Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads

Definitions

• This invention relates to methods for assay of inhibin, and in particular to methods for immunoassay of inhibin.
• 31-32kD 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) .
• 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 ELlSAs, immunoassays based on fluorescence detection, and related assays relying on polyclonal and -monoclonal antibodies against inhibin.
• an immunoassay for the estimation of inhibin in an inhibin-containing sample which comprises the step of using an antibody directed against inhibin.
• 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.
• antigens include preparations containing inhibin, purified bovine 58kD inhibin. 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.
• a monoclonal antibody or an IgG directed against any of the aforesaid inhibins may be used.
• the antibody is capable of neutralizing inhibin bioactivity.
• the immunoassay is further characterized by the step of using labelled 58kD or 31kD inhibin as tracer. More preferably said tracer is labelled with iodine ( I) with an enzyme, or with a fluorescent marker.
• the assay is a radioimmunoassay or an enzyme-liked immunosorbent assay (ELISA) , or a fluorescence- based immunoassay.
• the invention provides a method for measuring inhibin in samples such as follicular fluid or serum from various species (including humans) wherein concentrations of inhibin in standards are used to derive the concentration of inhibin in the follicular fluid or serum by competitive binding of 125I labelled inhibin and inhibin from test samples with bovine 58kD inhibin antiserum, followed by precipitation and- Counting of bound 125I labelled inhibin.
• the preferred specific radioimmunoassay system for inhibin of the invention is applicable to bovine and human follicular fluid and serum, and can employ an antiserum against (purified bovine) 58kD inhibin with iodinated 31kD or 58kD inhibin as tracer.
• the affinity f actionation step uses Matrex Red A.
• the purification procedure additionally comprises a gel filtration step.
• an assay standard selected from the group consisting of naturally-occurring or recombinant inhibin, or fragments or derivatives thereof.
• the standard displays parallelism in the assay with the samples under test.
• Particularly preferred standards include bovine 31kD inhibin, and partially purified or purified human inhibin.
• the conditions of the assay in particular incubation times, may be varied in order to attain desired levels of sensitivity.
• a form of the radioimmunoassay modified for increased sensitivity comprises: incubating sample and antiserum for 4 days at 4 C, followed by the addition of 1251-3lkD inhibin tracer, incubating for 3 days at 4°C and then adding second antibody, precipitating, and counting bound 125I labelled inhibin.
• the tracer is 1251-3lkD inhibin, and incubation with tracer is performed at elevated temperature (30 C) in the presence of inhibin-free serum, in order to minimize non-specific effects.
• Suitable sources of inhibin-free serum include steers or other castrated male animals, oophorecto ized women, women with
• Figure 1 shows the fractionation of I-58kD and 125 1-3lkD inhibin on analytical SDS-PAGE under reducing conditions.
• Figure 2 shows the time course of immunization of a rabbit with 58kD inhibin.
• FIG. 3 shows 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 ggrraainulosa cell culture medium (BGCM) using either 1251-3lkD or 125. I-58kD inhibin as tracers.
• Figure 5 shows the profile of inhibin in vitro bioactivity and immunoactivity following fractionation of bFF through the various steps of the inhibin purification procedure of Robertson et al (1986).
• Figure 6 shows non-reduced SDS-PAGE profiles of
• Figure 7 shows the effect of temperature on the binding of 1251-3lkD inhibin to the antiserum.
• Figure 8 shows logit-log dose response lines of bovine and human serum, in the plasma RIA system employing
• Figure 9 shows the ovulation induction regime and serum levels of FSH, LH, inhibin and oestradiol (E-) in twenty-six women involved in an In Vitro Fertilisation (IVF) programme and one normal woman (FL 27).
• Figure 10 is a comparison of plasma E- and inhibin levels plotted for some of the data in Figure 9.
• Figure 11 shows the correlation between the number of ova produced and E_ or inhibin levels in serum.
• Figure 12 shows the correlation between the numbers of ovarian follicles detected ultrasonically and peak inhibin levels in serum.
• Figure 16 shows inhibin, FSH, LH, oestradiol and progesterone concentrations in the sera of normal women during the menstrual cycle, assayed using anti-31kD inhibin.
• bFF bovine follicular fluid hFF human follicular fluid
• Peak I (58kD inhibin) and Peak II (3lkD inhibin) fractions from (b) were fractioned on an RPSC Ultrapore column (0.46 x 7.6 cm, Beckman) using a
• Hatched area denotes inhibin bioactivity. o o RIA with 125 I.-58kD inhibin as tracer. 125 o o RIA with 1-3lkD inhibin as tracer.
• Vo void volume
• BSA bovine serum albumin (mol. wt 67,000).
• OVA ovalbumin (mol. wt 43,000).
• the purified inhibin was stored in SDS electroelution buffer (approx. 3% SDS in 10 mM NH.HCO-,) prior to iodination.
• samples were methanol precipitated at -20°C in order to remove SDS and solubilized by heating at 37°C for 1 hour and sonication. Similar profiles of both bio- and immunoactive inhibin were observed at each stage of the inhibin purification procedure.
• the biological to immunological activity ratios for a number of purified 31kD and 58kD inhibin preparations using both tracers in the radioimmunoassay ranged from 0.30 - 0.43.
• Human follicular fluid was obtained at oocyte collection in the in vitro fertilisation programme at the Queen Victoria Medical Centre/Epworth Hospital, Melbourne. It was charcoal treated (100 mg/ml dextran-coated charcoal for 1 hour at 4°C) , lyophilised, stored at -20°C and resolubilized prior to assay by sonication in assay buffer or culture medium.
• Ovine follicular fluid oFF
• Ovine rete testis fluid (oRTF) is a lyophilised inhibin preparation (Baker et al 1985).
• Rat ovarian extract was a charcoal-treated rat ovarian cytosol preparation.
• Testes from four bulls were decapsulated and homogenised in equal w/v Dulbecco's phosphate buffer using an Ultra-Turrax tissue disperser (Janke and Kunkal KS, Staufen FRG) and centrifuged at 100,000g x 1 hour at 4°C and stored at -20°C. Prior to assay the supernatants were charcoal treated with an equal volume of 1% Norit A in Dulbecco's phosphate buffer and incubated at 4 C for 30 minutes prior to centrifugation and bioassay (Au et al 1983).
• Ultra-Turrax tissue disperser Janke and Kunkal KS, Staufen FRG
• Analytical SDS Polyacrylamide Gel Electrophoresis Sera and bFF were incubated at various temperatures in an equal volume of 100 mM phosphate buffer pH 7.4 containing 0.15M NaCl, 0.1% Triton X-100 and either 0.5% BSA ffor studies with 125 or I-31kD inhibin or 0.5% Polypep for 125 I.-58kD inhibin.
• Equal volumes (5ul) of sample and 10% SDS and Dulbecco's Phosphate buffer pH 7.4 (30 ⁇ l ) were placed in a boiling water bath for 2.0 minutes then in ice.
• Inhibin activity was determined using an in vitro bioassay based upon the dose-dependent suppression of FSH cell content in rat pituitary cell cultures utilizing a parallel line 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 e_t al 1980).
• Serum FSH was measured by RIA (Amerlex-M, Amersha , USA) using 2nd IRP FSH as standard with an interassay CV of 7.0% from 31 assays.
• LH was measured by RIA (LH RIA, Diagnostic Products Corp., L.A., USA) using the 2nd IRP LH as standard with an interassay CV of 10.1% from 31 assays.
• Both oestradiol and progesterone were measured using RIA (Coat-a-Count, Diagnostic Products Corp., L.A. ) with interassay CVs of 8.7% and 8.1% respectively from - * 150 assays.
• Serum beta subunit of hCG was measured by RIA (B-hCG RIA-Quant, Mallinckrodt Inc. St. Louis, USA) using the hCG 2nd IS as standard with an interassay CV of 10.4% from 30 assays.
• the RIA dose-response curves were linearised using a logit-log dose transformation. Parallelism was assessed from a comparison of slope values of dose-response curves using the multiple range test for groups of unequal size (Kramer, 1956) or by paired t-test. Potency estimates were determined using standard parallel line bioassay statistics. In situations • where non-parallelism was observed between dose response lines of unknown and standard preparations, potency estimates were determined from the ratio of their ED 50 values. The sensitivity (ED.-) was defined as the mass of hormone required to give 10% displacement in the assay whilst ED 5Q corresponded to the mass required for 50% displacement.
• the index of precision (Gaddura (1933); Finney (1964)) was used to describe assay precision.
• the between assay variation was calculated from the coefficient of variation of the repeated measurement of a partially purified inhibin preparation.
• the dissociation constant (K . ) was determined by Scatchard analysis using 125I-hormone and increasing amounts of unlabelled hormone. The mass of
• I-hormone used in the analysis was determined from its specific activity ( j Ci/ ⁇ g).
• Antisera against 58kD and 3lkD inhibin were character ⁇ ized by showing that following immunization, parallel changes in plasma FSH and inhibin antibody titre were observed, indicating inhibin neutralization in vivo.
• the antisera neutralized bFF, hFF and purified 3lkD and 58kD inhibin activity in an in vitro bioassay. The results described below refer to anti-58kD inhibin, but similar results were obtained using anti-31kD inhibin.
• Inhibin from ovine sources (follicular fluid, rete testis fluid) showed 8 and 6% cross-reactivity respectively. This antiserum, at a maximum non-toxic level of 4 ul per well, did not neutralise 2 units of inhibin activity in rat ovarian cytosol extracts. TABLE 1 Cross-reactivity of inhibin from various sources as assessed by inhibin neutralization in vitro and radioimmunoassay
• Ovine 1040* > 6.0 (1) ⁇ 6.0 > 52 ⁇ 2 >52 ⁇ 2
• the void volume fractions were pooled, made up to 20 ml and applied to a column of 200 j ⁇ l
• I-inhibin was eluted with 1M KC1/4M urea in phosphate buffer- The iodinated inhibin was further gel filtered on a Sephadex G25 column (PD10) with the appropriate
• I-inhibin as assessed by its molecular weight on SDS-PAGE, was found in this fraction.
• the specific activity of the iodinated preparations was assessed in the radioimmunoassay using a self-displacement procedure (Marana et al 1979) with the hormone used for iodination as standard. Specific activities of 50-60 juCi/ ⁇ g for 58kD inhibin and 24 ⁇ Ci/ ⁇ q for 31kD inhibin were obtained, with recoveries ranging from 5-25%.
• 125 I-58kD inhibin following fractionation on SDS-PAGE was similar to purified non-iodinated inhibin under both non-reducing (58kD) and reducing (43kD and 15kD) conditions except that a 58kD material of unknown identity was observed in relatively low proportions (18%) under reducing conditions (Fig. 1) .
• the molecular weight markers employed were BSA (bovine serum albumin) 67,000; OV (ovalbumin) 43,000; CA favor (carbonic anhydrase) 29,000; GL (goose egg lysozyme) 20,300; and.GL (chick egg lysozyme) 14,300.
• the arrow, in figure 1, refers to the point of sample application.
• the assay buffer used was 10 mM phosphate, 0.15 M NaCl, 0.5% BSA, pH 7.2.
• a delayed tracer addition, second antibody assay system was employed. The sample and antiserum were incubated in a volume of 300 ⁇ l for 16 hours at room temperature following which 125I-inhibin (10,000 cpm, 100 ⁇ l ) was added and the incubation continued either overnight at room temperature or for 48 hours at 4 C.
• Radioimmunoassay procedures were established using both 31 and 58kD inhibin tracers. Following a logit-log dose transformation of the response curves, linear displacement of each tracer was observed for a range of inhibin preparations, 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/Bo) 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. Scatchard analysis revealed similar affinities for the antiserum of either inhibin form.
• Non-parallel dose response lines were observed between bFF and either 3lkD inhibin with 125 I-31kD inhibin as tracer of 58kD inhibin with l25 I-58kD inhibin as inhibin tracer.
• the sensitivity (ED, Q ) and ED gQ values were comparable in each assay with either hormone.
• FIG. 7 This figure demonstrates the temperature dependence following a 16 hour incubation in the presence of various serum and inhibin preparations (bFF, 31kD inhibin, steer serum
• SS cow serum
• CS cow serum
• PMS human post-menopausal serum
• NFP human female serum pool
• Antiserum 100 ul, final dilution 1:8000
• samples 200 jul
• Second antibody was added and the tubes were incubated for 24 hours at 4°C, following which 2 ml 0.15M NaCl was added and the tubes were centrifuged.
• 3lkD bovine inhibin is favoured in view of its stability in serum.
• 3lkD bovine inhibin may be used as the standard in the RIA of human serum inhibin.
• the partially purified hFF inhibin preparation described above is preferred, and purified hFF inhibin, when available, would be the most preferred standard.
• the detectable levels of inhibin immunoactivity in serum from women under going ovarian stimulation with exogenous gonadotrophin is analogous to the findings of Lee et al (1982), where circulating levels of inhibin activity were detected in PMSG treated immature female rats, particularly directed against 58kD and 3lkD inhibin. - 21a - Individual antisera may behave differently in the assay, and assay parameters may have to be determined for each case. Considerable variations in sensitivity between antisera have been observed, particularly between antisera directed against 31kD and 58kD inhibin. Anti-31kD inhibin appeared to give greater sensitivity than anti-58kD inhibin in the samples tested so far.
• Example 7 Improved Sensitivity RIA for human serum
• the assay procedure above was modified as follows: the total volume of the assay was reduced from 400 to 300 ⁇ l (comprising 200 ⁇ l sample, 50 ⁇ l tracer and 50 ⁇ l antiserum).
• the assay buffer was 150 mM phosphate, 0.2% BSA pH 7.4, and the incubation of sample and antiserum was 4 days at 4°C followed by the addition of tracer and a further 3 days at 4°C prior to the addition of second antibody. Using this method, a 2.5 fold increase in sensitivity was achieved.
• This modified assay procedure has been applied to the measurement of human plasma inhibin. The modified assay allows the quantification of plasma inhibin in normal male plasma and in plasma throughout the normal menstrual cycle.
• the tracers were incubated overnight at either 4 or 30°C with either bFF, • steer serum (SS) or human post-menopausal serum (PMS). Incubation of either tracer in RIA 'buffer alone gave similar profiles to the bFF incubation shown.
• Molecular weight markers are described in Figure 1.
• the application of the inhibin RIA to serum from cattle resulted in parallel logit-log dose response lines of BS with either bFF or 31kD inhibin as standards (Fig. 8).
• the 5 response shown in figure 8 is for bovine and human serum, diluted in steer serum or post-menopausal serum respectively, in the plasma RIA system employing 125I3lkD inhibin as tracer.
• Cow serum shows a minimal detectable immunological response.
• the immunoactivity was expressed in terms of 3lkD inhibin standard the level of
• Example 11 Radioimmunoassay of inhibin in human serum The method was applied to:
• hMG human menopausal gonadotrophin
• hCG human chorionic gonadotrophin
• Inhibin immunoactivity in the plasma samples showed a highly significant correlation with 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.
• Example 12 hFF inhibin as standard for radioimmunoassay Human follicular fluid (hFF) obtained at oocyte collection in the IVF programme was prepared for use as the radiummunoassay (RIA) standard by two gel chromatographic steps and reversed phase HPLC as described for bFF inhibin (Robertson et al, 1985) . This material yielded parallel dose reppnse lines to human female serum inhibin obtained from women undergoing ovarian hyperstimulation for in vitro fertilisation.
• RIA radiummunoassay
• hFF human follicular fluid
• the unitage of the hFF inhibin standard was calibrated in terms of an ovine testicular lymph standard preparation of defined unitage 1 U/mg using the inhibin - 26 - bioassay.
• the RIA was specific to bovine and human inhibin and cross-reacted less than 0.3% with a range of glycoproteins and growth factors.
• inhibin-related peptides cross-reacted as follows: porcine transforming growth factor ⁇ ⁇ 0.9%, bovine Mullerian Inhibitory Substance 0.3%, purified bovine inhibin B subunit dimer ⁇ 1% and the subunits of 31kDa bFF inhibin 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.
• Inhibin levels were determined at 1 dilution level against the partially purified hFF standard preparation using an iterative curve-fitting procedure (Burger et al., 1973). In the calculation of results, a lognormal distribution of individual observations (Gaddum et al; 1933) was assum ed, i.e. all calculations were performed using logarithmically transformed values to give geometric means and 67% confidence intervals. Statistical comparison between pregnant and non-pregnant groups was performed using the unpaired t-test.
• Example 13 Inhibin levels during luteal phase and early pregnancy
• the protocol of ovulation induction has been described elsewhere (Wood and Trounson, 1984). Briefly, all subjects received clomiphene citrate (Clo id, Merell Dow, Sydney) 100-150 mg daily between days 5 and 9 of the cycle and HMG (Pergonal, Serono, Rome) 75-225 units daily from day 6.
• HMG HMG
• HCG Pregnyl, Organon, Oss
• 5,000 IU intramuscularly was administered to induce ovulation, and oocyte retrieval was undertaken 36 hours later.
• Embryo transfer was performed as described by Wood and Trounson (1984). Blood was taken on day 1 post laparoscopy and every second day from day 2 to day 14 and sera stored for measurement of FSH, LH, / subunit hCG, ' oestradiol, progesterone and inhibin. Three of the 19 women became pregnant.
• the number of subject serum samples per day was 13-16 except at day one when only eight were available. Results are expressed as the geometric mean ⁇ 67% confidence intervals. The broken line indicates the limit of sensitivity of the inhibin radioimmunoassay. The number of subjects showing non-detectable inhibin values is shown in parentheses. Non-detectable values are not included in the mean ⁇ . confidence intervals.
• inhibin levels were similar to non-conception cycles between days 2 and 8, increasing thereafter and becoming significantly higher (p 0.001) than in the non-pregnant group by day 12 post-laparoscopy.
• Figure 14 shows these results, expressed as the geometric mean ⁇ 67% confidence intervals. The broken line indicates the sensitivity of the inhibin radioimmunoassay. *p ⁇ _0.05, **p-c0.01, ***p ⁇ 0.001 comparing hormone values for the pregnant and non-pregnant groups on the same day. Significance values in the second panel refer to serum FSH.
• the late luteal phase rise in serum inhibin in the - 28 - pregnant patients coincided with both the rise in serum ⁇ hCG and with the decline in serum FSH to values below those seen in the non-pregnant group.
• luteal phase inhibin levels did not show significant correlations with either progesterone or oestradiol.
• Serum LH levels fell sharply from day 1 (21.0 [17.0-26.1] mlU/ l) to a nadir (3.5 [1.2-9.8] mlU/ml) on day 8.
• the assay may be used for determining inhibin concentration in a wide range of biological samples, such as serum, plasma, urine, follicular fluid, tissue homogenates, and culture fluids.
• the assay may be used to monitor the purification of inhibin from tissue, biological fluids, or culture medium, or to monitor transfection studies.
• Inhibin levels may be used as a marker of parameters of reproductive function, such as granulosa cell function, follicular development, number of ovarian follicles following ovarian hyperstimulation, and foetal well-being during early pregnancy, and Sertoli cell function.

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