQ Standard preparation Assay type

Laboratory code

hI90

ISI

ISP

Bioassays RPT RPTF RPT RPTM_F

01 06 09 10

6·28 0·37 0·97 5·31

0·98 0·04 0·15 0·64

35·70 6·97 7·15 55·82

01 01 04 04 04 06 09 10 14 14

40·24 — 0·4 0·28 20·39 3·14 2·57 4·90 13·58 2·40

— — 0·01 0·04 5·65 0·45 0·38 0·34 3·16 0·36

2948 8·76 2·34 11·96 135·8 93·71 29·17 23·84 — 20·47

Immunoassays ELA ELB IRMA_N IRMA_O RIA RIA RIA RIA CK:CK RIA

Table 5c. Potency of the immunoaffinity purified human inhibin preparation 92/650 (ipIR) relative to the rDNA-derived human inhibins and the IS for porcine inhibin expressed as ampoules of standard equivalent in activity to 100 ampoules of ipIR Standard preparation Assay type

Laboratory code

hI90

ISI

ISP

Bioassays RPT RPTF RPT RPTM_F RPT K562 RPTM RPTF_FN

01 06 09 10 12 14 14 15

1·16 0·21 0·67 1·72 0·14 2·69 1·32 0·61

0·18 0·02 0·10 0·21 0·03 0·52 0·14 0·10

6·57 3·96 4·43 18·08 1·95 100·9 — 14·02

01 01 04 04 04 06 09 10 14 14

52·27 — 0·76 1·04 16·80 7·68 8·56 19·92 10·48 5·36

— — 0·14 0·14 4·65 1·11 1·28 1·39 2·44 0·81

3829 300·4 37·97 45·05 111·9 229·0 97·11 96·85 — 45·66

Immunoassays ELA ELB IRMA_N IRMA_O RIA RIA RIA RIA CK:CK RIA

in vitro bioassays and immunoassays

15

Table 5d. Relative potency of the follicular fluid preparation 92/650 and 92/640 (ipIR and hffQ respectively) expressed as ampoules of preparation 92/696 (hffP) equivalent in activity to one ampoule of ipIR or hffQ, and of 92/646 (hffQ) expressed as ampoules of 92/650 (ipIR) equivalent to one ampoule of hffQ Assay type

Laboratory code

ipIR ampoules

hffQ of hffP

hffQ ampoules of ipIR

01 06 09 10 12

8·35 18·72 32·95 11·88 19·73

45·35 32·94 47·68 36·70 —

543·2 176·0 144·7 308·8 —

01 01 04 04 04 06 09 10 11 14 14

33·70 2460 586·8 124·7 17.20 58·81 88·03 63·09 333·5 26·65 72·74

24·65 24·92 25·00 33·10 20.88 24·06 26·44 15·53 29·64 34·53 32·60

73·15 1·01 5·96 26·55 121·4 40·92 30·04 24·62 8·89 129·5 44·82

Bioassays RPT RPTF RPT RPTM_F RPT Immunoassays ELA ELB IRMA_N IRMA_O RIA RIA RIA RIA IFMA CK:CK RIA

nius equation are less than 0·1% per year at 220°C, using overall estimates of activity or estimates for bioassay or immunoassay separately. (Since no loss of activity was detected in samples stored at 120°C, these predictions are likely to overestimate, rather than underestimate, losses).

Relative potency of thermally accelerated degradation samples of ISI. Estimates of the relative potency of the samples stored for 440 days at 14°C or 120°C did not differ significantly from estimates of potency for the coded duplicates, in terms of mean estimates or within or between laboratory variability of estimates. The overall mean estimate of the relative potency for the samples stored at 137°C did not differ significantly from 1. However for both bioassays and immunoassays the between laboratory variability was significantly larger than that for the coded duplicates, with some assay systems, most notably some bioassay systems, apparently detecting up to a 40% loss of activity. Samples stored at 145°C showed a significant loss of activity in most bioassays and in about 50% of the immunoassays. Predicted losses of activity (assumptions as above) are less than 0·1% per year at 220°C for assay systems in which loss of

activity was detected and sufficient data were obtained to permit prediction. Discussion The biological and immunological activity of both rDNA-derived preparations, hI90 and ISI, was confirmed by all laboratories. All three preparations of highly purified rDNA-derived human inhibin, hI90, ISI and hIL were broadly consistent relative to one another across the range of assay systems. The biological activity relative to the immunological activity is slightly greater for ISI than hI90 excluding bioassays from laboratory 4 and immunoassays from laboratory 10; i.e. approximately 6 ampoules of hI90 has equivalent biological activity to 1 ampoule of ISI whereas about 5 ampoules of hI90 has equivalent immunoreactivity to 1 ampoule of ISI. The reason for this is unknown. However, it is possible, as for other glycoprotein hormones, that relative biological activity may be a reflection of molecular heterogeneity. It is not clear whether differences in slopes for the candidate preparations hI90 and ISI reflect differences between these preparations which are detected

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M. P. Rose and R. E. Gaines Das

by some assay systems and not others or whether they are related to some aspect(s) of assay design. It has been reported that recombinant-DNAderived human inhibin A exhibits heterogeneity.31 The intact dimer was observed as two molecular weight forms of Mr 34 kDa and Mr 31 kDa and both the α subunit and β subunit were observed to exist as two molecular weight forms. The molecular weight variants of the dimer were suggested to be attributable to variation in glycosylation of the α subunit, which may also give rise to charge isomers observed on isoelectric focusing. Porcine 32 kDa inhibin is known to exhibit similar heterogeneity.30 Two molecular weight forms of the α subunit were observed on polyacrylamide gel electrophoresis (PAGE) and charge isoforms were observed on two dimensional-PAGE. The molecular weight heterogeneity was attributed to variations in glycosylation of the α subunit whereas the charge heterogeneity only partly so. The glycosylation of the α subunit which may be a unique structure, may account for up to one fifth of the molecular mass whereas the β subunit is not glycosylated. The physiological role of glycosylation of the α subunit is not clear since deglycosylation did not appear to affect in vitro biological activity. However, by analogy with other glycoprotein hormones, glycosylation may influence in vivo biological activity by affecting rate of clearance from the circulation. It is also known that natural human inhibin exists in multiple forms. Two species of inhibin β subunit have been identified which give rise to Inhibin A and Inhibin B in combination with the α subunit. Additionally, human follicular fluid has been found to contain biologically and immunologically active inhibin species of Mr 30, 35, 53, 65 and 125 kDa, whilst circulating inhibin, under gonadotrophic stimulation were identified as Mr 28, 34, 42, 58 and 70 kDa.32 It is possible, therefore, that different preparations of purified rDNA-derived human Inhibin A preparations might exhibit different relative biological activities. A future phase for the standardization of inhibins may be to evaluate reference preparations for isoforms of clinical (or therapeutic) importance once these have been identified and for other related proteins such as activins and follistatin. Direct calibration of the rDNA-derived inhibins in terms of the ISP was not possible. The ISP is not a suitable standard for preparations of human inhibin although the greater consistency of estimates in terms of the ISP by bioassay than by immunoassay

for either hI90 or ISI supports its use in bioassays when human inhibin was not available. Dose–response relationships do not, in most cases suggest consistent differences between the ISI and hI90. Differences in dose–response relation and in slope between the ISI and other preparations of human inhibin were noted but these may be due to the specificity of the assay system, some aspect of assay design or relatively low concentration of inhibin in the natural human inhibin preparations included in these assays. Both the hffQ and hffP and ISP are impure preparations which may also affect estimates of activity by some assay systems. The observation that the ISP was poorly detected in many immunoassay systems suggests that these systems show greater cross-reactivity with human than with porcine inhibin. It would not seem likely that this is due to loss of activity of ISP since this preparation showed biological activity during in vitro bioassays and an earlier study indicated that it was stable.9 However, because of its relative impurity it is possible that contaminants may interfere in the binding of inhibin to the antibody in these assays. It is intended that possible interfering molecules such as follistatin will be investigated in a future study. There is a slight suggestion that the similarity between hI90 and the ISP is greater than the similarity between ISI and the ISP, and that the similarity between ISI and hffP is greater than the similarity between hI90 and hffP. Three preparations of natural human inhibin were included in the study. These were two preparations of human follicular fluid and a preparation of immunoaffinity purified human inhibin. In human follicular fluid the level of inhibin is quite low and therefore the nominal content of ampouled materials, even of undiluted hff, (hffP) is only 5 ng/ampoule. However, in order to provide two different levels of inhibins, a relatively high content of inhibin and a relatively low content, hffQ was prepared from follicular fluid which was diluted with buffer. Immunoaffinity purification of inhibin also has the effect of concentrating the active principle and therefore preparation ipIR contained more inhibin per ampoule than hffP. The between laboratory agreement for estimates of hffQ in terms of hffP may reflect the similar nature of these preparations. However, it is not clear why estimates for hffQ and ipIR in terms of ISI, hI90 or the ISP vary widely whereas that for hffP is relatively consistent. Although hffQ and hffP are similar in nature the estimated nominal content of hffP is approximately 2·5–5 times that of hffQ. This may

in vitro bioassays and immunoassays

have a bearing on the precision of estimates of activity for these two preparations. Additionally both hffQ and hffP are pooled ffs collected from a number of patients undergoing therapeutic treatments. The possible effects of patient variation and treatment protocols on the qualitative and quantitative levels of inhibin are not yet fully defined. There are similar sources of difference with ipIR which, in addition, has also been selected by an antibody. The good agreement between laboratories of estimates for hffQ relative to hffP suggests both that hffP and hffQ are similar, i.e. have similar actions in these assay systems, and that there may be an element of non-specificity in these assay systems for whatever components hffP and hffQ have in common. Clearly there are other dissimilarities and non-specificities as shown by the between laboratory variability of estimates for ipIR relative to hffP. Assessment of the thermally accelerated degradation samples gave predicted losses of activity of less than 0·1% per year at 220°C for both candidate preparations and indicated that both were sufficiently stable to serve as standards. On the basis of the results of this study, and with the agreement of the participants, the preparation in ampoules coded 91/624 and referred to as ISI in this report was established by ECBS of WHO as the First International Standard for rDNA-derived human inhibin since this preparation appeared to have relatively greater biological activity and contained larger concentrations of inhibin per ampoule than hI90. The International Standard was assigned a unitage of 150 000 IU per ampoule which maintains an approximate continuity of units of biological activity with the International Standard for porcine inhibin in the bioassays using adult rat pituitary cells which reflect the FSH inhibition which has defined this material. The standard is available from NIBSC and further details are available by writing to NIBSC at: PO Box 1193, Potters Bar, Herts, EN6 3QH, U.K. (telephone and fax 0707 646977).

Acknowledgements Grateful acknowledgements to: Biotech Australia Pty Limited, Roseville, NSW, Australia and Genentech Inc., South San Francisco, California, U.S.A. for generously donating recombinant DNA-derived human inhibins; Medgenix Diagnostics, Fleurus, Belgium and The Middlesex Hospital, London, U.K. for generously donating natural human inhibins, including ffs; Dr J. M. Zanelli for arranging ampouling and preliminary assessment of hI90; Dr P. Dawson and

17

staff of the Standards processing division (NIBSC) for ampouling; Dr D. M. Robertson and colleagues (Melbourne), Dr J. Mather (Genentech), Dr M. Wheeler and Miss L. Simmons (St. Thomas’s Hospital, London) for preliminary characterization of ampouled materials and all participants in the international collaborative study. References 1. McCullagh DR. Dual endocrine activity of the testes. Science 1932; 76: 19–20. 2. Robertson DM, Foulds LM, Levershen L et al. Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Comm 1985; 126: 220–226. 3. Forage RG, Ring JM, Brown RW et al. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Nat Acad Sci USA 1986; 83: 3091–3095. 4. Mason AJ, Niall HD, Seeburg PH. Structure of two human ovarian inhibins. Biochem Biophys Res Comm 1986; 135: 957–964. 5. Stewart AG, Millborrow HM, Ring JM, Crowther CE, Forage RG. Human inhibin genes: genomic characterization and sequencing. FEBS Letters 1986; 206: 329–334. 6. Yu J, Shao L-E, Lemas V et al. Importance of FSHreleasing protein and inhibin in erythrodifferentiation. Nature 1987; 30: 765–767. 7. Burger HG. Inhibin as a tumour marker. Clin Endocrinol 1994; 41: 151–153. 8. Burger HG. What do inhibin measurements tell a clinician today? Ann Med 1992; 24: 419–421. 9. Gaines Das RE, Rose M, Zanelli JM. International collaborative study by in vitro bioassays of the first International Standard for porcine inhibin. J Reprod Fertil 1992; 96: 803–814. 10. World Health Organization Expert Committee on Biological Standardization. WHO Technical Report Series No. 800, Annex 4: 181–213. 11. Scott RS, Burger HG, Quigg H. A simple and rapid in vitro bioassay for inhibin. Endocrinology 1993; 107: 1536–1542. 12. Henderson KM, Franchimont P. Regulation of inhibin production by ovarian cells in vitro. J Reprod Fertil 1981; 63: 431–442. 13. Rivier J, Spiess J, McClintock R, Vaughn J, Vale W. Purification and characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Comm 1985; 133: 120–127. 14. Grootenhuis AJ, Steengergen J, Timmerman MA et al. Inhibin and activin-like activity in fluids from male and female gonads: different molecular weight forms and bioactivity/immunoactivity ratios. J Endocrinol 1989; 122: 293–301. 15. Miyamato K, Hasegawa Y, Fukuda M et al. Isolation of porcine follicular fluid inhibin 32 k Daltons. Biochem Biophys Res Comm 1985; 129: 396–403. 16. Sasamoto KT, Arakawa H, Kishi H. Inhibin secretion and suppression of the FSH surge in superovulating animals. Serono Symposoa Publications 1987; 42: 219–232.

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17. Parte P, Juneja HS. Assessment of bioactivity of WHO/NIH research standard for inhibin (Code 86/690) using two separate pituitary cell-culture systems. Horm Metab Res 1993; 25: 356–359. 18. Muttukrishna S, Knight PG. Effects of crude and highly purified bovine inhibin (Mr 32 000 form) on gonadotropin production by ovine pituitary cells in vitro: inhibin enhances gonadotropin-releasing hormone-induced release of LH. J Endocrinol 1990; 127: 149–159. 19. Smyth CD, Miró F, Whitelaw PF, Howles CM, Hillier SG. Ovarian thecal/interstitial androgen synthesis is enhanced by a follicle-stimulating hormone-stimulated paracrine mechanism. Endocrinology 1993; 133: 1532–1538. 20. Schwall RH, Lai C. Erythroid bioassays for activin. In: Barnes D, Mather JP, Sato G, eds. Peptide growth factors Part C. Methods in Enzymology 1991;198: 340–346. 21. Robertson DM, Tsonis CG, McLachlan RI et al. Comparison of inhibin immunological and in vitro biological activities in human serum. J Clin Endocrinol Metab 1988; 67: 438–443. 22. Hamada T, Watanabe G, Kohuho T et al. Radioimmunoassay of inhibin in various mammals. J Endocrinol 1989; 122: 697–704. 23. Knight PG, Muttukrishna S, Groome N, Webley GE. Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of non-dimeric form. Biol Reprod 1992; 47: 554–560. 24. Poncelet E, Delacroix DL, Franchimont P. Abstract from the 9th International Congress of Endocrinology, Aug 30–Sep 5, Nice, France.

25. Baly DL, Allison DE, Krummen LA et al. Development of a specific and sensitive two-site enzyme-linked immunosorbent assay (ELISA) for measurement of inhibin A in serum. Endocrinology 1993; 132: 2099–2108. 26. Groome N, O’Brien M. Immunoassays for inhibin and its subunits. Further applications of the synthetic peptide approach. J Immunol Meth 1993; 165: 167–176. 27. Knight PG, Groome N, Beard AJ. Development of a twosite immunoradiometric assay for dimeric inhibin using antibodies against chemically synthesised fragments of the α and β subunit. J Endocrinol 1991; 129: R9–R12. 28. Knight PG, Muttukrishna S. Measurement of dimeric inhibin using a modified immunoradiometric assay specific for oxidized (Met 0) inhibin. J Endocrinol 1994; 141: 417–425. 29. Keelan J. Inhibin: its measurement by immunoassay and production by the human placenta. PhD Thesis, University of Auckland, New Zealand. 30. Tierny ML, Goss NH, Tomkins SM et al. Physicochemical and biological characterization of recombinant human inhibin A. Endocrinology 1990; 126: 3268–3270. 31. Moore KH, Dunbar BS, Bousfield GR, Ward DN. The heterogeneity of porcine 32 000 Mr inhibin α subunit. A gel electrophoresis and immunoblot study. Endocrinology 1990; 127: 1477–1486. 32. Robertson DM, Sullivan J, Watson M, Cahir N. Forms of inhibin: biological and immunological charact erization. In: Burger HG, ed. Inhibin and inhibinrelated proteins. Frontiers in Endocrinology 1994; 3: 25–32.