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1.
莱克多巴胺单克隆抗体的研制及阻断ELISA检测方法的建立   总被引:5,自引:1,他引:4  
用混合酸酐法将莱克多巴胺(RAC)偶联于牛血清白蛋白(BSA),合成免疫原BSA-RAC,并用紫外和凝胶电泳鉴定;用BSA-RAC免疫Balb/C小鼠,细胞融合技术建立抗RAC单克隆抗体(mAb)杂交瘤细胞株,体内诱生腹水法制备RAC mAb。应用RAC mAb研制RAC残留快速检测阻断ELISA试剂盒(RAC-Kit),并测定其性能。结果表明,BSA-RAC偶联成功,分子结合比为1∶24.5;筛选出3株杂交瘤细胞,其中最好的4D8株的亲和常数Ka为1.65×1010L/mol。RAC-Kit的标准曲线呈典型的S型,符合4参数logit曲线拟合,线性范围为1~170μg/L,灵敏度为0.52μg/L,检测限为1μg/L,饲料样、猪尿样的平均添加回收率分别为91.1%和89.2%,平均批内和批间变异系数<15%,与多巴酚丁胺的交叉反应率为22.3%,与其他化合物无交叉反应,RAC-Kit在4℃可保存180d。  相似文献   

2.
用1×10~5、3×10~5、5×10~5和10×10~5 Gy的电子束和电子束加4%NaOH复合预处理稻麦秸秆。预处理稻秆200目以上粉末随辐照剂量的增加而增中。用1%纤维素酶水解电子束预处理稻麦秸秆48小时,葡萄糖得率随辐照剂量的增加而增加,10×10~5 Gy照射的比未预处理的增加了70%—80%;而复合预处理稻麦秸秆的葡萄糖得率在5×10~5 Gy以内,随辐射剂量增加而增加,分别为36%和35%,比未处理的10.2%增加了约2.5倍,而用10×10~5 Gy的反而下降。通过辐射聚合在纱布表面覆盖高分子poly(HEA)、poly(HEMA)、poly(HPMA)、poly(A-4G)和poly(A-TMPT),并以此作为固定化里氏木霉细胞的载体。这些载体均能较好地固定化里氏木霉细胞,固定化细胞的滤纸活性(FPA)均高于游离细胞,其中覆盖poly(HPMA)的载体固定化细胞的效果最好,FPA为3.5U/ml,比游离细胞增加了近40%。用这些固定化细胞的酶液水解NaOH和电子束复合预处理的稻麦秸秆,其葡萄糖得率随辐照剂量和水解时间的增加而增加,其中4%NaOH和10×10~5 Gy处理的稻麦秸秆第6天的葡萄糖得率为19%和22%。  相似文献   

3.
用EDC一步法和戊二醛法将SM偶联于载体蛋白牛血清白蛋白(BSA)和OVA,合成免疫原BSA-SM和包被原OVA-SM。用紫外线扫描、SDS-PAGE进行鉴定;用BSA-SM免疫BALB/C小鼠,用杂交瘤技术建立分泌SM mAb的细胞株3F9-C4;对SM mAb的效价、亲和性、敏感性和特异性等免疫学特性进行鉴定。结果表明,BSA-SM人工抗原分子结合比为1∶25;筛选出3F9-C4敏感特异的杂交瘤细胞1株,间接ELISA测定细胞培养上清,效价为1∶3.2×102,同种型为IgG2a/κ;腹水的亲和常数(Ka)为8.4×1011L/mol;IC50为8.99μg/L,与双氢链霉素交叉反应为109.6%,与其他SM结构相似物和功能近似物无交叉反应性。本试验获得了抗SM高价、敏感、特异的mAb,可用于SM的残留检测。  相似文献   

4.
本试验用放射免疫分析(RIA)药盒测定了沙能,杂交1代(沙能×成都麻羊)和杂交2代〔沙×(沙×成)〕共23头山羊发情周期中及妊娠期中奶孕酮和毛孕酮浓度的变化。试验结果表明,应用RIA药盒可测出毛申孕酮。配种后1—28天,孕羊奶和毛中孕酮含量,非孕羊奶和毛中孕酮含量均呈极显著相关(分别为r=0.5458,p<0.01和r=0.7832,p<0.01)。配种后22天,以每毫升奶含孕酮3.9ng为判斯孕否标准,孕与非孕羊确诊率分别为82.4%和100%;以每50mg毛含3.7ng孕酮为判断孕否标准,孕与非孕羊确诊率分别为77.8%和100%。妊娠20天起,毛中孕酮含量(5.67±0.98ng/50mg毛)逐渐上升,120天达到高峰(9.85±1.20ng/50mg)。产羔前8天起,毛中孕酮含量(7.73±1.91ng/50mg)下降较快,产羔当天下降到4.93±0.25ng/50mg。  相似文献   

5.
磺胺二甲氧嘧啶单克隆抗体的制备及其免疫学特性鉴定   总被引:1,自引:1,他引:0  
用重氮化法将SDM(磺胺二甲氧嘧啶)偶联于载体蛋白BSA和OVA,合成免疫原BSA-SDM和包被原OVA-SDM,并用紫外扫描(UV)、SDS-PAGE进行鉴定;用BSA-SDM免疫BALB/C小鼠,间接ELISA和阻断ELISA选择细胞融合备用鼠;应用杂交瘤技术建立分泌SDM mAb细胞株,用体内诱生腹水法制备SDM mAb;对SDM mAb的效价、亲和性、敏感性和特异性等免疫学特性进行鉴定。结果表明,BSA-SDM人工抗原制备成功,分子结合比约为1∶12.1;筛选出1B43、D9、4E1共3株敏感特异的杂交瘤细胞,间接ELISA效价细胞培养上清分别为1∶3.2×102、1∶5.12×102、1∶8.1×102,腹水分别为1∶2.56×105、1∶2.56×1051、∶5.12×105,4E1亲和常数(Ka)为1.25×1010L/mol;4E1株对SDM的IC50为16.64ng/ml,与磺胺-6-甲氧嘧啶交叉反应率为5%,与其他磺胺类药物无交叉反应性。本试验获得了抗SDM高价、敏感、特异的mAb,可用于SDM残留检测的免疫学试验。  相似文献   

6.
本研究调查了秦山核电站站址周围50km半径范围内土壤、水和农作物的总α、总β放射性水平,土壤中天然放射性核素含量,以及γ外照射积累剂量。结果表明:土壤中总α、总β值分别为0.54—1.48×10~(-8)Ci/kg和1.90—2.96×10~(-8) Ci/kg,低于全国一般地区平均水平,且0—100cm土层变化不大。土壤中~238U、~226Ra、~232Th、~40k、~137Cs的放射性活性分别为0.67—5.00×10~(-9)、4.49—11.4×10~(-10)、0.88—1.69×10~(-9)、0.39—2.09×10~(-8)和0.89—4.32×10~(-10)Ci/kg。河水、湖水以及核电站废水排放口处海水总β值分别为1.32—8.62、3.6和2.55×10~(-12)Ci/L。大米和油菜叶中的总β值分别为2.53和3.0×10~(-9)Ci/kg。室内外月平均γ照射量各为10.6—14.7和9.16—13.7mR。  相似文献   

7.
家鸡催乳素放射免疫测定法的建立   总被引:3,自引:0,他引:3  
本文建立了测定鸡血浆样品中催乳素(chPRL) 的放射免疫测定方法。所用试剂为标准chPRL( AFP 10328B) ,碘标chPRL( AFP 4444B) ,一抗为免抗chPRL(AFP 151040789) 抗血清,二抗驴抗免IgG 抗血清。碘标采用氯胺T 改进法,减少了氯胺T 的用量,并省略了偏重亚硫酸钠的使用,碘标chPRL 比放射性为29μCi/ug 。测定的灵敏度为0-34ng/ ml, 标准曲线的ED75 、ED50 和ED25 分别为1-30 ,3-71 和10-60ng/ ml。样品批内和批间变异低于15 % 。母鸡血浆样品倍比稀释产生的结合抑制曲线与标准曲线平行;测定不同繁殖状态母鸡血浆样品chPRL 显示出显著差异且变化规律符合繁殖活动的变化。证明本方法可用于测定家鸡血浆chPRL 浓度。  相似文献   

8.
利用合成的完全抗原(BSA-ENR)免疫小鼠,通过细胞融合技术和间接竞争ELISA筛选出了能分泌恩诺沙星单克隆抗体的杂交瘤细胞株,以此为基础研制恩诺沙星残留检测的竞争ELISA试剂盒(competitive ELISA ENR-Kit),并测定其性能。结果表明:筛选出的2株杂交瘤细胞,单抗亚类均为IgG1型,其中4G1-B3株的ENR mAb间接ELISA效价为1:1.024×106,亲和常数(Ka)为9×1010L/mol;竞争ELISA试剂盒的线性检测范围为0.05~101.6μg/L、灵敏度0.05μg/L、半数抑制浓度(IC50)1.1μg/L,与其他化合物的交叉反应率(CR%)均小于0.01%,鸡肉样、鸡肝样、鱼肉样和牛奶样的平均添加回收率分别为81.5%8、7.6%、84.3%和95%,不同基质添加恩诺沙星标准品做竞争ELISA对ENR-Kit检测结果影响小,试剂盒保存期6个月以上。结果说明该竞争ELISA试剂盒具有快速、敏感、特异、简便等特点,适用于ENR残留的快速检测。  相似文献   

9.
用 2MeV~ 9MeV 7个能量 ,5C~ 1 60C 1 2个剂量的质子处理 5个品种 ,研究质子对小麦的诱变效应。结果表明 ,M1代的生物损伤随剂量的提高而加重 ,在一定能量范围内 ( 6MeV以下 ) ,随能量的增加而加大 ,超过一定能量 ,生物损伤有所减轻 ;M1代较易出现γ辐照中少见的叶绿素条状缺失损伤。M2 代诱变效果显著 ,突变谱宽 ,有益突变频率明显高于γ射线 ,较易诱发早抽穗性状变异 ;明确诱变小麦的适宜能量为4MeV ,适宜剂量为 1 4× 1 0 10 ~3 7× 1 0 10 P cm2 。  相似文献   

10.
应用放射免疫分析和竞争蛋白结合分析法测定了成都麻羊、莎能羊及其杂交一代母羊产后四月内的乳中孕酮浓度变化。供试动物在试验期间的孕酮浓度一般保持基础水平,约有87.2%(成都麻羊)、94.4%(莎能羊)和93.9%(杂交羊)的测值在10ng/ml以下。孕酮浓度升高发生1—5次,较行为发情次数为多。第一次孕酮浓度升高发生在产后26—67天(成都麻羊)、19—52天(莎能羊)和39—122天(杂交羊)期间。产后五天内初乳中的孕酮含量平均为每日2—4ng。  相似文献   

11.
克百威残留放射免疫分析方法研究   总被引:4,自引:0,他引:4  
通过活化酯法与牛血清蛋白联接 ,制备出较高活性的人工抗原 ,以此来免疫兔子制备抗克百威的多克隆抗体。选择适宜的反应条件 ,以14 C 克百威建立了克百威放射免疫测定法。该方法对克百威标准品的最低检测量为 0 1 75ng ml,线性检测范围为 0 2 56~ 40 0 0 0ng ml,I50 值为 650 0ng ml。在线性检测范围内 ,添加不同浓度克百威的蔬菜样品检测的批内、批间变异系数均小于 1 0 %。在甘蓝菜样品中的添加回收率试验回收率为 93 0 %~ 1 0 4 0 % ,变异系数为 4 3 %~ 1 1 5%。所得克百威抗体特异性强 ,与丁硫克百威的交叉反应率为 9 1 5% ,与呋喃酚的交叉反应率为 5 2 9% ,与供试的其它 3种氨基甲酸酯类的交叉反应率均小于 0 5%。  相似文献   

12.
放射免疫法测定畜产品中的己烯雌酚   总被引:2,自引:0,他引:2  
黄世乐  陈祖义  成冰 《核农学报》1991,5(3):153-157
本文采用DES-MCME-BSA复合物免疫家兔,制备兔抗DES抗血清。其滴度为1:1250,亲和常数为1.1×10~9L/mol,与己烷雌酚和双烯雌酚的交叉反应分别为4.58%和2.14%,与17β-雌二醇、孕酮、甲地孕酮、睾酮和丙酸睾酮的交叉反应均<0.01%。应用制得的抗血清,建立了DES的放射免疫测定法。其最小检测量为2pg/管,平均回收率90.4±7.6%,批内和批间变异系数分别为5.4%和10.4%,表明本法灵敏、可靠,适于大批样品的常规筛检。  相似文献   

13.
A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.  相似文献   

14.
A rapid and sensitive colloidal gold immunochromatography test strip based on one monoclonal antibody with broad-specificity, which can detect 12 fluoroquinolones (FQs), was developed. Antigen and goat anti-mouse IgG were respectively drawn on NC membrane as test line and control line. Gold-labeled antibody was added on a pad and put on one end of the membrane. Fluoroquinolones in sample solution compete with antigen combined on NC membrane for the gold-labeled antibody. When enough fluoroquinolone exists, the test line vanishes as there are no spare gold-labeled antibodies that can bind with antigen on the membrane. The control line always exists when the antibody is activated. The lowest detection limits of the FQs in spiked chicken muscle and chicken liver samples were 25 ng mL(-1) for norfloxacin and pefloxacin. The lowest detection limit for the other 10 FQs (enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, ofloxacin, lomefloxacin, enoxacin, danofloxacin, amifloxacin, oxolinic acid, and marbofloxacin) was 50 ng mL(-1). The whole process involving sample preparation and detection can be finished in <10 min. The results demonstrate that the developed method can be potentially used as a screening tool for the determination of 12 FQ residues in a large amount of samples on site.  相似文献   

15.
An immunoenzymatic method for the quantitative determination of dietary lectin activities employing immobilized glycoproteins was studied. Lectins from wheat germ (WGA), peanut (PNA), and jack bean (ConA) were added to microtiter plates coated with ovalbumin or asialofetuin and quantified by enzyme-linked immunosorbent assay (ELISA) with lectin-specific antibodies. ELISA responses for lectin activity were dose-dependent in the concentration range 30-1000 ng/mL for WGA and 80-1000 ng/mL for both PNA and ConA. Inhibition assays carried out with different saccharides confirmed that the binding of lectins to immobilized glycoproteins was specific. The proposed method is specific and sensitive, allowing the quantitative determination of lectin activities on raw samples by simple dilution of the extracts. Examples of application to wheat germ and roasted peanut extracts are reported.  相似文献   

16.
To detect the organophosphorus (OP) pesticide pirimiphos-methyl in grain samples, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed and optimized. By the active esters method, pirimiphos-methyl hapten A was conjugated to keyhole limpet hemocyanin to be used as the immunogen for the production of monoclonal antibodies, and pirimiphos-methyl hapten B was conjugated to ovalbumin to be used as coating antigen. By using the monoclonal antibody and the coating antigen, an IC-ELISA has been developed. Under the established optimized conditions, the IC-ELISA showed an IC50 of 4.2 ng/mL with a detection limit of 0.07 ng/mL. The IC-ELISA showed negligible cross-reactivity with other OP pesticides except with pirimiphos-ethyl. Recoveries of pirimiphos-methyl from spiked grain samples ranged from 83 to 96%.  相似文献   

17.
伏马菌素B1人工抗原的合成及鼠源多克隆抗血清的制备   总被引:1,自引:0,他引:1  
本研究合成并鉴定伏马菌素B1(FB1)人工抗原,通过动物免疫制备敏感性高、特异性好的鼠源FB1多克隆抗血清。采用碳二亚胺(EDC)法将FB1分别与载体蛋白BSA和OVA偶联,合成免疫抗原FB1-BSA和检测抗原FB1-OVA,经鉴定后,分别按10和30μg/只的剂量免疫BALB/c小鼠,共免疫4次,每次间隔3周,最后1次免疫30d后,断尾采血,制备多抗血清。利用间接ELISA方法测定抗血清效价,间接竞争ELISA测定敏感性和特异性。结果表明,免疫的6只小鼠效价均达1∶104以上,3号小鼠多抗血清敏感性最好,半数抑制浓度IC50为61.3ng/ml,且具有良好的特异性。本试验成功合成了FB1人工抗原,并制备了敏感性高、特异性好的鼠源多克隆抗体血清,为FB1单克隆抗体制备及其免疫学快速检测方法的建立奠定了基础。  相似文献   

18.
血清雌二醇放射免疫试剂盒的研制   总被引:3,自引:1,他引:2  
张丁联  潘家荣  王斌 《核农学报》2003,17(4):304-307
制备了雌二醇抗体 (抗E2 6 CMO BSA)和12 5I放射配体E2 6 CMO histamine 12 5I,建立了血清雌二醇放射免疫分析方法 ,其特点是通过微量标记法提高标记物比活度 ,不用乙醚萃取直接测定血清样本 ,简化了测量步骤 ,缩短了测量时间 ,且试剂稳定时间可延长至 2个月以上。应用本法可以快速测量动物及人的血清样本 ,2 5~ 3h即可得出结果。  相似文献   

19.
仿刺参体腔液补体类似物化学发光免疫检测   总被引:1,自引:0,他引:1  
张峰  王海峰  宫晶  常少杰 《核农学报》2007,21(4):413-416
首次应用酶联化学发光免疫检测(Chemiluminesent Immunoassay,CLIA)技术测定仿刺参体腔液补体类似物AjC3和AjC4。羊抗人C3、C4抗体吸附到经过紫外线处理的聚苯乙烯管内,采用辣根过氧化物酶(HRP)标记抗体。过氧化氢和鲁米诺为辣根过氧化物酶的底物。捕获抗体包被最适浓度为1μg/ml,免疫反应20℃孵育2h达到平衡。HRP-IgC3、IgC4抗体复合物适宜稀释度为1:2000,HRP-IgC3I、gC4抗体复合物4℃下保存8d性能稳定,室温下5d内性能稳定。标准品浓度在0.1~10ng/ml范围内时与化学发光值之间具有良好的线性相关性,检测灵敏度为0.1ng/ml。结果表明应用酶联化学发光免疫检测技术能够检测到仿刺参体腔液中含有补体类似物,AjC3含量为6.58±1.4μg/ml,AjC4含量为0.67±0.3μg/ml。  相似文献   

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