Note: Descriptions are shown in the official language in which they were submitted.
i3~73~
METHOD OF ASSAY OF INHIBIN
This invention relates to methods for assay of
inhibin, and in particular to methods or immunoassay o
inhibin .
Two forms of inhibin from bovine follicular fluid
10 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 subunit~ with
molecular masses of 43kD and 15kD, and 20kD and 15kD
15 respectively. Their primary amino acid structures have been
~k
q~
0~'736
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. 31-3lkD inhibin molecules with similar subunit
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 Ç~ 31 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).
Summaxy_of the Invention
The present invention relates to more convenient assays
for the estimation of inhibin than have heretofore 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, immunoassay
based on fluorescence detection, and related assays relying on
polyclonal and monoclonal antibodies against inhibin.
Prefe~ed Embodiments of the Invention
According to one aspect of the present invention there is
provided a radioimmunoassay for determining inhibin suspected
to be present in a biological sample, comprising the steps of:
(a) contacting the sample with an antibody directed against
inhibin and incubating at 4- to 30-C for a period of 4 hours
to 4 days, (b) adding 125I-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-C or 16
hours at 30-C, (c) adding a second antibody and incubating for
30 minutes to 24 hours at 4-C, (d) separating precipitated
material, and (e) measuring bound 125I-labelled inhibin in
the precipitate.
13~)'77~
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 preparation including
inhibin, purified bovine 58kD inhibin,
,~
130'7736
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 neutraliz-
ing 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
(125I) or with an enzyme, or with a fluorescent marker.
Preferably the assay is a radioimmunoassay or an enzyme-
liked immunosorbent assay (ELISA), or a fluorescence-base
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 stan-
dards 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 and counting of
bound 125I labelled inhibin.
The preferred specific radioimmunoassay syste~ 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
5I-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.
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According to a third aspect of the invention there
is provided an assay standard selected from the group
con~isting of naturally-occurring or recombinant inhibin, or
fragments or derivatives thereof. Preferably the ~tandard
S display~ 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
10 levels of sensitivity.
A form of the radioimmunoassay modified for
increased sensitivity compri~es:
incubating s~mple and antiserum for 4 days at 4C,
followed by the addition of 125I-31kD inhibin tracer,
15 incubating for 3 days at 4C and then adding second antibody, ~'
precipitating, and counting bound 125I labelled inhibin.
According to one particularly preferred embodiment,
suitable for measurement of inhibin in human serum samples,
the tracer is 125I-31kD inhibin, and incubation with tracer i5
20 performed at elevated temperature (30C) in the presence of
inhibin-free serum, in order to minimize non-~pecific effects.
Suitable sources of inhibin-free serum include steers or other
castrated male animals, oophorectomized women, women with
premature ovarian failure, and post-menopausal women.
25 Brief Description of the Drawings
Figure 1 shows the fractionation of 125I-58kD and
125I-31kD inhibin on analytical SDS-PAGE under reducing
conditions.
Figure 2 shows the time course of immunization of a
30 rabbit with 58kD inhibin.
Figure 3 ~hows the in vitro neutralization of bFF
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
35 granulosa cell culture medium (BGCM) using either 125I-31kD or
125I-58kD inhibin as tracers.
13~7~
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Pigure S ~ho~s the profi~e of inhi~in in vitro
bioactivity and immunoactiv~ty follo~ing f~actionation of bFF
through the various ateps of the inhibi~ purification
procedure of Robert~on et al tl986~.
S Figure 6 shows non-reduced SDS-PAGE profile~ of
125I-58kD and l25r-3lkD inhibin following incubation wi~h bPP
and serum under condition~ used in the RIA of ser~m inhib~n.
Figure 7 shows the effect of temperature on the
binding of 125I-3lkD inhibin to the AntiSerum.
Figure 8 shows logit-log dose re~ponse lines of
bovine and human serum, in the plasma RIA system employing
5~-31kD inhibin as tracer.
F~gure 9 ~hows the ovulation induction regime and
~erum level~ of FS~, ~H, inhi~in and oestradiol (E23 in
15 twenty-six women involved in an In Vitro Fertilisation ~IVF)
programme and one normal ~oman (FL#27).
Figure 10 is a compari~on of plasma E2 and inhibin
levels plo~ted for some of the data in Fig~re g.
Figure 11 shows the correlation between the number
20 of ova produced and ~2 or ;nhibin levels in ~erum.
Figure 12 shows the corxelation bet~een the number~
of ovariAn follicle~ detected ultrasonioally and peak inhibin
level8 ~n ~erum.
Figure 13 shows inhibin, FS~, proge~terone and
25 oeatradiol concen~rations in the ser~ of non-pregnant subject~
~n ~ 16) on the days following oocyte r~trieval.
Figure 14 shows inhibin, FSH, progesterone ~nd
oe~tr~diol concentra~ion~ in the ~era of pregnant subj~cts (n
~ 3) on ~he days ollowing oocyte retrieval.
Figure 15 shows the relationship between serum
inhlbin and PSH duxing the lu~eAl phAse o~ non-pregnant
subjoct~. For thi~ analysis, non-detectable inhibin values ~n
29) were asaigned the ll~it of a3say sensitivity.
Figure 16 show~ inhibin, FS~, LH, oestradiol and
progesterone conoentrations in the ~era of normal women during
the menstrual cyole, as~a~ed u~ing anti-31kD inhibln.
13~7'~6
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-- 6 --
Detailed Description of_the Invention
Abbreviations
bFF : bovine follicular fluid
hFF : human follicular fluid
oFF : ovine follicular fluid
oRTF : ovine rete testis fluid
HPLC : high performance liquid chromatography
SDS-PAGE: sodium dodecyl sulphate
polyacrylamide gel electrophoresis
RIA : radioimmunoassay
SS : steer serum
~S : bull serum
CS : cow ~erum
~MS : human po~t-menopausal ~erum
HFP : human female plasma
Preparations
(a) Purificition of bFF inhibin
~he purification of bFF 31kD and 58kD inhibin was
based on the procedures described previously (Robert~on et al
- 20 1985, 1986)(Figure 5):
(a) bFF was fractionated on a Sephacryl S200 (9 x 90 cm)
gel filtration column in 0.05M ammonium acetate pH
7 Ø
~b) The void volume fraction from (a) was precipitated
at pH 4.75 and ractionated on Sephadex G100 (9 x 90
cm) in 4M acetic acid.
(c) and ~d)
: Peak I tS8kD inhibin) and Peak II ~31kD inhibin)
frActions from ~b) were fractioned on an RPSC
Ultrapore column ~0.46 x 7.6 cm, Beckman) using a
0-50~ acetonitrile gradient in 0.1% trifluoracetic
acid. In Figure 5 the continuous line indicates
optical density at 280 nm (a) and (b) and 254 nm (c)
and (d).
Hatched area denoteu inhibin bioactivity.
o---o RIA with 125I-58k~ inhibin as tracer.
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-- 7 --
o _ o RIA with 125I-31kD inhibin as tracer.
Vo = void volume.
BSA - bovine serum albumin (mol. wt 67,000).
OVA ~ ovalbumin (mol. wt 43,000).
The purified inhibin wa~ stored in SDS
electroelution buffer (approx. 3~ SDS in 10 mM NH4Hco3) prior
to iodination. For bioassay, sample~ were methanol
precipitated at -20C in order to remove SDS and solubilized
by heating at 37C 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
15 from 0.30 - 0.43.
(b) 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
20 was charcoal treated ~100 mg/ml dextran-coated charcoal for 1
hour at 4C), lyophillsed, stored at -20C and resolubilized
prior to assay by æonication in assay buffer or culture
medium. Ovine follicular fluid (oFF) was obtained by
aspiration of ovaries collected at a local abattoir. Ovine
25 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 inhi~in preparations are outlined in
Table 1.
Blood was collected in lithium heparin tubes from 40
women undergoing ovulation induction-therapy ~clomiphene
citrate and human menopausal gonadotrophin treatment) with
plasma estradiol level~ at time of plasma collection ranging
from 40-2,900 pg/ml. Equal aliquots of serum from each
35 subject were combined to produce a plasma pool designated as
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-- 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).
30vlne blood, ovaries and testes were collected on
S ice from a local abattoir and processed within one hour. All
samples were stored at -20C after snap freezing in solia
C02/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 4C prior to centrifugation and
10 storage. Bovine ovarian follicles were hemisected and
granulosa cells were collected by aspiration and cultured for
40 hours at a concentration of 105 viable cells per well
(Costar 48 well plate) in 400 ~1 DMEM/F12 complete medium.
Media were stored frozen at -20C prior to assay. Testes from
15 four bulls were decapsulated and homogenised in equal w/v
Dulbecco's phosphate buffer using an Ultra-Turrax tissue
disperser ~Janke and Xunkal XS, Staufen FRG) and centrifuged
at lOO,OOOg x 1 hour at 4C and stored at -20C. Prior to
assay the supernatants were charcoal treated with an equal
20 volume of 1% Norit A in Dulbecco's phosphate buffer and
incubated at 4C for 30 minutes prior to centrifugation and
bioassay ~Au et al 1983).
Immunizatlon Procedure
Purified 58kD inhibin ~14 ,ug in 500 ~1 Dulbecco's
25 phosphate buffer) was emulsified in an equal volume of
adjuvant ~Marcol 52 [Esso, Australia]: Montanide 888
[S.E.P.P.I.C., 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 ~g under the
30 same conditions were given at aix weeks and one year. Serum
was collected throughout for assessment of its in vitro
neutr~lization 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.
l~R.ll '87 07:57 CLEMENT HPCK ~IUST +613 5Z9 6Z96 ~ P.12
Analytical SDS Polyacr~lamide 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 NaCl, 0.1~ Tr~ton X-100 and either 0.5~ BSA
5 for studies with 125I-31kD inhibin or 0.5% Polypep for
125I-58~D inhibin. Equal volumes (5~1) of ~ample and 10~ SDS
and Dulbecco's Phosphate buffer pH 7.4 (30 ~ul) 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
10 H2O) was then added and the mixture centrifuged prior to
electrophoresis on 12.5~ slab gels (3 hours, 20-30 mA).
Protein molecular weight markers were either
co-electrophoresed with the iodinated sample in the absence of
bPF and serum or on a separate track in their presence. The
15 gels were fixed and stained overnight in ethanol: H2O
formaldehyde (180: 420: 100) containing 0.1% Coomassie
Brilliant Blue. Each track was divided into 50 2mm slice~ and
counted in a gamma counter.
Reversed-phase HPLC
2 1-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 o 0.50~ acetonitrile in 0.1
trifluoroacetic acid at 1 ml/min and 0.5 ml fractions using
Waters HP~C apparatus (model 6000A pumps and a model 660
25 Programmer, Nilford, Mass., USA).
In Vitro Bioassay
Inhibin activity was determined using an in vitro
bioassay based upon the dose-dependent suppression of ~SH cell
content in rat pituitary cell cultures utilizing a parallel
30 line bioassay 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
1;~0~36
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(i) 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 a~say was
O.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 mea6ured by a specific radioimmunoassay using
reagents supplied by the NIAMDD. 125I-rat FSH (I5)
was used as tracer and FSH RP-2 used as standard.
The within-assay coefficient of variation was 7%.
(iii) RIA of hum~n hormones
Serum PSH 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 2IA ~LH RIA, Diagnostic Products Corp.,
~.A., USA) using the 2nd IRP LH as standard with an
interassay CV of 10.1~ from 31 aCsays. Both
oestradiol and progesterone were measured using RIA
(Coat-a-Count, Diagnostic Products Corp., ~.A.) with
interassay CVs of 8.7~ and 8.1~ respectively from
150 assays. Serum beta subunit of hC~ was measured
by RIA (B-hCG RIA-Quant, Mallinckrodt Inc. St.
Louis, USA) using the hCG 2nd IS as standard with an
intera~say CV of 10.4% from 30 assays.
Calculations
The RIA dose-response curves were lineari~ed using a
logit-log dose transformation. Parallelism was asse~sed from
a comparison of slope values of dose-response curves using the
multiple range test for groups of unequal size (Kramer, l9S6)
or by paired t-test. Potency estimates were determined using
35 standard parallel line bioa~ay statistics. In situations
where non-parallelism was observed between dose response lines
1hF~. 12 !~37 1~:30 rLEMENT HI~CK hlJST +613 5Z9 ~2g~ P.05
of unknown and standard preparation,s, potency estimates were
determined ro~ the rat~o of their ~D50 values. The
sensitivity (~Dlo) was deined as the mass of hormone required
to give lO~ diçplacement in the as~ay whil6t ED50 corresponded
5 to the mass required for 50~ displacement.
The index of precision (Gaddum (l933~; F~nney
(19641) w~s used ~o describe assay precision. The between
assay variati~n was calculated from the coef~icient of
variation o~ the repeated meaaurement of a partia~ly purified
lO inhibin pr~paration. The dissociation cons~ant (KdiS) was
determined by scatchard anal~sis using 125I-tlvrmor~e and
incre~sin~ ~mounts of unlabelled ho~monc. ~ e mass of
5I-hormone l~sed in the analy~is was determined from its
~pecific activity (~ g).
The invention will now be illustrAted by reerence
to thc following non-limitin~ examples.
~xample 1 Antiserum Char~cterizat~on
Antisera against 58kD and 31kD inhibin were character~
~zed by showing that following immunization, parall-l changes in
plasma FS~ and inhib~n an~ibody titre were ob~erved, indicating
inhibin neutralization in vivo. ~he antisera neutralized bFF,
hFF and purified 31kD and 58kD inhibin activity in an in vitro
bioassay. The results des~ribed below refer to anti-58kD
inhlbin, but similar result~ wers obtained using anti-31kD
inhibin.
(a) Response In Vitro to ~mmunization
Following the fir~t booster (Fig. 2a) the antibody titre
was assessed by the ability of the antibody to neutralize inhibin
bioactivity in vitro and its capacity to bind l25I-58kD inhi~in.
A sharp pa~allel elevation in these activitieS was ob-~erved
between 1-8 weeks ~ost-boo~ter in~ection. During ~his period
significant (P 0.05) elevation in serum PSH ~6.~3 + 0.95 ng/ml,
n ~ 5 vs 4.97 ~ 0.87 ng~ml, n ~ 6 (mean + l S~)) was noted.
Following the second boostor ~Fig. 2b), an immediate and
sustained elevation of serum 125-31kD inhibin-binding capacity
and serum PS~ was observed.
1RR.11 '87 013:01 CLEI`1ENT H~CK ~UST +613 5~9 6296 7~6 P.15
- 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
5 bovine follicular fluid c~n be u~ed 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 pea~
antiserum titre ~as assessed by iodinated inhibin binding and
10 in vitro neutralizing capacity) indicate that the antibodies
produced are capable of neutralizing endogenous inhibin. The
combination of the neutralization of inhibin both in vivo and
in vitro and the clo~e relationship of the neutralizing
activity and iodinated inhibin binding capacity of the
15 antiserum provides convincing evidence o its specificity.
tb) Inhibin Neutralisation In Vitro
Bovine follicular fluid inhibin (2 units) was
quantitatively neutralised by 1 ~1 antiserum per culture well
while 75% inhibition of bioactivity was achieved with 0.35 ~1
20 per well (Fig. 3, Table 1). In Flgure 3, the vertical dotted
line indicates the volume of antiserum required to achieve 75%
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 ~1
25 respectively. In comparison, neutralisation of hFF inhibin
bioactivity required 1.32 ~1 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 ~ 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.
~3~','736
1~R. 11 '87 08:0ZCLEMENT HRCK fWST +613 529 6296 P, 16
- 13 --
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O _ _ O
a O a ::~ o ,~, A
O ~i
~: O a~ ~ O v
o.~ 0 ~( V
o C ~ o ~
a~
V X O N 0~ 0 1-
~ ~1
O +
~1
.C ~.
O ~ O O O
~U O V V V
'J
_ O~ "
~ ~:
e C ~ ~ Oo O o ` c
~¦ a --o e 2 2 i 2 ~ ; .
736
- 14 -
Example 2 Iodination of inhibin
Iodination of either 58kD or 31kD inhibin has been
achieved using a conventional Chloramine T iodination
procedure and was a~ociated with considerable iodination
5 damage. Purification of the tracer was therefore nece~ary
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
10 inhibin form thus purified had the physico-chemical properties
of its non-iodinated form.
~ lternative iodination procedures using Iodogen,
Iodobeads, lactoperoxidase, or Bolton-Hunter reagent were
found to cause les~ damage to the inhibin molecule, but
15 re~ulted in poorer incorporation of radioactivity.
Consequently iodination using Chloramine T was preferred.
Purified 58kD or 31kD inhibin (1-2 pg in 25 ~1
electroelution buffer was added to 25 ul 0.5 M phosphate -
buffer, pH 7,2. Nal25I ~0.5 mCi, 5 ,ul; Amersham, Bucks" UK)
20 was added. Chloramine T (40 ~1) was added at a ratio of 8:1
Chloramine T to hormone. The reaction proceeded for 60
second3 at room temperature with stirring and was terminated
with 20 Jul sodium metabisulphite (3 mg~ml). The reaction
mixture was made up to 50 yl in 20mM phosphate buffer 0.~ BSA
25 or 0.5~ Polypep (Sigma, St. Louis, Mo., USA) pH 6.0 and gel
filtered on a Sephadex G25 column tPD10, Pharmacia, Uppsala,
Sweden) to remove 125I. The void volume fractions were
pooled, made up to 20 ml and applied to a column of 200 pl
Matrex Red A (Amicon, Danvers, Ma~s., USA) and then washed
30 with phosphate buffer containing 400 mM KCl, the eluted counts
being di3carded. 125I--inhibin was eluted with lM XCl/4M urea
in phosphate buffer. The iodinated inhibin was further gel
filtered on a Sephadex G25 column (PD10) with the appropriate
RIA buffer t~ee below~ to remove the KCl/urea.
Following iodination of 58kD and 31~ inhibin, 60
~Ci and 25 ,uCi reipectively were recovered in the void volume
fractions following gel chromatography on Sephadex G25.
* TRADE MARK
.~
,J
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-- 15 --
Approximately 30~ was eluted with the lM KCl/4M urea buffer.
125I-inhibin, as assessed by its molecular weight on SDS-PA~E,
was found in this fraction.
The specific activity of the iodinated preparations
5 was asse~sed 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 ~Ci/~g
for 58kD inhibin and 24 ~uCi/~g for 31kD inhibin were obtained,
with recoveries ranging from 5-25%.
10 Example 3 Characterisation of iodinated inhibin
The physico-chemical char~cteristics of 125I-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
15 preparations (data not shown). The molecular mass of
125I-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
20 in relatively low proportions (18%) under reducing conditions
(Fig. 1). The molecular weight markers employed were BSA
(bovine serum ~lbumin) 67,000; OV tovalbumin) 43,000: CA
(carbonic anhydrase) 29,000; GL (goose egg lyso2yme) 20,300;
and CL (chick egg lysozyme) 14,300. The arrow, in figure 1,
25 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 5I-58kD material is not an iodinated contaminant.
30 In support, 125I-58kD inhibin was ~ractionated 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-PAGF each of these peaks showed persistence of
35 125I-58kD material. The results suggest that the presence of
130~736
~1RR.11 '87 08:05 CLEI`1ENT H~CK l:lUST +513 529 6296 P.l9
-- 16 --
the 125I-58kD material is attributable to difficulties in
reduction of the iodinated hormone rather than to the
iodination of a contaminating protein.
Practionation of the 125I-31kD inhibin on SDS-PAGE
5 revealed molecular weights of 30,200 under non-reducing
conditions and 20,000 and 15,000 subunits following reduction;
these values are si~ilar to those for the non-iodinated
hormone. A second antibody RIA system using either tracer
yielded a parallel displacement between purified 31kD and 58kD
10 inhibin.
Example 4 Radioimmunoassay Procedure
The assay buffer used was 10 mM phosphate, 0.15 M
NaCl, O.5~ BSA, pH 7.2. A delayed tracer addition, second
antibody assay system was employed. The sample and antiserum
15 were incubated in a volume of 300 ~1 for 16 hours at room
temperature followinq which 125I-inhibin (10,000 cpm, 1OOJU1)
was added and the incubation continued either overnight at
room temperature or for 48 hours at 4C. second antibody
(goat antiserum to rabbit IgG, 100 ~1) was added and incubated
20 for 1 hour at 4C 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 4C, decanted and
counted. The inclusion of Triton X-100 (final concentration
0.025%) in the assay buffer reduced non-specific binding from
25 4 to 0.5~.
Radioimmunoassay procedures were established using
both 31 and 58kD inhibin tracers. FollowLng a logit-log dose
transformation of the response curves, linear displacement of
each tracer was observed for a range of inhibin preparations,
30 with the exception of 31kD inhibin when using 125I-58kD
inhibin as tracer, in which a deviation from linearity below
logit -0.5 (38~ B/~o) was seen (Fig. 4). In figure 4, each
value represents the mean ~ SD of triplicates. The
characteristics of each assay are outlined in Table 2.
35 Scatchard analysis revealed similar affinities for the
antiserum of either inhibin form. Non-parallel dose response
13V'7736
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- 17 -
lines were observed between bFF and either 31kD inhibin with
125I-31kD inhibin as tracer of 58kD inhibin with 125I-58kD
inhibin as inhibin tracer. ~he sen~itivity (EDlo) and ED50
values were comparable in each assay with either hormone.
13~7~36
11:1R.11 '87 013:08 CLE11ENT H~CK FIUST +613 529 6296 P.21
- 18 --
TABLE 2
Character~stics of the two radioimmunoassaY sv6tems w{th
L ~ ~ ~2 __
I-31kD ~nhibin and r-58kD inhibin as tracers
5I-31kD inh~bin25I-58kD inhibin
Antibody Dilution 1: 8000 1: 4000
Tracer binding (8Ol 30~ 18%
Affinity (Kdis) x 10 lOM 0.66 0.72
20C
10 EDlo (ng, fml
3 lkD inhibin 0.10, 3 . 0 0 . 13, 4 . 4
58kD inhibin 0.07, 1.2 0.13, 2.2
ED50 ~ng )
31kD inhibin 0.30, 10.1 O.Sl, 17.1
58kD inhibin 0.26, 4.3 0.43, 7.0
Slope*
b~F 1-37 1 O.O9a(8)1.47 ~ 0.09C(8)
31kD inhibin 1.53 ~ o.09b(5)1.68 + 0.08 (3)
58kD inhibin 1.50 ~ 0.07 (3)1.73 ~ 0.14d(5)
20 Precision**, 0.036 (5) 0.038 (5)
Between Assay Variation** 14 (5) 8. 5~ (5)
Bio/Imm Ratio
31kD inhibin 0.34 * 0.09 (16)0.43 ~ 0.13 (4)
58kD inhibin 0.30 ~ 0.12 (7)0.37 t 0.12 (5)
BGCM 0.25
a vs b P~ 0.05)
) as assessed by paired t-test
a vs c P ~0.01)
30 Mean + SD
~ Number in brackets: number of preparations
*~ Number in bracXets: number of assays
BGCM - bovine granulosa cell culture medium
~or details see text.
1307~
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-- 19 --
Example S Specificity of the assay
The specificity was asqessed on the following
grounds. First, a similar hierarchy of cross-reaction of
inhibin from various species in the RIA using either tracer
5 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%, hF~ 30% ovine FF 1% and rat ovarian extract
non-detectable. With respect to this antiserum it is apparent
10 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 tO.5%~ occurred for a range of purified
15 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.
Alternatively, medium from the bovine granulosa cell culture
(Fig. 4), and bovine testis extract (data not shown), both
20 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,
25 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 ~PLC. However, in the 40-60k~ molecular
30 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
35 as standard was observed following fractionation of bFP
inhibin on gel filtration and RP-HPLC ~Fig. S) and between
purified inhibin preparations ~Table 2J. The ratios ranged
13~'7736
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-- ZO --
from 0.02-2.09 in fractions obtained during the purification
procedure and ~rom 0.30-0.43 with the purified inhibin
preparations.
It is concluded that the ~IA procedures are not
5 detecting molecular entities devoid of biological activity and
VlCe ~ers~ except in the lower ~olecular weight region
(40-60kD) of the Sephacryl S200 chromatogram. Whether this
lower molecular weight material represents a protein distinct
from inhibin ~hich cros~-reacts in the RIA or inhi~in devoid
10 of bioloqical activity ~as not been established.
The cross-reactivities of inhibin-r~lated prot~ins
in the RlA relative to 31 kDa bFF inhibin were as follows:
porcine transforming growth factor-beta ~R & D Systems, Minn.
~SA) ~0.9~, bovine Mullerian inhibitory substance (kindly
15 provided by Dr. J. Hutson, Royal Children's Ho~pital,
Melbourne) ~0.3~ purified bovine inhi~in B subunit dimer ~2
and the subunits of 31 kDa bFF inhibin obtained ~ollowing
reductio~ and alkylation ~0.1~. A range of glycoproteins and
growth factors hAve been previously tested (MeLachlan et al,
20 1986) and showed cros8 reactivities against anti-58kD inhibin
of less than 1.0~. The specii~i~y of anti-31kD i~hibin wan
similar, with Cros--reactiviti-- Of less than 1. 0~ .
Examp~e 6 Application of the Radioimmunoassay to Serum
The RIA in its appllca~ion to serum required
substantlal modification. Firstly lOOmM phogphate ~uffer pl~
7.4 containing 0.15M NaCl, 0.5~ BSA was used, and, b~cause of
25 its stability in gerum, 125I-31~P inhibin was preferred to
5I-58kP inhibin as RIA tracer. Sccondly, a
tempcrature~dependent interference of steer serum with
125I-31kD in~ibin binding to the antiserum was observed, with
an increase in binding (B/Bo) from 57~ at 4C to 94~ at 37C
30 ~Fig. 7). This figure demonstrates the temperature dependence
following a 16 hour incubation in the presence of various
serum an~ inhibin preparations ~bF~, 31k~ inhlbin, steer serum
~SS), cow serum ~CS), bull serum (BS), human post menopausal
serum ~PMS), human female serum pool (~FP)). Tracer binding
35 ~/T) was maxim~l at 30~ in the presence of steer serum,
being 87.1 ~ 3.4% ~n ~ 5). Displacement of 125I-31kD inhibin
13~)~7736
R. 12 ~7 13: 31 CLEMENT H~CK hU~T +~.13 5~ ~Z96 P. 07
by bFF or 31~D inhibin was largely unaffected by temperature
Human PMS showed no temperature related interference upon
binding although the bindin~ w~ elevated (B/~o 110-120~).
Ba~ed on these data, conditions for the as~ay of
5 elther bov~ne or human serum inhibin were established. T~ese
involved using 125I-31kD inhibin as tracer in an overnight
30C incubation ~nd, in order to compen~ate for the low level
of interference ~y SS or PMS (presumed to contain no inhibin1
standards and samples were diluted in SS or PMS accordingly.
No detectable activity was determined in ~teer or in
human post-menopaus~l serum, whil~t bull and human female
serum ~howed parallel dose-response curvefi to their re3pective
follicular fluid standards, with circ~lating levels of 0.9 and
1.1 ng re~pectively.
Inhibin preparation~ or ~erum samples were diluted
in SS or PMS to a sample volume of 200 ~1. Anti~erum (100 ~1,
final dilution 1:8000) and samples t200 ~1) were incubated for
4 hour~ at 30C, followed by a further incubation of 16 hours
at 30C in the presence of trace~. Second antibody was added
20 and the tubes were $ncubated for 24 hours at ~~, following
which 2 ml 0.15M NaCl was added and the tubes were
aentrifug-d.
Thirdly, with respect to a choice of stAndard in the
RIA of bovine serum inhibin, purified 31kD inhibin i~ faYoured
25 in view of its stability in serum. In ~he absence of a
p~rified human inhibin preparation, 8lkD bovine inhibin may be
used as the ~tandard in the ~IA of human serum inhibin.
However, the partially purified hFF inhibin preparation
described above i8 preferred, and purified h~P inhibin, when
30 available, would be the most pre~erred standard. The
detectable levels of inhibin $mmunoactivity in serum from
women under going ovarian stimulation with exogenOus
gonadotrophin is analogous to the findings o Lee et al
(lg82~, where circulatinq levels of inhibin activity were
35 detected in PMSG treated immat~re female rats, particularly
directed against 58kD and 31k~ inhlbin,
13~ 36
lf:ll~'. 1~ '87 13: 3Z CLE~`lEl`lT HRCI< fl~lST +61_: 5~g 6zg6 P.
- 21a -
Individual anti3era may behave differently in the
~s6~y, and assay parameters may have to be determined for each
casc. Considerable variation~ in 6ensitivity between antisera
have been ob~erved, particularly between antisera directed
against 31kD and 5akD lnhibin. Anti-31kD inhibin appeared to
glve greater ~ensitivity than anti-58kD inhibin ~n the samples
tested 80 far,
13~7736
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Exampls 7 Improved Sen~itivit~c RIA for human ~erum
In order to improve as~ay sensitivity, the assay
procedure above wa~ modified as follows: the total volume of
the assay was reduced from 400 to 300 ~1 (comprising 200 ~1
5 sample, 50~ul tracer and 50 ~1 anti~erum). The assay buffer
was 150 mM phosphate, 0.2~ ~SA pH 7.4, and the incubation of
sample and antiserum was 4 days at 4C followed by the
addition of tracer and a further 3 days at 9C prior to the
addition of second antibody. Using this method, a 2.5 fold
10 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.
.
15 Example 8 Stability of 125I-31k~ and 125I-58kD Inhibin in
Serum
SDS-PAGE profiles of 125I-58kD inhibin following
overnight incubation with serum (SS and PMS) showed an
increased formation of an 125I-30k~ component (12~ of
20 recovered activity at 4C; 17~ at 30C) in comparison with
either buffer or bFF (6% 4C and 30C; (Fig. 6). ~he tracers
were incubated overnight at either 4 or 30C with either bFF,
steer serum (SS) or human post-menopau~al serum ~PMS).
Incubation of either tracer in RIA buffer alone gave ~imilar
25 profiles 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
30 contrast, SDS-PA~E profiles of 125I-31kD 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.
1RR, 11 '87 08:14 CLE11ENT H~CK ~UST +613 525 62961 P.26
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Example 9 Radioimmunoassay of inhlbin 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 b~F or 31kD inhibin as standards (Fig. 8). The
5 response shown in figure 8 is for bovine and human ~erum,
diluted in steer serum or post-menopausal serum respectively,
in the plasma RIA system employing 12sI3lkD inhibin as tracer.
Potential RIA standards (bFF, 31kD inhibin, hFF) were assayed
in the presence of either phosphate buffer ~200 ~ul, o) or
10 steer serum (200~ ) or post-menopausal serum (200~ ) 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 31k2 inhibin standard the level of
15 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
20 (0.1 ng/ml).
Example 10 Radioimmunoassay of inhibin in infertile human
The radioimmunoassay previously described was
applied to pla~ma and serum from post-menopausal subjects
25 (presumed to be inhibin free, n = 8) and young women with
ovsrian failure (premature ovarian failure n - 2, Turner's
~yndrome n - 1, ovariectomized n ~ 1). There was no
difference in 125I-31k2 inhibin binding in the assay between
these two groups and therefore post-menopausal serum was used
30 as a diluent in the assay.
Example 11 Radioimmunoassav of inhibin in human serum
The method was applied to:
(a) Plasma from normal women during ~pontaneous
menstrual cycles from the early follicular phase until the
3S time of ovulation (n = 2, example 'FL' #27, Fig. 9). Inhibin
~3~'736
1FIR. 11 '~37 0~: lS CLEMENT H~CK flUST +613 529 6296 P.27
-- 2g --
immunoactivity was below the limit of assay detection prior to
day 13 and its increase correlated with the increa3e in
circulating level~ of oestradiol (E2), LH and FS~ on day 13
and 14.
(b) Inhibin immunoactivity in serum from normal
men (n e 7) was at the lower limit of assay detection.
(c) Plasma was obtained from 26 unselected women
undergoing their fourth cycle of ovulation induction in the in
vitro fertilization programme at the Epworth Hospital,
10 Richmond, Victoria. Briefly this involved the administration
of clomiphene citrate 100-150 mg da~ly 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
15 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
20 ovulation see 'BU' # 11, Fig. 9k and for an hCG-induced
ovulation sample see '~O' 1, Pig. 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
25 plasma 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 sign~ficant
30 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
35 detected ultrasonically prior to oocyte aspiration (Fig. 12).
Tt is therefore apparent that both plasma inhibin and plasma
13~7736
~l~R. 11 '87 0S: 16 CLEMENT Hf:~CK ~UST +613 529 6296 P.26
-- 25 --
oestradiol are parameters of follicular development and
health, and in the majority of cases these show a close
correspondence.
In certain examples ('BY' # 6, 'BR' # 10, Fig. 9h
5 and j), 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
10 theca cell product, the assay of plasma inhibin is the first
direct plasma parameter of granulosa cell/oocyte health and
maturation. The di~sociation of plasma inhibin and E2 may
therefore be of therapeutic importance in that the plasma
inhibin is a direct measure of follicular development, and its
15 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 a~ the
20 radiummunoassay (RIA) standard by two gel chromatographic
steps and reversed phase HP~C as described for bFF inhibin
(Robert~on et al, 1985). This material yielded parallel dose
reponse lines to human female serum inhibin obtained from
women undergoing ovarian hyperstimulation for in vitro
25 fertilisation. This partially purified human follicular fluid
(hFF) inhibin standard preparation was defined in term~ of its
in vitro inhibin bioactivity using an inhibin bioassay ba~ed
on the dose-related suppresion of FSH cell content (Scott et
al, 1980). This material was used as the RIA standard and
30 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
35 preparation of defined unitage 1 U/mg using the inhibin
'lQR. ll '87 139 le CLE~`lENT HflCK flU5T +613 529 6296 13~77~6 P.29
-- 26 --
bioassay. The RIA has an interassay coefficient of variation
(CV) of 6.4% (n = 5 assay) and the sensitivity (logit + 2~ was
0.37 U/ml.
The RIA was specific to bovine and human inhibin and
5 cross-reacted less than 0.3S with a range of glycoproteins and
growth factors. In addition, inhibin-related peptides
cross-reacted as follows: porcine transforming growth factorp c
0.9%, bovine Mullerian Inhibitory Substance 0.3~, purified
bovine inhibin 8 subunit dimer ~1~ and the subunits of 31kDa
10 bF~ inhi~in following reduction and alkylation ~0.1~. 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 = S assays), and a sensitivity of 0.37
15 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
20 individual observations (Gaddum et al 1933) was assummed,
i.e. all calculations were performed using logarithmically
transformed va~ues to give geometric means and 67% confidence
intervals. Statistical compar~son between pregnant and
non-pregnant groups was performed using the unpaired t-test.
25 Example 13 Inhibin levels during luteal phase and early
pregnancy
Nineteen women pre~enting consecutively for
treatment in the Monash university IVF program were studied.
Clinically their infertility resulted from tubal disease (n =
30 7), endometriosis ~n = 6), unkown causes (n 5 5) or poor semem
quality (n = 4). The protocol of ovulation induction has been
described elsewhere (Wood and Trounson, 1984). Briefly, all
subjects received clomiphene citrate (Clomid, Merell Dow,
Sydney) 100-150 mg daily between days 5 and 9 of the cycle and
35 HMG (Pergonal, Serono, Rome) 75-225 units daily from day 6.
The dosage and duration of HMG therapy were optimized
- 13(~7736'~R . 11 ' 57 08: 19 CLEMENT Hl:~CK RUST +613 529 6296 P. 30
-- 27 --
according to daily plasma oestradio1 concentrations and
follicular size as a~sessed by ovarian ultrasound. HCG
(Pregnyl, Organon, OS8) 5, OOO IU intramuscularly was
administered to induce ovulation, and oocyte retrieval was
5 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
10 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
15 who did not conceive, luteal phase inhibin levels rose to a
peaX 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.
20 ~esults are expressed as the geometric mean + 67~ confidence
intervals. The broken line indicates the limit of sensitivity
of the inhibin radioimmunoassay. The number of sub~ects
showing non-detectable inhibin values is shown in parentheQes.
Non-detectable values are not included in the mean
25 confidence intervals.
In three subjects who conceived, inhibin levels were
similar to non-conception cycles between days 2 and 8,
increaslng thereafter and becoming significantly higher (p
0.001) than in the non-pregnant group by day 12
30 po8t-laparoscopy. Figure 14 shows these results, expressed as
the geometric mean l 67~ confidence intervals. The broken
line indicates the sensitivity of the inhibin
radioimmunoassay. ~pc0.05, **p~0.01, ~**p~0.001 comparing
hormone values for the pregnant and non-pregnant groups on the
35 same day. Significance values in the second panel refer to
serum FSH. The late luteal phase rise in serum inhibin in the
7~6
R. 11 '~37 03:20 CLE11E~T H~CK RUST +613 529 6296 P.31
-- 28 --
pregnant patients coincided with both the rise in 5erum ~hCG
and with the decline in serum FSH to values below thoQe seen
in the non-pregnant group.
Serum FSH showed a significant inverse correlation
5 with inhibin in the luteal phase of the non-conceptlon cycles
(r = 0.51, n = 113, p ~0.001) (Fig. 15). Similar Qignificant
inverse relationships were observed between FS~ and inhibin
when the data were analysed wcording to whether the
progesterone concentrations were in the normal ovulatory
10 luteal phase range (25-100) nm) or greater, (r = 0.38, n =
76, p ~0.001 vs r = 0.37, n = 37, p c0.05, 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 -
15 0.52, n ~ 114, p ~0.001).
Plasma inhibin and progesterone concentrations weresignificantly correlated in the luteal phase of cycles in
which pregnancy did not occur (r = 0.81, n - 85, pcO.001), as
were plasma inhibin and oestradiol concentrations (r - 0.65, n
20 ~ 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 (3.5
~1.2-9.8~ mIU/ml) on day 8.
ln 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 menstrùal
cycles and during pregnancy.
130~;'736
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EXAm~1e 4 Inhibin Levels in the Nor~al Men5trUal ~ele
In a further study~3erum inhibin was determined
daily in 6 normal wo~en throughout the menstrual cycle,
using a rad~ OimmunoasSAy smploylng an antiser~m directed to
31kD inhibin. ~he normalcy of the menstrual oycle was a~oessed
from the serum profiles of ~S~, ~H, progesterOne, and oestradlol.
The increAse in ~en~it~vity of the as6ay using this antiserum
permitted the detectlon of inh~bln in over 97~ of sAmples~ The
results are shown in Flg. 16.
13~ 36
. lc '~7 1:~: 33 CLEMENT H~U< ~UST +~13 529 6296 P. 1
-- 30 --
10 Advant~es and applications of the assaY accordihq to the
invention.
1 The assay may be u~ed for determining inhibin
concentration in a wide range of biological samples, such as
serum, plasma, urine, follicular fluid, tissue homogenates,
15 and culture fluids.
2. The as~ay may be used to monitor the purification of
inhibin from tissue, biological fluids, or culture medium, or
to moni~or transfection studies.
3. Inhibin levels may be u~ed as a marker of parameters
of reproductive function, ~uch as granulosa cell function,
fol~ieular development, number of ovarian follicles following
ovarian hyper~timulation, and foetal well-being during early
pregnancy, and Sertoli cell function.
lt will be clearly understood that the invention in
25 its general a~pects i5 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, Polyp~p,
30 Pregnyl, RIA-Quant, Sephacryl, Sephadex, Triton X-100,
Ultrapore, and Ultra-Turrax.
Refqrences citcd herein are listed on the following
paqe~,
1RR 11 '87 08 24 CLE~ENT HPCK RUST +613 529 6296 1307736 P 34
- 31 -
REFERENCES
Au, C.L., Robertson, D.M. and de Kretser, D.M. ~1983)
Endocrinology 112, 239-244.
Baker, H.W.G., Eddie, L.W., Higginson, R~Eo~ Hudson, B.,
5 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, H.W.G., Eddie, L.W., Hudson, B. and Niall, H.D. (1985)
Australian J. Biological Sciences (in Press).
10 Burger, H.G., Lee, V.W.K., and Rennie, L.C. ~1983) J. Lab.
Clin. Med. 80 302-308.
Erickson, G.F., and Hsueh, A.J.W. (1978) Endocrinology 103,
1960-1963.
Finney, D.J. (1971) Statistical Method in Biological Assay,
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