Development and application of a two-site enzyme immunoassay for the determination of 'total' activin-A concentrations in serum and follicular fluid

1996 ◽  
Vol 148 (2) ◽  
pp. 267-279 ◽  
Author(s):  
P G Knight ◽  
S Muttukrishna ◽  
N P Groome
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Follicular Fluid ◽  
Biological Fluids ◽  
Activin A ◽  
Β Subunit ◽  
Serum Samples ◽  
Response Curves ◽  
Inhibin A ◽  
Α Subunit

Abstract The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and α2 macroglobulin (α2M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit ∼10 pg/well), precise (mean within- and between-plate coefficients of variation 4·9 and 9·1% respectively) and accurate (activin-A recovery values of 102 ± 3 and 96 ± 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and α2M (100 μg/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all <0·5%), bovine pro-αC and follistatin (both <0·1%). Response curves parallel to the activin-A standard curve were obtained for a variety of test samples including bovine, human, ovine and porcine FF, human sera and conditioned medium from cultured bovine and human granulosa cells. Fractionation of bovine FF by SDS-PAGE confirmed assay specificity since only one peak of activin-A immunoreactivity was detected (Mr ∼25 k) in eluted gel slices. However, gel-permeation chromatography showed that under physiological conditions all of the detectable activin-A in bovine FF eluted with apparent Mr values of >700 and 60–200 k reflecting its association with binding protein(s). Analysis of bovine FF samples (n=76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r=+0·54; P<0·001) and total β subunit immunoreactivity (r=+0·32; P<0·005) but not with total α subunit immunoreactivity (r= −0·09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total β subunit levels were highest in oestrogen-inactive follicles (P<0·01) whereas total α subunit levels were lowest in these follicles (P<0·001). Activin-A levels were measurable in all human serum samples analysed, ranging from 128 pg/ml during the normal menstrual cycle, 210 pg/ml in women undergoing ovarian hyperstimulation and ∼ 500 pg/ml in postmenopausal women to over 4000 pg/ml during pregnancy. In conclusion, the present assay provides a reliable method for quantitating total (i.e. bound+free) activin-A concentrations in a variety of biological samples and should prove useful for further in vivo and in vitro studies in a range of species including man. Journal of Endocrinology (1996) 148, 267–279

Determination of free follistatin levels in sera of normal subjects and patients with various diseases

1996 ◽  
Vol 135 (3) ◽  
pp. 345-351 ◽  
Author(s):  
Yukihiro Sakamoto ◽  
Yasumi Shintani ◽  
Kazuyo Harada ◽  
Masahiro Abe ◽  
Keiji Shitsukawa ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Activin A ◽  
Solid Cancer ◽  
Serum Samples ◽  
Response Curves ◽  
Standard Curve ◽  
Human Sera

Sakamoto Y. Shintani Y, Harada K, Abe M, Shitsukawa K, Saito S. Determination of free follistatin levels in sera of normal subjects and patients with various diseases. Eur J Endocrinol 1996:135:345–51. ISSN 0804–4643 We developed an assay system for measuring free follistatin by using an anti-follistatin mouse monoclonal antibody and [125I]activin A. The sensitivity of this assay was 0.5 μg/l and crossreactivities with inhibin. luteinizing hormone, follicle-stimulating hormone and growth hormone were all less than 0.5%. The dose-response curves of human sera and follicular fluid were parallel to the standard curve, and the follicular fluid contained a large amount of follistatin (6.4 ± 0.5 mg/l, mean ± SEM; N = 13). The within- and between-assay coefficients of variation calculated from the analysis of serum samples of four different concentrations were 3.3–7.8% and 3.9–11.0%, respectively. The recovery rates of free follistatin at five different doses were 86.4–102.4%. When activin A was added to the same sample, free follistatin recovery rate declined dose-dependently. Gel filtration analyses of human serum and follicular fluid resulted in a single peak corresponding to authentic follistatin. Using this assay, free follistatin concentrations in sera were measured in normal, pregnant and diseased subjects. The free follistatin level in serum of normal adults was 3.5 ± 0.2 μg/l (N = 60), which was significantly elevated in pregnant women (16.7 ± 1.3 μg/l, N = 56), and in patients with chronic liver disease (8.1 ± 1.1 μg/l, N = 20), chronic renal failure (6.7 ± 0.9 μg/l, N = 42), advanced solid cancer (8.5 ± 1.0 μg/l, N = 39) and hematological malignancies (6.8 ± 1.0 μg/l, N = 18). These data indicated that the free follistatin concentration in serum is detectable and varies during pregnancy and in various diseased states. Yukihiro Sakamoto, First Department of Internal Medicine, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770, Japan

scholarly journals Development of a two-site solid-phase immunochemiluminescent assay for measurement of dimeric inhibin-A in human serum and other biological fluids

1996 ◽  
Vol 42 (8) ◽  
pp. 1159-1167 ◽  
Author(s):  
D S McConnell ◽  
V Padmanabhan ◽  
T B Pollak ◽  
N P Groome ◽  
J J Ireland ◽  
...  
Keyword(s):  
Human Serum ◽  
Follicular Fluid ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Biological Fluids ◽  
Cross Reactivity ◽  
Inhibin A ◽  
Vitro Fertilization ◽  
Related Proteins

Abstract Inhibin is a heterodimeric glycoprotein that inhibits the secretion of follitropin from the pituitary and has been isolated in two distinct forms composed of a common alpha subunit and either a beta A or beta B subunit. Utilizing paired monoclonal antibodies specific to the alpha and beta A subunit, we have developed an immunochemiluminescent assay for dimeric inhibin-A. The assay is capable of quantifying free and bound inhibin-A in human serum and follicular fluid. The limit of detection is 10 ng/L. Related proteins exhibit little cross-reactivity or interference. Recovery is excellent. Whereas samples from men and postmenopausal women are near the detection limit of the assay, inhibin-A is higher in the luteal than the follicular phase of normally cycling women, 20-fold higher during in vitro fertilization treatment, and approximately 200-fold greater in pregnancy. The assay measures inhibin-A in follicular fluid from a variety of other species.

Molecular analysis of inhibin A and activin A subunit gene loci in epithelial ovarian cancer

2002 ◽  
Vol 12 (5) ◽  
pp. 443-447
Author(s):  
S. Depasquale ◽  
G. Lambert-Messerlian ◽  
M. R. Quddus ◽  
I. Campbell ◽  
M. Steinhoff ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Epithelial Ovarian Cancer ◽  
Loss Of Heterozygosity ◽  
Gene Locus ◽  
Elevated Serum ◽  
Activin A ◽  
Subunit Gene ◽  
Inhibin A ◽  
Α Subunit ◽  
Preoperative Serum

Inhibin A (α-βA) and activin A (βA-βA) are biochemically similar proteins that generally have opposite biologic functions. For example, while inhibin (α subunit) is proposed to be a tumor suppressor in some types of ovarian cancer, activin appears to stimulate tumor development. Previous reports suggest that a loss of α inhibin subunit expression and elevated serum activin levels are associated with human epithelial ovarian cancer (EOC). Our objective was to examine the α inhibin subunit gene locus on chromosome 2q for evidence of loss of heterozygosity (LOH) in cases of EOC and to correlate these results with serum activin A levels measured in the same patients. Ovarian tumor and matched healthy tissue samples were collected from 22 women with EOC. DNA was extracted and subjected to PCR analysis using 10 primers, seven from chromosome 2q (α inhibin subunit locus) and, as a control, three from chromosome 7p (inhibin/activin βA subunit). In addition, each patient had a preoperative serum activin A measurement using an ELISA assay. One (1/22) case of EOC demonstrated LOH for one microsatellite marker at the α inhibin gene locus. Thirty-six percent (8/22) of patients had an activin A level that was increased above the normal range.We conclude that loss of heterozygosity at the inhibin/activin α subunit locus is not frequently associated with EOC. More direct molecular analyses of the inhibin and activin genes are warranted to rule out mutations in cases of epithelial ovarian cancer.

scholarly journals In vivo effects of pregnancy-associated plasma protein-A, activin-A and vascular endothelial growth factor on other follicular-fluid factors during follicle deviation in mares

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 489-496 ◽  
Author(s):  
O J Ginther ◽  
E L Gastal ◽  
M O Gastal ◽  
M A Beg
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Follicular Fluid ◽  
Protein A ◽  
Activin A ◽  
Control Group ◽  
Vascular Endothelial ◽  
Inhibin A ◽  
Igf I ◽  
Endothelial Growth Factor

During a follicular wave in mares, the two largest follicles (F1 and F2) begin to deviate in diameter when F1 is a mean of 22.5 mm. The intrafollicular effects of pregnancy-associated plasma protein-A (PAPP-A), IGF-I, activin-A and vascular endothelial growth factor (VEGF) on other follicular-fluid factors during deviation were studied. In four treated groups (n= 7/group), a single dose of one of the four factors was injected into F2 when F1 was ≥20.0 mm (expected beginning of deviation). In a control group (n= 7), F2 was injected with vehicle. One day after treatment, a sample of follicular fluid was taken from F1 and F2 of the control group and from F2 of the treated groups and was assayed for free IGF-I, oestradiol, androstenedione, activin-A, inhibin-A, follistatin and VEGF. In the control group, the means for all end points were significantly greater in F1 than in F2, except that concentrations of androstenedione were lower in F1 than in F2. The treatment effects for F2 were significant as follows: PAPP-A increased the concentrations of free IGF-I, inhibin-A, follistatin and VEGF and decreased the concentrations of androstenedione; IGF-I increased the concentration of inhibin-A and decreased the concentration of androstenedione; activin-A decreased the concentrations of follistatin and androstenedione and increased the diameter of F2; and VEGF increased the concentration of IGF-I and decreased the concentration of androstenedione. These results support the hypotheses that during deviation in mares PAPP-A increases the follicular-fluid concentrations of free IGF-I, follistatin responds to changes in follicular-fluid concentrations of activin-A, and VEGF affects the concentrations of other follicular-fluid factors.

Effects of porcine follicular fluid, inhibin-A, and activin-A on goldfish gonadotropin release in vitro.

Endocrinology ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 1922-1929 ◽  
Author(s):  
W Ge ◽  
J P Chang ◽  
R E Peter ◽  
J Vaughan ◽  
J Rivier ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Activin A ◽  
Inhibin A ◽  
Gonadotropin Release ◽  
Release In Vitro

scholarly journals Nanoparticle-Aided Detection of Colorectal Cancer-Associated Glycoconjugates of Extracellular Vesicles in Human Serum

2021 ◽  
Vol 22 (19) ◽  
pp. 10329
Author(s):  
Rufus Vinod ◽  
Randa Mahran ◽  
Erica Routila ◽  
Janne Leivo ◽  
Kim Pettersson ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Human Serum ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Late Stage ◽  
Early Stage ◽  
Biological Fluids ◽  
Serum Samples ◽  
Benign Condition ◽  
Hek 293

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEACon-A, CA125WGA, CA 19.9Ma696, and CA 19.9Con-A further provided some complementation to the CD151CD63 assay. These results indicate the potential application of CD151CD63 assay for early detection of CRC patients in human serum.

scholarly journals SAT-043 Utility of Ultrasensitive Inhibin B Measurement for the Management of Men with Non-Obstructive Azoospermia

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Anne-Laure Barbotin ◽  
Ajay Kumar ◽  
Bhanu Kalra ◽  
Gopal Savjani ◽  
Valarie Mitchell ◽  
...  
Keyword(s):  
Activin A ◽  
Inhibin B ◽  
University Hospital ◽  
Testicular Sperm Extraction ◽  
Obstructive Azoospermia ◽  
Serum Samples ◽  
Inhibin A ◽  
Testosterone Levels ◽  
Medical Centers ◽  
Serum Lh

Abstract Inhibin B measurement by conventional assay(s) may be useful in the assessment of spermatogenesis in infertile male patients, especially in cases of azoospermia. Indeed, numerous previous studies have shown that Inhibin B could be helpful to predict a positive testicular sperm extraction (TESE). However, an undetectable Inhibin B concentration (&lt;10pg/mL) does not predict a TESE failure in all cases. These findings explained that most medical centers have precluded the use of Inhibin B assay in the pre-operative hormonal assessment of azoospermic men. Recently, an ultrasensitive Inhibin B assay has been developed allowing the measurement of concentrations below 10pg/mL. The current study aims to assess the clinical relevance of this new assay in men with azoospermia with undetectable Inhibin B levels by conventional assay(s). Methods: This retrospective study included 71 non-obstructive azoospermic men who had undetectable Inhibin B levels (i.e. &lt;10pg/mL by Gen II ELISA from Beckman Coulter, USA) and who underwent a TESE procedure between 2013 and 2019 in the Lille University Hospital. Serum LH, FSH and testosterone levels were systematically measured by routine immunoassays. Cryopreserved serum samples were used to perform ultrasensitive Inhibin B assay (Ultrasensitive Inhibin B, AL-195, Ansh Labs, USA). Additional hormonal assays including Inhibin A, Activin B and Activin A were performed on available subset of samples. Results: The TESE was successful, allowing sperm cryopreservation in 32.5 % (25/71) of the cases. No significant statistical difference was found in FSH, LH, or testosterone levels between patients with positive or negative TESE. By contrast, men with positive TESE had more than twice higher serum ultrasensitive Inhibin B levels (median 5.03pg/mL [1.93-8.5] vs. 2.19pg/mL [0.2-4.72], p=0.006). An ultrasensitive Inhibin B serum level &gt;3.67 pg/mL (determined by ROC analysis) was associated with increased odds ratio (OR= 4.82; 95% CI: 1.647-12.93) for positive TESE. Inhibin A, Activin B and Activin A serum concentrations did not differ significantly between the two groups. Conclusion: FSH measurement which is routinely performed in men with azoospermia was not predictive of successful TESE whereas, Inhibin B was found to be a valuable marker in predicting TESE success in this population using ultrasensitive Inhibin B assay.

Validation of an autotaxin enzyme immunoassay in human serum samples and its application to hypoalbuminemia differentiation

2008 ◽  
Vol 388 (1-2) ◽  
pp. 51-58 ◽  
Author(s):  
Kazuhiro Nakamura ◽  
Koji Igarashi ◽  
Kazufumi Ide ◽  
Ryunosuke Ohkawa ◽  
Shigeo Okubo ◽  
...  
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Serum Samples ◽  
Human Serum Samples

Inhibin A, Inhibin B and Activin A in Follicular Fluid of Infertile Women with Tubal Damage, Unexplained Infertility and Endometriosis1

2000 ◽  
Vol 43 (2) ◽  
pp. 61-69 ◽  
Author(s):  
A.V. AKANDE ◽  
S.D. KEAY ◽  
D.J. CAHILL ◽  
P.G. WARDLE ◽  
J. ASSELIN ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Activin A ◽  
Unexplained Infertility ◽  
Inhibin B ◽  
Inhibin A ◽  
Infertile Women

scholarly journals Second trimester levels of maternal serum total activin A and placental inhibin/activin alpha and betaA subunit messenger ribonucleic acids in Down syndrome pregnancy

1998 ◽  
pp. 425-429 ◽  
Author(s):  
GM Lambert-Messerlian ◽  
S Luisi ◽  
P Florio ◽  
V Mazza ◽  
JA Canick ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Placental Tissue ◽  
Normal Karyotype ◽  
Activin A ◽  
Maternal Serum ◽  
Mrna Levels ◽  
Serum Samples ◽  
Inhibin A ◽  
Ribonucleic Acids ◽  
Gapdh Mrna

OBJECTIVES: Previous data have shown that inhibin A (alpha/betaA) is increased about twofold in maternal serum samples from Down syndrome pregnancy. Our objectives were to determine whether activin A (betaA/betaA) was similarly increased in maternal serum from pregnancies affected with fetal Down syndrome, and to investigate whether increased expression of each inhibin/activin subunit occurred in placental tissue from cases of fetal Down syndrome. DESIGN AND METHODS: Maternal serum total activin A levels were measured in 20 cases of fetal Down syndrome and 100 unaffected pregnancy samples. In addition, analysis of inhibin/activin alpha and betaA subunit mRNA levels was performed in placental tissue extracts from six cases of fetal Down syndrome and six tissues with a normal karyotype. RESULTS: The median total activin A level in the Down syndrome cases was 0.82 MoM (multiples of the median); values did not differ significantly (P = 0.36, Mann-Whitney U analysis) from those in unaffected pregnancies. The inhibin alpha subunit/GAPDH mRNA ratio, but not that of betaA subunit/GAPDH mRNA, was significantly greater (P < 0.01, ANOVA) in placental tissue from Down syndrome than in control placental tissue. CONCLUSIONS: Unlike inhibin A, activin A is not significantly increased in Down syndrome relative to unaffected pregnancy. Furthermore, increased amounts of maternal serum inhibin A in Down syndrome pregnancy probably result from increased placental expression of inhibin alpha, but not betaA, subunit.

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