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.

Evaluation of a solid-phase enzyme immunoassay for human choriogonadotropin beta subunit.

1983 ◽  
Vol 29 (11) ◽  
pp. 1964-1966 ◽  
Author(s):  
D D Davey ◽  
M M Sample ◽  
T O Oei
Keyword(s):  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Beta Subunit ◽  
Neoplastic Disease ◽  
Standard Curve ◽  
Human Choriogonadotropin ◽  
Beta Hcg ◽  
Substantial Problem ◽  
Sample Dilution

Abstract We evaluated a quantitative solid-phase enzyme immunoassay for human choriogonadotropin beta subunit (beta-HCG) with anti-beta-HCG:horseradish peroxidase conjugate, recently marketed by Abbott Laboratories. We compared results on 56 patients' serum specimens, obtained mostly for followup of neoplastic disease, with those by a competitive radioimmunoassay kit. The correlation was good, the differences being of little clinical significance. Linear regression in the low and intermediate ranges gave a slope of 0.93, a y-intercept of 0.34, and a correlation coefficient of 0.97. Precision studies yielded an interassay CV of 6.4% in the intermediate range and 13% in the low range. Sensitivity was 0.69 int. unit/L. Cross reactivity was 1 to 2% with specimens fortified with lutropin or follitropin. The only substantial problem was with linearity in the upper part of the standard curve, especially in the interval, 100-200 int. units/L. This problem is obviated by adequate sample dilution.

New separation method for monoclonal immunoradiometric assays and its application to assays for thyrotropin and human choriogonadotropin.

1984 ◽  
Vol 30 (9) ◽  
pp. 1457-1461 ◽  
Author(s):  
S J Rattle ◽  
D R Purnell ◽  
P I Williams ◽  
K Siddle ◽  
G C Forrest
Keyword(s):  
Monoclonal Antibodies ◽  
Magnetic Particles ◽  
Solid Phase ◽  
Reaction Times ◽  
Fluorescein Isothiocyanate ◽  
High Sensitivity ◽  
Separation Method ◽  
Separation Procedure ◽  
Human Choriogonadotropin ◽  
Capture Antibody

Abstract We describe a novel separation procedure for immunoradiometric assays involving monoclonal antibodies in which both radiolabeled and capture antibodies are used in solution, the capture antibody being labeled with fluorescein isothiocyanate (FITC). Separation is achieved by incubation with anti-FITC antibodies on magnetic particles. This technique enhances reaction kinetics relative to those of assays in which a solid-phase capture antibody is used, thus allowing faster reaction times and more economic use of the monoclonal antibodies. The use of anti-FITC magnetic solid phase produces an assay having a highly specific separation method, minimal nonspecific binding, and high sensitivity. The method is illustrated by application to assays for thyrotropin and human choriogonadotropin.

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.

Ultrasound-accelerated immunoassay, as exemplified by enzyme immunoassay of choriogonadotropin.

1984 ◽  
Vol 30 (9) ◽  
pp. 1446-1451 ◽  
Author(s):  
R Chen ◽  
L Weng ◽  
N C Sizto ◽  
B Osorio ◽  
C J Hsu ◽  
...  
Keyword(s):  
Solid Phase ◽  
Test Strip ◽  
Immobilized Antibodies ◽  
Human Choriogonadotropin ◽  
Rate Limiting Step ◽  
Slow Kinetics ◽  
Assay Time ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Enhance Mass

Abstract The rate-limiting step in many solid-phase immunoassays is associated with the slow kinetics of binding of macro-molecular antigen and conjugate to the immobilized phase. We demonstrate that the use of ultrasonic energy to enhance mass transport across liquid/solid interfaces can dramatically accelerate antigen binding to immobilized antibodies. We use an ultrasound-accelerated procedure with an enzyme-channelling test strip containing glucose oxidase and specific antibody to the alpha-subunit of human choriogonadotropin (HCG) co-immobilized onto a cellulose support. A horseradish peroxidase conjugate of monospecific antibody to the beta-subunit of HCG is used in the liquid phase to complete the immune "sandwich." Use of ultrasound to accelerate binding and of enzyme channelling to eliminate wash steps result in a simple two-incubation protocol by which 25 int. units of urinary HCG per liter can be detected visually in less than 20 min of assay time.

Simple enzyme immunoassay for the simultaneous measurement of whole choriogonadotropin molecules and free beta-subunits in sera of women with abnormal pregnancies or tumors of the reproductive system

1991 ◽  
Vol 37 (5) ◽  
pp. 673-677 ◽  
Author(s):  
M J Choi ◽  
I S Choe ◽  
H K Kang ◽  
J S Lee ◽  
T W Chung
Keyword(s):  
Enzyme Immunoassay ◽  
Reproductive System ◽  
Solid Phase ◽  
High Specificity ◽  
Beta Subunits ◽  
Human Choriogonadotropin ◽  
Enzyme Substrate ◽  
Specificity And Sensitivity ◽  
Enzyme Substrates ◽  
Selection Of

Abstract A multiple enzyme immunoassay (multi-EIA) was developed to quantify whole-molecule human choriogonadotropin (w-hCG) and free hCG beta-subunits (hCG-beta) simultaneously. A clone of a specific monoclonal antibody was coupled to solid phase; two other clones of different monoclonal antibodies were conjugated to horseradish peroxidase (HRP; EC 1.11.1.7) and alkaline phosphatase (AP; EC 3.1.3.1), respectively. These two enzyme conjugates were mixed together to measure w-hCG or hCG-beta, depending on the selection of the enzyme substrate. To measure w-hCG and hCG-beta simultaneously, both enzyme substrates were used with the blended enzyme conjugates. The assay is simple and reproducible, and can be completed within 2 h with high specificity and sensitivity. We measured w-hCG and hCG-beta in the sera of women with abnormal pregnancies and in patients with tumors of the reproductive system, and observed different hCG-beta/w-hCG ratios in patients with various types of trophoblastic tumors.

scholarly journals Enzyme Linked Immunosorbent Assay (ELISA)

2021 ◽  
Vol 1 (2) ◽  
pp. 29-38
Author(s):  
Maya Chandra Dita
Keyword(s):  
Quality Control ◽  
Enzyme Immunoassay ◽  
Immunosorbent Assay ◽  
Solid Phase ◽  
Control Measures ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Binding ◽  
Fluorogenic Substrate ◽  
Specific Antibodies

Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are both widely used as diagnostic tools in medicine and as quality control measures in many industries. ELISA has been the system of choice when testing soluble antigens and antibodies. EIA / ELISA uses the basic immunological concept of antigen binding to specific antibodies, which allows the detection of small amounts of antigens such as proteins, peptides, hormones or antibodies in fluid samples. In all protocols, solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled with the enzyme. The unbound conjugate is washed and a chromogenic or fluorogenic substrate is added.

Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells

2021 ◽  
Vol 66 (6) ◽  
pp. 353-357
Author(s):  
S. S. Vetchinin ◽  
Anton Georgievich Shevyakov ◽  
A. E. Khomyakov ◽  
R. I. Mironova ◽  
A. N. Mokrievich ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Magnetic Particles ◽  
Solid Phase ◽  
Test System ◽  
Diagnostic Tools ◽  
Continuous Control ◽  
Negative Bacterium ◽  
Tularensis Subsp ◽  
Gram Negative ◽  
Microbial Cells

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

Incidence and specificity of interference in two-site immunoassays.

1986 ◽  
Vol 32 (8) ◽  
pp. 1491-1495 ◽  
Author(s):  
L M Boscato ◽  
M C Stuart
Keyword(s):  
Binding Site ◽  
Antibody Binding ◽  
Assay System ◽  
Antigen Binding ◽  
Immunoradiometric Assay ◽  
Serum Samples ◽  
Antigen Binding Site ◽  
Human Choriogonadotropin ◽  
A Site

Abstract Using a modified "two-site" immunoradiometric assay, termed an "interference assay," we have demonstrated the occurrence of non-analyte antibody-binding substances in approximately 40% of serum samples. These substances multivalently bind antibodies from any of several species at a site other than the antigen-binding site. Using a two-site immunoradiometric assay for human choriogonadotropin, we have investigated their effect on analyte quantification. In this system these antibody-binding substances mimic the presence of analyte; when analyte is actually present, they can also cause over- or underestimates. Addition of excess nonspecific immunoglobulin from an appropriate species eliminates this interference. However, the amount of nonspecific immunoglobulin added to an assay system may not always be enough to block interference in all samples.

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