小鼠神经生长因子直接竞争酶联免疫检测方法的建立

目的:利用小鼠神经生长因子(mNGF)和纯化的兔抗mNGF IgG,建立mNGF直接竞争酶联免疫检测方法。方法:以亲和纯化兔抗mNGF IgG作为包被抗体,封闭、洗涤后加入用稀释液稀释的不同浓度的标准mNGF或样品液和生物素标记的mNGF混合液,于37℃孵育80 min或室温孵育120 min,洗涤后加入辣根过氧化物酶标记的链酶亲和素,洗涤后加入显色液显色,测定D450nm值,通过标准曲线计算待测样品中mNGF含量。结果与结论:建立了mNGF直接竞争酶联免疫检测方法;该方法的灵敏度约20 ng,变异系数为批内≤10%、批间≤15%,回收率为85%～115%;利用该方法检测了3批样品中mNGF的含量。     

快速同时检测玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇的研究

目的：利用稀土离子作为示踪剂,建立DON/ZEN双标记间接竞争时间分辨荧光免疫分析方法同时检测DON、ZEN。 方法：以DON BSA、ZEN-BSA共包被于固相微孔板,与DON/ZEN标准或样品中的DON、ZEN竞争结合抗DON多抗、抗ZEN单抗,然后分别用稀土离子Eu3+-羊抗兔IgG及Sm3+ 羊抗鼠IgG进行示踪检测,并对建立DON/ZEN-双标记TRFIA进行方法学的考核。结果：DON/ZEN-双标记TRFIA检测灵敏度,DON为0.2 ng/ml、ZEN为0.7 ng/ml,检测范围为：DON 0.2~100 ng/ml,ZEN 0.7~50 ng/ml,批内、批间变异率均小于10%。不同样品添加回收实验表明玉米、小麦样品中DON平均回收率分别为102.8%、98.8%,ZEN平均回收率分别为94.2%、95.7%。DON/ZEN-双标TRFIA检测时,DON与ZEN不相互干扰,该方法特异性好。玉米样品检测结果表明,DON/ZEN双标记TRFIA与单标记DON -TRFIA、ZEN-TRFIA试剂盒结果高度相关,具有较好的一致性,两者检测DON的结果相关系数为0.9760,检测ZEN结果的相关系数为0.9695,结论：DON/ZEN-双标记TRFIA灵敏度高,检测范围宽,重复性、稳定性好,一次检测可同时得到DON、ZEN两个结果,是一种简便、快速、经济、稳定、可进行大批量样品筛查的检测方法。     

食品中农药残留的高通量微生物检测方法研究

目的甲胺磷的微生物高通量定量分析法,并在待检蔬菜样品进行初步的探索。方法以短双歧杆菌LJM-006作为测试菌,选择浓度为0、0.25、0.5、1.0、2.0和4.0 mg/L的甲胺磷标准品溶液20μL加入所述检测管内,37℃厌氧培养8～10 h,分别测定反应管和阴性对照反应管的A500值,B值为样品反应管的A500值,B0值为阴性对照反应管的A500值;以B/B0为纵坐标Y,甲胺磷浓度的对数值为横坐标X,绘制标准曲线,建立甲胺磷含量与B/B0的线性回归方程;以此方法进行模拟食品样品中所含甲胺磷检测,进行准确度、精密度分析。结果双歧杆菌菌株LJM-006对甲胺磷敏感性高,与其他有机磷农药的交叉反应率均小于2%;在0.01～100 mg/L浓度范围内B/B0与甲胺磷浓度对数值的线性关系良好,甲胺磷检测的标准曲线为Y=-24.68X＋66.72,R2为0.9902,IC50=0.25 mg/L,检测下限达到0.1 mg/L,回收率为78.5%～105.7%,批内变异系数≤12%,批间变异系数≤8.2%。结论该方法具有操作简便,具备一定灵敏度和特异性等优点,可作为一种食品中甲胺磷残留筛检方法。     

表皮生长因子的发光免疫测定法——吖啶酯标记抗体的应用

介绍了吖啶酯标记抗体应用于表皮生长因子的发光免疫测定法.吖啶酯类发光剂标记抗体的优点是标记方法简便、抗体用量少、发光强度大.所述方法其标准曲线范围是400pg/ml—25ng/ml.表皮生长因子浓度与发光值之间的线性关系良好.批内及批间变异系数分别为7.3%及11%.灵敏度与使用 ¹²⁵I 标记同一抗体的放免法同属 pg 水平.加入保护剂低温保存,使标记物的使用期由原来的1个月延长到6个月.     

阿特拉津高灵敏酶联免疫吸附检测法的建立

目的：建立高灵敏度的阿特拉津酶联免疫吸附检测法。方法：将间接竞争ELISA进行条件优化以提高检测灵敏度,包括包被抗原与一抗的最佳工作浓度筛选、选择一抗的最佳稀释度对包被抗原进行细化筛选、不同有机溶剂对竞争结合反应的影响、酶标二抗稀释度筛选等。用建立的酶联免疫检测法检测实际样品,再与高效液相色谱法（HPLC）检测进行比较。结果：利用优化后条件建立了阿特拉津间接竞争ELISA检测曲线,标准曲线的相关系数R²=0.9958,相关性较好。另由此标准曲线可得LOD （最低检出限）为1.972 ng/ml。用于检测实际样品,回收率在80%-120%之间。当添加样品浓度为（0~6） ng/ml时,该法的检测灵敏度高于HPLC。结论：新建立的阿特拉津ELISA特异性好、精密度高,可代替大型仪器用于阿特拉津实际样品检测。     

胃蛋白酶原双标记时间分辨荧光免疫分析及其初步临床应用

采用钐(Sm3 )标记抗胃蛋白酶原Ⅰ单克隆抗体(PG Ⅰ)及铕(Eu3 )标记抗胃蛋白酶原Ⅱ单克隆抗体(PGⅡ),建立了双标记时间分辨荧光免疫分析法(TRFIA),同时检测人血清PGⅠ和PGⅡ.将抗PG Ⅰ单克隆抗体8003#、抗PGⅡ单克隆抗体8101#以一定比例共包被于96孔微孔板上,用Sm3 标记抗PGⅠ单抗8016#、Eu3 标记抗PGⅡ单抗8102#,采用双抗体夹心法建立PGⅠ/PGⅡ的双标记TRFLA.标准曲线由TRFLA检测仪自带的Log-LogB函数处理.PGI可测范围为0.2～300μg/L,批内和批间变异系数(CV%)分别为5.2%和8.1%,平均回收率为96.9%;PGⅡ可测范围为0.05～55μg/L,批内和批间变异系数(CV%)分别为7.1%和11.7%,平均回收率为103.7%;双标记PGⅠ/PGⅡ-TRFLA与PGⅠ-ELISA法、PGⅡ-ELISA法的测定结果高度相关,具有较好的一致性,相关系数分别为0.9426和0.9396.检测300例健康人员的PGⅠ为(157.3±51.0)μg/L,PGⅡ为(10.6±5.9)μg/L,PGⅠ/pGⅡ比值为(14.8±4.3)μg/L.PGⅠ的正常参考值范围在55.3～259.3μg/L之间,PGⅡ正常参考值为<23μg/L,PGⅠPGⅡ>6.双标记PGI/PGⅡ-TRFLA方法的研究在国内外尚未见报道,是一种灵敏、简便、快速、经济并可用于大批量样品筛查的方法,有利于各种胃病的大规模普查筛选及患者病程监测.     

重组碱性成纤维细胞生长因子游离巯基的测定分析

建立了一种紫外-可见分光光度检测法,可以快速、简便、稳定地测定重组碱性成纤维细胞生长因子的游离巯基含量。将检测试剂、标准品及待测样品一起反应后,在412 nm波长处读取OD值,以标准品浓度为横坐标,以吸光值为纵坐标,绘制标准曲线,根据标准曲线计算待测样品中游离巯基含量;同时对该方法进行了方法学验证。结果表明:该方法专属性强,游离巯基含量测定范围为20~80 μg/ml,线性相关系数在0.999以上,回收率范围为91.7%~107.4%;而且该法精密度较好,精密度的变异系数范围为5%~8%。测得的分子表面和总游离巯基含量与理论一致,可用于蛋白质游离巯基检测,以监测分子结构的正确性与均一性。     

抗体夹心酶联免疫吸附法测定重组溶葡萄球菌酶研究

用重组溶葡萄球菌酶免疫家兔获得抗血清,经亲和层析纯化后用HRP标记,以双向免疫扩散法确定抗血清效价,以Westernblot鉴定抗体的特异性,建立双抗夹心法标准曲线,鉴定其最小检出限、精确度、回收率。实验显示多克隆抗体能与溶葡萄球菌酶特异性结合,双抗夹心ELISA法检测抗原的最小检出限为0·98ng/mL,标准曲线在0·98~500ng/mL范围内线性良好。3份同批样本分别重复6次测定,平均批内变异系数为6·4%;3份不同批样本分别重复6次测定,平均批间变异系数为6·5%。血清中加入已知量的标准抗原,测得平均回收率为98·6%。此法检测重组溶葡萄球菌酶的可测范围广,灵敏度和精密度高,变异系数较小。结果证实建立的检测血清中重组溶葡萄球菌酶含量的双抗夹心酶联免疫吸附测定法(Enzyme-linkedimmunosorbentassay,ELISA)灵敏、准确、可靠。     

ELISA试剂盒检测Sabin株脊髓灰质炎灭活疫苗Vero细胞宿主蛋白残留量的验证

目的 验证ELISA试剂盒检测Sabin株脊髓灰质炎灭活疫苗(Sabin strain inactivated poliovirus vaccine, sIPV)中Vero细胞宿主细胞蛋白(host cell protein, HCP)残留量的适用性。方法 用同一批ELISA试剂盒检测sIPV中Vero细胞HCP残留量,验证其专属性、重复性、中间精密度、准确度、线性、范围、定量限和耐用性等指标。结果 将样品用2种不同稀释液(样品稀释液和疫苗稀释液)稀释后,检测Vero细胞HCP残留量结果均<12.5 ng/mL,表明该方法专属性强;同一检验人员检测同一样品6次,Vero细胞HCP残留量结果均<12.5 ng/mL;不同检验人员检测同一样品6次,Vero细胞HCP残留量结果均<12.5 ng/mL,表明具有良好的重复性和中间精密度;准确度试验中回收率均在99%～106%,CV为2%;在12.5～400.0 ng/mL的线性范围内,标准曲线线性良好(R²>0.98);定量限为50.0 ng/mL;显色时间在25～35 min内对实验无影响,显色...     

云南省O139霍乱弧菌引起的一次食源性霍乱爆发

霍乱是由O1霍乱弧菌(Vibrio cholerae O1)或O139霍乱弧菌(Vibrio cholerae O139)引起的一种肠道传染病,是发展中国家的一个主要公共卫生问题,2000年以来世界卫生组织每年报告霍乱病例数范围是101383～184311例,其中非洲报告病例数占86.8%～96.9%,亚洲占3.1%～8.2%,拉丁美洲占0.0%～2.3%,非洲、亚洲和拉丁美洲报告病死率范围分别是1.7%～3.3%、0.3%～0.6%和0.0%[1].     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Indomethacin inhibits cholera toxin-induced cyclic AMP accumulation in Chinese hamster ovary cells

Abstract Indomethacin was examined for its capacity to inhibit increases in adenosine-3',5'-monophosphate (cAMP) concentrations in Chinese hamster ovary (CHO) cells treated with cholera toxin. When added to the culture medium 1 h prior to cholera toxin (100 ng/ml), indomethacin (500 μg/ml) exhibited maximum protection against the typical increase in cAMP. Application of indomethacin at the same time as cholera toxin or up to 3 h after the toxin progressively decreased the drug's capacity to block further increases in cAMP. The drug appeared to block adenylate cyclase activity because addition of forskolin to drug-treated cells did not elicit a cAMP response. Binding of ¹²⁵I-labeled cholera toxin to indomethacin-treated cells was also reduced by at least 50%. These data indicate that indomethacin's inhibitory effect on cAMP formation in cholera toxin-treated cells could be explained by its capacity to alter adenylate cyclase activity and cholera toxin binding.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Effects of cholera toxin on vasoconstriction and cyclic AMP content of the isolated rabbit ear artery

We have studied the effect of cholera toxin on the constrictor responses of the isolated, perfused rabbit ear artery to nerve stimulation and to norepinephrine infusion. We found that when we perfussed arteries with cholera toxin (1–9 μg/ml) for five minutes or longer, the toxin gradually inhibited the responses to intermittent stimulation of the adrenergic nerves and to brief infusion of norepinephrine. The constrictor responses began to decrease between one and two hours after we added cholera toxin, and the responses were still depressed after 24 hours. Cholera toxin inhibited both the rapid, initial phase and the slower, sustained phase of the biphasic response of the ear artery to nerve stimulation. Propranolol and indomethacin did not block the effect of cholera toxin on vasoconstriction. However, when we mixed the toxin with antitoxin or G_M1 ganglioside, we prevented the inhibitory effect on vasoconstriction. Levels of adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in arteries treated with cholera toxin were greater than levels of cyclic AMP in untreated arteries. The cyclic AMP content increased and the constrictor responses decreased with a similar time course after the arteries were exposed to the toxin. Thus an increase in cyclic AMP may be involved in the relaxation of vascular smooth muscle induced by cholera toxin.     

Inhibitory effect of ibuprofen on the cholera toxin-induced cyclic AMP response in Chinese hamster ovary cells

Abstract Ibuprofen, an inhibitor of prostaglandin synthesis in eukaryotic cells, was shown to inhibit the accumulation of 3',5'-cyclic adenosine monophosphate (cyclic AMP) in Chinese hamster ovary (CHO) cells exposed to cholera toxin. The inhibition was dose dependent, with a dose of 100 μg/ml reducing the cholera toxin response by approximately 50%, and maximal inhibition was observed when the drug was applied to the cells simulataneously with or 1 h before the toxin. Although ibuprofen also inhibited adenylate cyclase stimulation by forskolin, suggesting a nonspecific effect, the drug had no effect on cholera toxin-induced cyclic AMP accumulation when added to the culture medium 15 min or more after the toxin.     

Charge heterogeneity of cholera toxin and its subunits

Abstract Analytical isoelectric focusing (IEF) in thin layers of polyacrylamide gels resolved cholera toxin into 3 isomeric forms differing in charge (isoelectric points 6.80, 6.65 and 6.55). All these forms had identical molecular weights, and were also antigenically similar, as demonstrated by their reactivity to antisera to cholera toxin. Both the B and A subunits possessed charge heterogeneity. The B subunit was detected in a free form when a solution of cholera toxin was aged for a few days. Antisera to cholera toxin, irrespective of mode of immunisation, contained antibodies to both the intact cholera toxin and the free B subunit as demonstrated by the immunoblotting technique based on IEF.     

Inhibition by cholera toxin of parathyroid hormone-induced calcium release from bone in culture.

Cholera toxin was added to the culture of fetal rat limb bone and its effect on calcium release as well as on adenylate cyclase activity was examined. Cholera toxin increased the content of adenosine 3′:5′-monophosphate (cAMP) in bone. The effect on cAMP was of slower onset and of longer duration as compared with parathyroid hormone (PTH) effect. PTH added to the tissue which had been stimulated by cholera toxin increased cAMP further but the effect was partially additive. In contrary to PTH which caused a clear calcium mobilization, cholera toxin by itself had no effect or rather inhibited the release of ⁴⁵Ca from the prelabeled bone. When the toxin (0.1–1 μg/ml) was combined with PTH (10 U/ml), calcium release stimulated by PTH was completely abolished.     

Inhibition of lipolysis and cyclic AMP accumulation by adenosine analogues in hamster epididymal adipocytes exposed to cholera toxin

The effects of adenosine, N6-phenylisopropyl adenosine and 2',5'-dideoxyadenosine on lipolysis and cyclic AMP accumulation, in hamster adipocytes treated with cholera toxin, were studied. Cholera toxin caused an increase in lipolysis and cyclic AMP accumulation that was dependent upon the concentration of toxin and the length of time cells were exposed to the toxin. When N6-phenylisopropyl adenosine or 2',5'-dideoxyadenosine were present, the lipolytic and cyclic AMP responses to cholera toxin were inhibited. The adenosine analogues were equally effective inhibitors of lipolysis and cyclic AMP accumulation, when they were added 1 or 2 h after exposure to the toxin. Enzymatic removal of endogenously produced adenosine with adenosine deaminase potentiated both the lipolytic and cyclic AMP responses to cholera toxin. In addition, the inhibitory effects of N6-phenylisopropyl adenosine, 2'5'-dideoxyadenosine and clonidine on lipolysis and cyclic AMP were enhanced consequent to enzymatic removal of adenosine. These data show responses of intact fat cells to N6-phenylisopropyl adenosine, 2',5'-dideoxyadenosine or removal of endogenous adenosine and provide evidence for an adenosine sensitivity of fat cells exposed to cholera toxin.     

Effects of cholera toxin and guanosine 5'-[betagamma-imido]triphosphate on beta-adrenergic-receptor affinity.

A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.