ABSTRACT
T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.
ABSTRACT
Objective To analyze the correlation between LC-MS/MS method and enzymatic cycling assay for determination of homocysteine concentration in human serum,and the application of two methods in the determination of homocysteine concentration.Methods Homocysteine concentrations of 63 serum samples were collected and determined by LC-MS/MS method and enzymatic cycling assay,respectively.The correlation between the concentrations by different methods was analyzed and evaluated.Results The concentrations were(19.11 ± 15.69) μmol/L by LC-MS/MS method and(16.95 ± 14.41) μmol/L by enzymatic cycling assay,the P value evaluated by paired-samples T test showed that there was statistical difference among the concentrations determined by two different methods (t =6.25,P ＜ 0.05).The conversion formula was YLC-MS/MS method =1.074Xenzymatic cycling assay + 0.892,R =0.987.Conclusion There is good correlation between LC-MS/MS method and enzymatic cycling assay for the determination of homocysteine concentration in serum,providing a theoretical basis for estimating the concentrations in the same serum sample by the two methods.
ABSTRACT
In order to evaluate the pharmacokinetic profile of cefotiam hexetil hydrochloride tablet in Chinese healthy volunteers, a sensitive, specific and rapidprotein precipitation-liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma. Chromatographic separation was achieved on a Waters Symmtry-C18 column (50 mm × 4. 6 mm, 5 μm), using a gradient mobile phase consisting of methanol and 1 mmol/ L ammonium acetate in water at a flow rate of 1. 0 mL/ min. Cefotiam and diazepam (internal standard) were detected without interference in the multiple reaction monitoring (MRM) mode with positive electrospray ionization. The calibration curve was linear from 5. 0 ng / mL to 5000 ng / mL (r>0. 99) with limit of quantitation of 5. 0 ng / mL. The assay met the published acceptance criteria. This rapid, sensitive and reproducible method was successfully applied to the pharmacokinetic study of cefotiam hexetil hydrochloride tablet in healthy Chinese volunteers and therefore provided a considerable mirror for quantification of other cephalosporins in human matrix.
ABSTRACT
Oridonin is an en-kaurene diterpenoids compound which was found to have effect on tumor in vitro.But the clinical application has been limited because of poor water solubility,low bioavailability and oral absorption.Many scholars conducted a large number of structure modification researches of oridonin to search for new diterpenes with higher bio-activities and better bio-availabilities,and to develop new antitumor drugs.In this paper,studies on this aspect of oridonin were re-viewed.
ABSTRACT
Objective To build a clinical key disciplines evaluation index system for county level hospitals in Chengdu city.Methods Literature meta analysis, focus group discussion, expert consultation method, boundary value method, brainstorming and hierarchy analysis method were comprehensively used.Results The clinical key disciplines evaluation index system for county level hospitals in Chengdu city comprises 5 level-1 indexes,1 6 level-2 indexes,47 level-3 indexes.Among the level-1 indexes,service capacity,medical quality,technical personnel,scientific research and education, and foundation of specialty was 0.474 6,0.202 7,0.148 2,0.097 7,0.076 8 respectively.Conclusion The clinical key disciplines evaluation index system for county level hospitals in Chengdu city is scientific, guiding and practical,which can be used to evaluate the status of the clinical key disciplines for county level hospitals in Chengdu city.