蟹类食物过敏症的ELISA检测试剂的研究

目的研制蟹类食物过敏症的ELISA检测试剂。方法以纯化的中华绒螯蟹主要过敏原蛋白为抗原包被酶标反应板,对包被浓度和包被时间、封闭液的种类与封闭时间、酶标二抗稀释度等ELISA条件进行优化。结果中华绒螯蟹过敏原蛋白的最适包被浓度为100ng/孔,最适包被条件为37℃孵育3h、4℃过夜;酶标反应板的封闭以20g·L-1脱脂奶粉、37℃孵育3h、4℃封闭过夜为最适条件;HRP标记羊抗人IgE抗体以1∶2 500为最适稀释度。结论以中华绒螯蟹的主要过敏原蛋白为诊断抗原制备ELISA检测试剂,适用于临床蟹类食物过敏症的实验诊断。     

虾过敏症特异性IgE的ELISA检测方法的研究

目的研制虾过敏症特异性IgE的ELISA检测试剂。方法以纯化的刀额新对虾的2种主要变应原30 kD和33 kD蛋白为抗原分别包被酶标反应板,对抗原的包被浓度和时间、封闭液的选择与封闭时间、酶标二抗稀释度等ELISA条件进行优化。结果 30 kD和33 kD变应原的最适包被浓度分别为200 ng/孔和12.5ng/孔,最适包被条件为37℃孵育3 h,4℃过夜;酶标反应板的封闭以1.0%BSA、4℃封闭过夜为最适条件;HRP标记的羊抗人IgE抗体以1∶1 500为最适稀释度。结论以刀额新对虾的33 kD主要变应原为诊断抗原研制特异性IgE的ELISA检测试剂,适用于临床虾过敏症的实验诊断。     

间接抗体夹心酶联免疫吸附法测定人红细胞生成素(EPO)体外活性

目的　介绍一种经济、实用的红细胞生成素(EPO)体外活性检测方法—间接抗体夹心酶联免疫吸附法。方法　采用捕捉抗体(单克隆抗体:鼠抗EPO)包被酶标板,然后用封闭液封闭酶标板,加入稀释成合适浓度的EPO样品进行温育,再加入EPO第二抗体(多克隆抗体:兔抗EPO)温育,然后加入酶连抗体(HRP -羊抗兔)温育,最后加入底物缓冲液显色,用酶标仪读数,计算出EPO体外效价。结果　该方法重复性好(CV <5 %),与ELISA试剂盒比较,两法测定的结果相符( P >0 . 5 )。结论　该方法是一种经济、实用、准确、可靠的红细胞生成素(EPO)体外活性检测方法,与EPOELISA试剂盒比较,大大降低了实验费用     

甲基苯丙胺半抗原设计与抗体间接ELISA检测方法的优化

目的:提供一种有效的甲基苯丙胺(methamphetamine,MA)半抗原设计,并对MA抗体ELISA检测方法进行优化。方法:将合成的MA半抗原与破伤风类毒素(tetanus toxoid,TT)和牛血清白蛋白(bovine serum albumin,BSA)分别偶联(TT/BSA-SMA)作为免疫抗原和包被抗原,通过梯度稀释法优化ELISA反应体系中抗原最佳包被浓度和最佳酶标抗体浓度(1∶2000-1∶6000)等工作条件,并进行方法的稳定性、特异性验证。结果:优化后最佳的包被缓冲液为磷酸缓冲液(phosphate buffer solution,PBS),2μg·ml-1的包被抗原在37℃2 h后4℃包被过夜为最佳包被反应条件,最佳封闭条件为5%脱脂奶粉封闭120 min,最佳酶标抗体稀释度为1∶3000(1.33μg·ml-1),最佳孵育时间为60 min,最佳底物显示时间为15 min。该方法重复性实验变异系数均在5%以内,并且只能检测出MA抗体,特异性良好。结论:本研究中的半抗原结构能诱导MA抗体的产生,并成功建立间接ELISA检测MA抗体的最佳工作条件,可为MA疫苗临床前研究奠定基础。     

Western blot在梅毒螺旋体特异性抗体检测结果可疑样本复核中的应用价值

目的 探讨对ELISA和梅毒螺旋体颗粒凝集试验(TPPA)检测抗梅毒螺旋体(TP)特异性抗体结果不一致的样本行Western blot检测的必要性.方法 对我院检验科抗TP抗体检测结果进行回顾性分析.对酶标板有显色的S/CO＜1的ELISA阴性样本和S/CO≥1的ELISA阳性样本以TPPA复检结果,对ELISA和TPPA结果不一致的样本行Western blot检测.结果 采用ELISA初筛检测抗TP抗体样本106 757例,3972例需行TPPA复检,其中酶标板有显色的ELISA阴性样本29例和ELISA阳性样本3943例.TPPA复检与ELISA结果不一致的血清样本163例,其中酶标板有显色的ELISA阴性样本29例、阳性样本134例;TPPA阴性样本76例、阳性样本23例、保留样本64例;Western blot阴性样本93例、阳性样本31例、可疑样本39例.TPPA与ELISA检测结果分别与Western blot检测结果比较,差异均有统计学意义(P＜0.05).结论 TPPA是抗TP抗体检测的有效复检手段;ELISA与TPPA检测抗TP抗体结果不一致时,有必要采用Western blot进行确认.     

用阴道毛滴虫可溶性抗原和单克隆抗体进行ELISA试验研究

目的　探讨用阴道毛滴虫可溶性抗原和单克隆抗体进行ELISA试验的条件。方法　选用不同抗原浓度(50μg／ml～400μg／mg)包被酶标板,选用不同孵育时间(15～120)、不同封闭剂(脱脂奶粉、吐温20/PBS、牛血清白蛋白、小牛血清)和不同酶标板(国产和进口)。结果　以200μg／ml抗原量,用1%牛血清白蛋白封闭,37℃孵育1小时为本试验最佳条件。结论　检测10例滴虫性阴道炎患者血清,阳性率为90%,与多种寄生虫感染血清或单克隆抗体均无交叉反应。X     

不同波长选择对酶标比色计检测结果的影响

目的:探讨不同波长选择对酶标比色计检测结果的影响.方法:将20份吸光度A450-620nm-B均值为0.523(0.307～0.625)的阳性样本及经过预稀释的临界值样本HBsAg试剂盒检测,显色完毕后在4种不同波长下比色,连续检测5天,观察20份样本的最大A值、最小A值、吸光度A均值以及临界值样本的最高、最低吸光度A值、阴阳性样本的例数.4种不同波长选择分别为:A450,A450nm-B,A450-620nm,A450-620nm-B.结果:20份样本在4种波长下的A值比较接近,最小A值均＞0.105.而对于临界值样本,不同波长选择导致了部分检测孔阴阳性结果发生改变.结论:不同波长选择对CELISA检测较高HBsAg浓度的样本时几无影响,而对于临界值样本的影响较大,可至结果误判,应尽可能选择A450-620nm的比色方式.     

血管内皮生长因子定量检测试剂盒的研制

 目的   研制血管内皮生长因子（vascular endothelial growth factor,VEGF）检测试剂盒（酶标法）。方法  以一株特异性抗VEGF单克隆抗体作为捕获抗体,另一株单克隆抗体作为检测抗体,通过棋盘滴定法确定两抗体的最佳浓度组合。制备冻干VEGF165参考品,并对样本稀释液、封闭液等实验条件进行优化。检测自制试剂盒的特异性并对其进行初步应用。结果  棋盘滴定法确定捕获抗体的质量浓度为10 μg/ml,检测抗体的稀释倍数为1:20 000。冻干VEGF165的质量浓度为720~880 pg/ml,样本间变异系数＜10%。以5%牛血清白蛋白作为封闭液和样本稀释液最为合适。自制试剂盒与10种细胞因子均无交叉反应,具有较高的特异性和灵敏度。结论  成功研制出VEGF检测试剂盒。     

抗中性粒细胞胞浆抗体、抗核抗体联合检测

目的 探讨抗中性粒细胞胞浆抗体(ANCA)与抗核抗体联合检测的临床意义.方法 应用间接免疫荧光法(IIF)检测疑似自身免疫性疾病患者血清中的ANCA和抗核抗体(ANA),并用ELISA法进行抗丝氨酸蛋白酶3(PR3)和抗髓过氧化物酶(MPO)的定量检测.结果 (1)在ANA阳性组和ANA阴性组中均检出ANCA,但ANA阳性组的阳性率(15.64%)高于ANA阴性组(3.25%)(P＜0.01).(2)ANCA阳性组以抗MPO和抗PR3升高为主,与ANCA阴性组比较有统计学差异(P＜0.01).结论 同时进行ANCA、ANA检测,有助于提高自身免疫性疾病的诊断.     

几种不同方法学检测胃蛋白酶原 I，II 临床应用探讨

目的 使用化学发光法(CLIA),流式荧光发光法(FFIA),酶联免疫法(ELISA)三种不同的方法检测血清胃蛋白酶原I(PGI)、胃蛋白酶原II(PGII),探讨三种检测方法用于胃癌辅助诊断的临床价值。方法 通过方法学比对与相关分析,比较三种方法的一致性与相关性。通过检测确诊为胃癌患者的临床血清样本,利用ROC曲线确定PGI与PGI/PGII(PGR)临界值,评价三种方法的灵敏度与特异度。结果 三种方法测试不同组别血清样本中PGI、PGII,根据PGI、PGII结果计算PGR。发现胃癌组与非胃癌组间PGI、PGR两个指标差异有统计学意义,PGII差异无统计学意义。方法学比对发现,不同方法学之间PGI、PGII相关性良好,相关系数r为：0.9612~0.9789,不同方法学之间PGR相关系数为：0.8602~0.9109。对三种方法进行ROC曲线分析,确定ROC曲线最佳临界值后,分析PGI、PGR组合用于胃癌诊断,化学发光法、流式荧光发光法、酶联免疫法灵敏度分别为67.6%,61.8%,58.8%,特异度分别为96.6%、94.7%、96.6%。 结论 化学发光法,流式荧光发光,酶联免疫法检测PGI、PGII存在较好的相关性。通过临床评估,PGI结合PGR可以有效提高胃癌的诊断特异度与阳性预测值,具有较好的临床价值。     

乳酶生片及原料质量标准乳酸鉴别试验研究

摘 要 目的:选用３种市售纯牛奶和２种脱脂奶粉,优选乳酶生质量标准鉴别中乳酸鉴别试验培养基。方法: 分别用伊利、银桥、蒙牛3种纯牛奶,完达山和伊利2种脱脂奶粉制备牛奶培养基,比较不同牛奶培养基进行乳酸鉴别试验的结果,为乳酶生质量标准提高提供参考。结果:牛奶培养基对照管均表现为黄色,供试品管表现有黄色、橙黄、淡橙红、橙红与红色,该结果与乳酶生现行标准不符。结论:采用完达山脱脂奶粉制备的牛奶培养基在乳酸鉴别试验中灵敏度最高,其次为伊利脱脂奶粉,3种纯奶在乳酸鉴别中的灵敏度均较低。     

乳酶生片及原料鉴别中牛奶凝固力培养基制备方法的优选

目的优选乳酶生片及原料鉴别中牛奶凝固力培养基的制备方法。方法以伊利、银桥、蒙牛3种纯牛奶,完达山和伊利2种脱脂奶粉为样本,比较低温离心和煮沸2种脱脂方法。结果低温离心和煮沸2种脱脂方法均可,经脱脂处理的牛奶培养基表面无多量乳清分出,凝块均匀稠密,无气体产生,无消化现象。结论用煮沸脱脂方法简便易操作,经脱脂的纯牛奶比脱脂奶粉凝固较好。     

应用微生物法试剂盒检测奶粉中维生素B6的含量

目的建立应用微生物法试剂盒检测奶粉中维生素Bs含量的分析方法。方法采用维生素Be微生物检测试剂盒定量检测奶粉中添加维生素B6的含量。结果维生素B6的线性范围为0．002～0．012mg]100g（ml）,检出限为0．002mg／100g（ml）,其标准曲线的相关系数为0．9987,相对标准偏差均〈10％,其回收率达到90．0％～108％。结论微生物法试剂盒具有灵敏度高,检出限低,线性范围宽,操作方法简便、快捷,结果准确可靠等优点,可用于奶粉中维生素B6的检测工作。     

金黄色葡萄球菌α-毒素溶血活性测定方法优化及初步应用

 目的  优化金黄色葡萄球菌α-毒素（α-toxin,AT）溶血活性测定方法,用于无毒突变AT（non-toxic mutant AT,mAT）特异性血清抗体体外功能性评价和中和抗体滴度测定。方法  使用AT裂解兔红细胞,释放血红蛋白,通过分光光度法检测溶血活性,并对该方法中的孵育时间、兔红细胞终浓度、Triton X-100浓度进行优化,同时验证方法重复性。对mAT特异性血清抗体进行体外功能性评价,绘制AT溶血曲线,计算AT 溶血率50% 时的浓度,检测血清半数中和抗体滴度。结果  AT溶血活性最适检测条件为：孵育时间90 min,兔红细胞终浓度为2%（V/V）,Triton X-100浓度为0.25%。此条件下,分别加入mAT和含mAT的多价抗原血清抗体,对AT溶血活性均起到明显的抑制作用。AT 溶血率50%时浓度为1.75 μg/ml,变异系数3.75%。mAT和含mAT的多价抗原血清抗体的半数中和抗体滴度初步结果均为1∶64。结论  优化的AT溶血活性检测方法重复性良好,且检测时间较短,可用于特异性血清抗体体外功能性评价和中和抗体滴度测定。     

新型食物不耐受sIgG定量检测方法的构建

【摘要】目的以检测鸡蛋特异性免疫球蛋白G（sIgG）为例,探讨新型食物不耐受的定量检测方法。方法选择行食物过敏原sIgG检测的患者173例,另择健康体检者78例为阴性对照。制备生物素化牛血清白蛋白-链酶亲和素酶标反应板,同时加入生物素化鸡蛋抗原和待检血清,洗涤后再加入酶标记抗人IgG,建立一种抗原间接包被-液相反应模式的酶联免疫方法;优化生物素化过敏原和酶标记抗体的浓度,确定反应条件。应用此方法检测血清标本中的sIgG。结果选用生物素化蛋清作为抗原,其最适稀释比例是1∶2000,酶标抗体的最适稀释比例是1∶12000。此方法批内及批间变异系数分别为4.83%~8.55%,4.88%~7.93%;与蟹、牛奶、羊奶等的交叉反应率均<10%;与临床现用食物不耐受sIgG检测法有较好的相关性（=0.977X+8.45,r=0.961, P<0.05）。结论本检测方法可用于检测鸡蛋不耐受患者血清sIgG,操作简便,具有较高的准确性和特异性,重复性好,并显示较好灵活性潜能,为今后研制“个性化”随机组合食物品种的检测试剂盒奠定基础。     

Microencapsulation of water-soluble isoflavone and physico-chemical property in milk

This study was carried out to investigate the addition of water-soluble isoflavone into milk by means of microencapsulation technique. The yield of microencapsulation, sensory attributes, and capsule stability of water-soluble isoflavone microcapsules in milk were measured. Coating materials used was polyglycerol monostearate (PGMS), and core material was water-soluble isoflavone. The encapsulation yield of water-soluble isoflavone with PGMS was 67.2% when the ratio of coating material to core material was 15:1. The rate of water-soluble isoflavone release from capsules was 18, 19, and 25% when stored at 4, 20, and 30 degrees C for 12 days in milk, respectively. In sensory evaluation, beany flavor and color of microencapsuled water-soluble isoflavone added milk were significantly different from uncapsuled water-soluble isoflavone added milk, however, bitterness was not significantly different. In vitro study, micro-capsules of water-soluble isoflavone in simulated gastric fluid with the range of 3 to 6 pHs were released 3.0-15.0%, however, the capsules in simulated intestinal fluid with pH 7 were released 95.7% for 40 min incubation time. In conclusion, this study provided that PGMS as coating materials was suitable for the microencapsulation of water-soluble isoflavone, and the capsule containing milk was almost not affected with sensory attribute.     

Factors influencing the protein binding of YH-439 using an equilibrium dialysis technique. A new hepatoprotective agent

Various factors influencing the plasma protein binding of YH-439 to 4% human serum albumin (HSA) were evaluated using the equilibrium dialysis method at the initial YH-439 concentration of 2 μg mL^?1. It took approximately 12 h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% of dextran (‘the buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off, 12 000–14 000) in a water bath shaker kept at 37. C and at a rate of 50 oscillations min^?1. YH-439 was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24 h incubation. The binding of YH-439 to 4% HSA was constant (97.4 ± 0.55%) at YH-439 concentrations ranging from 0.5 to 10 μg mL^?1. However, the extent of binding was dependent on HSA concentrations: the values were 90.7, 94.7, 96.7, 97.0, 97.0, 97.1, and 97.5% at HSA concentrations of 0.5, 1, 2, 3, 4, 5, and 6%, respectively. The plasma protein binding decreased with increasing incubation temperature: the binding values were 98.2, 97.6, 97.2, and 96.8% when incubated at 10, 21, 26, and 37°C, respectively. The binding of YH-439 was also influenced by the chloride concentration in the buffer: the binding values were 94.5, 97.0, and 96.8% for the chloride concentrations of 0, 0.249, and 0.546%, respectively. The binding of YH-439 was also dependent on the buffer pH: the percentages of free fraction were 6.0, 4.1, 3.8, 2.8, 2.7 and 2.8% for the buffer pHs of 5.0, 6.0, 6.5, 7.0, 7.4, and 8.0, respectively. The free fraction of YH-439 was slightly increased by the addition of heparin (up to 40 U mL^?1), sodium azide (NaN₃, up to 0.5%), and its metabolites. The protein binding of YH-439 was influenced neither by AAG, acetylsalicylic acid, or sulphisoxazole, nor by the addition of citrate or EDTA. The free fractions of YH-439 in rabbit (4.2%) and dog (4.7%) plasma seemed to be higher than in rats (2.9%) and humans (3.1%).     

Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

Factors influencing the protein binding of a new phosphodiesterase V inhibitor, DA-8159, using an equilibrium dialysis technique

Various factors influencing the protein binding of DA-8159 to 4% human serum albumin (HSA) were evaluated using an equilibrium dialysis technique at an initial DA-8159 concentration of 5 microg/mL. It took approximately 8 h incubation to reach an equilibrium between 4% HSA and an isotonic phosphate buffer of pH 7.4 containing 3% of dextran ('the buffer') using a Spectra/Por 2 membrane (mol. wt. cut-off: 12,000--14,000) in a water bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. The extent of binding was dependent on DA-8159 concentrations, HSA concentrations, incubation temperature, buffer pH, and alpha-1-acid glycoprotein (AAG) concentrations. The binding of DA-8159 in heparinized human plasma (93.9%) was significantly higher than in rats (81.4%), rabbits (80.4%), and dogs (82.2%), and this could be due to differences in AAG concentrations in plasma.