Dual-label time-resolved fluoroimmunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A

A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA–bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu³⁺- and Sm³⁺-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 μg/L (range 0.02–100 μg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 μg/L (range 0.05–50 μg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA–TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA–TRFIA and the single-label AFB1–TRFIA or OTA–TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA–TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.     

吗啡对原代培养海马神经元内Gi2蛋白水平的影响

目的 观察吗啡对原代培养海马神经元内Ⅱ型抑制性鸟苷酸结合蛋白（Gi2蛋白）水平的影响。方法 取培养7天的成熟海马神经元，随机分为吗啡处理4h组（M4）、8h组（M8）、16h组（M16）、24h组（M24）、48h组（M48）和空白对照组（C），每组6孔。吗啡处理组加入终浓度为10μmol／L的吗啡，C组加入生理盐水。免疫荧光技术检测神经元内的Gi2蛋白水平。结果 与C组比较，M16组、M24组和M48组海马神经元内Gi2蛋白水平显著降低（P〈0．01），而M4和M8组无明显变化（P〉0．05）。M48组海马神经元内Gi水平明显低于M16、M24组（P〈0．05）。结论 吗啡可降低原代培养的海马神经元内Gi2蛋白水平，降低程度与吗啡处理时间密切相关。     

血清半乳糖凝集素3对胰腺癌的诊断价值

目的 运用时间分解免疫荧光(TRFIA)法检测血清半乳糖凝集素3(Galectin-3,Gal-3)水平,并探讨Gal-3对胰腺癌的诊断价值.方法 采用固相双抗体夹心法建立检测血清Gal-3的TRFIA,探讨最佳实验条件.在最适条件下检测胰腺癌、胰腺良性占位、胰腺炎患者及健康对照者血清Gal-3水平,并联合检测血清CEA及CA19-9水平.结果 TRFIA法检测血清Gal-3的线性为0～100μg/L,批内变异系数(CV)≤6.45％,批间CV≤8.68％,平均回收率为106.6％.胰腺癌患者血清Gal-3水平为4.93(0.85～23.80) μg/L,明显高于胰腺良性占位者的2.83(2.17～4.06) μg/L、胰腺炎患者的2.62(0.55 ～9.76) μg/L和健康者的1.88(0.59 ～ 3.94) μg/L(P值均＜0.05).以3.77 μg/L为界,其诊断胰腺癌的敏感性为75.5％,特异性达90.9％.Gal-3与CEA及CA19-9水平均无相关性(r=0.1321,P=0.3761；r =0.0920,P=0.5384),Gal-3联合CEA或CA19-9检测,对胰腺癌的诊断敏感性可提高到92％.结论 TRFIA法检测血清Gal-3具有较好的敏感性和稳定性；Gal-3有望成为新的胰腺癌标记物.     

血清透明质酸时间分辨荧光免疫分析法的反应条件优化及其临床应用

目的优化血清透明质酸(HA)时间分辨荧光免疫分析法(TRFIA)的反应条件,并探讨其临床应用价值。方法采用TRFIA检测血清中HA的含量,确定参与HA-TRFIA的各反应成分的浓度。结果最佳反应条件为包被浓度10 mg/L、HA结合蛋白(HABP)1∶500稀释、铕标记HA(Eu3+-BSA-HA)1∶200稀释。敏感性为5.17μg/L,批内变异系数(CV)为2.21%~3.78%,批间CV为3.73%~5.40%,平均回收率为100.4%。该法HA测定值与放射免疫测定值显著相关,且与临床结果一致。结论本研究建立的血清HA-TRFIA是一种灵敏准确的分析方法,适合临床应用。     

AFP/CEA双标记时间分辨荧光免疫分析试剂的研制及性能鉴定

目的研制甲胎蛋白（AFP）及癌胚抗原（CEA）的双标记时间分辨荧光免疫分析（TRFIA）试剂,并鉴定其性能。方法将抗AFP单克隆抗体（E014）与抗CEA单克隆抗体（3E6）混合包被96孔板,用钐标记抗AFP单克隆抗体（E010）,铕标记抗CEA单克隆抗体（S001）,用双抗体夹心一步法建立AFP/CEA TRFIA试剂,测定其线性相关性、灵敏度、精密度、回收率、特异性,并与对应的单标记进口试剂进行比较。结果 AFP分析灵敏度为0.3 U/ml,线性范围为0.3~800 U/ml,平均回收率为99.8%,分析内和分析间变异系数分别为2.1%~5.8%、4.1%~8.7%;CEA分别为0.2 ng/ml、0.2~400 ng/ml、100.3%、2.4%~5.6%、4.8%~9.8%。该试剂与对应的单标记进口试剂的相关系数分别为0.99、0.98。结论成功研制了AFP/CEA双标记TRFIA试剂,其性能均达到临床检测要求,可替代国内外的单标记试剂。     

大鼠胃肠道肌间神经丛铺片的制备和染色法的研究

目的：对传统的消化道全层铺片技术和染色方法加以改进,以提高实验结果的准确性.方法：运用改进的方法制作消化道全层铺片,运用免疫荧光染色法显示胃肠道肌间神经丛,并以第I组代谢型谷氨酸受体mGluR1,mGluR5和神经型一氧化氮合酶（nNOS）为例进行染色,显示大鼠胃和小肠肌间神经丛.结果：采用免疫荧光染色并运用荧光显微镜进行观察,得到了较好的结果,可以更加清楚的显示肌间神经丛的整体结构以及单个神经元及神经纤维.结论：胃肠道肌间神经丛铺片结合免疫荧光染色方法是研究胃肠道肌间神经丛的理想方法,精确度较高,为实验结果的准确性打下了良好的基础.     

胎盘活性因子体外抗单纯疱疹病毒作用

目的：探讨胎盘活性因子（ＰＡＦ） 在细胞培养中对单纯疱疹病毒（ＨＳＶ）起抑制作用。方法：运用间接免疫荧光法检测药物抗病毒作用。结果：ＰＡＦ对病毒吸附细胞无干扰作用，而对病毒有直接的杀伤及抑制作用。以抑制浓度为１００ μｇ／ｍｌ，作用时间为８ ｈ 效果最好。结论：该药对细胞无毒无害，无副作用，是较为理想的免疫调节剂。     

大鼠脑缺血时碱性成纤维细胞生长因子表达的变化

目的:观察局灶性脑缺血时,大鼠脑内碱性成纤维细胞生长因子(bFGF)表达分布的改变。方法:用荧光免疫组化方法观察bFGF的表达。用MCAO模型造成大鼠局灶性脑缺血损伤。结果:缺血后,bFGF在纹状体及额顶叶皮质均显著升高。在纹状体梗塞核心及周边区,bFGF主要在星状胶质细胞内表达;在邻近梗塞的区域,bFGF灰度比率从98％±10％增至125％±6％,神经元内也少量表达。在额顶叶皮质,bFGF灰度比率从104％±11％增至132％±28％。bFGF主要分布于神经元内。结论:局灶性缺血大鼠脑内不同区域bFGF表达的增加有所差异,提示纹状体内,可能主要由星状胶质细胞起神经保护作用;而在皮质,可能由神经元起自我保护作用。     

Comparison of the determination of chloramphenicol residues in aquaculture tissues by time-resolved fluoroimmunoassay and with liquid chromatography and tandem mass spectrometry

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of chloramphenicol (CAP) residues in aquaculture tissues based on monoclonal antibodies. The limits of detection (LOD) were determined to be 0.04 ng g^?1 and the limits of quantification (LOQ) were less than 0.15 ng g^?1. The intra-assay variations were below 10% and the interassay variations ranged between 9.7 and 13.3%. The mean recoveries established at six concentration levels varied from 83.7–109.6% and the coefficient of variation was from 8.3–11.5%. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.     

新型铕络合物用于HBsAg的时间分辨荧光免疫检测

利用稳定的新型铕荧光络合物BHHCT与Eu3+标记羊抗人HBsAb和Eu3+标记BSA-SA,建立定量测定血清HBsAb的Eu3+-BHHCT-HBsAb-TRFIA法和Eu3+-BHHCT-BSA-SA-TRFIA法.结果表明,这两种方法最低检出值分别为0.2ng/mL和0.05ng/mL,标准曲线范围均为0-100ng/mL,批内变异系数CV均小于10%,后者回收率为85%-115%.用Eu3+-BHHCT-BSA-SA-TRFIA法与ELISA法同时检测118份血清样品,结果表明前者的检出率比后者高.