Graphical determination of specific activity, binding constants, and antibody-site concentrations for radioimmunoassays, with application to thymopoietin.

1980 ◽  
Vol 26 (6) ◽  
pp. 718-723 ◽  
Author(s):  
F H Verhoff ◽  
P J Lisi ◽  
C D Fischer ◽  
J W Teipel ◽  
G Goldstein ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Binding Constants ◽  
Experimental Information ◽  
Antibody Binding ◽  
Displacement Analysis ◽  
Additional Information ◽  
Graphical Technique ◽  
Materials Used

Abstract A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.

Graphical determination of specific activity, binding constants, and antibody-site concentrations for radioimmunoassays, with application to thymopoietin.

1980 ◽  
Vol 26 (6) ◽  
pp. 718-723
Author(s):  
F H Verhoff ◽  
P J Lisi ◽  
C D Fischer ◽  
J W Teipel ◽  
G Goldstein ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Binding Constants ◽  
Experimental Information ◽  
Antibody Binding ◽  
Displacement Analysis ◽  
Additional Information ◽  
Graphical Technique ◽  
Materials Used

Abstract A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.

Determination of specific activity of labeled ligands by nonlinear regression analysis

1993 ◽  
Vol 265 (5) ◽  
pp. E728-E735 ◽  
Author(s):  
J. A. Durr
Keyword(s):  
Regression Analysis ◽  
Nonlinear Regression ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Nonlinear Regression Analysis ◽  
Independent Variables ◽  
Lysine Vasopressin ◽  
Dependent Variables

The specific radioactivity (SA) of 125I-lysine vasopressin (LVP) was determined by analyzing the binding B (cpm/tube) of variable amounts of tracer T (cpm/tube) to a constant amount of an LVP antibody, in the presence of known quantities L (mol/tube) of LVP standards. The parameters of the equations B = f(F) and B/F = g(T), describing B as a function of free F (cpm/tube) tracer or the ratios B/F as a function of T, were first calculated by nonlinear regression analysis of the results obtained with tracer alone. Then the dependent variables B or B/F were measured in the presence of LVP and analyzed with the same equations by substituting the independent variables F or T with (F + alpha FL) and (T + alpha L), respectively, where alpha (cpm/mol) represents a measure of the SA and FL (FL = L.F/T), free LVP, respectively. The SA was thus treated as an unknown parameter to be calculated by nonlinear regression. This method was compared with the traditional interpolation of the SA from the self-displacement and standard curves. Tracer and ligand were found to have the same affinity for the binding sites, since the set of equations B = f(F + alpha FL) and B/F = g(T + alpha L), describing the binding of the tracer in the presence of LVP and equations B = f(F) and B/F = g(T) to which these equations reduce in the absence of LVP (L = 0), had identical binding parameters. To be valid, any method based on self-displacement requires that the tracer and standards have the same affinity for the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals An Isotopic Method for the Determination of Vitamin B12 Levels in Blood

Blood ◽  
1963 ◽  
Vol 21 (1) ◽  
pp. 70-79 ◽  
Author(s):  
RUSSEL M. BARAKAT ◽  
ROGER P. EKINS
Keyword(s):  
Vitamin B12 ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Binding ◽  
Normal Subjects ◽  
Free Form ◽  
Protein Binding Sites ◽  
Isotopic Method ◽  
Specific Binding Protein

Abstract Vitamin B12 is normally present in plasma mainly bound to a specific binding protein. Addition of exogenous vitamin ultimately results in saturation of protein binding sites and excess vitamin remains in free form. The ratio of free to bound fractions thus quantitatively depends upon the concentration of exogenous compound. This observation has been utilized to determine amounts of vitamin B12 extracted from the blood of normal subjects and of patients with certain pathologic conditions. The method is simple and reproducible. The sensitivity of the method is such that vitamin levels down to roughly 20 µµg./ml. may be evaluated using labeled vitamin B12 of a specific activity of about 1 µc./µg. Repeated assays on identical specimens of normal plasma have shown a reproducibility of about 5-6 per cent. Results on 39 normal subjects gave a range of 330-1070 µµg./ml. with an average of 611 ± 167. Values observed in plasma taken from patients suffering from pernicious anemia were around 100 µµg./ml. or less. Results on subjects with other pathologic conditions are also presented and the limitations of the method are discussed.

Determination of the binding sites and binding constants between Pb(ii) and DNA using capillary electrophoresis combined with electrothermal atomic absorption spectrometry

2015 ◽  
Vol 30 (4) ◽  
pp. 903-908 ◽  
Author(s):  
Yuying Liang ◽  
Biyang Deng ◽  
Caiying Shen ◽  
Xiangdong Qin ◽  
Shaojun Liang
Keyword(s):  
Capillary Electrophoresis ◽  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Binding Sites ◽  
Electrothermal Atomic Absorption Spectrometry ◽  
Binding Constants ◽  
Absorption Spectrometry ◽  
New Method

A new method for studying the interaction between lead(ii) and DNA was developed using capillary electrophoresis electrothermal atomic absorption spectrometry.

Mucosal gastrin receptor. II. Physical characteristics of binding.

1979 ◽  
Vol 237 (3) ◽  
pp. E295
Author(s):  
K Takeuchi ◽  
G R Speir ◽  
L R Johnson
Keyword(s):  
Binding Sites ◽  
Low Temperatures ◽  
Specific Activity ◽  
Specific Binding ◽  
Cooperative Effect ◽  
Trypsin Treatment ◽  
Single Class ◽  
Gastrin Receptor ◽  
Protein Nature

Gastrin binding to rat gastric mucosal membrane preparations revealed a linear Scatchard plot, suggesting the existence of a single class of binding sites. Further studies revealed the absence of a positive cooperative effect. Because the system appeared to satisfy the conditions for Michaelis-Menten type kinetics, the determination of a Kd of approximately 3.6 X 10(-10) M was justified. These studies also demonstrated the efficacy of the specific activity of our iodinated gastrin. Trypsin treatment abolished specific binding of gastrin to the membrane preparation, indicating the protein nature of the receptor. The importance of maintaining extremely low temperatures during the incubation and wash period is demonstrated by the decrease in both the rate and the amount of dissociation at 0 degrees C compared with 30 degrees C.

scholarly journals Determination of the Binding Parameters between Proteins and Luminol by Chemiluminescence Using Flow Injection Technique

2013 ◽  
Vol 2013 ◽  
pp. 1-5
Author(s):  
Jie Guo ◽  
Donghua Chen ◽  
Zhenghua Song
Keyword(s):  
Bovine Serum Albumin ◽  
Flow Injection ◽  
Binding Sites ◽  
Bovine Serum ◽  
Binding Constants ◽  
Injection Technique ◽  
Binding Parameters ◽  
Hydrophobic Force ◽  
Number Of Binding Sites

The interaction behavior of bovine serum albumin (BSA), lysozyme (LYS), myoglobin (MB), and catalase (CAT) with luminol, respectively, was first studied by chemiluminescence (CL) using flow injection (FI) technique based on the fact that the studied proteins can enhance the CL intensity of luminol. A FI-CL model of protein-luminol interaction, lg[(I0−I)/I]=1/nlg[P]+1/nlgKa+2lgn, was constructed, and the interaction parameters of BSA, LYS, MB, and CAT with luminol were determined accordingly. The binding constants Ka are in the descending order of CAT > MB > LYS > BSA at the level of 105 to 107 L mol−1, and the number of binding sites n of luminol to BSA or LYS is around 2 and to MB or CAT is around 1. The results of thermodynamic parameters (ΔH, ΔS, and ΔG) showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobic force.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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