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Clinical Aspects of Acid–Base Control
Published in Peter Kam, Ian Power, Michael J. Cousins, Philip J. Siddal, Principles of Physiology for the Anaesthetist, 2020
Peter Kam, Ian Power, Michael J. Cousins, Philip J. Siddal
In vivo titration curves are derived from the collation of changes of pH, and during acute and chronic disorders. The main advantage is that this accounts for changes in and pH due to the interaction of buffers in different compartments and also changes secondary to normal compensatory mechanisms.
Technetium-Labeled Compounds
Published in Garimella V. S. Rayudu, Lelio G. Colombetti, Radiotracers for Medical Applications, 2019
Suresh C. Srivastava, Powell Richards
Details of these methods are as follows. All tin(II) stock solutions are prepared inside a nitrogen glove box starting with high purity tin wire as described in an earlier section. Aliquots of this stock tin(II) in HCl are added to a 20-fold excess of various ligands of interest and the pH adjusted to 7. The tin(II) is then determined using iodate as follows. Aliquots are transferred into an airtight titration vessel containing 10 mℓ 1 N HCl inside a nitrogen glove box. The vessel is taken out and the mixture titrated poten-tiometrically under a nitrogen gas cover. Aliquots containing 15 μg to 4 mg tin(II) routinely produce a 300 mV response at the endpoint. A typical titration curve is shown in Figure 6. Representative data on the analysis of tin(II) in various media are described in Tables 8 and 9.157
Experimental Strategies
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
When adding small (μl) volumes of ligand from a fixed-volume pipette, errors can accumulate and give inaccurate results. It is better to add the ligand with a microsyringe whose total volume delivers a saturating amount of ligand. In this way, any slight overshoot on addition of one aliquot will be compensated for in the next addition. Indeed, if the microsyringe is driven by a precision pump, the whole process can be automated. With the pump under computer control, it is relatively simple to devise an instrument that will add a small volume of ligand, activate a stirrer, read the signal, and log it when it is stable and then add a further aliquot of ligand and so on. Such systems can be made “intelligent” such that if little signal change is incurred, the next aliquot added will be doubled in volume, whereas if a significant change occurs, it will halve the aliquot [356]. In this way, a typical titration curve will be efficiently tracked with a large number of points in the early stages of the titration (Figure 6.5). Autotitrators are also available commercially.
Generation of robust bispecific antibodies through fusion of single-domain antibodies on IgG scaffolds: a comprehensive comparison of formats
Published in mAbs, 2023
Andreas V. Madsen, Peter Kristensen, Alexander K. Buell, Steffen Goletz
The FIDA experiments were performed as previously described.16 In brief, we used a FIDA One instrument employing light-emitting diode-induced fluorescence detection using an excitation wavelength of 480 nm and an emission wavelength of >515 nm (Fida Biosystems). The PD-L1 (ACRO Biosystem, #PD1-H5229) and HER2 (ACRO Biosystems, #HE2-H5225) antigens were fluorescently labeled with DY-490 (EMP Biotech, #MKD0125) and the assay buffer was PBS+0.1% bovine serum albumin (BSA). Affinity measurements were performed in capmix mode by initial loading of the capillary with antibody solution at varying concentrations (3500 mbar, 20s), then injecting 20 nM fluorescent indicator ± equimolar unlabeled antigen (50 mbar, 10s), and finally a second loading step with antibody (400 mbar, 180-200s). Sizing of antibody complexes was performed in a complex dissociation mode by loading buffer (3500 mbar, 20s), then injecting a pre-mixed (>15 min) solution with 32 nM fluorescent antigen and 40 nM antibody ±80 nM unlabeled antigen (50 mbar, 10s), followed by a second buffer loading (400 mbar, 180-200s). A flush with assay buffer (3500 mbar, 120s) was performed between each measurement. Rh values were obtained by Taylorgrams to FIDA Software v2.3 (Fida Biosystems) with a Taylorgram fraction of 75%. The titration curves were fitted to a simple 1:1 binding using our in-house Python script.
Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice
Published in mAbs, 2022
Jerome Egli, Stefan Heiler, Felix Weber, Guido Steiner, Timo Schwandt, Katharine Bray-French, Christian Klein, Sebastian Fenn, Gregor P. Lotz, Eugenia Opolka-Hoffmann, Thomas E. Kraft, Laetitia Petersen, Rebecca Moser, Jonathan DeGeer, Michel Siegel, Daniela Finke, Juliana Bessa, Antonio Iglesias
The titration curve was fitted using the 5th degree polynomial and intersected with the baseline to obtain titer values. If no intersection occurred within the range of applied dilutions and the titration curve stayed consistently below baseline, we fixed the titer at the value corresponding to an intersection at the lowest dilution (1:50). If the intersection was not reached until the highest dilution, we allowed extrapolation of the titration curve up to a dilution of 1:328050, i.e., three times as high as the actual highest one used, applying a more stable 2nd degree polynomial fit. In any case, the titer was bounded by a maximum value corresponding to a 1:328050 dilution, and a flag was retained to indicate whether the result was obtained by proper interpolation or whether extrapolation/bounding was applied instead. Titers above the arbitrary threshold of 200 are considered ADA positive results.
Combination of two novel blocking antibodies, anti-PD-1 antibody ezabenlimab (BI 754091) and anti-LAG-3 antibody BI 754111, leads to increased immune cell responses
Published in OncoImmunology, 2022
Markus Zettl, Melanie Wurm, Otmar Schaaf, Sven Mostböck, Iñigo Tirapu, Ilse Apfler, Ivo C. Lorenz, Lee Frego, Cynthia Kenny, Michael Thibodeau, Elisa Oquendo Cifuentes, Markus Reschke, Jürgen Moll, Norbert Kraut, Anne Vogt, Jonathon D. Sedgwick, Irene C. Waizenegger
hPD-1-expressing CHO cells were plated in a 96-well round-bottom plate. Primary antibodies (ezabenlimab, BI 905725 or respective isotype controls) were added in multiple concentrations, and the plate was incubated for 1 h at 4°C. Corresponding secondary FITC-labeled antibody was added in fivefold excess (compared with the highest primary antibody concentration) without wash, and the plate was incubated for 30 min at 4°C. Cells were washed and analyzed by flow cytometry. For analysis, moles of FITC molecules per well were calculated based on detected FITC molecules per cell. The moles of bound antibody per well [B] were calculated by dividing the resulting value by the fluorescence-to-protein ratio (F/P ratio) of the secondary antibody. Moles of free antibody per well [F] were calculated by subtracting [B] from the initial concentration of antibody that was added into the well. In the titration curve, the MFI of the different wells were plotted against the logarithms of the respective initial antibody concentrations. Three or more points in the linear range of the sigmoidal titration curve were chosen. Of these, [B] (x-axis) versus [B/F] values (y-axis) were plotted as a Scatchard plot. The slope of a fitted line in the Scatchard plot and its intercept with the y-axis were calculated with integrated formulas in Microsoft Excel. KD was calculated with the following formula: −1/slope = KD (moles/L).