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1.
苏丹红Ⅰ号多克隆抗体的制备与特异性鉴定   总被引:4,自引:4,他引:0  
采用琥珀酸酐法,分别以BSA、OVA为载体蛋白合成了免疫抗原和包被抗原并通过SDS-PAGE凝胶电泳和紫外扫描法鉴定了合成的抗原为载体蛋白和苏丹红I号的复合物.将合成的免疫抗原用弗氏佐剂乳化后免疫新西兰大白兔,制备出多克隆抗体,经辛酸纯化,间接ELISA法测得有高效价血清抗体;进一步采用琼脂双扩散试验和免疫金试纸法鉴定出血清中含有苏丹红Ⅰ号特异性抗体.  相似文献   

2.
拟除虫菊酯类农药单克隆抗体的制备及鉴定   总被引:1,自引:0,他引:1  
以间苯氧基苯甲酸(PBA)为半抗原,与牛血清白蛋白(BSA)偶联后作为抗原,免疫Balb/c小鼠,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,使用PBA与鸡卵清白蛋白(OVA)偶联物作为包被物,间接ELISA法筛选到4株特异性针对PBA的杂交瘤。其中两株细胞2F7和7B1制备腹水并用ProteinG柱纯化抗体,抗体效价均达到1:625000。单抗7B1用于间接竞争ELISA法,PBA检测下限为0.025μg·mL-1,IC50为0.57μg·mL-1,对溴氰菊酯、甲体氯氰菊酯、甲氰菊酯、氰戊菊酯均有特异性识别,IC50分别为0.79μg·mL-1、0.74μg·mL-1、0.63μg·mL-1、0.8μg·mL-1,对联苯菊酯识别弱,IC50为99.7μg·mL-1。该单抗可用于多种菊酯类农药的检测。  相似文献   

3.
目的针对可食包装纸原料中的玉米赤霉烯酮进行检测,采用以酶联免疫法为主,胶体金试纸条检测法为辅的免疫分析方法,为实时监控食品的包装、包装存储条件等提供有效技术手段。方法实验主要选取可食包装纸原料玉米粉作为检测对象,为提高酶联免疫法检测结果的准确性,通过添加回收实验,以最高的回收率为判断标准对应得到最优的前处理条件;先通过标准曲线的封闭温度和温育时间的优化,再进行玉米赤霉烯酮的前处理方式(包括对提取液浓度、稀释液的pH值、稀释液中有机溶剂的浓度等3个方面进行优化,以添加回收结果作为判断依据)进行测试。同时,将优化后的实验结果组装到胶体金试纸条,并对掺杂玉米赤霉烯酮的玉米粉进行快速检测。结果前处理最适条件,甲醇溶液的体积分数为50%,缓冲液pH值为8.0且含甲醇的体积分数为10%,在该条件下测得的回收率最高。在该优化条件下,组装的胶体金试纸条其检测灵敏度可达0.75ng/mL。结论该检测结果与添加标品所测实验结果基本一致,可以确定该胶体金试纸条是一种快速有效的检测方法。  相似文献   

4.
目的对抗氯霉素单克隆抗体4D10进行纯化与特性鉴定,以获得可用于检测氯霉紊残留试剂盒的单克隆抗体。方法用两步硫酸铵法和G蛋白亲和层析法纯化单克隆抗体,用间接酶联免疫吸附试验、竞争ELISA等方法测定抗体效价、特异性、敏感性与亲和性以及抗体与其它氯霉素结构类似物的交叉反应率。结果纯化后抗体的纯度高达95%,对抗体进行相关性质的鉴定得出:4D10效价为1:2.56×105,其亲和力常数可达2×108M-1,该抗体与CAPBSA,CAPOVA有反应,与BSA、OVA无反应。与氯霉素琥珀酸钠、氯霉素反应,而与其它氯霉索结构类似物无交叉反应。结论抗氯霉索单克隆抗体4D10效价高、特异性强、亲和力高,可利用该抗体制备检测氯霉索残留的ELISA试剂盒。  相似文献   

5.
以鳗弧菌M3和SMP1为细菌抗原制备了油乳化二价疫苗,用饵料包埋后连续7天口服免疫大菱鲆鱼,评价鱼的免疫应答和疫苗的保护效果.结果显示,乳化疫苗在鱼后肠刺激产生的溶菌酶和抗蛋白酶活性以及抗体水平均高于未乳化疫苗(P<0.05);并且在乳化疫苗免疫的鱼血清检测到明显升高的M3抗体效价(P<0.05).原位杂交结果显示,乳化疫苗免疫鱼的后肠IgM的产生水平高于未乳化疫苗免疫鱼.攻毒实验显示,乳化疫苗免疫的鱼对M3和SMP1的感染分别获得100%和50%的免疫保护率,而未乳化疫苗获得的免疫保护率分别为57.9%和0%.结果表明,鳗弧菌油乳化二价口服疫苗能引起大菱鲆后肠的非特异性和特异性免疫应答并有效地抵抗鳗弧菌的感染,适合用作水产口服鱼用疫苗.  相似文献   

6.
陶新民  杜宝祥  徐勇  吴志军 《振动与冲击》2008,27(2):120-124,136
针对轴承故障检测系统中异常样本数据不易收集以及异常样本数据分布不均导致传统分类算法出现过适应现象等现实应用问题,提出了一种基于自回归(AR)模型自相关系数峰态特征的一类故障检测方法.该方法利用正常样本生成AR模型参数,其他样本在该模型的投影形成残差序列,计算残差序列的自相关系数并取其峰态特征作为相似性的度量.实验结果表明该方法能有效地克服以AR模型参数为特征计算复杂度高且检测性能易受样本大小影响的不足.同时,文章给出了单一故障诊断模型并提出基于粒子群优化算法的阈值设定决策方法.实验中将本方法同其他以AR模型为特征的多层感知机(MLP)及自组织映射(SOM)方法进行比较,实验结果验证了本文建议方法的正确性和有效性.  相似文献   

7.
三聚氰胺人工抗原及多克隆抗体的制备   总被引:1,自引:0,他引:1  
采用戊二醛法将三聚氰胺(Melamine,MEL)偶联到牛血清白蛋白(BSA)上合成免疫原三聚氰胺-牛血清白蛋白(MEL-BSA).三聚氰胺甲醛树脂法对三聚氰胺衍生化,再偶联到鸡卵清蛋白(OVA)合成包被原三聚氰胺-鸡卵清蛋白(MEL-OVA).对纯化后的合成抗原进行紫外扫描、红外扫描和SDS-PAGE电泳鉴定.免疫新西兰大白兔,检测抗体的生成.结果表明,免疫兔血清的效价达到了1∶20 000,从而为进一步建立三聚氰胺的免疫检测方法奠定了基础.  相似文献   

8.
《中国测试》2015,(9):51-55
在模拟生理p H条件(p H=7.40)下,用荧光光谱和分子模拟法研究β-榄香烯与牛血清白蛋白(BSA)的相互作用。在308 K和318 K温度下,激发波长(λex)为280 nm,测定BSA在340 nm的内源性荧光强度随着β-榄香烯浓度增加的变化,用分子对接方法研究β-榄香烯与牛血清白蛋白(BSA)的相互作用。β-榄香烯与牛血清白蛋白的反应机制为静态猝灭,作用力类型为疏水作用。分子模拟结果表明:β-榄香烯与牛血清白蛋白亚结构域A结合,二者之间有疏水作用和静电作用,且以疏水作用为主,这与荧光光谱结果一致。β-榄香烯与BSA具有较强的相互作用,以血清白蛋白为载体,β-榄香烯作为药物可通过血液循环到达病变部位,发挥药效。  相似文献   

9.
针对无线电信号的攻击愈来愈频繁的情况,本文在数据流形理论基础上,使用深度神经网络(DNN)检测无线电信号对抗样本及其攻击方法。首先使用5种不同攻击方法对无线电信号进行攻击产生对抗样本,其次使用3种不同的神经网络检测对抗样本,最后用残差神经网络(ResNet)检测对抗样本的攻击方法。在信噪比(SNR)为30 d B和20 dB的无线电信号数据上的实验结果表明,本文所使用的残差神经网络检测精度接近100%,在信噪比为10 dB的无线电信号数据上的检测精度仍然在90%以上。结果表明本文所用的残差神经网络能有效检测无线电信号的对抗样本及其攻击方法。  相似文献   

10.
免疫层析试纸(ICTSs)是基于免疫层析检测技术发展起来的一种新型检测试纸。ICTSs将免疫技术的高度特异性和层析法优越的分离能力进行了有效结合,具有携带便利、测试所耗时间较短、测试结果相对稳定、价格便宜等诸多优点,目前普遍应用于各种生物分子、化学污染物和侵染因子的检测。近些年,由于ICTSs的灵敏度、检测限度和特异性低等缺点,使其发展受到了很大的限制。而影响试纸性能的主要因素为用作探针的标记物。为了提高试纸的性能,研究者们主要从两个方面进行探究:(1)通过向试纸上添加增强剂,进行信号放大等;(2)除了常用的胶体金、量子点和乳胶颗粒之外,尝试使用上转换荧光颗粒、碳纳米颗粒、脂质体、磁性纳米颗粒以及纳米硒等新型标记物来提高试纸性能。目前,这两方面的研究均取得了丰硕的成果。其中胶体金作为ICTSs最常用的探针之一,虽然其检测范围广、操作方便、快捷、特异性强,但其灵敏度较低。通过在胶体金ICTSs上添加增强剂(即HAuCl_4和NH_2OH·HCl)可以明显提高其检测限度和灵敏度。在相同的生物条件下进行分析时,用作ICTSs的上转换荧光颗粒与胶体金或彩色乳胶珠相比,也可以将检测的灵敏度提高10~100倍;此外,纳米硒颗粒由于具有良好的生物相容性且成本较低,作为ICTSs免疫探针的研究和开发具有广阔的前景。本文介绍了ICTSs的基本结构和两种检测方法(竞争型和三明治型),并对近些年关于ICTSs的不同设计(同一试纸增加多条检测线;不同形状的试纸以及微阵列试纸芯片等)进行了阐述。从不同探针标记物对ICTSs的影响入手,重点介绍了几种近年来研究较为广泛的标记物,分析了不同标记物的优缺点以及应用现状。最后综述了ICTSs在人类医学疾病、农业生产和食品安全等领域的应用,并指出今后的研究重点应放在进一步深入探索复合探针的研究上,以满足在检测领域所追求的"快速、便捷、特异、灵敏"的目标。  相似文献   

11.
Liu BH  Tsao ZJ  Wang JJ  Yu FY 《Analytical chemistry》2008,80(18):7029-7035
A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.  相似文献   

12.
Chen  Yanni  Liu  Liqiang  Xu  Liguang  Song  Shanshan  Kuang  Hua  Cui  Gang  Xu  Chuanlai 《Nano Research》2017,10(8):2833-2844
A gold immunochromatographic sensor (GICS) was developed for the rapid detection of 26 sulfonamides in honey samples.The sensor was based on a group-specific monoclonal antibody (mAb) that can recognize all 26 sulfonamides.Three haptens (hapten 1 with a thiazole ring,hapten 2 with a benzene ring,and hapten 3 with a straight carbon chain) were used for antigen preparation.With hybridoma technology,a group-specific mAb was screened with a 50% maximal inhibitory concentration (IC50) against sulfathizole (STZ) and the other 25 analogues ranging from 0.08 to 90.18 ng/mL.Mono-dispersed gold nanoparticles were conjugated with the mAb to develop the lateral immunochromatographic strip.A labeled antibody concentration of 0.1 μg/mL and a coating antigen concentration of 0.2 μg/mL in the test line were chosen for strip preparation.Under optimized conditions,the visual limits of detection (vLOD) for the concentrations of STZ,sulfamethoxazole,sulfamethizole,sulfadiazine,sulfamerazine,sulfadimethoxine,sulfamonomethoxine,sulfameter,sulfamethoxypyridazine,and sulfachloropyridazine were 5,0.25,0.25,10,5,10,25,2.5,5,0.25,and 10 μg/kg,respectively.Scanner analysis in honey samples revealed good performance for detection of the 26 sulfonamides.Commercial honey samples were tested with the sensor and positive results were confirmed with high-performance liquid chromatography.The proposed strip sensor provides a convenient method for the rapid and reliable determination of sulfonamides pollutants in honey samples.  相似文献   

13.
Liu G  Lin YY  Wang J  Wu H  Wai CM  Lin Y 《Analytical chemistry》2007,79(20):7644-7653
We describe a disposable electrochemical immunosensor diagnosis device that integrates the immunochromatographic strip technique with an electrochemical immunoassay and exploits quantum dot (QD, CdS@ZnS) as labels for amplifying signal output. The device takes advantage of the speed and low cost of the conventional immunochromatographic strip test and the high sensitivity of the nanoparticle-based electrochemical immunoassay. A sandwich immunoreaction was performed on the immunochromatographic strip, and the captured QD labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable screen-printed electrode, which is embedded underneath the membrane on the test zone. The new device coupled with a portable electrochemical analyzer shows great promise for in-field and point-of-care quantitative testing of disease-related protein biomarkers. The parameters (e.g., voltammetric measurement of QD labels, antibody immobilization, the loading amount of QD-antibody, and the immunoreaction time) that govern the sensitivity and reproducibility of the device were optimized with IgG model analyte. The voltammetric response of the optimized device is highly linear over the range of 0.1-10 ng mL(-1) IgG, and the limit of detection is estimated to be 30 pg mL(-1) in association with a 7-min immunoreaction time. The detection limit was improved to 10 pg mL(-1) using a 20-min immunoreaction time. The device has been successfully applied for the detection of prostate-specific antigen (PSA) in human serum sample with a detection limit of 20 pg mL(-1). The results were validated by using the commercial PSA enzyme-linked immunosorbent assay kit and showed high consistency. The new disposable electrochemical diagnosis device thus provides a rapid, clinically accurate, and quantitative tool for protein biomarker detection.  相似文献   

14.
A colloidal gold immunochromatographic assay based on a generic monoclonal antibody is developed for the simultaneous detection of benzimidazoles and metabolite residues in milk samples. The monoclonal antibody is prepared using 2‐(methoxycarbonylamino)‐3H‐benzimidazole‐5‐carboxylic acid as the hapten, and it can recognize 11 types of benzimidazoles simultaneously. The immunochromatographic strip is assembled and labeled using gold nanoparticles. This strip can detect 11 benzimidazoles including albendazole, albendazole s‐oxide, albendazole sulfone, fenbendazole, fenbendazole sulfone, flubendazole, mebendazole, parbendazole, oxfendazole, oxibendazole, and carbendazim within 15 min in milk samples. Results are obtained visually with the naked eye, and the cutoff values and the visual limit of detection values for these benzimidazoles are 25, 6.25, 12.5, 12.5, 50, 25, 50, 50, 50, 6.25, and 25 ng mL?1, and 6.25, 3.125, 3.125, 1.56, 12.5, 6.25, 12.5, 12.5, 6.25, 0.78, and 12.5 ng mL?1, respectively. Results are also obtained using a hand‐held strip scan reader, with calculated limit of detection values for these benzimidazoles of 0.83, 0.77, 1.83, 0.98, 7.67, 3.50, 3.96, 5.71, 0.92, 0.59, and 1.69 ng mL?1, respectively. In short, the developed paper sensor is a useful tool for rapid and simple screening of residues of benzimidazoles in milk samples.  相似文献   

15.
This work presented a rapid, inexpensive, reliable, and flexible quantitative immunoassay for cardiac troponin I (cTnI). The assay was based on the concepts of one-step dual monoclonal antibody "sandwich" principle, the low density protein array, the nanogold probe, and the silver enhancement on the gold particles. The capture antibody (IgG1) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu-IgG2) were prepared before the detection. The detection procedure involved two steps, i.e., immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients, The whole detection procedure of the assay could be fulfilled within 40 min (much faster than the routine enzyme-linked immunosorbent assay (ELISA) that takes usually at least 3 hours for a turnaround test). The detection results could be easily imaged with a simple flatbed scanner or even observed with the naked eye. The assay showed good specific response to cTnI with very little cross-reactivity to the skeletal isoforms of troponin I (sTnl), cardiac troponin T (cTnT), and myoglobin (Mb). A cut-off value of 0.3 ng/ml was obtained from a reference control group (200 normal serum samples). 588 patients' serum samples were assayed simultaneously by routine ELISA and this colloidal gold method to test the validity of the method. The data were analyzed using the statistical package SPSS version 11.0 (SPSS Inc.) There was no significant difference between these two assays (P = 0.66 > 0.05). The agreement between this method (> or < 0.3 ng/ml) and ELISA was 86%.  相似文献   

16.
Abstract

The concentration of salivary secretory immunoglobulin A (sIgA) is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA) for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2) containing non-ionic surfactant (3 wt% Tween 20). The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.  相似文献   

17.
Paper‐based assays for detection of physiologically important species are needed in medical theranostics owning to their superiorities in point of care testing, daily monitoring, and even visual readout by using chromogenic materials. In this work, a facile test strip is developed for visual detection of a neurotransmitter dopamine (DA) based on dual‐emission fluorescent molecularly imprinted polymer nanoparticles (DE‐MIPs). The DE‐MIPs, featured with tailor‐made DA affinity and good anti‐interference, exhibit DA concentration‐dependent fluorescent colors, due to the variable ratios of dual‐emission fluorescence caused by DA binding and quenching. By facile coating DE‐MIPs on a filter paper, the DA test strips are obtained. The resultant test strip, like the simplicity of a pH test paper, shows the potential for directly visual detection of DA levels just by dripping a tiny amount of biofluid sample on it. The test result of real serum samples demonstrates that the DA strip enables to visually and semiquantitatively detect DA within 3 min by using only 10 µL of serum samples and with a low detection limit ((100–150) × 10?9m ) by naked eye. This work thus offers a facile and efficient strategy for rapid, visual, and on‐site detection of biofluids in clinic.  相似文献   

18.
Du D  Wang J  Wang L  Lu D  Lin Y 《Analytical chemistry》2012,84(3):1380-1385
An integrated lateral flow test strip with an electrochemical sensor (LFTSES) device with rapid, selective, and sensitive response for quantification of exposure to organophosphorus (OP) pesticides and nerve agents has been developed. The principle of this approach is based on parallel measurements of postexposure and baseline acetylcholinesterase (AChE) enzyme activity, where reactivation of the phosphorylated AChE is exploited to enable measurement of the total amount of AChE (including inhibited and active) which is used as a baseline for calculation of AChE inhibition. Quantitative measurement of phosphorylated adduct (OP-AChE) was realized by subtracting the active AChE from the total amount of AChE. The proposed LFTSES device integrates immunochromatographic test strip technology with electrochemical measurement using a disposable screen printed electrode which is located under the test zone. It shows a linear response between AChE enzyme activity and enzyme concentration from 0.05 to 10 nM, with a detection limit of 0.02 nM. On the basis of this reactivation approach, the LFTSES device has been successfully applied for in vitro red blood cells inhibition studies using chlorpyrifos oxon as a model OP agent. This approach not only eliminates the difficulty in screening of low-dose OP exposure because of individual variation of normal AChE values but also avoids the problem in overlapping substrate specificity with cholinesterases and avoids potential interference from other electroactive species in biological samples. It is baseline free and thus provides a rapid, sensitive, selective, and inexpensive tool for in-field and point-of-care assessment of exposures to OP pesticides and nerve agents.  相似文献   

19.
Retinol binding protein 4 (RBP4) is a useful biomarker in the diagnosis of type 2 diabetes since its level in the serum is higher in insulin-resistant states. Accurate measurement of the serum RBP4 levels is hampered by conventional immunologic methods, such as enzyme-linked immunosorbent assay (ELISA). In this study, therefore, we have developed an aptamer-based surface plasmon resonance (SPR) biosensor that can be used to sense for RBP4 in serum samples. A single-stranded DNA (ssDNA) aptamer that showed high affinity (Kd = 0.2 +/- 0.03 microM) and specificity to RBP4 was selected. This RBP4-specific aptamer was immobilized on a gold chip and used in a label-free RBP4 detection using SPR. Analysis of RBP4 in artificial serum using SPR was compared with ELISA and Western blot analysis. Our results indicated that the RBP4-specific aptamer-based SPR biosensor gave better dose-dependent responses and was more sensitive than ELISA assays. As such, this RBP4 aptamer-based SPR biosensor can be potentially used to monitor the RBP4 levels within the serum as an indicator of type 2 diabetes.  相似文献   

20.
Celiac disease is a condition associated with the ingestion of gluten by genetically susceptible individuals. Measurement of serum antigliadin antibodies is a diagnostic tool also used as a means of monitoring a patient's compliance to a gluten-free diet. In this work, we demonstrate the applicability of an electrochemical supramolecular platform based on cyclodextrin-modified gold surfaces to detect antigliadin antibodies in real serum samples. Several support layer-biorecognition element combinations were tested in order to maximize the electrochemical response, and the assay was optimized in terms of incubation times and resistance to nonspecific interactions. The developed supramolecular biosensor was then applied to the amperometric detection of antigliadin IgA and IgG autoantibodies in real samples of celiac disease patients under follow-up treatment; the results were compared with a commercial enzyme linked immunosorbent assay (ELISA) test, and an excellent correlation was observed between both methods.  相似文献   

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