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Diagnostic applications of immunology
Published in Gabriel Virella, Medical Immunology, 2019
Ajay Grover, Virginia Litwin, Gabriel Virella
The principles of quantitative immunofluorescence are similar to those described for indirect fluorescence assays: antigen is bound to a solid phase, exposed to a serum sample containing specific antibody, unbound immunoglobulins are rinsed off, and a fluorescein-labeled antibody is added to reveal specific antibody. A fluorometer is used to measure the amount of fluorescence emitted by the second antibody. Since the amount of fluorescent antibody added to the system is fixed, the amount that remains bound is directly proportional to the concentration of antibody present in the sample. Thus, a quantitative correlation can be drawn between the intensity of fluorescence and the concentration of antibody. These quantitative tests have been adapted to microbiological assays and can identify IgG and IgM antibodies.
The Fluorochromasia Cell Mediated Lympholysis Assay (CML)
Published in Soldano Ferrone, B. G. Solheim, HLA Typing: Methodology and Clinical Aspects, 2019
J. W. Bruning, J. J. van der Poel, M. J. Kardol
Apart from being nonradioactive, the fluorochromasia method spares cells and materials and is fast (short labeling time, rapid reading procedure: 1 min/tray). In addition, the automated fluorometer is a multipurpose instrument which can read C-de-pendent cytotoxicity assays, mitogen and antigen lymphocyte transformation assays, fluid fluorescence and light absorption assays — all with the same speed.
Experimental Strategies
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
In the case of interaction between two macromolecules, aside from any changes in spectral properties, the complexes may show increased light scattering which is sufficient to provide a titration signal [351,352]. Such studies can be carried out in a conventional fluorimeter with the wavelengths of excitation and emission channels set to the same value. Turbidity (apparent absorption) may also be detected in a conventional spectrophotometer. The signal changes are likely to be smaller than for light scattering, but the results are less sensitive to particulate matter in the sample. The best signal will be achieved at the shortest wavelength, which is clear of true absorption effects (typically >300 nm for proteins lacking chromophores). Light scattering and turbidity often provide useful signals to study macromolecular polymerization reactions (Section 5.2). The latter may also be probed using other assays such as dynamic light scattering, fluorescence correlation spectroscopy, or dye-binding [203,353]. Because polymerization reactions give a distribution of product sizes, different assays are likely to give different weighting to the different sizes present. This will affect the apparent kobs values when modeled as a simple reaction mechanism, such as monomer nucleus polymer (Section 5.2).
Effects of platelets activated by different agonists on fibrin formation and thrombin generation
Published in Platelets, 2023
Ivan A. Muravlev, Anatoly B. Dobrovolsky, Olga A. Antonova, Svetlana G. Khaspekova, Alexey V. Mazurov
Platelets prepared in the same way as for PRA were tested in TGT. All reagents used in TGT were from Diagnostica Stago (France). A total of 80 µl citrated plasma and 20 µl trigger PRP reagent (tissue factor and minimum amount of phospholipids) were added to immobilized platelets. After incubation (10 min, 37 С) the reaction was started by the addition of 20 µl Fluo-Buffer (thrombin fluorogenic substrate and CaCl2). Final concentration of tissue factor was 0.5 pM. In control samples plasma thrombin generation was monitored with the use of “PPP reagent” that provided a final concentration of 5 pM for tissue factor and 4 µM for phospholipids. For calibration of fluorescent signal Thrombin Calibrator (750 nM Thrombin-α2-macroglobulin complex) was added instead of trigger reagent. Measurements were performed in a Fluoroscan Ascent plate fluorimeter (ThermoLab Systems, Finland). Thrombin generation curves were analyzed using Thrombinoscope software (Thrombinoscope BV, Netherlands).
Human umbilical cord-derived mesenchymal stem cells conditioned medium ameliorates CCl4-induced liver fibrosis through regulation of expression and activity of liver lysyl oxidase
Published in Toxin Reviews, 2021
Mahdi Bahmani, Nasrin Ziamajidi, Mohammad Hashemnia, Roghayeh Abbasalipourkabir
LOX activity was measured by a commercial kit (Kiazist life science, Iran). At first, 10–20 mg of liver tissue was lysed by the buffer. Then, the lysate was centrifuged. 50 µl of each supernatant was loaded in black microplate wells and a 50 µl master mix was added. The master mix contained LOX substrate, LOX probe, and HRP. H2O2 is produced by the lysyl oxidase activity of the sample. The LOX probe of the master mix is oxidized by the HRP in the presence of H2O2. The automated microplate fluorometer was adjusted on 537/585 nm (excitation/emission wavelength) and the readings were done every 10 min once for up to 50 min. Then, the curve was plotted for each sample reaction and two points were selected in the linear zone of the curve. Δ570 was calculated by the following formula:
Dendrimer-based posaconazole nanoplatform for antifungal therapy
Published in Drug Delivery, 2021
Shengzhuang Tang, Jesse Chen, Jayme Cannon, Zhengyi Cao, James R. Baker, Su He Wang
Posaconazole release profiles in human plasma were monitored by measuring an increase in fluorescence intensity. Fluorescent spectra were recorded in a Fluoromax-2 fluorimeter (Horiba Scientific, Piscataway, NJ). To 2.4 mg of conjugate II-5 dissolved in 0.5 mL of phosphate buffer (pH 7.4) was added 0.5 mL of plasma from human. The mixture was immediately transferred to a dialysis bag (MWCO = 10 kDa). It was immersed in 19 mL of phosphate buffer (pH 7.4) in a shaking water bath at 37 °C. At predetermined time points, t = 0, 0.5, 1, 2, 6, 12, 24, 48, and 72 h, 1.5 mL of the external buffer was withdrawn and replenished with an equal volume of fresh medium. The amount of released POS was analyzed with fluorescence spectrophotometer with the excitation at 260 nm and detection using emission scan (320–480 nm). Conjugate II-5 in PBS and plasma in PBS were conducted under the same conditions, for comparison.