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Jeffrey J. Talbot Dominador Calamba Melody Pai Mark Ma Theingi M. Thway 《The AAPS journal》2015,17(6):1446-1454
Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of Kon, Koff, and KD were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC50, a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (Koff) from TPx and that capture and detection antibodies had significantly higher binding affinities (KD) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.KEY WORDS: affinity and kinetics, association and dissociation, free versus total, interference, ligand binding assay 相似文献
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Displacement of endogenous enterokinase into portal venous blood and bile following luminal perfusion of proximal small intestine in guinea pigs 总被引:2,自引:0,他引:2
R. W. Talbot FRCS D. A. W. Grant PhD Professor J. Hermon-Taylor MChir FRCS 《Digestive diseases and sciences》1984,29(11):1009-1014
The displacement of endogenous enterokinase into portal venous blood or bile was studied in conscious guinea pigs both with the small intestine undisturbed and during gentle, intermittent luminal perfusion of a 25-cm segment of duodenum and proximal jejunum. Perfusates tested included water, 150 mM saline, 5% (v/v) ethanol, 0.2% (w/v) lysolecithin, and mixtures of ethanol and lysolecithin. Enterokinase activity was absent from portal venous blood of control guinea pigs with the intestine undisturbed but perfusion with luminal saline or water was consistently associated with substantial levels of active enterokinase in portal venous blood. Similar concentrations of enterokinase in portal blood were also detected in response to luminal ethanol and lysolecithin. The capacity of the normal liver rapidly to clear the enzyme from portal blood was demonstrated. Of the estimated total endogenous enterokinase displaced, 0.2–0.4% was recovered in catalytically active form from the pooled bile of luminally perfused but not control animals. The readiness with which enterokinase was displaced into the circulation in the absence of mucosal damage raises the unexpected possibility that the event may be physiological. Induced penetration of the mucosa and absorption of luminal components is clearly different from the release into portal venous blood of endogenous mucosal macromolecules. 相似文献
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E A Talbot P Jensen H J Moffat C D Wells 《The international journal of tuberculosis and lung disease》2002,6(8):738-741
The recommended role of ultraviolet germicidal irradiation (UVGI) is to reduce the risk of tuberculosis (TB) transmission in health care facilities. However, excess exposure may result in dermatosis and photokeratitis. In one hospital setting in Botswana, two nurses and one housekeeper complained of eye discomfort, 'like sand in the eyes', after working in an administrative office. The following day, one employee noted facial skin peeling. All symptoms resolved over 2-4 days without sequelae. Six weeks later, the syndrome recurred for all three employees. A workplace investigation revealed that the office had been converted from a hospital sputum induction room, and that an unshielded 36-W UVGI lamp was still installed and operational. The on/off switch for the UVGI lamp was immediately adjacent to the fluorescent bulb on/off switch, and did not have a locking mechanism. The US National Institute for Occupational Safety and Health recommends that exposure to UVGI (254 nm) be less than 6000 microJ/cm2 (6000 microW approximately = sec/cm2) over a daily 8-hour period on unprotected skin or eyes. In the office, UVGI measurements at eye level and looking directly at the UVGI lamp ranged from a low of 20.0 microW approximately = sec/cm2 when seated to a high of 49.9 microW approximately = sec/cm2 when standing. These irradiance levels result in allowable exposure times of 300 and 120 seconds, respectively, and are the most likely cause of the clinical syndrome described. 相似文献
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