乙肝患者血清的HBV-DNA含量与ALT水平的关系研究

目的:探讨和研究乙型肝炎患者乙型肝炎病毒(HBV)DNA复制水平与丙氨酸氨基转移酶(ALT)水平之间的关系,为临床中乙肝患者的诊治提供参考。方法:对240例慢性乙型肝炎患者采用荧光标记定量PCR方法测定血清HBV-DNA含量,采用全自动生化分析仪测定血清ALT水平,比较和分析HBV-DNA含量与ALT水平之间的关系。结果:慢性乙肝轻、中、重度患者的HBV-DNA含量三组之间差异有统计学意义(p0.01),肝脏的损害程度与HBV-DNA含量之间具有一定的关联,等级相关系数为0.162(P=0.012);ALT水平也与HBV-DNA载量之间存在关联,等级相关系数为0.371(P0.0001)。结论:肝损伤程度与HBV-DNA含量有显著相关性;同时血清ALT水平与HBV-DNA含量呈正相关。检测血清中的HBV-DNA含量和ALT水平为指导乙肝患者HBV感染、复制、传染性的判断、治疗方案的选择和疗效评定提供有一定的依据。     

双波长微孔板赖氏法检测人血浆丙氨酸氨基转移酶含量的方法学验证

目的采用双波长(492 nm/630 nm)微孔板赖氏法检测人血浆中丙氨酸氨基转移酶(alanine aminotransferase, ALT)的含量,并对该方法进行系统性的评价。方法按照ALT测定试剂盒的要求加样,应用双波长(492 nm/630 nm)微孔板赖氏法测定人血浆中的ALT含量,并对检测系统的专属性、线性范围、准确度和精密度进行验证。结果专属性分析结果显示,血浆样品与不同比例的抗凝剂混合,其中的ALT均能有效被检出,回收率分别为100%、95%和90%;对28和150个单位丙酮酸钠标准溶液进行倍比稀释5个梯度以制作标准曲线,采用多项式对标准曲线进行拟合,结果显示丙酮酸钠标准溶液分别在9.375～150、1.75～28和1.75～150个单位范围内线性关系均良好,相关系数r均达到0.99以上;准确度检测结果显示,已知含量的厂家质控血清和国家能力验证样品的实测值和理论值相比,回收率均在90%～110%之间;同一样品,重复加样18孔,重复性检测结果变异系数(CV)为5.29%;两位试验人员在不同日期对同一样品重复测定6次,中间精密度检测结果变异系数为5.49%。结论该检测方法专属性强、检测结果稳定可靠、线性良好、重复性好、检测通量大,可以有效降低成本,提高工作效率,适用于大规模检测人血浆中ALT的含量。     

新疆维吾尔族与汉族乙型肝炎患者HBVDNA 及肝功能检测结果的比较

目的:探讨新疆乌鲁木齐地区伴有肝功能指标:丙氨酸氨基转移酶(ALT)浓度异常的维吾尔族(维族)及汉族HBeAg阳性乙型肝炎初次就诊患者,乙型肝炎病毒DNA复制载量及ALT浓度是否存在差异及其对患者诊断、预后的意义。方法:回顾性选取门诊伴有ALT浓度异常的汉族、维族初次就诊患者并筛选出HBeAg阳性患者汉族、维族共373例。采用实时荧光定量聚合酶链反应、生化测定及酶联免疫吸附试验法分别测定HBV DNA、ALT浓度及乙肝HBeAg。结果:(1)汉族HBV DNA组秩和8869,维族HBV DNA组秩和10359.36,经Mann-Whitney Test检验两组间尚不能肯定HBVDNA分布有统计学意义,即伴有肝功能损害的汉族、维族初次就诊HBeAg阳性患者HBV DNA复制程度没有差异。(2)汉族ALT组秩和26818.50,维族ALT组秩和22009.50,经Mann-Whitney Test检验两组间ALT分布有统计学意义,即伴有肝功能损害的初次就诊HBeAg阳性患者汉族肝功能损害程度高于维族。(3)HBVDNA低复制组(103-104copy/mL):汉族秩和3771.46,维族秩和4993.2;中复制组(104-106copy/mL):汉族秩和6412.4,维族秩和5088.2;高复制组(>106copy/mL):汉族秩和929.04,维族秩和666.96,经Mann-Whitney Test检验在低复制组两民族间ALT分布无统计学意义,在中、高复制组两民族间ALT具有统计学意义。即:伴有肝功能损害的初次就诊HBeAg阳性患者在HBV DNA低复制组两民族间肝功能损害程度无差异,但在中、高复制组汉族肝功能损害程度高于维族。结论:新疆乌鲁木齐地区伴有肝功能损害的初次就诊的HBeAg阳性的汉族与维族之间HBV DNA的病毒复制无统计学意义(P>0.05),但两民族间的ALT具有统计学意义,可能跟维族的民俗、饮食习惯及生存环境、免疫相关基因HLA基因频率分布差异等因素有关。     

乙肝病毒载量与血清标志物及ALT相关性研究

探讨了乙型肝炎病毒核酸(HBV-DNA)水平与乙型肝炎免疫标志物(HBVM)和丙氨酸氨基转移酶(ALT)的关系。分别采用实时荧光定量聚合酶链反应(FQ-PCR),酶联免疫法和连续监测法检测了345例血清标本HBV-DNA含量,HBVM(HBsAg、HBsAb、HBeAg、HBeAb、抗HBcIgM)表达及ALT水平。HBsAg、HBeAg(和抗HBcIgM)阳性患者HBV DNA阳性率要明显高于HBsAg、HBeAb(和抗HBcIgM)阳性患者、仅HBsAg阳性患者及HBsAb、HBeAb阳性患者(P<0.01)。血清HBeAg阳性标本HBV-DNA阳性率为98.7%,明显高于HBeAg阴性标本的61.6%(P<0.01),并且血清HBeAg阳性标本HBV-DNA含量(log值,7.42±1.43)也明显高于HBeAg阴性标本(4.36±1.73)(P<0.01);在HBV-DNA含量小于107copy/mL的标本中,ALT与HBV-DNA含量呈正相关(P<0.01)。血清中HBV DNA含量与乙型肝炎免疫标志物以及肝细胞损伤三者之间存在密切的关系,在临床工作中应对血清HBVM、ALT和HBV-DNA含量联合检测,这样才能更准确地判断患者病情、预后及指导抗病毒药物的应用。     

ERCP术与腹腔镜手术治疗胆总管结石的疗效对比及对血清AST、ALT、ALP的影响

目的:探讨内镜逆行胰胆管造影术(ERCP)与腹腔镜手术治疗胆总管结石的疗效对比及对血清门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALP)的影响。方法:选择2017年4月到2018年4月我院接诊的胆总管结石患者50例作为研究对象,以随机数表法分为观察组(n=26)和对照组(n=24),对照组使用腹腔镜手术治疗,观察组采用ERCP取石术治疗。比较两组手术时间、术中出血量、术后下床时间、住院时间、血清AST、ALT、ALP水平变化情况、术后胃肠道恢复时间及术后不良反应发生情况。结果:观察组患者手术时间高于对照组,(P0.05),术中出血量、术后下床时间及住院时间均显著低于对照组(P0.05);手术前,两组患者血清AST、ALT、ALP水平无明显差异;治疗后,两组患者血清AST、ALT、ALP水平均明显上升(P0.05),且观察组患者血清AST、ALT、ALP水平显著低于对照组(P0.05);观察组患者肠鸣音恢复时间、排气时间、排便时间均明显低于对照组(P0.05);治疗期间,观察组患者不良反应总发生率为7.69%,显著低于对照组的29.17%(P0.05)。结论:在胆总管结石患者中应用ERCP取石术效果显著,对患者血清血清AST、ALT、ALP影响较小,值得推广与运用。     

家蚕丙氨酸转氨酶理化性质的研究

丙氨酸转氨酶(ALT)是家蚕丝心蛋白合成中的关键酶,为了阐明丝心蛋白的合成机理及利用ALT活力调控丝物质的形成,有必要深入了解ALT的分子性质。作者在分离纯化家蚕后部丝腺ALT的基础上,应用聚丙烯酰胺凝胶电泳等生化方法进一步研究了该酶的若干理化性质。结果表明,家蚕ALT由两种不同亚基组成,分子量分别是54kd和21kd;最适底物是丙酮酸;最适温度为50℃;最适DH为8．5;钾、钠、钙、镁等离子对酶有激活作用,而锰、铜、锌等离子对酶有抑制作用。研究还表明,该酶是一种依赖于金属离子的酶类,活性中心含有巯基或咪唑基。     

游离细胞法与固定化细胞法生产L-丙氨酸的比较

游离细胞法与固定化细胞法生产Ｌ－丙氨酸的比较徐虹王雪根范伟平欧阳平凯（南京化工大学生物工程与科学系,南京２１０００９）摘要利用假单胞菌的Ｌ－天冬氨酸－β－脱羧酶从Ｌ－天冬氨酸生产Ｌ－丙氨酸,具体方法有两种,一种为游离细胞法,另一种为固定化细胞法。本文...     

紫外分光光度计法与酶标仪微量法测定酚氧化酶蛋白含量及活力的比较

分别采用紫外分光光度计比色法与酶标仪微量法测定菜青虫Pieris rapae(L.)体内酚氧化酶蛋白含量和活力,以水杨醛缩对硝基苯胺为抑制剂,对2种方法的结果进行比较。结果表明,微量法和比色法在检测酚氧化酶(PO)蛋白含量、酶活力和抑制剂对PO抑制作用时结果无显著性差异。因此,微量法可以替代比色法,使用试剂量大大降低,重复性好,操作简单、快捷。     

游离细胞法与固定化细胞法生产L—丙氨酸的比较

利用假单胞菌的Ｌ－天冬氨酸－β－脱羧酶从Ｌ－天冬氨酸生产Ｌ－丙氨酸,具体方法有两种,一种为游离细胞法,另一种为固定化细胞法。本文比较这二种方法的生产工艺、转化率、提取收率和生产成本后得出结论：采用游离细胞法生产Ｌ－Ａｌａ优于固定化细胞法。     

Prophylactic effect of vitamin E against hepatotoxicity,nephrotoxicity, haematological indices and histopathology induced by diazinon insecticide in mice

Considering that the involvement of reactive oxygen species(ROS)has been implicated in the toxicity of various pesticides,this study was designed to study the ameliorative effect of Vitamin E(100 mg/kg body weight)on mice(25-30 mg)treated with diazinon(32.5 or 16.25 mg/kg body weight)organophosphate insecticide for 14 days.Subchronic DZN exposure and the protective effects of vitamins E(vitE)were evaluated for their effects on haematological indices,the enzymes concerning liver damage [plasma alanine aminot...     

An electrophoretic mobility shift assay for the identification and kinetic analysis of acetyl transferase inhibitors

Histone acetyl transferases are important regulators of cellular homeostasis. This study describes a sensitive acetyl transferase electrophoretic mobility shift assay applicable both for kinetic analysis of acetyl transferase inhibitors and for high-throughput testing. Application of the assay for human GCN5L2 enabled dissection of inhibitor competition with respect to acetyl coenzyme A. Furthermore, we demonstrated that the assay can detect time-dependent inhibition of human GCN5L2 by reactive inhibitors.     

Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases

A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high K_M and low k_cat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.     

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that “free” circulating antibodies are the pharmacologically active form needed to determine the PK/ pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, “total” drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand /shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.     

Correlation of zona-binding with oocyte maturation and sperm motility in rhesus monkeys by hemizona assay

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PBI). The mean numbers of sperm bound to hemizona for PB1, PVS, GV, and MC groups were 132.9 ± 12.0, 71.5 ± 10.1, 36.1 ± 4.0, and 20.1 ± 2.9 (Mean ± SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 ± 2.2 and 25.3 ± 2.9 (Mean ± SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1) The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the “zona maturation” hypothesis. © 1994 Wiley-Liss, Inc.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

A simple,real-time assay of horseradish peroxidase using biolayer interferometry

ABSTRACT

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.     

Noninstrumented enzyme-linked immunosorbant assay for detection of early pregnancy in macaques

A practical, noninstrumented enzyme-linked immunosorbant assay (NELISA) for the measurement of urinary monkey chorionic gonadotropin (mCG) has been developed for the detection of early pregnancy in macaque monkeys for use in both the laboratory and the field. Five rhesus monkeys (Macaca mulatta) and six crab-eating monkeys (Macaca fascicularis) were tested for the presence of mCG in urine on gestational days (GDs) 12 to 35. The mCG NELISA detected pregnancy as early as GD 14, with an average earliest detection at GD 16.5 +/- 1.4 (n = 11). Out of 90 tests, 27 false-negative and zero false-positive tests were obtained, for an accuracy of 70.0%. Without the aid of a spectrophotometer, the presence of mCG in pregnant monkey samples was indicated by a dark green color change. Nonpregnant monkey urine samples, on the other hand, exhibited no color change. These findings suggest that the simple, economical, and reliable urinary mCG NELISA may be useful for diagnosing early pregnancy in these and related species. Because capture and restraint are unnecessary for collecting urine samples, the mCG NELISA has widespread potential for confined and free-ranging animals.