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.

scholarly journals Characterization and quantitation of the circulating forms of serum transferrin receptor using domain-specific antibodies

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 234-238
Author(s):  
YJ Shih ◽  
RD Baynes ◽  
BG Hudson ◽  
JD Cook
Keyword(s):  
Amino Acids ◽  
Sickle Cell ◽  
Transferrin Receptor ◽  
Normal Subjects ◽  
Extracellular Domain ◽  
Total Serum ◽  
Intracellular Domain ◽  
Domain Specific ◽  
Human Transferrin Receptor ◽  
Human Sera

Abstract To characterize the nature of the immunoreactive transferrin receptor in human serum, antisera were developed to peptide sequences of the extracellular domain of human transferrin receptor between amino acids 107 and 120 and the intracellular domain between amino acids 40 and 54. Antisera against the extracellular domain exhibited reactivity against both purified intact receptor and immunopurified circulating receptor, whereas antisera against the intracellular domain reacted only with intact receptor. Using competitive binding enzyme-linked immunosorbent assays, transferrin receptor in ultracentrifuged sera from normal subjects and patients with sickle cell anemia could be detected with antisera against the extracellular but not the intracellular domain. When the pellet obtained by ultracentrifugation of these sera was assayed after solubilization in 1% teric (polyoxyethylene-9-lauryl ether), only 0.6% of total serum receptor was detected in normal subjects and 3.8% in subjects with sickle cell disease. Roughly equal amounts of this pelleted immunoactivity were detected with antibodies against the extracellular and intracellular domains. These results indicate that less than 1% of transferrin receptor in normal human sera is intact receptor consistent with an exosomal origin and that virtually all circulating transferrin receptor is in the form of a truncated extracellular domain.

scholarly journals Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1971 ◽  
Vol 122 (5) ◽  
pp. 623-631 ◽  
Author(s):  
Anne M. S. Marr ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Chemical Composition ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Subunit Structure ◽  
Terminal Amino

1. Tamm–Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm–Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000±6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm–Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm–Horsfall antiserum.

Characterization and quantitation of the circulating forms of serum transferrin receptor using domain-specific antibodies

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 234-238 ◽  
Author(s):  
YJ Shih ◽  
RD Baynes ◽  
BG Hudson ◽  
JD Cook
Keyword(s):  
Amino Acids ◽  
Sickle Cell ◽  
Transferrin Receptor ◽  
Normal Subjects ◽  
Extracellular Domain ◽  
Total Serum ◽  
Intracellular Domain ◽  
Domain Specific ◽  
Human Transferrin Receptor ◽  
Human Sera

To characterize the nature of the immunoreactive transferrin receptor in human serum, antisera were developed to peptide sequences of the extracellular domain of human transferrin receptor between amino acids 107 and 120 and the intracellular domain between amino acids 40 and 54. Antisera against the extracellular domain exhibited reactivity against both purified intact receptor and immunopurified circulating receptor, whereas antisera against the intracellular domain reacted only with intact receptor. Using competitive binding enzyme-linked immunosorbent assays, transferrin receptor in ultracentrifuged sera from normal subjects and patients with sickle cell anemia could be detected with antisera against the extracellular but not the intracellular domain. When the pellet obtained by ultracentrifugation of these sera was assayed after solubilization in 1% teric (polyoxyethylene-9-lauryl ether), only 0.6% of total serum receptor was detected in normal subjects and 3.8% in subjects with sickle cell disease. Roughly equal amounts of this pelleted immunoactivity were detected with antibodies against the extracellular and intracellular domains. These results indicate that less than 1% of transferrin receptor in normal human sera is intact receptor consistent with an exosomal origin and that virtually all circulating transferrin receptor is in the form of a truncated extracellular domain.

Immunoradiometric assay of parathyrin with polyclonal and monoclonal region-specific antibodies

1990 ◽  
Vol 36 (2) ◽  
pp. 271-276 ◽  
Author(s):  
R Bouillon ◽  
W Coopmans ◽  
D E Degroote ◽  
D Radoux ◽  
P H Eliard
Keyword(s):  
Primary Hyperparathyroidism ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Immunoradiometric Assay ◽  
Healthy Elderly ◽  
Normal Individuals ◽  
Undetectable Serum ◽  
Capture Antibody ◽  
Carboxyl Terminal ◽  
Serum Pth

Abstract In this immunoradiometric assay (IRMA) of parathyrin (PTH) a polyclonal anti-amino-PTH(1-34) is the capture antibody and a radiolabeled monoclonal anti-hPTH(44-68) is the second antibody. Gel filtration of serum from a hyperparathyroid patient yielded only a single peak of PTH, corresponding to the elution position of synthetic PTH(1-84). Healthy elderly individuals (ages 78 +/- 5 y, mean +/- SD, n = 45) had PTH concentrations (21 +/- 13 ng/L) not significantly higher than those from healthy younger (38 +/- 11 y) adults (20 +/- 8 ng/L, n = 94). Assay results agreed well with those obtained with a carboxyl-terminal PTH assay both in normal subjects (r = 0.63, P less than 0.001) and in patients with primary hyperparathyroidism (r = 0.59, P less than 0.001). Both assays equally discriminated patients with surgically confirmed primary hyperparathyroidism from normal individuals, but the PTH(1-84) IRMA also allowed a nearly absolute discrimination between normal subjects and patients with primary hypoparathyroidism (undetectable serum PTH in 18 of 21 cases) and secondary hypoparathyroidism (caused by hypercalcemia that was caused by a malignant tumor, PTH 1.3 +/- 1.3 ng/L, n = 32). Moreover, the PTH(1-84) IRMA is more sensitive (detection limit in serum, 0.8 ng/L) and easier and quicker to perform than the carboxyl-terminal assay.

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

IMMUNOLOGICAL REACTIVITY OF INSULIN IN HUMAN SERUM

1966 ◽  
Vol 53 (4) ◽  
pp. 673-680 ◽  
Author(s):  
Torsten Deckert ◽  
Kai R. Jorgensen
Keyword(s):  
Normal Subjects ◽  
The Other ◽  
Human Pancreas ◽  
Porcine Pancreas ◽  
Crystalline Insulin ◽  
Endogenous Insulin ◽  
Significant Difference ◽  
Human Sera ◽  
Normal Human ◽  
The One

ABSTRACT The purpose of this study was to investigate whether a difference could be demonstrated between crystalline insulin extracted from normal human pancreas, and crystalline insulin extracted from bovine and porcine pancreas. Using Hales & Randle's (1963) immunoassay no immunological differences could be demonstrated between human and pig insulin. On the other hand, a significant difference was found, between pig and ox insulin. An attempt was also made to determine whether an immunological difference could be demonstrated between crystalline pig insulin and crystalline human insulin from non diabetic subjects on the one hand and endogenous, circulating insulin from normal subjects, obese subjects and diabetic subjects on the other. No such difference was found. From these experiments it is concluded that endogenous insulin in normal, obese and diabetic human sera is immunologically identical with human, crystalline insulin from non diabetic subjects and crystalline pig insulin.

Biochemische Aspekte des Aminosäuren-Stoffwechsels bei Psoriasis vulgaris / Biochemical Aspects of Amino Acid Metabolism in Psoriasis vulgaris

1973 ◽  
Vol 28 (7-8) ◽  
pp. 449-451 ◽  
Author(s):  
G. Peter ◽  
H. Angst ◽  
U. Koch
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Free Amino Acids ◽  
Free Amino ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Psoriasis Vulgaris ◽  
Normal Subjects ◽  
Pathological Process

Free and protein-bound amino acids in serum and scales were investigated. In serum the bound amino acids of psoriatics are significantly higher with exception of Pro, Met, Tyr and Phe in contrast to normal subjects. For free amino acids the differences between normal subjects and psoriatics found in serum and scales are not significant. Results are discussed in relation to the single amino acids and the biochemical correlations are outlined which takes the pathological process as a basis.

Tissue Deposition of Zinc from a Zinc Chelate and from Inorganic Zinc in Rats

1994 ◽  
Vol 1994 ◽  
pp. 171-171 ◽  
Author(s):  
Ronan Power ◽  
Kevin Cashman ◽  
Albert Flynn
Keyword(s):  
Amino Acids ◽  
Tissue Distribution ◽  
Wistar Rats ◽  
Gel Filtration ◽  
Normal Diet ◽  
Farm Animals ◽  
Trace Minerals ◽  
Inorganic Salts ◽  
Male Wistar Rats ◽  
Differential Tissue

Some reports have suggested differential tissue deposition of dietary trace minerals such as Zinc (Zn) when supplied to farm animals either chelated to amino acids or as inorganic salts. To test this hypothesis, an experiment was conducted to determine the ultimate tissue distribution of Zinc in rats fed either a radioactively-labeled 65Zn-chelate or 65ZnSO4. The 65Zn-chelate was prepared by heating a solution of 65ZnSO4 and an equimolar mixture of glycine and methionine for 5 minutes at 90°C. The resulting chelate was then separated from unincorporated 65ZnSO4 by gel filtration chromatography. Ten 25-d old male wistar rats (mean weight 34.5 g) were randomized by weight into two groups (n = 5/group), fasted for 18 hours and given 0.4 ml (8 μg Zn, 1 μCi65Zn) of one or other labelled solution by gavage. Four hours later, animals were returned to their normal diet for the duration of the experiment. The 65Zn activity of the animals was determined two hours after administration and daily thereafter for 7 days.

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.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

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