Immunoassay Classification and Commercial Technologies

Deshpande, S. S.

doi:10.1007/978-1-4613-1169-0_8

Cited by 5 publications

(3 citation statements)

References 42 publications

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“…An immunoassay in a reagent excess format , (also known as immunometric, two-site noncompetitive, or sandwich assay) generally involves the use of two antibodies targeting different epitopes (one for antigen capture and another labeled for detection) that can be added in excess as compared to the analyte. At low analyte concentration, unoccupied capture binding sites are always available, but as signal measurement occurs only at the occupied binding sites, the signal is directly proportional to the amount of analyte present.…”

mentioning

confidence: 99%

“…At low analyte concentration, unoccupied capture binding sites are always available, but as signal measurement occurs only at the occupied binding sites, the signal is directly proportional to the amount of analyte present. In general, a reagent excess immunoassay , provides considerable benefits in terms of assay robustness, sensitivity, specificity, kinetics, and extended working range as compared to the reagent limited competitive assays. In competitive format, the analyte and the labeled tracer analyte compete for a limited number of binding sites of a single-type antianalyte antibody used, and the signal decreases with increased analyte concentration.…”

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confidence: 99%

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Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

et al. 2016

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A broad-spectrum noncompetitive immunoassay allowing sensitive and simple detection of a group of similar compounds would be an ideal tool for screening low-molecular weight analytes (<2000 Da) having many variants. However, the development of an essential antibody pair capable of sandwich-type recognition of the analytes' small generic core structure is a demanding task due to limited space available for simultaneous binding of two different antibodies. We report here a generic noncompetitive assay for cyanobacterial microcystins (MCs) and nodularins (Nod), a group of structurally related small cyclic peptides (∼1000 Da) with more than 100 naturally occurring analogs. The assay is based on the unique combination of a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domain (scFv) and a monoclonal antibody capable of binding to an Adda-group (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) present in all MCs/Nod. The anti-IC scFv was isolated from a large synthetic antibody library with phage display and used to develop a single-step sandwich-type noncompetitive immunocomplex assay. The sensitive time-resolved immunofluorometry-based assay is capable of detecting all the 11 tested commonly occurring hepatotoxins (MC-LR, -dmlR, -RR, -dmRR, -LA, -LY, -LF, -LW, -YR, -WR, and Nod-R) at concentration below 0.1 μg/L in a 1 h assay. Using MC-LR, the most studied toxic and widely distributed of the toxins, the calculated detection limits (based on blank + 3SD response) are ∼0.026 μg/L in 1 h and ∼0.1 μg/L in 10 min assay time. This is by far the fastest reported immunoassay for MCs and Nod with a detection limit far below the World Health Organization's guideline limit (1 μg/L of MC-LR equivalent in drinking water). The assay was validated with spiked tap and lake water as well as with environmental surface water samples. The developed assay provides a simple, rapid, and highly sensitive tool for the quantitative detection of MCs/Nod with the additional benefit of automation and high-throughput possibilities for large scale screening of drinking and environmental surface water samples. Furthermore, the study describes the first demonstration of the assay intended for the detection of an analyte group comprising similar low-molecular weight compounds exhibiting the benefits of a reagent excess type assay.

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Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

et al. 2016

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“…One of the most common methods to group the different EIAs is by the process of detection; either direct or indirect detection. The difference is the method in which the antigen (molecule of interest) is bound to the antibody of the plate and then detected [288]. In the direct EIA method, the antigen and the other constituents of the sample adsorb onto the base of a micro-well.…”

Section: Enzyme Immunoassaymentioning

confidence: 99%

Development of METx: A Vasopressin Agonist

Patel¹

2016

Preprint

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Vasopressin (AVP) was one of the first peptides to be extracted and produced synthetically in the early 1950s. Since then AVP and its analogues have been increasingly researched as both AVP agonists and antagonists have therapeutic uses. Although much time and effort has been used to design potent selective AVP agonists and antagonists, very few have reached the market. AVP agonist analogues have only been approved for diabetes insipidus and variceal bleeding. Current main stay treatment for diabetes insipidus is desmopressin, a synthetic analogue of AVP. Desmopressin is well tolerated by the majority of patients, however severe adverse affects, such as hyponatraemia and water intoxication require a strict fluid intake, restricting fluid intake. Our aim is to develop METx for the treatment of diabetes insipidus to help overcome some of the limitations in lifestyle. The solubility and aggregation of METx was measured using the shake flask and pyrene assay respectively. The activity of METx was determined by measuring cAMP production by MDCK cells. The activity of METx was compared to that AVP, the natural agonist of the vasopressin receptor. Formulation studies were performed using GCPQ to create a formulation that could be used subcutaneously or orally. The stability of GCPQ formulations was determined in plasma, simulated gastric fluid (SGF) and rat intestinal wash (RIW). The activity of METx was measured in vivo by monitoring the urine production of rats upon administration of METx intravenously. METx was practically insoluble in aqueous solvents such as water and PBS, with increased solubility in organic solvents. Using TEM imaging aggregation of METx of was seen, the pyrene assay determined the critical micelle concentration to be 27.4mg l-1. METx was found to be a partial agonist of the vasopressin 2 receptor naturally expressed on the MDCK cells, it exhibited a similar EC 50 to AVP. Formulating with GCPQ a ratio of 1:5 (METx:GCPQ) was found to be the most stable in plasma, SGF and RIW. In vivo studies found METx to have a dose dependent reduction in urine production in rats, with 40mg kg-1 rats not producing any urine. The experiments suggest the development of a novel partial agonist of the vasopressin 2 receptor in vitro. The addition of GCPQ results in the formation of nanoparticles, with a ratio of 1:5 (METx:GCPQ) most stable in plasma, SGF and RIW. In vivo experiments resulted in reduced urine production by rats, in a dose responsive manner.

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E

Gressner

Arndt²

2007

Lexikon Der Medizinischen Laboratoriumsdiagnostik

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Immunoassay Classification and Commercial Technologies

Cited by 5 publications

References 42 publications

Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

Development of METx: A Vasopressin Agonist

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