Sensitive Analyte Detection and Quantitation Using the Threshold Immunoassay                                         System

Dill, Kilian

doi:10.1021/bk-1996-0646.ch009

Cited by 4 publications

(2 citation statements)

References 13 publications

(22 reference statements)

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“…For the detection of nucleic acids in pharmaceutical samples, hybridization techniques such as the dot blot or immunoligand assay (ILA) are often used. The ILA (a.k.a “Threshold Assay”) reliably detects very small amounts of DNA and impurities in liquid solution [ 102 ]. This assay employs a biotinylated single-stranded binding (SSB) protein and general anti-ssDNA antibody to complex with any host ssDNA available in the sample.…”

Section: Methods For Iimi Detectionmentioning

confidence: 99%

“…Streptavidin filtration then captures any biotinylated complexes on a specialized matrix-embedded silicon chip, after which the DNA is quantified via enzymatic hydrolysis and subsequent light-addressable potentiometric sensor (LAPS) detection [ 99 ]. This method has been shown to be 10–100 times more sensitive than traditional colorimetric or ELISA assays, with a detection range of 5–40 ng/mL [ 99 ], requires only small amounts of sample, removes steric binding or stability issues inherent in solid-phase systems, and comes in two formats (sandwich or competitive) depending on the size of the analyte being detected [ 102 ] though optimal ssDNA fragments tend to be larger than 600 base pairs [ 99 ]. However, this method has reduced specificity due to its sequence-independent binding by general ssDNA antibodies.…”

Section: Methods For Iimi Detectionmentioning

confidence: 99%

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Innate Immunity Modulating Impurities and the Immunotoxicity of Nanobiotechnology-Based Drug Products

Holley

Dobrovolskaia

2021

Molecules

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Innate immunity can be triggered by the presence of microbial antigens and other contaminants inadvertently introduced during the manufacture and purification of bionanopharmaceutical products. Activation of these innate immune responses, including cytokine secretion, complement, and immune cell activation, can result in unexpected and undesirable host immune responses. These innate modulators can also potentially stimulate the activation of adaptive immune responses, including the formation of anti-drug antibodies which can impact drug effectiveness. To prevent induction of these adverse responses, it is important to detect and quantify levels of these innate immunity modulating impurities (IIMIs) that may be present in drug products. However, while it is universally agreed that removal of IIMIs from drug products is crucial for patient safety and to prevent long-term immunogenicity, there is no single assay capable of directly detecting all potential IIMIs or indirectly quantifying downstream biomarkers. Additionally, there is a lack of agreement as to which of the many analytical assays currently employed should be standardized for general IIMI screening. Herein, we review the available literature to highlight cellular and molecular mechanisms underlying IIMI-mediated inflammation and its relevance to the safety and efficacy of pharmaceutical products. We further discuss methodologies used for direct and indirect IIMI identification and quantification.

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Section: Methods For Iimi Detectionmentioning

confidence: 99%

Section: Methods For Iimi Detectionmentioning

confidence: 99%