ABC-ELISA检测抗精子抗体方法的探讨

本文探讨ABC-ELISA检测抗精子抗体的实验条件。结果提示:最适包盖精子浓度为10×10~9/ml;最适血清稀释度为1:400;甲醛与戊二醛固定效果无显著性差异(P>0.05),10％FCS-PBS封板并不显著降低非特异反应(P>0.05);批内和批间误差分别为2.1％～4.3％和6.4％～9.4％;比较检测男,女阳性血清灵敏度,AEC-ELISA较普通ELISA敏感4倍,较Franklin-Dukes试验敏感100倍;比较检测兔抗人精子免疫血清灵敏度,则分别敏感8倍和400倍。本法具有灵敏度高、特异性强和稳定性好等优点,作者认为,不仅可用于循环抗体的检测,而且在局部抗体检测上也具有应用前景。     

静脉麻醉药物群体药代学、药效学研究近况

群体药代学与药效学研究即药代动力学和药效动力学的群体分析法,它定量地考察患者群体中药物浓度的决定因素,常采用的统计学方法为非线性混合效应模型。对几种常用的静脉麻醉药物药动学和药效学进展一并综述。     

静脉麻醉药物群体药代学、药效学研究近况

群体药代学与药效学研究即药代动力学和药效动力学的群体分析法，它定量地考察患者群体中药物浓度的决定因素，常采用的统计学方法为非线性混合效应模型。对几种常用的静脉麻醉药物药动学和药效学进展一并综述。     

两种舒芬太尼靶控输注系统的准确性

目的 评价两种舒芬太尼靶控输注系统的准确性.方法 择期手术患者18例,年龄21～64岁,ASA Ⅰ或Ⅱ级,均采用舒芬太尼、异丙酚及维库溴铵行麻醉诱导和维持.随机选择6例患者行体重修正舒芬太尼Gepts药代动力学参数研究,靶控输注舒芬太尼(血浆靶浓度0.8 ng/ml)10 min,输注异丙酚(血浆靶浓度3～4 mg/L),意识消失后静脉注射维库溴铵0.1 mg/kg,靶控输注舒芬太尼(血浆靶浓度0.2～0.8 ng/ml),术毕前30 min停止输注.分别于靶控输注舒芬太尼前、输注舒芬太尼1、3、5、10、20、40、60、90、120和150 min时取桡动脉血3 ml/次,采用ELISA法测定舒芬太尼血药浓度.计算偏离度、准确度,中央室容积(V1)与体重(m)作直线回归分析,并修正药代动力学参数.余12例患者选用上述体重修正后药代动力学参数行临床麻醉,计算舒芬太尼靶控输注系统的偏离度、准确度、分散度、摆动度.结果 采用舒芬太尼Gepts药代动力学参数靶控输注舒芬太尼时,偏离度为16.7%、准确度为42.0%;体重修正后参数为:V1(L)=0.147 m+2.82,K10=0.064 5 min-1、K12=0.108 6 min-1、K21=0.024 5 min-1、K13=0.022 9 min-1、K31=0.001 3 min-1;采用体重修正后药代动力学参数靶控输注舒芬太尼时,偏离度、准确度分别为4.0%、22.3%,较Gepts药代动力学参数靶控输注舒芬太尼时小(P＜0.05),分散度、摆动度分别为-4.4%/h、20.4%.结论 舒芬太尼Gepts药代动力学参数的中央室容积偏大,体重修正后嵌入靶控输注系统,可提高靶控输注的精确度及稳定性,可维持较准确的血药浓度.     

胃癌患者手术前后血清可溶性白介素2受体动力研究的临床意义

通过对胃癌患者手术前后血清ＳＩＬ－２Ｒ变化的研究，探讨手术前后机体免疫功能的变化及ＳＩＬ－２Ｒ变化的临床意义。双抗体夹心酶联免疫吸附法同批检测胃癌患者手术前后血清ＳＩＬ－２Ｒ水平。     

重型颅脑损伤患者心肌损害的早期诊断

目的 探讨重型颅脑损伤患者心肌损害的早期诊断及心脏型脂肪酸结合蛋白(h-FABP)对患者预后的影响.方法 血清h-FABP采用双抗体夹心酶联免疫一步法定量检测,cTnI采用固相酶联免疫吸附试验(ELISA),CK-MB采用免疫抑制法.结果 重型颅脑损伤患者组血清h-FABP、cTnI及CK-MB水平分别显著高于健康对照组(P<0.01),重型颅脑损伤患者血清h-FABP阳性率显著高于血清cTnI、CK-MB或心电图的阳性率(P<0.01),47例重型颅脑损伤患者治疗后死亡8例,病死率为17.02%(8/47),血清h-FABP、cTnI及CK-MB异常组的病死率显著高于正常组(P<0.01);重型颅脑损伤患者血清h-FABP预测发生死亡具有高的敏感性和阴性预测值,但特异性和准确度较低.结论 血清h-FABP定量测定作为一种对微小心肌损伤高敏感的指标,可作为判断重型颅脑损伤急性期病情轻重、评价发生意外的一项客观指标.     

丹参素在大鼠骨组织中分布动力学研究

目的研究大鼠静脉注射复方丹参液后丹参素在骨骼中的分布动力学,同时探讨药效学部分相关组织中药物分布的相互关系。方法采用液液萃取法对血清、肾和骨样品进行预处理,以对羟基苯甲酸为内标,甲醇-1%冰醋酸(8∶92)为流动相,用高效液相色谱法于280nm处检测,液质联用进行定性分析。结果大鼠静脉注射复方丹参液后在血清、肾和骨中检测到丹参素,用所建立的方法绘制了其经时血药浓度曲线、肾药浓度和骨药浓度柱形图。结论大鼠静脉注射复方丹参液后,丹参素可以在其抗骨质疏松相关组织中快速分布,其血药动力学类型属二室模型。     

5-氟尿嘧啶不同容量腹腔化疗的药代动力学及活性炭应用对其的影响

目的　探讨不同药物浓度及缓释剂应用对 5 氟尿嘧啶 (5 Fu)药代动力学的影响。方法　以家兔为模型 ,用高效液相色谱法 ,检测相同 5 Fu剂量下 (10 0mg/kg) ,大容量腹腔化疗、小容量腹腔化疗、小容量缓释腹腔化疗 (加入活性炭 )在不同时间点的药物浓度 ,计算药代动力学参数。结果　大容量腹腔化疗峰浓度 69.75 3mg/L ,半衰期 0 .80 1h。小容量腹腔化疗峰浓度略低大容量腹腔化疗 60 .3 65mg/L ,半衰期 1.10 8h ,作用时间较长。小容量缓释腹腔化疗 ,虽然浓度较小2 0 .693mg/L ,但半衰期 19.10 2h ,作用时间最长。 结论　从药代动力学的角度 ,小容量腹腔化疗是一种较为理想的化疗方式。加入活性炭以后可以更进一步增加药物的作用时间。     

联合检测AFP、CA199和GGTⅡ对诊断原发性肝细胞癌的临床价值

目的探讨甲胎蛋白(AFP)、癌抗原125(CA125)及ɑ-谷氨酰转肽酶同功酶Ⅱ(GGTⅡ)三种标志物联合检测对原发性肝细胞癌(hepatocellular carcinoma,HCC)患者的诊断价值。方法 HCC未手术组患者80例,肝良性病变组患者54例,HCC手术组35例。采用双抗体夹心原理(ELISA)检测人血清中的AFP和CA125,特异免疫膜吸附法检测血清中的GGTⅡ水平。结果 HCC未手术组血清中AFP、CA125及GGTⅡ水平高于肝良性病变组,差异有统计学意义(P﹤0.01)。HCC手术组术后三者的血清水平均低于HCC未手术组,差异有统计学意义(P﹤0.01)。3种指标联合检测对于HCC的诊断,灵敏度和准确度有所提高,但特异性有所下降。结论联合检测AFP、CA125及GGTⅡ可以提高肿瘤标志物对HCC的临床诊断价值。     

基于直接标记法尿微量白蛋白检测抗体微阵列的构建

目的 探讨基于直接标记法的尿微量白蛋白检测抗体微阵列（也称抗体芯片）构建方法,并评价其技术可行性。 方法 通过玻片处理、活化、固定等程序构建白蛋白抗体芯片。摸索用生物素标记标准品白蛋白及尿液样本的最佳条件。经封闭后加入生物素标记的标准品白蛋白,孵育,洗涤后加入链酶亲和素-Cy3,再经过孵育、洗涤后,用共聚焦激光扫描仪获取荧光强度值。根据不同浓度标准品白蛋白的荧光信号强度值做出浓度-信号强度曲线,并评价其检测灵敏度、特异性、重复性。收集临床糖尿病患者尿液标本,对尿液样本同时行免疫比浊法和抗体芯片法检测白蛋白值。 结果 生物素标记标准品白蛋白和样本的最佳总蛋白/生物素质量比为2∶1。根据标准品白蛋白所做出的标准曲线其R2 > 0.99;捕获抗体浓度为1 g/L时,最低白蛋白检测浓度为0.0617 mg/L。阵列内变异系数为3.35%~7.59%;阵列间变异系数为6.78%~9.22%。抗体芯片检测值与免疫比浊法检测值呈正相关（R2 = 0.9199,P ＜ 0.01）。 结论 成功构建基于直接标记法的抗体芯片,其具有简单、快速、高效、高灵敏度和重复性好等特点;具有开发应用于微量白蛋白尿大规模人群筛查的潜力。     

Cytokeratin shedding in urine as a biological marker for bladder cancer: monoclonal antibody-based evaluation.

Cytokeratin shedding into urine was measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) in 282 individuals. Samples included urine from normal controls, patients with urogenital conditions and bladder cancer patients. A monoclonal antibody prepared against cytokeratins extracted from a hyperkeratotic low grade squamous cell carcinoma (UNME/K1) was used in the assay. The results indicated reasonable levels of sensitivity (83%), specificity (67%) and overall accuracy (70%) in the detection of bladder cancer. The levels of sensitivity in detecting squamous and transitional cell carcinoma patients were 87 and 73% respectively. The low level of specificity was due to a high frequency of false positive results (55%) within the urogenital controls; this suggests that further immunochemical and immunohistopathological analyses of associated urothelial cytokeratins are required.     

Fibulin-1蛋白的真核表达与ELISA检测方法的建立

目的建立检测血清中Fibulin-1蛋白浓度的ELISA方法。方法构建Fibulin-1蛋白真核表达载体,以人Fibulin-1基因编码序列为模板,将目的片段定向克隆入载体并转入毕赤酵母菌,进行Fibulin-1蛋白的真核表达。表达产物纯化后,Westernblotting检测证明纯化的重组蛋白的抗原特异性。以该蛋白为定标绘制标准曲线,建立人血清ELISA检测方法并检测其精密度和回收率,最后初步分析人血清中Fibulin-1的含量。结果从真核表达体系获得了纯化的Fibulin-1蛋白,其具有良好的抗原特异性;建立的ELISA方法线性相关系数W=0．98,线性范围为12．5—800ng／mL,检测灵敏度为6．25ng／mL,蛋白回收率在94．33％～109．33％之间,CV均〈10％,具有良好的重复性;检测30例正常人血清样本中Fibulin-1的含量约为（31±8）μg／mL。结论建立的ELISA方法可用于检测血清中的Fibulin-1蛋白含量。     

高尔基体糖蛋白73检测对原发性肝癌的诊断价值探讨

目的：研究高尔基体糖蛋白73（GP73）、甲胎蛋白（AFP）、血管内皮生长因子（VEGF）在原发性肝癌（PHC）患者血清中的表达意义。方法43例PHC及9例肝良性肿瘤患者接受双抗体夹心ELISA法检测血清GP73、VEGF浓度及电化学发光法检测血清AFP浓度。结果 GP73在不同年龄、性别、肿瘤大小间的表达差异无统计学意义。伴乙肝病毒携带的患者其GP73表达水平高于不伴有乙肝病毒携带的患者（P＜0.05）,伴有肝硬化的患者其GP73表达水平高于不伴有肝硬化的患者（P＜0.05）。PHC组高于肝良性肿瘤组[（241.413±77.079）μg/L vs（101.866±74.192）μg/L,P＜0.01]。手术前GP73、AFP和VEGF表达水平均高于手术后[（674.176±1090.083）μg/Lvs（178.560±289.330）μg/L,（256.666±164.760）μg/L vs（149.072±158.643）μg/L,P＜0.01]。AFP、GP73、VEGF在PHC鉴别诊断的ROC曲线下面积分别为0.894、0.791、0.612,GP73面积为最大。GP73诊断PHC组敏感性及特异性为88.4%、77.7%,高于AFP。将AFP及GP73并联检测后发现,敏感度为96.8%,特异度为63.7%,与单项检测相比,GP73联合AFP可明显提高PHC诊断的准确性。结论 GP73是一种灵敏度、特异度更高的PHC诊断的血清标志物,而GP73、AFP联合检测可提高PHC诊断准确性。     

Simplifying sampling for African swine fever surveillance: Assessment of antibody and pathogen detection from blood swabs

African swine fever (ASF ) is a notifiable disease with serious socio‐economic consequences that has been present in wild boar in the Baltic States and Poland since 2014. An introduction of ASF is usually accompanied by increased mortality, making fallen wild boar and hunted animals with signs of disease the main target for early warning and passive surveillance. It is difficult, however, to encourage hunters and foresters to report and take samples from these cases. A pragmatic and easy sampling approach with quick‐drying swabs could facilitate this. In this study, we further evaluated the use of dry blood swabs for the detection of ASFV antibody and genome with samples from animal trials and diagnostic submissions (blood, bone and organs) from Estonia. Compared to serum samples, dried blood swabs yielded 93.1% (95% confidence interval: [83.3, 98.1]) sensitivity and 100% [95.9, 100.0] specificity in a commercial ASFV antibody ELISA . Similarly, the swabs gave a sensitivity of 98.9% [93.4, 100.0] and a specificity of 98.1% [90.1, 100.0] for genome detection by a standard ASFV p72 qPCR when compared to EDTA blood. The same swabs were tested in a VP 72‐antibody lateral flow device, with a sensitivity of 94.7% [85.4, 98.9] and specificity of 96.1% [89.0, 99.2] compared to the serum ELISA . When GenoTube samples tested in ELISA and LFD were compared, the sensitivity was 96.3% [87.3, 99.5] and the specificity was 93.8% [86.0, 97.9]. This study demonstrates reliable detection of ASFV antibody and genome from swabs. A field test of the swabs with decomposed wild boar carcasses in an endemic area in Estonia also gave promising results. Thus, this technique is a practical approach for surveillance of ASF in both free and endemic areas.     

Detection of Antibodies Against Capripoxviruses Using an Inactivated Sheeppox Virus ELISA

An indirect ELISA was developed to detect antibodies specific for capripoxviruses in goat, sheep and cattle sera. Heat‐inactivated Nigerian sheeppox virus was used as the ELISA antigen. Sera obtained from sheep and goats that were experimentally infected with different capripoxvirus isolates were used to develop and evaluate the sensitivity of the ELISA. Virus neutralization indexes were determined for the experimental sera in OA3.Ts cells. The specificity of the ELISA was determined using 231 sera from capripoxvirus naïve sheep and goats from Canada. In addition, the ELISA was tested for cross‐reactivity to anti‐orf virus antibodies using orf‐reactive sera and no cross‐reactivity was observed. Using experimentally generated sera obtained from animals infected with virulent sheeppox or goatpox virus isolates, the diagnostic sensitivity of the ELISA was 96% with a diagnostic specificity of 95%, where the diagnostic sensitivity of the virus neutralization assay was 96% with a diagnostic specificity of 100%. Further evaluation of this ELISA, using 276 cattle serum samples that were positive by virus neutralization assays, revealed a diagnostic sensitivity of 88% with a specificity of 97%. These results indicated that the inactivated capripoxvirus ELISA can detect capripoxvirus‐specific antibodies in sheep, goats and cattle that have been infected with virulent capripoxvirus isolates. Non‐virulent capripoxvirus isolates, in contrast, did not elicit positive (≥1.5 Log₁₀ neutralization index) antibody responses.     

流式细胞术-群体反应性抗体的检测在临床器官移植中的应用

目的 比较流式细胞术检测群体反应性抗体(FLOW-PRA)的方法与传统的酶联免疫吸附检测群体反应性抗体(ELIA-PRA)的方法在临床器官移植中应用的相关性、灵敏性、准确性及实用性.方法 采用FLOW-PRA和ELISA-PRA两种方法对212份等待肾移植患者的血清进行检测,对两种方法的检出结果进行比较分析.结果 FLOW-PRA和ELISA-PRA两种方法各耗时1.5h和3 h.两种方法所得结果有很好的相关性,PRA Ⅰ类和PRAⅡ类检测结果的相关系数分别为0.94和0.89.采用FLOW-PRA法检出PRA Ⅰ类和Ⅱ类阳性率分别为24.5%和18.4%,采用ELISA-PRA法检出PRAⅠ类和Ⅱ类的阳性率分别为17.9%和14.6%,FIOW-PRA法的阳性检出率明显高于ELISA-PRA法.FLOW-PRA法能准确检测到低浓度抗体及其抗体特异性.结论 与ELISA-PRA法相比.FLOW-PRA法简单快捷,有更高的敏感性和准确性,对低致敏患者有更高的阳性检出率,更适合临床器官移植对PRA的检测.     

PLA2R、 THSD7A与IgG亚型在特发性膜性肾病中的诊断价值

目的探讨特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者血清抗磷脂酶A2受体(phospholipase A2 receptor,PLA2R)抗体水平、肾小球PLA2R、1型血小板反应蛋白7A域(thrombospondin type I domain containing 7A,THSD7A)、免疫球蛋白G(immunoglobulin G,IgG)亚型的表达及其在IMN中的诊断价值。方法选取2016年1月至2019年6月在雅安市人民医院肾内科经肾活检并确诊的IMN患者72例,以同期72例非IMN肾小球疾病患者为对照。采用酶联免疫吸附法检测血清抗PLA2R抗体滴度,免疫荧光法检测肾小球PLA2R、THSD7A及IgG亚型表达。采用受试者工作特征(ROC)曲线分析血清抗PLA2R抗体、肾小球PLA2R、THSD7A、IgG4诊断IMN的价值。结果血清抗PLA2R抗体、肾小球PLA2R、IgG4、THSD7A诊断IMN的灵敏度分别为61.11%、80.56%、97.22%、8.33%,特异度分别为97.22%、100.00%、97.22%、100.00%。血清抗PLA2R抗体和肾小球PLA2R任一指标阳性即诊断IMN的敏感性为83.33%;肾小球PLA2R、THSD7A和IgG4中任一指标阳性即诊断IMN的敏感性为97.22%。结论血清抗PLA2R抗体、肾小球PLA2R、THSD7A及IgG4亚型对于IMN诊断具有较高的临床价值。血清抗PLA2R抗体及肾小球PLA2R抗原的联合检测,肾小球PLA2R、THSD7A与IgG4的联合检测可以增加IMN诊断的敏感性。     

Reappraisal of HLA Antibody Analysis and Crossmatching in Kidney Transplantation

Enzyme-linked immunosorbent assay (ELISA) and flow cytometric techniques have been introduced to overcome the limited sensitivity and specificity of the CDC assay. This retrospective study used lambda antigen tray-mixed screening and Luminex HLA class I and II specificity assays to re-examine: (1) the accuracy with which detection of HLA antibody and specificity by ELISA predicts pretransplantation National Institutes of Health (NIH)/Centers for Disease Control and Prevention (CDC) crossmatch; and (2) a comparison of Luminex and ELISA methods to detect HLA antibodies. Sera from 481 patients awaiting kidney transplantation were tested using the ELISA method lambda antigen tray-mixed and using NIH-CDC to determine how well HLA antibodies detected using ELISA predicted crossmatches using CDC. Pretransplantation sera from 48 patients with follow-up data were retested using both ELISA lambda antigen tray-mixed and Luminex to compare the efficacy of the 2 methods.     

荧光PCR技术在粪便幽门螺杆菌ureA基因检测中的应用

目的 评价荧光PCR技术检测粪便幽门螺杆菌（Helicobacter pylori,HP）urea基因的价值.方法 采用荧光PCR技术检测50例消化内科住院患者的粪便样本,其中23例通过胃黏膜活检组织尿素酶及病理组织染色确诊为HP感染.同时进行HP培养和血清HP尿素酶抗体检测.四格表x2检验比较3种方法的敏感性、特异性、阳性预测值和阴性预测值.结果 荧光PCR技术检测粪便HP感染的敏感性、特异性、阳性预测值和阴性预测值分别为1.00、0.96、96%和100%,而HP培养检测的敏感性、特异性、阳性预测值和阴性预测值分别为0.78、1.00、100%和84%,其中敏感性和阴性预测值与荧光PCR法相比,差异具有统计学意义（X2=5.60和4.44,P值均〈0.05）.血清HP尿素酶抗体检测的敏感性、特异性、阳性预测值和阴性预测值分别为0.96、0.74、76%和95%,其特异性和阳性预测值与荧光PCR法比较,差异具有统计学意义（X2=5.28和4.08,P值均〈0.05）.结论 粪便HPureA基因检测对诊断HP感染具有较高的临床价值.     

Schmallenberg Virus Infection Diagnosis: Results of a German Proficiency Trial

Since Schmallenberg virus (SBV), an orthobunyavirus of the Simbu serogroup, was detected in Central Europe in 2011 for the first time, numerous diagnostic test systems for genome or antibody detection have been established. Therefore, a laboratory proficiency trial with 28 veterinary laboratories was initiated to allow performance evaluations of the different veterinary diagnostic laboratories and the performance of the used assays. A panel of selected sera and bovine semen samples for the analysis by real‐time PCR and an additional set of serum samples for serological analysis were provided. All participants were asked to investigate the samples with the test systems routinely used in their laboratory. While SBV‐genome was reliably detected in serum samples, the sensitivity in semen samples seems to depend on the application of the recommended optimized nucleic acid extraction method (TRIzol^® LS Reagent‐based, Hoffmann et al., 2013, Vet. Microbiol., 167, 289). SBV‐antibody‐positive samples and sera negative for antibodies against Simbu serogroup viruses were in most cases correctly classified by the participants with the used commercial ELISA kits. However, a serum of the panel which contained antibodies against Akabane and Aino viruses, which are closely related to SBV, was repeatedly tested positive by two of four used ELISA kits. However, an excellent diagnostic sensitivity and specificity was achieved using a serum neutralization test. In conclusion, the here described German SBV proficiency test demonstrated that the available test systems allowed reliable SBV diagnostics in standard veterinary laboratories when recommended and approved assays are used.