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.

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

Follicular fluid and assisted reproductive technology programs outcomes (literature review)

GYNECOLOGY ◽  
2020 ◽  
Vol 21 (6) ◽  
pp. 36-40
Author(s):  
Anna G. Burduli ◽  
Natalia A. Kitsilovskaya ◽  
Yuliya V. Sukhova ◽  
Irina A. Vedikhina ◽  
Tatiana Y. Ivanets ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Assisted Reproductive Technology ◽  
Follicular Fluid ◽  
Economic Cost ◽  
Reproductive Technology ◽  
Art Programs ◽  
Vitro Fertilization ◽  
Non Invasive ◽  
Established Fact

The review presents data on metabolites in the follicular fluid (FF) from the perspective of reproductive medicine and their use in order to predict outcomes of assisted reproductive technology (ART) programs. It considers various components of this biological medium (hormones, lipids, melatonin, etc.) with an assessment of their predictive value in prognosis of the effectiveness of in vitro fertilization (IVF) programs. The data on experimental directions in this field and the prospects for their use in clinical practice are presented. The article emphasizes that the growing clinical need and the unsolved problem of increasing the effectiveness of ART programs determine the need for further studies of the FF composition. Materials and methods. The review includes data related to this topic from foreign and Russian articles found in PubMed which were published in recent years. Results. Given the established fact of a direct effect of FF composition on growth and maturation of oocytes, and further, on the fertilization process, various FF metabolites are actively investigated as non-invasive markers of quality of oocytes/embryos. The article provides data on the experimental directions in this field and the prospects for their use in clinical practice. However, clinical studies of a relation between various FF metabolites levels and outcomes of IVF programs are contradictory. Conclusion. Owing large economic cost for treatment of infertility with IVF, there is need for expansion and intensification of studies to identify and use reliable predictors in prognosis of ART programs outcomes.

Calcium/Copper alginate framework doped with CuO nano-particles as a novel adsorbent for micro-extraction of benzodiazepines from human serum

2020 ◽  
Vol 16 ◽  
Author(s):  
Nadereh Rahbar ◽  
Fatemeh Ahmadi ◽  
Zahra Ramezani ◽  
Masoumeh Nourani
Keyword(s):  
Human Serum ◽  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Nano Particles ◽  
Limit Of Quantification ◽  
Analytical Methodology ◽  
Fiber Fabrication ◽  
Relative Standard ◽  
Green Procedure

Background: Sample preparation is one of the most challenging phases in pharmaceutical analysis, especially in biological matrices, affecting the whole analytical methodology. Objective: In this study, a new Ca(II)/Cu(II)/alginate/CuO nanoparticles hydrogel fiber (CCACHF) was synthesized through a simple, green procedure and applied for fiber micro solid phase extraction (FMSPE) of diazepam (DIZ) and oxazepam (OXZ) as model drugs prior to high-performance liquid chromatography-UV detection (HPLC-UV). Methods: Composition and morphology of the prepared fiber were characterized and the effect of main parameters on the fiber fabrication and extraction efficiency have been studied and optimized. Results: In optimal conditions, calibration curves were linear ranging between 0.1–500 µg L−1 with regression coefficients of 0.9938 and 0.9968. Limit of detection (LOD) (S/N=3) and limit of quantification (LOQ) (S/N=10) of the technique for DIZ and OXZ were 0.03 to 0.1 µg L−1. Within-day and between-day relative standard deviations (RSDs) for DIZ and OXZ were 6.0–12.5% and 3.3–9.4%, respectively. Conclusion: The fabricated adsorbent has been substantially employed to extraction of selected benzo-diazepines (BZDs) from human serum real specimens and the obtained recoveries were also satisfactory (82.1-109.7%).

scholarly journals 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Sinnes ◽  
Ulrike Bauder-Wüst ◽  
Martin Schäfer ◽  
Euy Sung Moon ◽  
Klaus Kopka ◽  
...  
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Solid Phase ◽  
Negative Influence ◽  
Binding Motif ◽  
Lncap Cells ◽  
Cell Binding ◽  
Binding Characteristics

Abstract Background The AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs. Results AAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay. Conclusions The AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

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