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1.
Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR), and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curve was established. This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml, with the half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml, respectively. The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested, and the IC50 values to enoxacin, ciprofloxacin, and pefloxacin were 3.1, 3.4, and 4.1 ng/ml, respectively. It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%. When spiked in milk at 5, 20, and 50 ng/ml, the recoveries for NOR, enoxacin, ciprofloxacin, and pefloxacin ranged 90.5%–98.0%, 84.0%–95.2%, 94.0%–106.0%, and 89.5%–100.0%, respectively. The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.  相似文献   

2.
A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2–4F, 7–3G, and 11–11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2–4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2–4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%–118% for an intra-assay and 96%–113% for an inter-assay. The coefficients of variation of the assays were 3.9%–13.9% and 5.5%–14.9%, respectively.  相似文献   

3.
Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method was employed to synthesize the artificial antigen of enrofloxacin (ENR), and New Zealand rabbits were used to produce anti-ENR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (ELISA) standard curve was established. This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg (R 2=0.9567), with the half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 9.4 μg/kg and 0.2 μg/kg, respectively. Of all the competitive analogues, the produced pAb exhibited a high cross-reactivity to ciprofloxacin (CIP) (87%), the main metabolite of ENR in tissues. After optimization, the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork. The overall recoveries and coefficients of variation (CVs) were in the ranges of 86%–109% and 6.8%–13.1%, respectively. It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.  相似文献   

4.
To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC50) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products.  相似文献   

5.
A protein conjugate of streptomycin (streptomycin-bovine serum albumin (BSA) conjugate) was prepared and used as immunogen to produce monoclonal antibodies (MAb). One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentration (IC50) value of 4.65 ng/ml and the IC20 value of 0.21 ng/ml in phosphate buffered saline (PBS). At optimum conditions, an indirect competitive enzyme-linked immunosorbent assay (ELISA) and a co...  相似文献   

6.
A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53?12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06?39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.  相似文献   

7.
A water-soluble adjuvant named QuickAntibody (QA) was introduced into the procedure of mouse immunization for the development of hapten-specific monoclonal antibodies (mAbs), using four kinds of pesticides as model compounds. Compared with conventional Freund;’s adjuvants, QA treatments offered relatively low but acceptable antiserum titers after three inoculations, gave little adverse effects to the experimental animals, and were preferable in harvesting splenocytes during the steps of cell fusion. Afterwards, hybridomas from the QA group were prepared and screened by both non-competitive and competitive indirect enzyme-linked immunosorbent assays (ELISAs). The efficiency of gaining immune-positive hybridomas was satisfactory, and the resultant mAbs showed sensitivities (half maximal inhibitory concentration (IC50)) of 0.91, 2.46, 3.72, and 6.22 ng/ml to triazophos, parathion, chlorpyrifos, and fenpropathrin, respectively. Additionally, the performance of QA adjuvant was further confirmed by acquiring a high-affinity mAb against okadaic acid (IC50 of 0.36 ng/ml) after three immunizations. These newly developed mAbs showed similar or even better sensitivities compared with previously reported mAbs specific to the corresponding analytes. This study suggested that the easy-to-use adjuvant could be applicable to the efficient generation of highly sensitive mAbs against small compounds.  相似文献   

8.
The enantioselective assay for S( )- and R(-)-propafenone (PPF) in human urine that developed in this work involves extraction of propafenone from human urine and using S( )-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-β-D-glucopranosyl isothiocyanate, and quantitation by an RP-HPLC system with UV detection (λ=220nm). A baseline separation ofpropafenone enantiomers was achieved on a 5-μm reverse phase ODS column, with a mixture of methanol:water:glacial acetic acid (25:12:0.02,v/v) as mobile phase. There was good linear relationship from 24.9 ng/ml to 1875.0 ng/ml for both of enantiomers. The regression equations of the standard curves based on Cs-PPF (or CR-PPF ) versus ratio of As-PPF/As (or AR-PPF/As ) were y=0.0032x-0.081, (r=0.999) for S-PPF and y=0.0033x 0.0039, (r=0.998) for R-PPF,respectively. The method's limit of detection was 12.5 ng/ml for both enantiomers, and the method's limit ofquantitation was 28.2±0.52 ng/ml for S-PPF, 30.4±0.53 ng/ml for R-PPF (RSD＜8%, n=5). The analytical method yielded average recovery of 98.9% and 100.4% for S-PPF and R-PPF, respectively. The relative standard deviation was no more than 6.11% and 6.22% for S-PPF and R-PPF, respectively. The method enabled study of metabolism of S( )- and R(-)-propafenone in human urine. The results from 7 volunteers administered 150 mg racemic propafenone indicated that propafenone enantiomers undergo stereoselective metabolism and that in the human body, S( )-propafenone is metabolized more extensively than R(-)propafenone.  相似文献   

9.
Development and evaluation of immunoassay for zeranol in bovine urine   总被引：1，自引：0，他引：1  
A high affinity polyclonal antibody-based enzyme linked immunosorbent assay （ELISA） was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1：5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml, and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation （CVs） were in the range of 82%-127% and 3.5%-8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol （ranging from 0.2 to 10 μg/ml） were detected by the ELISA and liquid chromatography （LC） method, and good correlations were obtained between the two methods （R^2=0.9643）. We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an altemative for the conventional LC method for zeranol in bovine urine.  相似文献   

10.
Ethanol extracts of brown seaweeds from Pakistan and China were isolated and compared for their antiallergenic activities.They included Sargassum tennerimum (ST) and Sargassum cervicorne (SC) from Pakistan,and Sargassum graminifolium turn (SG),Sargassum thunbergii (STH),and Laminariajaponica (LJ) from China.The ethanol extracts of these brown seaweeds were optimized at 85% (v/v) ethanol for the maximum yield of phlorotannin,an inhibitor against hyaluronidase.Total phlorotannins contained in the crude extracts were measured as 1.71% (SG),0.74% (STH),0.97% (LJ),3.30% (SC),and 5.06% (ST).The 50% inhibitory concentrations (IC50) of Pakistani SC and ST were 109.5 and 21 μg/ml,respectively,lower than those of Chinese SG,STH,and LJ (134,269,and 148 μg/ml,respectively).An antiallergic drug,disodium cromoglycate (DSCG),had an IC50=39 μg/ml,and a natural inhibitor of hyaluronidase,catechin,had an IC50=20 μg/ml.The IC50 of ST extract was found similar to that of catechin (21 vs 20 μg/ml) and lower than that of DSCG (21 vs 39 μg/ml).This suggests that ST is a potent inhibitor of hyaluronidase,indicating a promising future development of natural antiallergic medicines or functional foods.  相似文献   

11.
以固载于玻碳电极（GC）表面的壳聚糖／多壁碳纳米管（MWNT～CS）复合物膜为基底电聚合媒介体甲苯胺蓝（PTOB）,利用静电作用吸附纳米金（nano--Au）,再利用纳米金良好的生物兼容性、大的比表面积固定癌胚抗体（anti—AFP）,从而制得高灵敏、高稳定电流型癌胚抗原免疫传感器。多壁碳纳米管复合物膜促进了电子的传递．增大了电极的比表面积：纳米金的存在增加了抗体在电极表面的固定量,从而提高了免疫电极的灵敏度。在优化的实验条件下．该免疫电极在1．0～80．0ng／mL范围内,峰电流与CEA抗原的浓度呈良好的线性关系。检测线为0．4ng／mL。该免疫传感器制备方法简单,灵敏度高、选择性好。  相似文献   

12.
For preparing fluorinated quinolone antibiotic medicine locally used in stomatology, simultaneous determination of norfloxacin, ciprofloxacin, and enoxacin was carried out by multiphase ion chromatography with fluorescence detection. Quinolone antibiotics were separated by Dionex OmniPac PAX-500 column with an eluent of 15 mmol/L H2SO4 and 35% methanol （v/v） at a flow-rate of 1.0 ml/min and detected with fluorescence with excitation and emission wave lengths of 347 ran and 420 ran respectively. The detection limits （S/N=3） of norfloxacin, ciprofloxacin and enoxacin were 50, 105 and 80 ng/ml respectively. The relative standard deviations of retention time, peak area and peak height were less than 1.1% and good linear relationship resulted. The developed method was applied to pharmaceutical formulations and biological fluids.  相似文献   

13.
本文报道丹贝中生物活性物质大豆甙元同辣根过氧化物酶偶联及其酶活性、免疫活性和光谱分析鉴定。大豆甙元—辣根过氧化物酶标记物中两者摩尔比11：1，标记物工作浓度50ng／ml，3．5ng／ml大豆甙元能与氧化酶标记物发生50％左右竞争。  相似文献   

14.
Objective To investigate the relationship between serum resistin level and acute coronary syndrome (ACS) or stable angina pectoris (SAP). Methods Sixty-five patients, with coronary artery disease, were enrolled and divided into three subgroupsacute myocardial infarction (AMI), unstable angina pectoris (UAP) and SAP, and 26 healthy people were recruited as controls in the cross-sectional study. Serum resistin levels were determined by ELISA (enzyme-linked immunosorbent assay), and WBC (white blood cell count), hsCRP (high sensitive C-reaction protein), CKmax (maximum ofcreatinkinase), CK-MBmax (maximum of isozyme of creatinkinase) and cTnImax (maximum oftroponin) were measured by standard laboratory methods. Results The serum resistin levels were 4 folds higher in AMI patients, 2.43 folds in UAP patients and 1.12 folds in SAP patients than in the healthy controls (P＜0.05). The resistin levels were also significantly different between AMI [(8.16±0.79) ng/ml], UAP [(5.59±0.75) ng/ml]and SAP [(3.45±0.56) ng/ml] groups (P＜0.01); WBC, hsCRP, CKmax, CK-MBmax and cTnImax were significantly increased in AMI patients over UAP and SAP patients. Spearman analysis showed that serum resistin levels were positively correlated with WBC (r=0.412, P=0.046), hsCRP (r=0.427, P=0.037), CKmax, CK-MBmax and cTnImax (r=0.731, 0.678, 0.656; P＜0.01). ConclusionSerum resistin levels increased with inflammatory factors and myocardial impairment. The results suggest that human resistin might play an important role in the pathogenesis of atherosclerosis and AMI as an inflammatory factor.  相似文献   

15.
Potato virus S (PVS) often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS (PVSO) was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies (MAbs). Five highly specific and sensitive murine MAbs (1A3, 16C10, 18A9, 20B12, and 22H4) against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and double-antibody sandwich (DAS)- ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1:10 240 and 1:163 840 (w/v, g/ml) in phosphate buffer saline (PBS) by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.  相似文献   

16.
Objective: To evaluate the interaction between serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) andHelicobacter pylori (H. pylori) infection in patients with chronic gastritis and peptic ulcer. Methods: The serum levels of sICAM-1 in 205 patients with chronic gastric diseases were detected by ELISA method and the status ofH. pylori was determined by histologic examination, RUT,14C-UBT, and serology. The sera obtained from 18 healthy volunteers served as controls. Results: The serum levels of sICAM-1 were significantly higher in patients withH. pylori positive than those of H. pylori negative (889.43±32.52 ng/ml vs. 747.07±30.45 ng/ml,P<0.05). The serum levels of sICAM-1 in patients with mild, moderate and severe infection ofH. pylori were 841.68±72.36 ng/ml, 905.43±37.59 ng/ml and 1012.54±49.34 ng/ml, respectively (P<0.05). The serum levels of sICAM-1, proved to be significantly correlated with the density ofH. pylori colonization in gastric mucosa (r s=0.316,P<0.001). The serum levels of sICAM-1 in patients with chronic gastritis and peptic ulcer were significantly higher than those in healthy controls (P<0.05). Conclusions: These results indicated thatH. pylori infection up-regulates the expression of sICAM-1.  相似文献   

17.

Objective

The aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer (CIK) cells and then demonstrate the appropriate mechanism.

Methods

Tumor-derived exosomes (TEXs), which are derived from RBE cells (human cholangiocarcinoma line), were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting (FACS) was performed to determine the phenotypes of TEX-CIK and N-CIK (normal CIK) cells. The concentrations of tumor necrosis factor-α (TNF-α) and perforin in the culture medium supernatant were examined by using an enzyme-linked immunosorbent assay (ELISA) kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line.

Results

The concentrations of TNF-α and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml (P<0.01) and 3.39 ng/ml (P<0.05). The killing rate of the group TEX-CIK was 33.35%, lower than that of the group N-CIK (47.35% (P<0.01)). The population of CD3+, CD8+, NK (CD56+), and CD3+CD56+ cells decreased in the TEX-CIK group ((63.2±6.8)%, (2.5±1.0)%, (0.53±0.49)%, (0.45±0.42)%) compared with the N-CIK group ((90.3±7.3)%, (65.7±3.3)%, (4.2±1.2)%, (15.2±2.7)%), P<0.01.

Conclusions

Our results suggest that RBE cells-derived exosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3+, CD8+, NK (CD56+), and CD3+CD56+ cells and the secretion of TNF-α and perforin. TEX may play an important role in cholangiocarcinoma immune escape.
  相似文献   

18.
目的：研究人体片形吸虫病ELISA检测试剂盒的检测效果。方法：采用云南大理本地采集的牛体片形吸虫成虫制备可溶性粗抗原,包被反应板,采用人体片形吸虫病ELISA法（FAWA-ELISA）进行研究,分别检测26份片形吸虫患者血清、102份其他寄生虫病患者血清和25份健康人血清,评价检测试剂盒的检测效果。结果：检测试剂盒的敏感性为100.0%、特异性分别为95.28%（95%CI：98.9%～91.2%）,约登指数分别为0.95。结论：FAWA-ELISA试剂盒检测效果较好,且成本相对较低,可适宜用于云南片形吸虫病流行区流行病学筛查,减轻病原学检测的工作量。  相似文献   

19.
玉米中脱氧雪腐镰刀菌烯醇的气相色谱分析   总被引：2，自引：0，他引：2  
这个实验建立了一种简单、灵敏、高效的测定玉米中脱氧雪腐镰刀菌烯醇(DON)的电子捕获气相色谱方法.在该方法中,玉米样品通过氯仿-乙醇(8∶2)提取,经硅胶柱净化,先用甲苯-丙酮(8∶2)进行洗柱,最后用二氯甲烷-甲醇(95∶5)洗脱,洗脱液蒸干、衍生后气相色谱进行测定,检测器为电子捕获检测器.结果表明,该方法对玉米中DON的最低检测限为10ng/g,检测线性范围为50～500ng/g.平均回收率76.21%,变异系数0.10.该方法灵敏、简单、快速、重现性好,具有较高的实用价值.  相似文献   

20.
在碱性条件下,Ni(II)和1-(2-吡啶偶氮)-2-萘酚(PAN)形成聚合物,对PAN的共振光散射有增强作用,加入十二烷基苯磺酸钠(SDBS)进一步敏化该体系.共振光散射增强强度(ΔI)与Ni(II)浓度呈良好的线性关系,据此建立了测定Ni(II)的共振光散射分析方法.在优化的实验条件下,体系的最大散射波长位于545nm处,方法的线性回归方程为ΔIRLS=4502.9ρ(μg/mL)+271.82;线性范围为15.2500ng/mL;相关系数γ=0.9975;检出限为4.57 ng/mL.对Ni(II)分别为50ng/mL、200ng/mL、400ng/mL低、中、高三个浓度进行11次平行测定,其相对标准偏差分别为:3.5%、3.0%和1.7%.  相似文献   

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