Radioimmunoassay for Serum Triiodothyronine: Evaluation of Simple Techniques to Control Interference from Binding Proteins

1974 ◽  
Vol 20 (9) ◽  
pp. 1150-1154 ◽  
Author(s):  
Victor S Fang ◽  
Samuel Refetoff
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Cross Reactivity ◽  
Heat Inactivation ◽  
Nonspecific Binding ◽  
High Concentration ◽  
Assay Sensitivity ◽  
Thyroxine Binding Globulin ◽  
Fixed Concentration

Abstract Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (T3) have been evaluated for their efficacy, and their effect on assay sensitivity and on recovery of added T3. Ethanol precipitation of serum proteins decreased the assay sensitivity, nonspecific binding was increased, and recoveries of added T3 were inconsistent. Heat-inactivation of thyroxine-binding globulin or use of 8-anilino-1naphthalene sulfonic acid (ANS) to displace T3 from thyroxine-binding globulin produced comparable recovery rates. The heat-inactivation method slightly decreased the sensitivity of the assay and prolonged the procedure, whereas use of ANS is simple, and the assay sensitivity is maintained. When sera contain a high concentration of thyroxine-binding globulin, a fixed concentration of ANS (175 µg/100 µl of serum) might be too low to displace T3 from all its binding sites, but a concentration of ANS greater than 200 µg/100 µl of serum interferes with T3 quantitation by competitively binding to the antibodies. The cross-reactivity of thyroxine to T3-antibodies varies with the antiserum. Thyroxine-binding globulin appears to be the only protein in serum that competes with the antibody for T3 binding.

CORTICOSTERONE BINDING BY MOUSE SERA DURING FOETAL AND POST-NATAL DEVELOPMENT

1977 ◽  
Vol 84 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Lia Savu ◽  
Emmanuel Nunez ◽  
Max-Fernand Jayle
Keyword(s):  
Amniotic Fluid ◽  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Normal Adult ◽  
Scatchard Analysis ◽  
Mammalian Development ◽  
Single Class ◽  
Mean Values

ABSTRACT The binding properties of corticosterone binding globulin (CBG) of mouse sera have been studied by equilibrium dialysis and electrophoretic techniques, at different stages of foetal and post-natal development. Scatchard analysis has demonstrated in all cases a single class of high affinity saturable binding sites for corticosterone. Remarkable increases of the binding capacities were observed in the foetal and pregnant sera, as compared to normal adult and immature levels. The mean values of n1M1 × g−1 of serum proteins (concentration of binding sites, n1 × moles of binding proteins M1) were 21 10−8 in 14–19 day pregnant females, 17 10−8 in the amniotic fluid, 4.2 10−8 in 14–19 day embryos, and only 0.8 10−8 in the normal adult female. Neonatal mice, aged 0–6 days exhibited no CBG activities. The association constants showed values of 2.5–4.1 108 m−1 when measured with foetal sera, and of 1.2–2.1 108 m−1 with pregnant or control adult sera and with the amniotic fluid, at 25°C. Comparative electrophoretic, thermal denaturation and competition studies with foetal and pregnant plasma CBG's are also reported. The results are discussed in relation to the origin of CBG in the foetal serum, and also with respect to similar studies in the rat, guinea pig and man. The possible biological implications of serum steroid binding proteins in mammalian development are briefly outlined.

Effect of Sodium Ethylmercurithiosalicylate (Thimerosal) on Serum Binding of Thyroid Hormones

1973 ◽  
Vol 51 (2) ◽  
pp. 156-159 ◽  
Author(s):  
Diego Bellabarba ◽  
Raymonde Tremblay
Keyword(s):  
Thyroid Hormones ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Free Fraction ◽  
Inhibitory Effect ◽  
Free T4 ◽  
Thyroxine Binding Globulin ◽  
Free T3 ◽  
Serum Binding Protein ◽  
Barbital Buffer

Sodium ethylmercurithiosalicylate (Thimerosal, Merthiolate) has been found to interfere with the binding of thyroid hormones to serum proteins. Dialysis studies showed that this compound, added to serum in concentrations varying from 90 to 360 mg/100 ml, caused an increase of the dialyzable or free fraction of thyroxine (T4) and triiodothyronine (T3). The increase was higher for the free T4 (3.8- to 18-fold) than for the free T3 fraction (2.3- to 5-fold). Electrophoretic studies on the distribution of tracer amounts of labeled T4 among the serum binding proteins revealed that the inhibitory effect of sodium ethylmercurithiosalicylate was exerted mainly on thyroxine binding globulin (TBG). In presence of this compound (180 mg/100 ml of serum) the percentage of tracer T4 bound to TBG was reduced from 53% to 9%. These findings were also confirmed by examining the binding of tracer amounts of labeled T4 and T3 in a serum diluted in barbital buffer, which inhibits the hormonal binding to thyroxine binding prealbumin and albumin. In presence of sodium ethylmercurithiosalicylate a significant displacement of both T4 and T3 from the serum binding protein (TBG) was observed.

A Reassessment of 8-Anilino-1-Naphthalene Sulphonic Acid as a Thyroxine Binding Inhibitor in the Radioimmunoassay of Thyroxine

1977 ◽  
Vol 14 (1-6) ◽  
pp. 330-334 ◽  
Author(s):  
S. E. Evans ◽  
W. A. Burr ◽  
T. C. Hogan
Keyword(s):  
Polyethylene Glycol ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Sulphonic Acid ◽  
Thyroxine Binding Globulin ◽  
Double Antibody ◽  
Resin Separation

The effectiveness of 8-anilino-1-naphthalene sulphonic acid (ANS) in the radioimmunoassay (RIA) of thyroxine (T4) as an inhibitor of the binding of T4 to serum T4-binding proteins is assessed. The optimum ANS concentration is dependent upon the antiserum and the method used for separating free and bound T4. If T4 binding to serum proteins is not completely inhibited, resin separation methods may yield low values, while polyethylene glycol and double-antibody methods may produce high values for T4 concentration. Even with optimum ANS concentration gross errors in measurement of T4 may occur in patients with high thyroxine-binding globulin (TBG) concentrations.

scholarly journals Quantifying Spurious Free T4 Results Attributable to Thyroxine-Binding Proteins in Serum Dialysates and Ultrafiltrates

2007 ◽  
Vol 53 (5) ◽  
pp. 985-988 ◽  
Author(s):  
Kristofer S Fritz ◽  
R Bruce Wilcox ◽  
Jerald C Nelson
Keyword(s):  
Serum Protein ◽  
Total Protein ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Free T4 ◽  
Serum Total Protein ◽  
Semipermeable Membranes ◽  
Serum Free ◽  
Thyroxine Binding Globulin

Abstract Background: Direct equilibrium dialysis and direct ultrafiltration free thyroxine (T4) assays rely on semipermeable membranes to exclude T4-binding serum proteins from dialysates and ultrafiltrates. The presence of these proteins in dialysates or ultrafiltrates will yield spuriously high free T4 values when free T4 is quantified by RIA. Methods: We used a nonanalog free T4 RIA that detects and quantifies dialyzable and ultrafilterable serum free T4 to detect T4-binding serum proteins. Two equilibrium dialysis devices and 3 ultrafiltration devices were used to illustrate this application. Displacements of [125I]T4 from anti-T4 by various concentrations of T4-depleted thyroxine-binding globulin, albumin, and serum total protein were compared to displacements by various concentrations of free T4. Results: Both dialysis devices excluded detectable T4-binding serum proteins from dialysates. Two of 3 ultrafiltration devices excluded detectable T4-binding serum proteins from ultrafiltrates. One did not, and its ultrafiltrate yielded spurious free T4 values that correlated directly with serum protein concentrations. Conclusion: The presence or absence of T4-binding proteins in dialysates and ultrafiltrates and the spurious free T4 values that these proteins cause can be documented using a nonanalog free T4 RIA.

Identification of the serum binding proteins for iron, zinc, cadmium, nickel, and calcium.

1983 ◽  
Vol 29 (4) ◽  
pp. 629-633 ◽  
Author(s):  
B J Scott ◽  
A R Bradwell
Keyword(s):  
Binding Proteins ◽  
Serum Proteins ◽  
Radioactive Isotopes ◽  
Two Dimensional ◽  
High Concentration ◽  
Zinc Cadmium

Abstract The binding of five biologically important metals to serum proteins has been studied. After suitable radioactive isotopes were added to serum proteins separated and precipitated by two-dimensional immunoelectrophoresis, the sample plates were exposed to roentgenogram film. 59Fe bound to transferrin alone; 65Zn bound mostly to albumin, but also to another 12 proteins; 109Cd was mostly associated with alpha 2-macroglobulin, but was also present on albumin, immunoglobulins G and A, and prealbumin; 63Ni, added in high concentration, was associated with an alpha 2-mobility protein and albumin; and, finally, 45Ca was mostly bound to albumin, but seven other binding proteins were also identified, with transferrin predominant. The results are not quantitative, but the technique is simple and specific, and the information gained can direct further studies on isolated proteins.

ENDOCRINE CHANGES BEFORE AND AFTER THE MENARCHE

1973 ◽  
Vol 74 (4) ◽  
pp. 685-694 ◽  
Author(s):  
B.-A. Lamberg ◽  
R.-L. Kantero ◽  
P. Saarinen ◽  
O. Widholm
Keyword(s):  
Binding Proteins ◽  
Maturation Process ◽  
Hormone Binding ◽  
Maximal Level ◽  
Continuous Increase ◽  
Mean Values ◽  
Thyroxine Binding Globulin ◽  
Before And After ◽  
Free Thyroxine Index ◽  
Level 1

ABSTRACT In an endocrine survey of healthy girls aged 8 to 20 years before and after the menarche, the serum thyroxine (T4), uptake of triiodothyronine by Sephadex (T3U), and the binding capacities of thyroxine binding globulin (TBG) and pre-albumin (TBPA) were measured, and a free thyroxine index (FTI = T4 × T3U) was calculated. The subjects were grouped according to skeletal age (SA) until the menarche and after this in the post-menarcheal age (PMA), expressed in years. T4 and FTI increased concomitantly and reached peak values of 8.40 μg/100 ml and 8.40, respectively, at 2–3 years PMA. The corresponding mean values for post-menarcheal girls (7.74 μg/100 ml and 7.51) differed statistically significantly from the means before the menarche (7.03 μg/ 100 ml and 6.75). The TBG remained virtually unchanged during the whole period, whereas the TBPA showed a continuous increase and reached a maximal level 1–2 years after the menarche. The maturation process in girls in some way involves an increase in the total and free T4 level which is not dependent on hormone binding proteins.

Eliminating Nonspecific Binding Sites for Highly Reliable Immunoassay via Super-resolution Multicolor Fluorescence Colocalization

Nanoscale ◽  
2021 ◽  
Author(s):  
Shenfei Zong ◽  
Yun Liu ◽  
Kuo Yang ◽  
Zhaoyan Yang ◽  
Zhuyuan Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Super Resolution ◽  
Specific Adsorption ◽  
Nonspecific Binding ◽  
Nonspecific Adsorption ◽  
Multicolor Fluorescence

Non-specific adsorption in immunoassays has always been a major problem that affects the reliability of assay results. Despite the emergence of various methods which can reduce nonspecific adsorption, a universal...

scholarly journals Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

2015 ◽  
Vol 471 (3) ◽  
pp. 403-414 ◽  
Author(s):  
M. Florencia Rey-Burusco ◽  
Marina Ibáñez-Shimabukuro ◽  
Mads Gabrielsen ◽  
Gisela R. Franchini ◽  
Andrew J. Roe ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Ligand Binding ◽  
Tissue Distribution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Retinol Binding Protein ◽  
Internal Cavity ◽  
Binding Characteristics ◽  
Necator Americanus ◽  
Ligand Binding Sites

Necator americanus fatty acid and retinol-binding protein-1 (Na-FAR-1) is an abundantly expressed FAR from a parasitic hookworm. The present work describes its tissue distribution, structure and ligand-binding characteristics and shows that Na-FAR-1 expands to transport multiple FA molecules in its internal cavity.

scholarly journals Specific Binding of Rubidium in Chlorella

1962 ◽  
Vol 45 (5) ◽  
pp. 959-977 ◽  
Author(s):  
Dan Cohen
Keyword(s):  
Active Transport ◽  
Binding Sites ◽  
Specific Binding ◽  
High Concentration ◽  
External Concentration ◽  
Specific Binding Sites ◽  
Dense Suspension ◽  
Concentration Of Ions ◽  
The Individual ◽  
A Site

Specific binding sites for potassium, which may be components of the carriers for active transport for K in Chlorella, were characterized by their capacity to bind rubidium. A dense suspension was allowed to take up Rb86 from a low concentration of Rb86 and a high concentration of ions which saturate non-specific sites. The amount bound was derived from the increase in the external concentration of Rb86 following addition of excess potassium. The sites were heterogeneous. The average affinity of Rb and various other ions for the sites was determined by plotting the degree of displacement of Rb86 against log molar concentration of the individual ions. Interpolation gave the concentration for 50 per cent displacement of Rb, which is inversely related to affinity. The order of affinity was not changed when the cells were frozen, or boiled either in water or in 70 per cent ethanol. The affinity is maximal for ions with a crystalline radius of 1.3 to 1.5 A and a high polarizability, and is not related to the hydrated radius or valency. It is suggested that binding groups in a site are rigidly arranged, the irregular space between them being 2.6 to 3.0 A across, so that affinity is high for ions of this diameter and high polarizability.

scholarly journals Increase in alkaline phosphatase activity in calvaria cells cultured with diphosphonates

1979 ◽  
Vol 183 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R Felix ◽  
H Fleisch
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Heat Inactivation ◽  
High Concentration ◽  
Control Value ◽  
Km Value ◽  
Inhibitor Of Protein Synthesis

1. Dichloromethanediphosphonate and to a lesser degree 1-hydroxyethane-1,1-diphosphonate, two compounds characterized by a P-C-P bond, increased the alkaline phosphatase activity of cultured rat calvaria cells up to 30 times in a dose-dependent fashion. 2. Both diphosphonates also slightly inhibited the protein synthesis in these cells. 3. Thymidine, an inhibitor of cell division, did not inhibit the induction of the enzyme, indicating that the increase in enzyme activity was not due to the formation of a specific population of cells with high alkaline phosphatase activity. 4. The effect on alkaline phosphatase was suppressed by the addition of cycloheximide, an inhibitor of protein synthesis. 5. After subculturing the stimulated cells in medium without diphosphonates, the enzyme activity fell almost to the control value. 6. Bovine parathyrin diminished the enzyme activity of the control cells and the cells treated with dichloromethanediphosphonate; however, at high concentration the effect of parathyrin was greater on the diphosphonate-treated cells than on the control cells. 7. The electrophoretic behaviour, heat inactivation, inhibition by bromotetramisole or by phenylalanine, and the Km value of the induced enzyme were identical with that of the control enzyme.

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