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

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.

Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

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

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.

Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

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

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.

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

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.

Angiotensin-converting enzyme 2 alleviates liver fibrosis through the renin-angiotensin system

The present letter to the editor is related to the study titled‘Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells’.Angiotensin-converting enzyme 2 can alleviate liver fibrosis by regulating autophagy of hepatic stellate cells and affecting the renin-angiotensin system.

A NOVEL POLYMER ENZYME LINKED IMMUNOASSAY METHOD AND ITS APPLICATIONS

During the course of study, we found that both poly (N-isopropylacrylamide ) (PNIP) and PNIP-Ab (enzyme-labelled antibody)could be adhere tightly to a cellulose acetale-nitrate membrane, and that the retention of PNIP-Ab on the membrane increased over 30-fold when compared with the unconjugated Ab.Thus we used this characteristic to develop a novel immunoassay method-polymer enzyme linked immunoassay method: homogeneous antigen-antibody reaction and heterogeneous separation process. When applied for detection of human serum HBsAg, this immunoassay system can detect as little as 1 ng/ml of human serum HBsAg.

Determnation of ochratoxin A in grain by monoclonal antibody－based enzyme－linked immunosorbent assay

The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.

SERODIAGNOSIS OF CLONORCHIASIS BY ENZYME—LINKED IMMUNOSORBENT ASSAY WITH HRP—SPA

In thes paper the authors used the Horseradish peroxidase labelledstaphylococcal protein A(HRP—SPA)in ELISA,for the detection of Clo-norchis sinensis infection.Serum tests were made on 116 confirmed cases ofclonorchiasis,103(88.8%)of them showed positive,while only 6(4.4%)werepositive among 138 healthy people.Samples were collected on filter paperstrips,111(95.7%)cases were positive among 116 comfirmed cases tested,but only 2(1.5%)were positive out of 138 healthy persons.The resultswere similar to those obtained by sheep antihuman IgG.Animal experimentalso showed that the SPA—ELISA can be used for the diagnosis ofclonorchiasis.In an endemic area,stool egg positive rate was 8.8%(62/703).whenchecked with SPA—ELISA,the rate of conformity in both filter paperstrips and stool examinations was 90.3(56/62).Among 641 serum testsfrom individuals negative in stool examinations,only 35(5.5%)reactedpositively.The authors suggested—that SPA—ELISA with soluble Clo-norchis antigens could be used in a large scale seroepidemiological surveyin endemic areas.

In vitro cleavage of hepatitis B virus C mRNA by 10-23 DNA enzyme

BACKGROUND: 10-23 DNA enzyme is one kind of de-oxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2. 2. 15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A1816UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. RESULTS: Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km,Kcat and Kcat/ Km for Drz-HBV-C-9, were 1.4 ×10-9 mol, 1.6 min-1 and 1.1 × 109 mol-1 · min-1, respectively. CONCLUSIONS: 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

A new angiotensin-converting enzyme inhibitor from Peperomia pellucida(L.) Kunth

Objective:To isolate,identify,and evaluate a new angiotensin-converting enzyme inhibitor from Peperomia pellucida(L.)Kunth herbs.Methods:A dried sample of Peperomia pellucida herb was successively macerated with n-hexane and ethyl acetate.The ethyl acetate extract solution was evaporated to obtain the crude extract.Vacuum liquid column chromatography and thin layer chromatography were performed to obtain two pure compounds.Then,both compounds were elucidated and identified using the spectroscopic method.Angiotensin-converting enzyme inhibitory activity studies of both compounds were determined using angiotensin-converting enzyme kit WST-1 with spectrophotometer microplate reader 96-well at 450 nm wavelength.Results:Two bioactive compounds were successfully isolated from Peperomia pellucida herb,including a new compound of 2,3,5-trimethoxy-9-(12,14,15-trimethoxybenzyl)-1 H-indene and pellucidin A.Both compounds demonstrated angiotensin-converting enzyme inhibitory activity,with IC50 values of 72 μM(27.95 μg/mL)and 1 1μM(4.4 μg/mL),respectively.Conclusions:In the present study,two active angiotensin-converting enzyme inhibitors were successfully isolated and purified from Peperomia pellucida which is used as an antihypertensive in traditional medicine,and support its use as an angiotensin-converting enzyme-inhibiting drug.

Electrochemical Enzyme Immunoassay of Tumor Marker CA15-3 with Capillary Electrophoresis

Tumor marker CA15-3 was determined by using capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED). The method can be used to detect CA15-3 with a limit of 0.024 U/mL.

Effects of tachykinin neuropeptide NKB and A<i>β</i>(25-35) on antioxidant enzymes status in 17<i>β</i>estradiol treated aging female rats

Aging is the leading risk factor for neurodegenerative diseases and oxidative stress involved in the pathophysiology of these diseases. These changes increase during menopausal condition in females when the level of estradiol is decreased. The aim of the present study was to determine the effect of tachykinin neuropeptide, Neurokinin B (NKB) and Amyloid beta fragment Aβ (25 -?35) on 17β estradiol (E2) treated aging female rat synaptosomes of different age groups. Aging brain functions were assayed by measuring the activities of antioxidant enzymes—superoxide dismutase (SOD) and monoamine oxidase (MAO) with neuropeptides. An in-vitro incubation of Aβ (25 -?35) in E2 treated brain synaptosomes showed toxic effects on all the parameters. However, NKB and NKB combined with Aβ (25 35) showed stimulating effects in E2 treated rat brain synaptosomes. In the present study, an increase in activity of SOD and decrease in the level of MAO, in the presence of NKB and combined NKB and Aβ in E2 treated brain synaptosomes of aging rats. This study elucidates that treatment of NKB and Aβ with E2 incombination exerts more protective influence than their individual application, against excitotoxicity in age related changes.

Correlation of serum Aβ1-42 content with inflammatory factors and receptors as well as antioxidant enzymes in patients with Parkinson's Disease

Objective: To investigate the correlation of serum β-amyloid 1-42 (Aβ1-42) content with inflammatory factors and receptors as well as antioxidant enzymes in patients with Parkinson's Disease. Methods: A total of 48 patients with Parkinson's disease who were treated in this hospital between December 2014 and October 2017 were selected as Parkinson's disease group, and 50 healthy volunteers who received physical examination in this hospital during the same period were selected as normal control group. The differences in serum contents of Aβ1-42, inflammatory factors and receptors as well as antioxidant enzymes were compared between the two groups, and Pearson test was used to assess the correlation between serum Aβ1-42 content and illness in patients with Parkinson's disease. Results: Serum Aβ1-42 content of Parkinson's disease group was lower than that of normal control group;serum inflammatory factors and receptors IL-2, sIL-2R, IL-6 and sIL-6R contents were higher than those of normal control group;serum antioxidant enzymes SOD, GSH-Px, CAT and TPX contents were lower than those of control group. Pearson test showed that serum Aβ1-42 content of patients with Parkinson's disease was directly correlated with the contents of inflammatory factors and receptors as well as antioxidant enzymes. Conclusions: Serum Aβ1-42 content decreases in patient with Parkinson's disease, and the specific content is directly correlated with the condition of Parkinson's disease, and can be used as an important auxiliary indicator for diagnosis and judgment of Parkinson's disease.

急性胆囊炎患者胆囊切除术后血清CCK-8、TREM1水平与发生感染的关系

目的分析急性胆囊炎(AC)患者胆囊切除术后血清胆囊收缩素-8(CCK-8)、髓系细胞触发受体1(TREM1)水平与发生感染的关系。方法将该院2020年12月至2022年12月收治的70例胆囊切除术后发生感染的AC患者纳入研究组,66例胆囊切除术后未发生感染的AC患者纳入对照组。采用酶联免疫吸附试验检测血清CCK-8、TREM1水平。采用Pearson相关分析胆囊切除术后发生感染AC患者血清中CCK-8、TREM1水平与炎症因子水平的相关性。采用受试者工作特征(ROC)曲线分析血清CCK-8、TREM1水平对AC患者胆囊切除术后发生感染的诊断价值。采用多因素Logistic回归分析AC患者胆囊切除术后感染的影响因素。结果研究组与对照组有胆囊结石、胆囊周边积液比例比较,差异均有统计学意义(P<0.05)。与对照组比较,研究组血清CCK-8水平明显降低,TREM1水平明显升高,差异均有统计学意义(P<0.05)。研究组C反应蛋白(CRP)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)水平明显高于对照组,差异均有统计学意义(P<0.05)。胆囊切除术后发生感染的AC患者血清CCK-8水平与CRP、IL-8、TNF-α水平均呈负相关(P<0.05),TREM1水平与CRP、IL-8、TNF-α水平均呈正相关(P<0.05)。ROC曲线分析显示,血清CCK-8与TREM1联合检测诊断AC患者胆囊切除术后发生感染的曲线下面积(AUC)明显大于CCK-8、TREM1单独检测的AUC(Z=5.703,P<0.001;Z=4.584,P<0.001)。有胆囊结石、胆囊周边积液及血清CCK-8水平降低、血清TREM1水平升高均为AC患者胆囊切除术后发生感染的危险因素(P<0.05)。结论胆囊切除术后发生感染的AC患者血清CCK-8水平降低,TREM1水平升高,二者联合检测能够提高对AC患者胆囊切除术后发生感染的诊断价值。

S-烯丙基巯基半胱氨酸促进CD8+T细胞杀伤功能的机制研究

目的探究S-烯丙基巯基半胱氨酸(S-allylmercaptocysteine,SAMC)对CD8^(+)T细胞杀伤鼻咽癌细胞功能的影响及机制。方法将人鼻咽癌细胞HK-1和C666-1与SAMC共培养,分为0、25、50、100μmol/L SAMC组,Western blot检测肿瘤细胞程序性死亡配体-1(programmed cell death-ligand 1,PD-L1)表达。CD8^(+)T细胞分别与HK-1和C666-1细胞以10∶1的比例共培养并加入0、25、50、100μmol/L SAMC,检测CD8^(+)T对HK-1和C666-1的杀伤能力,酶联免疫法(ELISA)检测干扰素(INF-γ)和肿瘤坏死因子-α(TNF-α)浓度,构建鼻咽细胞HK-1的小鼠皮下移植瘤模型,分为对照组、CD8^(+)T细胞组、SAMC组、SAMC+CD8^(+)T细胞组,各组小鼠治疗期间测量瘤体积,治疗结束后取小鼠肿瘤组织,Western blot检测肿瘤组织中PD-L1表达,ELISA检测小鼠血清INF-γ和TNF-α浓度。结果相比于0μmol/L SAMC组,25、50、100μmol/L SAMC组HK-1和C666-1细胞PD-L1表达均显著下调(P<0.05),相比于0μmol/L SAMC+CD8^(+)T细胞组,25、50、100μmol/L SAMC+CD8^(+)T细胞组HK-1和C666-1细胞培养上清中INF-γ和TNF-α浓度均能显著增加,HK-1和C666-1细胞裂解率显著增加(P<0.01)。相比于对照组,CD8^(+)T细胞组和SAMC+CD8^(+)T细胞组小鼠瘤体积和瘤重显著下降(P<0.05),小鼠血清INF-γ和TNF-α浓度显著增加,相比于CD8^(+)T细胞组,SAMC+CD8^(+)T细胞组小鼠瘤体积和瘤重显著下降(P<0.05),小鼠血清INF-γ和TNF-α浓度显著增加(P<0.05),肿瘤组织PD-L1表达显著下调(P<0.01)。结论SAMC可通过抑制人鼻咽癌细胞PD-L1表达而促进CD8^(+)T细胞的杀伤功能。

基于卡尔曼滤波的Link-22数据链网络时间同步算法

为了维持网络正常运行,Link-22数据链所采用的时分多址接入协议必须要求高精度的网络时间同步,传统的RTT算法降低了系统的实时性,PTP算法增大了系统的网络负担,且2种算法都忽略了延迟抖动以及获取时间戳带来的误差,导致时间同步精度低。基于此,文中提出了一种基于卡尔曼滤波的RTT-PTP算法,该算法在不影响系统的实时性以及不增加网络开销的情况下,利用卡尔曼滤波有效地降低延迟抖动以及不同时间戳精度带来的误差。最后通过仿真验证新算法能够有效地提高系统的时钟精度。

不同品牌ELISA试剂盒检测细胞培养上清液中IL-8对比研究

目的 对比研究2种不同品牌ELISA试剂盒检测细胞培养上清液中人白细胞介素-8(IL-8)水平情况。方法 不同浓度聚肌胞苷酸(Poly I:C)刺激A549细胞10、24 h,收集细胞上清液,共获得40份样本,分为1~10组,每组4份。采用2种ELISA试剂盒(试剂盒A、试剂盒B)检测细胞培养上清液中IL-8水平,对比2个试剂盒检测得到的组间数据或同组数据的一致性或差异性。结果 2种试剂盒拟合曲线中相关系数值分别为:0.999 600、0.999 602,检测标准品结果相关性良好。试剂盒B检测样品1、2组中IL-8水平比较,差异有统计学意义(P<0.01),但试剂盒A检测样品1、2组中IL-8水平比较,差异无统计学意义(P>0.05);2种试剂盒检测样品8、9组及样品9、10组中IL-8水平比较,差异均有统计学意义(P<0.01)。仅试剂盒B检测样品1、5组中IL-8水平明显低于试剂盒A,差异有统计学意义(P<0.01)。结论 不同品牌ELISA试剂盒均可用于检测IL-8水平,但检测结果仍在一定程度上存在差异。为避免实验误差,研究中应尽量选择同一个厂家生产的同品牌试剂盒。