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Development of a competitive enzyme-linked immunosorbent assay for therapeutic drug monitoring of afatinib

Journal of Pharmaceutical Analysis 2019年第1期9卷 49-54页

作者： Rintaro Sogawa Tetsuya Saita Yuta Yamamoto Sakiko Kimura Yutaka Narisawa Shinya Kimura Masashi Shin Department of Pharmacy Saga University Hospital Applied Life Science Department Faculty of Biotechnology and Life Science Sojo University Division of Hematology Respiratory Medicine and Oncology Department of Internal Medicine Faculty of Medicine Saga University

Afatinib is an oral tyrosine kinase inhibitor(TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring(TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay(ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using(S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine(CTQD), which has the same substructure as afatinib, as a hapten. enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods(Y=0.976 X – 0.207, r=0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.

关键词： Afatinib enzyme-linked immunosorbent assay Therapeutic drug monitoring Tyrosine-kinase inhibitor

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Quantitative determination of erlotinib in human serum using competitive enzyme-linked immunosorbent assay

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Journal of Pharmaceutical Analysis 2018年第2期8卷 119-123页

作者： Yuta Yamamoto Tetsuya Saita Yutaro Yamamoto Masashi Shin Applied Life Science Department Faculty of Biotechnology and Life Science Sojo University

A selective and sensitive competitive enzyme-linked immunosorbent assay(ELISA) method was developed and validated for the quantification of erlotinib in 50 mL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N-succinimidyl ester method. enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity(6.7%) with a major metabolite, O-desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.

关键词： Erlotinib enzyme-linked immunosorbent assay O-desmethyl erlotinib Tyrosine-kinase inhibitor

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Cyanidin-horseradish peroxidase-hydroperoxide reaction system and its application in enzyme-linked immunosensing assays

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Science China Chemistry 2009年第8期52卷 1142-1147页

作者： GONG FuChun LI DingZhong YANG Rong WEI JianKe CAO Zhong TAN ShuZhen TAN YaFei College of Chemistry and Biological Engineering Changsha University of Science and TechnologyChangsha 410004China

A cyanidin-based horseradish peroxidase(HRP)-catalyzed reaction system was established in this work.In B-R buffer solutions(pH 6.8),a UV-visible absorbance peak of cyanidin(CAG) at 540 nm(Ap1) appeared.After the oxidation reaction of CAG catalyzed by HRP in the presence of H2O2,a significant absorbance peak at 482 nm(Ap2) occurred.The ratio R(AP2/AP1)was proportional to the HRP concentration.The application of CAG in the enzyme-linked immunosensing assays was investigated using food and mouth disease virus antigen(FMDVAg) as a model analyte.In sandwich immunoreaction,the analyte FMDVAg and food and mouth disease virus antibody(FMDVAb)-modified magnetic nanoparticles bound the supported conconvalina(Con A) bound with HRP-FMDVAb.After de-absorbing and separating,the HRP-FMDVAb-FMDVAg-FMDVAb-magnetic nanoparticles complexes were subject to enzymatic reaction and UV-visible absorbance measurements.The HRP moiety of the immunoreaction complexes can catalyze the oxidation reaction of CAG by H2O2,and the substrate CAG is converted to products.Based on this principle,a sandwich assay model has been employed to determine FMDVAg in rabbit serum samples with the aid of FMDVAb-Fe3O4 magnetic nanoparticles.The linear range of the FMDVAg determination is 1.5×10-8-2.7×10-6 g/mL with the relatively standard deviation of 3.7%(n = 11).The detection limit is 3.1×10-9 g/mL.Additional advantages of the typical substrate such as OPD,OAP and TMB are good water-solubility and stability.

关键词： cyanidin HRP substrate enzyme-linked immunosensing food and mouth disease virus

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A Comparison of enzyme-linked Immunosorbent Assay versus Multiplex Methodology Using an <i>in Vitro</i>Model of Pulmonary Hypertension and Inflammation

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Journal of Biomedical Science and Engineering 2014年第7期7卷 419-426页

作者： Yan Zhu Deepthi Alapati Joanna Costa Victoria L. Maduskuie Paul T. Fawcett Thomas H. Shaffer Center for Clinical Diagnostics Nemours Alfred I duPont Hospital for Children Wilmington USA Nemours Research Lung Center Biomedical Research Nemours Alfred I duPont Hospital for Children Wilmington USA

enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.

关键词： enzyme-linked Immunosorbent Assay (ELISA) Luminex Pulmonary Artery Smooth Muscle Cells (PASMC) Inflammation Bland-Altman Plot Analysis

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enzyme-linked Immunosorbent Assay for Detection of Anti-idiotype Antibodies to Antibodies to Ligand of Nicotinic Acetylcholine Receptor in Sera of Patients with Myasthenia Gravis

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Journal of Medical Colleges of PLA(China) 1990年第3期5卷 237-242页

作者：黄德仁涂来慧张仁琴周广智沈茜 Department of Neurology Changhai HospitalSecond Military Medical UniversityShanghai 200433 Clinical Laboratory Changhai Hospital

Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.

关键词： myasthenia gravis enzyme-linked immunosorbent assay nicotinic acetylcholine receptor ligand antibungarotoxin antiserum anti-idiotype antibodies

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Development of a sandwich ELISA for the detection of bovine herpesvirus type 1

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Asian Pacific Journal of Tropical Medicine 2011年第5期4卷 363-366页

作者： Shanaz Bashir Rashmi Singh Barkha Sharma Sharad K Yadav Department of Epidemiology and Vety.Preventive medicine College of Veterinary SciencesVeterinary UniversityMathura(U.P.)India

Objective:To develop a standard enzyme-linked immunosorbent assay（ELJSA） for the detection of bovine herpesvirus type 1（BHV-1）.Methods:The assay was based on hyperimmune rabbit and guinea pig antisera raised against purified BHV-1.Polyethylene glycol precipitation and sucrose density gradient methods were adopted for viral concentration and purification.Antisera were raised using Freund’s adjuvant followed by extraction of IgG of high purity.Results: Optimum antisera dilutions as determined by titrations were chosen as 14 000,whereas the conjugate was used at 1:2 000 dilution.Using 95 clinical specimens,the ELISA test showed a sensitivity and specificity of 91.90%and 93.10%,respectively when compared to PCR.The cutoff value was fixed at 0.15490) and a P/N ratio of>1.30 indicated a significant positive reaction. Conclusions:The results have demonstrated that this ELISA could efficiently detect BHV-1 and can be used as an important diagnostic tool.

关键词： Bovine herpesvirus type 1 Antisera enzyme-linked immunosorbent assay (ELISA)

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Epidemiologic characteristics of Helicobacter pylori infection in southeast Hungary

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World Journal of Gastroenterology 2019年第42期25卷 6365-6372页

作者： Lenke Bálint Andrea Tiszai Gábor Kozák Ilona Dóczi Veronika Szekeres Orsolya Inczefi Georgina Ollé Krisztina Helle Richárd Róka András Rosztóczy Division of Gastroenterology First Department of MedicineUniversity of SzegedSzeged 6720Hungary Department of Physiology University of SzegedSzeged 6720Hungary of Clinical Microbiology University of SzegedSzeged 6725Hungary Hungarian National Blood Transfusion Service Szeged 6722Hungary

BACKGROUND Epidemiologic studies have revealed a decrease in the prevalence of Helicobacter pylori(H.pylori)infection in Western Europe.AIM To obtain data regarding the prevalence of H.pylori in Csongrád and Békés Counties in Hungary,evaluate the differences in its prevalence between urban and rural areas,and establish factors associated with positive seroprevalence.METHODS One-thousand and one healthy blood donors[male/female:501/500,mean age:40(19–65)years]were enrolled in this study.Subjects were tested for H.pylori IgG antibody positivity via enzyme-linked immunosorbent assay.Subgroup analysis by age,gender,smoking habits,alcohol consumption,and urban vs nonurban residence was also performed.RESULTS The overall seropositivity of H.pylori was 32%.It was higher in males(34.93%vs 29.2%,P=0.0521)and in rural areas(36.2%vs 27.94%,P=0.0051).Agricultural/industrial workers were more likely to be positive for infection than office workers(38.35%vs 30.11%,P=0.0095)and rural subjects in Békés County than those in Csongrád County(43.36%vs 33.33%,P=0.0015).CONCLUSION Although the prevalence of H.pylori infection decreased in recent decades in Southeast Hungary,it remains high in middle-aged rural populations.Generally accepted risk factors for H.pylori positivity appeared to be valid for the studied population.

关键词： Helicobacter pylori Epidemiology Prevalence Central Europe Healthy volunteers enzyme-linked immunosorbent assay Differences in urban and rural population

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Fluorometric enzyme immunosensing system based on natural product resveratrol for horseradish peroxidase and Ag/SiO_2 nanoparticles

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Science China Chemistry 2008年第3期51卷 233-240页

作者： GONG FuChun, CAO Zhong, TAN ShuZhen, TAN YaFei & HE XiaoChun College of Chemistry and Environmental Engineering, Changsha University of Science and Technology, Changsha 410076, China College of Chemistry and Environmental Engineering Changsha University of Science and Technology Changsha China

The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO2 particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization reaction of RST by H2O2 forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation: 315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO2 particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 1.5×10-6-7.3×10-4 g/L and with a detection limit of 1.5×10-6 g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with satisfactory results.

关键词： resveratrol HRP fluorogenic substrate Ag/SiO2 nanoparticles enzyme-linked immunosensing Schistosomia japonicum

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PRODUCTION AND CHARACTERIZATION OF A MONOCLONAL ANTIBODY AGAINST TRICHOTHECENE MYCOTOXIN T-2

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Chinese Science Bulletin 1991年第21期36卷 1829-1833页

作者：阳传和罗雪云 Institute of Food Safety Control and Inspection Ministry of Public Health Beijing 100021 PRC

Trichothecene mycotoxin T-2 is a secondary metabolite of several Fusarium species, and is known as a natural contaminant of stored grain and other agricultural products. This compound is a potent inhibitor of protein synthesis and has been implicated as the causative agent of several mycotoxicoses. There is also a close relationship between T-2 toxin and esophageal carcinoma, Keshan, Kaschin-Beck diseases in China.

关键词： T-2 toxin monoclonal antibody enzyme-linked immunosorbent assay

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Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

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World Journal of Gastroenterology 2001年第1期7卷 22-27页

作者： Yu Cheng Tang Yu Li Guan Xiang Qian Department of Biochemistry, Shanghai Second Medical University, Shanghai 200025, China Department of Biochemistry Shanghai Second Medical University Shanghai 200025China. tangyc@***

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone.Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up. PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomicDNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgeneexpressing cells was 65 ± 10 ng / L per 106 cells,420 ± 45 ng/L per 106 cells in sense group and 485± 30 ng/L per 106 cells in the negative control group, (P＜0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S. C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells.CONCLUSION Expression of antisen

关键词： liver neoplasms endothelial growth factors gene therapy endothelium vascular enzyme-linked immunosorbent assay carcinoma, hepatocellular RNA, antisense

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