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Methods and Equipment for Quality Control of Radiopharmaceuticals
Published in Michael Ljungberg, Handbook of Nuclear Medicine and Molecular Imaging for Physicists, 2022
Rolf Zijlma, Danique Giesen, Yvette Kruiter, Philip H. Elsinga, Gert Luurtsema
The endotoxin quantification method used by the Endosafe testing module is based on the reaction cascade of the following proteins; Factor C (FC), Factor B (FB), clotting pro-enzyme and a chromogenic substrate (Figure 6.5). The cascade of endotoxin detection in the LAL assay starts when the endotoxin LPS reacts with FC, which activates FB. FB turns the clotting pro-enzyme into an enzyme. The resulting clotting enzyme is responsible for cleaving the chromogenic substrate, which produces a yellow colouring. The yellow colouring is detected by the Endosafe device by measuring the solution at 280 nano meters (nm). The resulting data is compared to a pre-programmed standard curve and a concentration is calculated.
Immunoglobulins
Published in Constantin A. Bona, Francisco A. Bonilla, Textbook of Immunology, 2019
Constantin A. Bona, Francisco A. Bonilla
ELISA is very similar to SPRIA except that one labels a reagent with an enzyme instead of with a radioactive isotope. The enzyme is quantified by measuring the amount of chromophore generated from a chromogenic substrate in a given period of time. One enzymesubstrate system often used is alkaline phosphatase and p-nitrophenyl phosphate. ELISA is also widely applied in clinical chemistry.
Testing strategies used in the diagnosis of rare inherited bleeding disorders
Published in Expert Review of Hematology, 2023
[17–20]Screening tests like the prothrombin time (PT) and activated partial thromboplastin time (APTT) are the most commonly ordered initial tests in evaluation of suspected bleeding disorders; the assays assess the different coagulation pathways (Figure 1). However, the PT and APTT have limitations in their sensitivity to mild coagulation factor deficiencies, which vary based on reagent/instrument combinations and may result in a missed diagnosis [21–26]. Laboratory professionals are generally aware of these limitations; however, the majority of clinicians are not aware of limitations of assay sensitivity. Examples of sensitivity of PT and APTT to commonly ordered coagulation factor assays are shown in Figure 2a–h. The most commonly performed coagulation factor assay is the one-stage factor assay [27], where the assay end point is the detection of a fibrin clot. Chromogenic assays, where the end point is the detection of the release of a chromogenic substrate, are more expensive, have limited availability, and are rarely performed except for the diagnosis of HA and HB [28] and to monitor selected extended half-life coagulation factor concentrates.
An in vitro intestinal model captures immunomodulatory properties of the microbiota in inflammation
Published in Gut Microbes, 2022
Jaclyn Y. Lock, Mariaelena Caboni, Philip Strandwitz, Madeleine Morrissette, Kevin DiBona, Brian A. Joughin, Kim Lewis, Rebecca L. Carrier
Endotoxin concentration in fresh and spent media was quantified using Pierce Limulus Amebocyte Lysate Chromogenic Endotoxin Quantification Kit (ThermoFisher Scientific). Briefly, a microplate was equilibrated on a heating block for 10 min at 37°C. 50 μl of each test medium (GIFU, pGIFU, GIFU-S, or pGIFU-S) or standard was added to a separate well, followed by addition of 50 μl of LAL solution and incubation for 10 min at 37°C. The chromogenic substrate solution (100 μl) was added to each well and incubated for 6 min at 37°C. Finally, 50 μl of the stop reagent (25% acetic acid) was added, and the absorbance was measured at 405 nm using a BioTek Powerwave XS spectrophotometer. The standard curve ranged from 0 to 1.0 endotoxin units (EU)/ml and was used to calculate the concentration of the samples that were run in triplicate.
Inhibition of bacterial and human zinc-metalloproteases by bisphosphonate- and catechol-containing compounds
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Fatema Rahman, Tra-Mi Nguyen, Olayiwola A. Adekoya, Cristina Campestre, Paolo Tortorella, Ingebrigt Sylte, Jan-Olof Winberg
The inhibitorial power of 100 μM of the eight catechol containing compounds was tested against the two human metalloproteases MMP-9(T) and MMP-14 and the three bacterial metalloproteases ALN, PLN and TLN (Figure 1). In order to detect putative slow and slow-tight binding, the catechol derivatives were first incubated along with the enzyme for 15 min at 37 °C. In controls without inhibitor present, enzymes and buffer were preincubated under identical conditions. Thereafter, the enzyme reaction was started by adding the relevant chromogenic substrate and the rate was followed continuously for 30 min. Except for BF486, which did not affect MMP-9, and BF482 which did not affect PLN, all cathecol containg compounds showed inhibition of the five proteases (Figure 1). Both BF471 and BF489 showed more than 50% inhibition of all five proteases, while ML32 reduced the activity more than 50% for four of the proteases (not for TLN). The activity was reduced with more than 50%, by MT336 for the two MMPs, by BF482 for MMP-9(T) and by ML33 for ALN (Figure 1). In all other inhibitory studies with catechol containing compounds, the activity was reduced between 0 and 45%.