Antigen Binding in the Two-Site Immunoradiometric Assay for Serum Ferritin: The Nature of the Hook Effect

1984 ◽  
Vol 21 (5) ◽  
pp. 393-397 ◽  
Author(s):  
Praemi Perera ◽  
Mark Worwood
Keyword(s):  
Monoclonal Antibody ◽  
Count Rate ◽  
Solid Phase ◽  
Specific Binding ◽  
Significant Proportion ◽  
Antigen Binding ◽  
Immunoradiometric Assay ◽  
Second Stage ◽  
Hook Effect ◽  
Two Stages

The two stages of a two-site immunoradiometric assay were investigated separately. In the first stage, the amount of ferritin bound to coated tubes initially showed a rapid increase with increasing concentration of added ferritin. This was followed by a plateau and then a further increase which appeared to be largely due to non-specific binding. During the second stage, a significant proportion of the bound ferritin dissociated from the solid phase and sequestered some of the labelled antibody in solution. Thus less antibody was available to bind to ferritin attached to the tube, causing a decrease in count rate at high ferritin concentrations. The use of a monoclonal antibody for coating the tubes did not eliminate this hook effect.

Characterization of Primate Antibody Responses to Administered Murine Monoclonal Immunoglobulin

1990 ◽  
Vol 5 (4) ◽  
pp. 177-187 ◽  
Author(s):  
D.E. Milenic ◽  
B. Detrick ◽  
J.C. Reynolds ◽  
D. Colcher
Keyword(s):  
Monoclonal Antibody ◽  
Antibody Response ◽  
Solid Phase ◽  
Hypervariable Region ◽  
Antigen Binding ◽  
Post Inoculation ◽  
Antigen Binding Site ◽  
Antibody Treatment ◽  
Cancer Management ◽  
Monoclonal Antibody Treatment

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.

Thermodynamic Stability of Ptal Thin Films on GaAs

1990 ◽  
Vol 181 ◽  
Author(s):  
Dae-Hong Ko ◽  
Robert Sinclair
Keyword(s):  
Thin Films ◽  
Solid Phase ◽  
Second Stage ◽  
Sequential Deposition ◽  
Low Temperature Annealing ◽  
Gaas Substrates ◽  
Transmission Electron ◽  
C 30 ◽  
Two Stages ◽  
Thermal Stability Of

ABSTRACTThe thermal stability of PtAl thin films on GaAs substrates has been studied using transmission electron microscopy and Auger electron spectroscopy. The PtAl thin films were formed by sequential deposition of discrete Pt and Al layers on GaAs by e-beam evaporation followed by subsequent annealing processes. Interfacial reactions in the Al/Pt/GaAs system proceed in two stages. Upon low temperature annealing Pt and GaAs react to form PtGa and PtAs2. Further high temperature annealing causes PtGa, PtAs2 and Al to react together producing the desired PtAl on GaAs. We observed solid-phase epitaxial regrowth of GaAs during the second stage of reaction. The PtAl/GaAs interface is determined to be thermally stable during an 800°C/30 min. anneal, while remaining morphologically uniform on GaAs.

Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes.

1985 ◽  
Vol 31 (9) ◽  
pp. 1449-1452 ◽  
Author(s):  
G A Robinson ◽  
H A Hill ◽  
R D Philo ◽  
J M Gear ◽  
S J Rattle ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzyme Immunoassay ◽  
Magnetic Particles ◽  
Solid Phase ◽  
Model System ◽  
Antigen Binding ◽  
Immunoradiometric Assay ◽  
Human Choriogonadotropin ◽  
Assay Time ◽  
Rapid Quantification

Abstract We describe an amperometric technique for quantification of an enzyme immunoassay in which we use a magnetic working electrode, both to separate bound and free analyte and to monitor the electrochemical response. We used a "two-site" immunometric assay with monoclonal antibodies for human choriogonadotropin (hCG) as a model system in which magnetic particles were used as the solid phase. Separation of bound and free label is readily achieved by localizing the particles at the electrode. Activity of the bound enzyme in the environment of the electrode is determined electrochemically, permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the system is 150 int. units of hCG per litre (1st Int. Ref. Preparation). Correlation between the amperometric measurement of urinary hCG and data for an immunoradiometric assay was r = 0.9. The assay is rapid, requiring a total assay time for each sample of 20 min, which includes 15 min for antibody/antigen binding.

A Rapid One-Step Immunoradiometric Assay for Factor VIII-Procoagulant Antigen Utilizing Monoclonal Antibodies

1983 ◽  
Vol 50 (04) ◽  
pp. 860-863 ◽  
Author(s):  
H V Stel ◽  
E C I Veerman ◽  
J G Huisman ◽  
M C Janssen ◽  
J A van Mourik
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Lower Limit ◽  
Solid Phase ◽  
Immunoradiometric Assay ◽  
Step Procedure ◽  
One Step

SummaryA two-site immunoradiometric assay for factor VIII-procoagulant antigen (VIIICAg) that relies completely on monoclonal antibodies has been developed. By selecting an appropriate combination of these antibodies, it was possible to develop an assay in which the radiolabelled monoclonal antibody did not inhibit the binding of antigen to the solid-phase monoclonal antibodies. Thus, the entire test could be carried out as a one-step procedure. With this one-step assay, an amount of 0.0025 U VIIICAg/ml plasma could be detected after 4 hr of incubation, whereas 18 hr of incubation resulted in a lower limit of sensitivity of 0.0005 U VIIICAg/ml. The use of a one-step assay provides a significant advantage over the conventional two-step assay by simplifying, shortening and rendering the performance of the assay more convenient.

An automated immunoradiometric assay for human thyrotropin.

1984 ◽  
Vol 30 (8) ◽  
pp. 1396-1398 ◽  
Author(s):  
R John ◽  
M K Jones
Keyword(s):  
Monoclonal Antibody ◽  
Thyroid Function ◽  
Solid Phase ◽  
Immunoradiometric Assay ◽  
First Line ◽  
Line Test ◽  
The Mean

Abstract In this two-site immunoradiometric assay for thyrotropin, developed for use in the "Kemtek 3000" automated radioimmunoassay system, commercially available monoclonal antibody to thyrotropin is labeled with 125I, and the solid-phase antibody is an IgG fraction of sheep antiserum to thyrotropin, covalently coupled to reprecipitated aminocellulose. There are two incubations, totalling 3 h, the sensitivity is 0.03 milli-int. unit/L. The mean thyrotropin value for 82 healthy euthyroid subjects was 1.7 milli-int. units/L (range 0.4-3.6). For 19 overtly clinically and biochemically hyperthyroid subjects the values ranged from undetectable to 0.2 milli-int. unit/L. In this assay, euthyroid and hyperthyroid subjects can be distinguished with assay of a single basal sample. The assay appears suitable for routine use as a first-line test of thyroid function.

The Application of a Monoclonal Antibody to Factor VIII Related Antigen (VIIIRAg) in Immunoradiometric Assays for Factor VIII

1985 ◽  
Vol 53 (01) ◽  
pp. 143-147 ◽  
Author(s):  
J E Thomas ◽  
I R Peake ◽  
J C Giddings ◽  
A N Welch ◽  
A L Bloom
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Inhibitory Effect ◽  
Immunoradiometric Assay ◽  
Strong Inhibitory Effect ◽  
Cofactor Activity ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

SummaryA two site solid phase immunoradiometric assay (IRMA) for VIIIRAg has been developed using a monoclonal antibody as both the solid phase ligand and the radiolabelled antibody. A range of normal plasmas and von Willebrand’s disease (vWd) plasmas has been assayed for VIIIRAg by this method and compared with VIIIRAg values measured by an IRMA using polyclonal antibodies and by immunoelectrophoresis and with ristocetin cofactor activity (VIIIRiCoF). It was found that the monoclonal IRMA correlated well with the polyclonal IRMA and the immunoelectrophoretic assay, but the correlation with the VIIIRiCoF assay was relatively poor, in spite of the strong inhibitory effect of the antibody on VIIIRiCoF activity.

A new monoclonal-antibody two-site solid-phase immunoradiometric assay for human thyrotropin evaluated.

1984 ◽  
Vol 30 (7) ◽  
pp. 1213-1215 ◽  
Author(s):  
A E Pekary ◽  
J M Hershman
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Polystyrene Bead ◽  
Good Precision ◽  
Immunoradiometric Assay ◽  
Serum Samples ◽  
Low Concentrations ◽  
The Mean ◽  
Source Of Error ◽  
Hypothyroid Patients

Abstract We compared results with a commercial solid-phase two-site immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used (TANDEM-R TSH; Hybritech Inc.) with those by our radioimmunoassay (J Clin Endocrinol Metab 41:676, 1975), which is optimized for measurement of low concentrations of thyrotropin. In the immunoradiometric assay a specific antibody to the beta subunit of human thyrotropin is immobilized on a polystyrene bead, and a radiolabeled monoclonal antibody directed against the alpha subunit provides a measure of bead-immobilized hormone. The mean thyrotropin concentrations in 70 euthyroid serum samples were similar in the two assays. Values for hypothyroid patients were clearly higher in both assays than values for euthyroid individuals. In commercial assays the major source of error in measurement of thyrotropin response to thyroliberin in terms of the increment over the basal concentration of thyrotropin has been systematic errors in the measurement of those basal concentrations. With the present assay, however, basal values are obtained with good precision and accuracy: CV = 2.9% at 6 milli-int. units/L (n = 48).

Immunofluorometry of choriogonadotropin by time-resolved fluorescence spectroscopy, with a new europium chelate as label.

1987 ◽  
Vol 33 (11) ◽  
pp. 1994-1999 ◽  
Author(s):  
M J Khosravi ◽  
E P Diamandis
Keyword(s):  
Monoclonal Antibody ◽  
Dynamic Range ◽  
Solid Phase ◽  
High Dose ◽  
Nitrogen Laser ◽  
Time Resolved ◽  
Hook Effect ◽  
Step Procedure ◽  
Sandwich Type ◽  
Europium Chelate

Abstract We describe a new "sandwich"-type non-isotopic immunoassay for human choriogonadotropin (hCG) in serum. In the assay, hCG is captured by a beta-subunit-specific monoclonal antibody, which is immobilized in a white microtiter well. The sandwich is completed by adding a second biotinylated monoclonal antibody specific for the whole hCG molecule. The degree of binding of biotinylated antibody, which is proportional to the amount of hCG present in the sample, is quantified by adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The fluorescent complex formed on the solid-phase [monoclonal antibody-hCG-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+] is measured by excitation at 337.1 nm with a nitrogen laser and monitoring the emission at 615 nm in a specially designed gated fluorometer working in a time-resolved mode. A two-step procedure is proposed for routine use to avoid the "high-dose hook effect" of the simpler and faster one-step procedure. The hCG assay described has a dynamic range of 1 to 500 int. units/L, and is precise and accurate. Results agree well with those obtained with a commercially available immunoradiometric and a time-resolved immunofluorometric procedure.

Altered Fine Structure of B Lymphocytes Correlated with Defective Terminal Differentiation

1973 ◽  
Vol 31 ◽  
pp. 386-387
Author(s):  
Dale E. Bockman ◽  
L. Y. Frank Wu ◽  
Alexander R. Lawton ◽  
Max D. Cooper
Keyword(s):  
Immune Deficiency ◽  
B Lymphocytes ◽  
Plasma Cells ◽  
Present Report ◽  
Specific Antigen ◽  
Terminal Differentiation ◽  
Second Stage ◽  
Two Stages ◽  
Deficiency Diseases ◽  
Cell Line Generation

B-lymphocytes normally synthesize small amounts of immunoglobulin, some of which is incorporated into the cell membrane where it serves as receptor of antigen. These cells, on contact with specific antigen, proliferate and differentiate to plasma cells which synthesize and secrete large quantities of immunoglobulin. The two stages of differentiation of this cell line (generation of B-lymphocytes and antigen-driven maturation to plasma cells) are clearly separable during ontogeny and in some immune deficiency diseases. The present report describes morphologic aberrations of B-lymphocytes in two diseases in which second stage differentiation is defective.

Distributed Entropy Energy-Efficient Clustering algorithm for cluster head selection (DEEEC)

2020 ◽  
Vol 39 (6) ◽  
pp. 8139-8147
Author(s):  
Ranganathan Arun ◽  
Rangaswamy Balamurugan
Keyword(s):  
Energy Efficient ◽  
Clustering Algorithm ◽  
Cluster Head ◽  
Residual Energy ◽  
Energy Utilization ◽  
Sensor Nodes ◽  
Second Stage ◽  
Energy Efficient Clustering ◽  
Two Stages ◽  
Ch Selection

In Wireless Sensor Networks (WSN) the energy of Sensor nodes is not certainly sufficient. In order to optimize the endurance of WSN, it is essential to minimize the utilization of energy. Head of group or Cluster Head (CH) is an eminent method to develop the endurance of WSN that aggregates the WSN with higher energy. CH for intra-cluster and inter-cluster communication becomes dependent. For complete, in WSN, the Energy level of CH extends its life of cluster. While evolving cluster algorithms, the complicated job is to identify the energy utilization amount of heterogeneous WSNs. Based on Chaotic Firefly Algorithm CH (CFACH) selection, the formulated work is named “Novel Distributed Entropy Energy-Efficient Clustering Algorithm”, in short, DEEEC for HWSNs. The formulated DEEEC Algorithm, which is a CH, has two main stages. In the first stage, the identification of temporary CHs along with its entropy value is found using the correlative measure of residual and original energy. Along with this, in the clustering algorithm, the rotating epoch and its entropy value must be predicted automatically by its sensor nodes. In the second stage, if any member in the cluster having larger residual energy, shall modify the temporary CHs in the direction of the deciding set. The target of the nodes with large energy has the probability to be CHs which is determined by the above two stages meant for CH selection. The MATLAB is required to simulate the DEEEC Algorithm. The simulated results of the formulated DEEEC Algorithm produce good results with respect to the energy and increased lifetime when it is correlated with the current traditional clustering protocols being used in the Heterogeneous WSNs.

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