Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.

Groome, Nigel P.; Illingworth, Peter; O’Brien, Martin; Priddle, J D; Weaver, Kristal; McNeilly, A. S.

doi:10.1210/jcem.80.10.7559876

Cited by 54 publications

(41 citation statements)

References 14 publications

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“…The most sensitive of these assays has a detection limit of 500 pg/ml follistatin. This is two orders of magnitude less sensitive than the inhibin enzyme-linked immunosorbent assays (ELISAs) from our laboratory (Groome et al 1994(Groome et al , 1995. These previous follistatin assays have other disadvantages, including the use of hazardous short-lived isotopic reagents.…”

Section: Introductionmentioning

confidence: 95%

“…The spleen was removed and the splenocytes were fused to Sp2/0 myeloma cells using polyethylene glycol following a standard fusion protocol (Galfre & Milstein 1981). Hybridoma supernatants were screened on a 96-well plate coated with rh-FS288 (0·2 µg/ml in 0·2 M sodium bicarbonate buffer, pH 9·4) following a screening protocol described elsewhere (Groome et al 1995). Positive clones were expanded and recloned in methyl cellulose (McCullough & Spier 1990).…”

Section: Antibody Productionmentioning

confidence: 99%

“…Positive clones were expanded and recloned in methyl cellulose (McCullough & Spier 1990). The supernatants from these single clones were titrated against rh-FS288 (0·2 µg/ml) under a standard ELISA protocol (Groome et al 1995). On the basis of these experiments two clones (29/9 and 17/2) were selected and isotyped with a commercial kit (Sigma Chemical Co., Poole, Dorset, UK).…”

Section: Antibody Productionmentioning

confidence: 99%

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Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Evans

Muttukrishna²,

Groome³

1998

Journal of Endocrinology

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Recent studies have found follistatin to be an important regulator of activin bioactivity. Whilst a number of assay formats have been described, all are of limited sensitivity and require the use of isotopes. Many use polyclonal antibodies. Furthermore, a wide range of follistatin preparations have been used as standards, complicating inter-laboratory comparison.We now describe an ultra-sensitive two-site enzyme immunoassay using a pair of mouse monoclonal antibodies raised against follistatin 288. The presence of sodium deoxycholate and Tween 20 in the diluent gave results for total (free and activin-dissociated) follistatin. The assay had a detection limit of <19 pg/ml and recovery of spiked follistatin 288 from amniotic fluid, serum, seminal plasma, human follicular fluid and granulosa cell conditioned medium averaged 100·7 7·5%, 89·1 5·5%, 98 4·9%, 96 7·2% and 123·9 11% respectively. The intra-and interplate coefficients of variation were <5%. An excess of activin-A (50 ng/ml) prior to assay did not affect follistatin recovery. Inhibin-A, inhibin-B, activin-A, activin-B and activin-AB had minimal cross-reactivity (<0·3%). However, follistatin 315 had a significant cross-reaction (9·9%).Serially diluted human samples gave dose-response curves parallel to the standard. Pooled human follicular fluid contained high concentrations of follistatin (242 ng/ml). Follistatin was also found in maternal serum during pregnancy (first trimester 0·8 ng/ml, third trimester 2·8 ng/ml), normal male serum (0·45 ng/ml), amniotic fluid (sixteen week 3·63 ng/ml, term 0·89 ng/ml), seminal plasma (2·4-30 ng/ml) and human granulosa cell conditioned media (0·44 ng/ml). Serial serum samples taken throughout the menstrual cycle of ten women showed fluctuating follistatin concentrations (0·62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmenopausal women appeared to have higher follistatin levels than any of the normal women (1·4 ng/ml).The possible presence in certain samples of mixtures of follistatin isoforms with different immunoreactivities poses major problems of interpretation in this and all other current follistatin immunoassays. Further work is needed to identify the major immunoreactive forms in different tissues and fluids. Nevertheless, the new assay has a number of advantages over previous assays and should prove a useful tool for various clinical and physiological studies.

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Section: Introductionmentioning

confidence: 95%

Section: Antibody Productionmentioning

confidence: 99%

Section: Antibody Productionmentioning

confidence: 99%

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Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Evans

Muttukrishna²,

Groome³

1998

Journal of Endocrinology

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“…Inhibin B (Groome et al, 1996), inhibin A (Groome et al, 1994) and Pro-alpha C (Groome et al, 1995) concentrations were measured using two-site ELISA as described previously. The CV were o8% within plate and o10% between plates for each of these assays with sensitivities of 7.8 pg ml À1 for inhibin B, 2 pg ml À1 for inhibin A and 5 pg ml À1 for Pro-alpha C.…”

Section: Hormone Assaysmentioning

confidence: 99%

Phase II trial of tamoxifen and goserelin in recurrent epithelial ovarian cancer

et al. 2005

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Endocrine therapy is a recognised option in the treatment of chemo-resistant ovarian cancer. We conducted a nonrandomised phase II evaluation of combination endocrine therapy with tamoxifen and goserelin in patients with advanced ovarian cancer that had recurred following chemotherapy. In total, 26 patients entered the study, of which 17 had platinum-resistant disease. The median age was 63 years and enrolled patients had received a median of three chemotherapy regimens prior to trial entry. Patients were given oral tamoxifen 20 mg twice daily on a continuous basis and subcutaneous goserelin 3.6 mg once a month until disease progression. Using the definition of endocrine response that included patients with stable disease (SD) of 6 months or greater, the overall response rate (clinical benefit rate) was 50%. This included one complete response (CR) (3.8%), two partial responses (PR) (7.7%) and 10 patients with SD (38.5%). The median progression-free interval (PFI) was 4 months (95% CI 2.4 -9.6) while the median overall survival (OS) was 13.6 months (95% CI 5.5 -30.6). Four patients received treatment for more than 2 years (range 1 -31) and one of them is still on treatment. In none of the four patients was there any evidence of recurrent or cumulative treatment related toxicity. Treatment-limiting toxicity was not seen in any of the study population. Endocrine data demonstrated a marked suppression of luteinising hormone (LH) and follicle-stimulating hormone (FSH) to less than 4% of baseline values. No consistent correlation could be established between LH/FSH suppression and tumour response. Likewise no relationship was observed between Inhibin A/B and pro-alpha C levels and tumour response. Inhibin is unlikely to be a useful surrogate marker for response in locally advanced or metastatic ovarian cancer. Combination endocrine therapy with tamoxifen and goserelin is an active regimen in platinum-resistant ovarian cancer patients. Hormonal therapy is advantageous in its relative lack of toxicity, ease of administration and tolerability, thus making it suitable for patients with heavily pretreated disease, compromised bone marrow function and other comorbid conditions that contraindicate cytotoxic therapy as well as in patients with indolent disease.

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“…L'isolement et la caract6risation des inhibines ont 6t4 laborieux [22], quelques m 6 t h o d e s de dosage peu sp6cifiques ont e n t r e n u des doutes sur la physiopathologie de cette h o r m o n e [7]. I1 a fallu a t t e n d r e 1993 pour disposer d' u n dosage sp6cifique des diff4rentes formes de l'inhibine (inhibine B, inhibine A, sous-unit6 de l'inhibine) [18,6,19]. Ces dosages p a r m 6 t h o d e ELISA, outre l e u r bonne sp6cificit6 se sont m o n t r 6 s tr6s fiables et ont p e r m i s de nomb r e u s e s 6tudes soit chez l'homme, soit chez la f e m m e [20].…”

Section: Age I N H I B I N E B F S H T E S T O S T 4 R O N E Testo Lunclassified

L'Inhibine B chez les blessés radiculo-médullaires. Résultats préliminaires

Mathian

Pallant

Pilonchery

et al. 1999

Androl.

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RESUM]~Les l~sions neurologiques constat~es chez les hommes blesses radiculo-m~dullaires entrainent souvent une alteration de la qualit~ du sperme et parfois une an~jaculation. Des biopsies testiculaires ont permis de mettre en ~vidence que ratteinte m~dullaire peut ~tre responsable de l'alt~ration des cellules de la lign~e germinale. Un bilan hormonal a ~t~ pratiqu~ chez 13 patients pr~sentant une absence de sperme au moment de rexamen.

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Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.

Cited by 54 publications

References 14 publications

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Phase II trial of tamoxifen and goserelin in recurrent epithelial ovarian cancer

L'Inhibine B chez les blessés radiculo-médullaires. Résultats préliminaires

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