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

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

Identification of N-Monoacetylcystine in Uraemic Plasma

1981 ◽  
Vol 60 (3) ◽  
pp. 331-334 ◽  
Author(s):  
F. Gejyo ◽  
G. Ito ◽  
Y. Kinoshita
Keyword(s):  
Plasma Levels ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Cysteic Acid ◽  
Homocysteic Acid ◽  
Normal Limits ◽  
Acidic Nature ◽  
Sulphur Amino Acids

1. An unidentified ninhydrin-positive substance of an acidic nature was detected in the plasma of uraemic patients. This substance was isolated from haemodialysate by ion-exchange chromatography and gel filtration, and identified as a sulphur-containing amino acid: N-monoacetylcystine. 2. The quantitative determination of sulphur amino acids in plasma revealed that the plasma levels of cysteic acid, homocysteic acid, taurine, cystine and cystathionine as well as N-monoacetylcystine in uraemic patients were markedly higher than in normal subjects (P < 0.001 for each). However, the plasma levels of methionine in uraemic patients were within normal limits.

Immunochemical Determination of Serum Albumin with a Centrifugal Analyzer

1975 ◽  
Vol 21 (2) ◽  
pp. 195-198 ◽  
Author(s):  
Mogens Blom ◽  
Niels Hjørne
Keyword(s):  
Polyethylene Glycol ◽  
Reaction Time ◽  
Serum Albumin ◽  
Human Serum ◽  
Albumin Concentration ◽  
Good Precision ◽  
Standard Curve ◽  
Immunochemical Determination ◽  
Human Sera

Abstract The turbidity resulting from the reaction between albumin and specific anti-human serum was measured with a good precision by using a GEMSAEC centrifugal analyzer. The reaction was enhanced by polyethylene glycol to shorten the reaction time (5 min) and to displace the point of equivalence between antigen and antibody to an albumin concentration unlikely to occur in human sera (about 100 g/liter). An additional program for the computer was necessary to fit the absorbance readings of individual sera to the nonlinear standard curve. Serum albumin values obtained by the described method correlated well with values obtained by the electroimmuno-technique. About 100 samples could be analyzed per hour, 500 µl of 100-fold diluted antiserum being used per specimen.

Measurement of serum bone gla-protein (BGP) in humans with an ovine BGP-based radioimmunoassay

1990 ◽  
Vol 36 (9) ◽  
pp. 1620-1624 ◽  
Author(s):  
P Pastoureau ◽  
P D Delmas
Keyword(s):  
Amino Acids ◽  
Primary Hyperparathyroidism ◽  
Renal Osteodystrophy ◽  
Clinical Investigation ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Conformational Epitope ◽  
Cross Reactivity ◽  
Bone Gla Protein ◽  
Human Sera

Abstract Most RIAs of serum bone gla-protein (BGP; also called osteocalcin) used for clinical investigation are based on bovine BGP for standard, tracer, and immunogen because of the homology between bovine and human BGP. However, ovine BGP differs from human BGP by only five amino acids, being identical from residues 11 to 49, as compared with homology at residues 20-49 between bovine and human BGP. In screening various anti-ovine BGP polyclonal anti-sera we selected one (R310) that exhibits apparently complete cross-reactivity with human BGP, as assessed by dilutions of 13 human sera from normal subjects and from patients with bone disease. This RIA gave a 42% binding at a 10,000-fold final dilution, with intra- and interassay variations less than 7% and 11%, respectively. Gel-filtration chromatography of human serum showed a single immunoreactive peak. Synthetic fragments of human BGP 1-10, 7-19, 25-37, and 37-49 were not recognized by R310, suggesting that either a mid-molecule region or a conformational epitope was its target. Using this RIA, we determined that serum BGP increased with age in women (P less than 0.02), by a mean of 90% from ages 30 to 70 years. Serum BGP was also increased in patients with primary hyperparathyroidism, renal osteodystrophy, and Paget's disease. In contrast with the "normal" concentrations of BGP detected with an anti-bovine BGP antiserum (R102), serum BGP was increased in patients with postmenopausal osteoporosis as measured with the R310 ovine assay, suggesting a greater sensitivity for the latter assay.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was &lt; or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually &lt; or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently &gt; 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

Determination of alpha-amylase activity: methods comparison and commutability study of several control materials

1995 ◽  
Vol 41 (3) ◽  
pp. 435-438 ◽  
Author(s):  
G Gubern ◽  
F Canalias ◽  
F J Gella
Keyword(s):  
Human Serum ◽  
Amylase Activity ◽  
Alpha Amylase ◽  
Human Origin ◽  
Serum Samples ◽  
Chromophore Group ◽  
Laboratory Control ◽  
Wide Range ◽  
Human Sera

Abstract Six different methods for alpha-amylase determination were compared by assaying human serum samples covering a wide range of alpha-amylase values. All the methods studied use as substrate a maltooligosaccharide with a chromophore group at the reducing end; some are chemically blocked at the nonreducing end. Intermethod comparison by regression and correspondence analyses showed significant differences for two methods. The commutability of 12 commercial control materials containing alpha-amylase was also assessed by the different methods in comparison with human serum specimens containing the pancreatic and salivary isoenzymes. We also studied the behavior of pancreatic and salivary materials prepared in our laboratory. Control materials with alpha-amylase of non-human origin were not commutable with the enzyme in human sera and should not be used for intermethod calibration.

IMMUNOREACTIVE GROWTH HORMONE IN HUMAN URINE

1972 ◽  
Vol 71 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Kristian F. Hanssen
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Linear Relationship ◽  
Human Urine ◽  
Gel Filtration ◽  
Human Growth ◽  
Normal Subjects ◽  
Normal Human ◽  
Radio Immunoassay

ABSTRACT By using a double antibody radio-immunoassay (pre-precipitation technique) for the determination of immunoreactive human growth hormone (IRHGH) in normal human urine concentrated by dialysis and lyophilization, a factor was revealed that displaces 125I-HGH from HGH antibodies. This displacement was neither due to salts nor to glucose; it is suggested that it is due to IRHGH in the urine. A linear relationship between dilution of urine and the measured IRHGH concentration was obtained. Recovery of exogenous HGH was between 70–105%. The recovery of IRHGH from different volumes of urine following dialysis and lyophilization was between 97–110%. Plasma IRHGH and urinary IRHGH was measured simultaneously after HGH injection in a normal subject. A correlation was shown between plasma IRHGH and urinary IRHGH. In 9 normal subjects, the urinary IRHGH ranged from 28–53 ng/24 h. The excretion of urinary IRHGH was increased in acromegaly and was diminished in some, but not in all patients with adult hypopituitarism. The urinary IRHGH was further studied by gel filtration. It was recovered in one peak corresponding to a molecular weight of approximately 20 000 – 30 000. However, in the present work it was not clarified whether the urinary IRHGH represents pituitary HGH excreted in the urine or a metabolite of high molecular weight with retained immunological properties.

scholarly journals Determination of diazepam and its metabolites in serum by the use of liquid chromatography: Mass spectrometry method

2007 ◽  
Vol 64 (10) ◽  
pp. 659-662 ◽  
Author(s):  
Snezana Djordjevic ◽  
Vesna Kilibarda
Keyword(s):  
Biological Fluids ◽  
Accurate Method ◽  
Reversed Phase ◽  
Active Metabolites ◽  
Serum Samples ◽  
Standard Curve ◽  
Routine Identification ◽  
Selective Ion Monitoring ◽  
Pharmacologically Active

Background/Aim. Diazepam is a benzodiazepine anxyolitic. Metabolism of diazepam takes place in liver which generates pharmacologically active metabolites N-desmethyldiazepam, temazepam and oxazepam. The aim of this study was to develop and validate the method of liquid chromatographymass spectrometry (LC-MS) for separation and determination of diazepam and its active metabolites in the serum of rats samples after i.p. application of diazepam in a dose of 10 mg/kg. Methods. The serum samples taken from Wistar rats, were used in LC-MS analysis after the application of 10 mg/kg of diazepam i.p. Results. After alkaline extraction from the serum samples with diethylether and separation on a C18 reversed-phase column by using mobile phase methanolglacial acetic acid-water (50:1:49 v/v), diazepam and its metabolites were quantified. Determination was performed in a selective ion monitoring (SIM) mode, thereby the other exogenous and endogenous compounds did not interfere with this assay. Diazepam, N-desmethyldiazepam, oxazepam and temazepam were eluted in 14 minutes. The standard curve was linear in the range from 10-2 000 ng/ml. The limits of detection for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 4.37, 3.13, 4.38 and 7.31 ng/ml, respectively. The limits of quantitation for diazepam, Ndesmethyldiazepam, oxazepam and temazepam were 14.58, 10.41, 14.59 and 24.36 ng/ml, respectively. Conclusion. The described LC-MS is a simple, sensitive, specific and accurate method and could be used for routine identification and quantification of small concentrations of diazepam and its metabolites in biological fluids.

Kinetic fluorometric determination of aluminum in serum.

1986 ◽  
Vol 32 (8) ◽  
pp. 1481-1483 ◽  
Author(s):  
P C Ioannou ◽  
E A Piperaki
Keyword(s):  
Atomic Absorption ◽  
Metal Chelate ◽  
Linear Regression Analysis ◽  
Spectrometric Method ◽  
Serum Samples ◽  
Standard Curve ◽  
Aluminum Ions ◽  
Standard Additions ◽  
Atomic Absorption Spectrometric

Abstract We describe a simple fluorometric method for determining aluminum in serum samples by monitoring the rate of reaction of 2-hydroxy-1-naphthaldehyde-p-methoxybenzoylhydrazone with aluminum ions. The emission of the resulting fluorescent metal-chelate formed is measured at 475 nm. Aluminum was measured in the supernate of serum after proteins were removed by precipitation with concentrated nitric acid, and calculations were based on the technique of standard additions. Within-run precision (CV) was 7.8% and 4.8% at mean aluminum concentrations of 7.7 and 60.7 micrograms/L, respectively (n = 10); between-run precision (CV) was 8.9% and 5.7% at mean aluminum concentrations of 23.3 and 46.8 micrograms/L, respectively (n = 10). The standard curve for the method is linear over the range of 0-250 micrograms of aluminum per liter. Samples from 49 patients were analyzed for aluminum by the proposed method (y) and by electrothermal atomic absorption spectroscopy (x). Linear regression analysis of the results yielded the equation y = 0.98x + 2.3 (r = 0.989, Syx = 6.7). The proposed method is comparable in sensitivity to the well-accepted atomic absorption spectrometric method but is simpler and less expensive.

Mechanized determination of the apparent unbound unconjugated bilirubin concentration in serum.

1979 ◽  
Vol 25 (8) ◽  
pp. 1444-1447 ◽  
Author(s):  
R P Wennberg ◽  
L F Rasmussen ◽  
C E Ahlfors ◽  
T Valaes
Keyword(s):  
Total Bilirubin ◽  
Gel Filtration ◽  
Operation Time ◽  
Serum Samples ◽  
Bilirubin Concentration ◽  
Coefficients Of Variation ◽  
Control Serum ◽  
Total Bilirubin Concentration ◽  
Unbound Bilirubin

Abstract The peroxidase method for determining the apparent unbound bilirubin concentration in serum has been automated by use of a programmable, computer-directed spectrophotometer. This mechanized assay determines the total bilirubin concentration and apparent unbound bilirubin concentration in serum samples and titrates the serum with bilirubin to estimate the effect of increasing total bilirubin concentrations on the apparent unbound bilirubin concentration. The entire analysis requires 0.1 mL of serum and 4 min operation time, as compared with about 30 min for the manual method. The coefficients of variation for determination of the apparent unbound bilirubin concentration in bilirubin-enriched commercial control serum were 2.8% within-day and 5.6% between-day. Bilirubin--albumin binding in serum samples from infants with severe hyperbilirubinemia was analyzed by the manual peroxidase method, the automated peroxidase method, and Sephadex gel filtration. Good correlation was found among all three methods.

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