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.

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.

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.

scholarly journals Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG

2021 ◽  
Author(s):  
Veronica Costantini ◽  
Kenny Nguyen ◽  
Zoe Lyski ◽  
Shannon Novosad ◽  
Ana C Bardossy ◽  
...  
Keyword(s):  
Immune Response ◽  
Enzyme Immunoassay ◽  
Limit Of Detection ◽  
Natural Infection ◽  
High Specificity ◽  
Cross Reactivity ◽  
Igg Antibodies ◽  
Antibody Isotype ◽  
Salivary Iga ◽  
Specificity And Sensitivity

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

scholarly journals COMPARATIVE ANALYSIS OF EFFECTIVENESS OF SOLID-PHASE METHODS OF IMMUNE DETECTION OF BOTULINIC TOXIN IN BLOOD SERA OF A PATIENT WITH BOTULISM DIAGNOSIS

2017 ◽  
pp. 64-70
Author(s):  
T. Yu. Zagoskina ◽  
E. Yu. Markov ◽  
E. A. Chaporgina ◽  
Yu. O. Popova ◽  
T. M. Dolgova ◽  
...  
Keyword(s):  
Solid Phase ◽  
Detection System ◽  
High Specificity ◽  
Test System ◽  
Detection Methods ◽  
Clinical Material ◽  
Specificity And Sensitivity ◽  
Botulinic Toxin ◽  
Immune Detection ◽  
Dot Immunoassay

Aim. Comparison of effectiveness of solid phase methods of immune detection of botulinic toxin in blood sera of a patient with botulism diagnosis: dot-immune assay using specific anti-botulinic antibodies (AT) labeled with nanoparticles of colloid silver, phosphorescent analysis (PHOSPHAN) using streptavidin label with platinum coproporphyrin (PtCP) and polystyrene nanoparticles, containing chelate complex of europium ions with naphthoyl trifluoroacetone (NA-Eu). Materials and methods. Silver nanoparticle labeled IgG isolated from a commercial diagnostic polyvalent sera against type А, В, С, E, F botulotoxins manufactured by SPA Allergen (Stavropol) with 5000 - 10000 IU activity and biotin conjugated commercial monoclonal antibodies against botulotoxin A, polyclonal mono-specific AB against botulotixin В and E and polyvalent immunoglobulin against botulotoxin А, В, С, E, F. Detection ofbotulotoxin in clinical material was carried out in dot-immunoassay on nitrocellulose membrane by PHOSPHAN method in an experimental test system using 2 detector systems based on streptavidin: PtCP and NA-Eu. Results. Botulotoxin was detected in blood sera of the botulism patient using both of the developed immune detection methods. PHOSPHAN method allowed to identify serotype В botulotoxin, that corresponded with the results obtained in botulotoxin biological neutralization reaction. Sensitivity of PHOSPHAN with NA-Eu luminescent nanoparticle based detection system was higher than with PtCP label. Conclusion. The developed methods (PHOSPHAN and dot-immunoassay) differ by high specificity and sensitivity and may be recommended for express detection of botulinic toxin in clinical material.

A Nylon Filter A-ELISA for Detecting Viruses in Water

1993 ◽  
Vol 27 (7-8) ◽  
pp. 135-141 ◽  
Author(s):  
Abidelfatah M. Nasser ◽  
Yehudit Elkana ◽  
Leon Goldstein
Keyword(s):  
Positive Result ◽  
Solid Phase ◽  
Enteric Viruses ◽  
High Specificity ◽  
Negative Results ◽  
Order Of Magnitude ◽  
Lower Background

This study was designed to develop a modification of A-ELISA performed in microtitre plates. Nylon filters have been utilized successfully as a solid phase for the performance of A-ELISA. The use of nylon filters resulted in lower background than nitro-cellulose and paper filters, indicating their suitability as a solid phase for developing A-ELISA. With enteric viruses, human rotaviruses and MS-2 coliphage, negative results were obtained, suggesting high specificity of the developed technique for poliovirus 1. The sensitivity of the developed A-ELISA has been shown to be at least one order of magnitude greater than ordinary ELISA. A positive result with the nylon A-ELISA can be obtained with samples containing 100-1000 pfu/ml of poliovirus. Up to date methods used for detecting viruses in water are elaborate, time consuming and costly. Applying the nylon A-ELISA may overcome some of these disadvantages.

Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

2020 ◽  
Vol 20 (10) ◽  
pp. 831-840
Author(s):  
Weibin Li
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Diagnosis ◽  
Bloodstream Infections ◽  
High Specificity ◽  
Peptide Sequence ◽  
High Morbidity ◽  
Specificity And Sensitivity ◽  
Prospective Application ◽  
Small Molecule Therapeutics

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

scholarly journals System-wide identification and prioritization of enzyme substrates by thermal analysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Amir Ata Saei ◽  
Christian M. Beusch ◽  
Pierre Sabatier ◽  
Juan Astorga Wells ◽  
Hassan Gharibi ◽  
...  
Keyword(s):  
Thermal Analysis ◽  
Thioredoxin Reductase ◽  
Structural Level ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Enzyme Substrate ◽  
Thioredoxin Reductase 1 ◽  
Enzyme Substrates ◽  
Substrate Proteins

AbstractDespite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

scholarly journals SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mikail Dogan ◽  
Lina Kozhaya ◽  
Lindsey Placek ◽  
Courtney Gunter ◽  
Mesut Yigit ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
High Specificity ◽  
Spike Protein ◽  
Humoral Immune ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Specific Igg ◽  
Negative Controls

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

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