石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应

目的 观察石蒜碱（LYC）对脂多糖（LPS）诱导的原代小胶质细胞炎症反应和表型变化的影响,并探讨其作用机制。方法 将培养的原代小胶质细胞分为对照组、LYC组（0.1、0.5、1和5 μmol/L LYC）、LPS组（0.1 mg/L LPS）和LPS（0.1 mg/L LPS）+LYC组（5 μmol/L LYC）。倒置显微镜观察细胞形态变化;流式细胞术检测原代小胶质细胞的纯度及LPS诱导原代小胶质细胞向两种表型转化的情况;CCK-8法检测细胞活性;实时荧光定量PCR（qPCR）检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）以及不同表型小胶质细胞表面标志物mRNA表达;Griess法检测一氧化氮（NO）含量;免疫印迹法检测细胞TLR4、p-NF-κB p65蛋白表达情况。结果 原代小胶质细胞纯度达95%以上。对照组及各浓度LYC组细胞活性差异无统计学意义。后续实验以5 μmol/L的LYC为药物浓度。与LPS组相比,LPS+LYC组的阿米巴样小胶质细胞数量明显减少,IL-1β、IL-6、TNF-α mRNA表达水平以及NO含量降低,CD86 mRNA表达水平和表型比例降低,CD206 mRNA表达水平和表型比例升高,TLR4和p-NF-κB p65蛋白表达水平降低（P<0.01）。结论 LYC通过TLR4/NF-κB信号通路抑制LPS诱导的原代小胶质细胞炎症反应,并促进其向“抑炎型”极化。     

白藜芦醇通过BRD4调控Wnt/β-catenin通路逆转胶质瘤细胞替莫唑胺耐药的机制研究

目的 探究白藜芦醇（RES）逆转胶质瘤细胞替莫唑胺（TMZ）耐药的作用是否与通过溴结合域蛋白4（BRD4）调控Wnt/β-链蛋白（β-catenin）通路有关。方法 取人神经胶质瘤TMZ低敏细胞株（U138）、高敏细胞株（U251）、耐药株（T98G）,Western blot法检测3种细胞株中BRD4、Wnt3a、β-catenin、TMZ耐药蛋白（MGMT）蛋白表达。取T98G细胞株分为对照1组（添加100 μmoL/L TMZ）、RES1组（添加50 μmoL/L RES）、RES+TMZ（添加100 μmoL/L TMZ和50 μmoL/L RES）组,用CCK-8法、流式细胞术检测各组细胞增殖、凋亡情况;Western blot法检测各组BRD4、Wnt3a、β-catenin、MGMT蛋白表达。为分析BRD4过表达对TMZ耐药性的影响,在添加100 μmoL/L TMZ的基础上,转染BRD4过表达质粒（pcDNA BRD4）或加入50 μmoL/L RES分别作为pcDNA NC组、pcDNA BRD4组、RES2组、RES+pcDNA BRD4组。为验证BRD4对Wnt3a/β-catenin通路的调控作用,在添加100 μmoL/L TMZ的基础上,加入BRD4抑制剂JQ1和Wnt3a/β-catenin通路激活剂LiCl,分为对照2组、JQ1组、JQ1+LiCl组。将T98G细胞接种于裸鼠左肩胛区,给予RES、TMZ和（或）JQ1治疗,检测瘤体中Ki67,BRD4、MGMT及Wnt3a/β-catenin蛋白表达。结果 与U251细胞相比,U138、T98G中BRD4、Wnt3a、β-catenin及MGMT表达均依次升高（P<0.05）。与对照1组相比,RES干预可抑制T98G细胞BRD4、Wnt3a/β-catenin、MGMT蛋白表达及增殖,促进凋亡,逆转细胞的耐药性（P<0.05）。pcDNA BRD4可逆转RES的抗增殖、促凋亡等上述作用。BRD4抑制剂JQ1可抑制T98G细胞BRD4、Wnt3a/β-catenin、MGMT蛋白表达及增殖,促进凋亡（P<0.05）;LiCl可逆转JQ1的抗增殖、促凋亡作用。RES单独或与JQ1联合治疗,可激活TMZ对瘤体内Wnt3a/β-catenin通路、MGMT表达和细胞增殖的抑制作用（P<0.05）。结论 RES可能通过下调BRD4,进而抑制Wnt3a/β-catenin通路活化,实现对胶质瘤T98G细胞TMZ耐药性的逆转。     

γ-分泌酶抑制剂在肺纤维化上皮间质转化中的作用

目的 检测体外转化生长因子（TGF）-β1诱导肺纤维化上皮间质转化（EMT）的形成情况并检测Notch信号特异性抑制剂γ-分泌酶抑制剂（DAPT）对其影响及可能机制。方法 将A549细胞分成对照组（RPMI 1640完全培养基培养）、TGF-β1组（在含有10 μg/L TGF-β1的RPMI 1640培养基中培养）、TGF-β1+DAPT组（在含有10 μg/L TGF-β1和2 μmol DAPT的RPMI 1640培养基中培养）、DAPT组（在含有2 μmol DAPT的RPMI 1640培养基中培养）。通过倒置显微镜观察细胞形态,实时荧光定量逆转录聚合酶链反应检测肺泡上皮细胞特异性蛋白E-钙黏合素以及间质细胞特异性蛋白α-肌动蛋白（α-SMA）mRNA相对表达水平,Western blot检测E-钙黏合素以及α-SMA蛋白表达水平。结果 倒置显微镜检查示,对照组细胞呈多边形,铺路石样,细胞间连接紧密;TGF-β1组细胞呈梭行,纺锤状,细胞间连接减少;TGF-β1+DAPT组仅有少量细胞呈梭形,多数细胞形态与对照组相似;DAPT组细胞形态大致同对照组。与对照组相比,TGF-β1组α-SMA蛋白及mRNA表达增加,而E-钙黏合素蛋白及mRNA表达减弱（P<0.05）;与TGF-β1组比较,TGF-β1+DAPT组α-SMA蛋白及mRNA表达水平降低,而E-钙黏合素蛋白及mRNA表达水平增加（P<0.05）;DAPT组与对照组2指标的蛋白与mRNA相对表达水平差异无统计学意义（P>0.05）。结论 TGF-β1可诱导肺上皮间质转化,而Notch信号抑制剂DAPT能够阻断、部分或全部逆转这一过程。     

内毒素血症通过下调HNF4α表达加剧肝损伤

目的 探讨内毒素血症下调肝细胞核因子4α（HNF4α）表达介导肝损伤进展的机制。方法 144只BALB/c小鼠采用随机数字表法分组进行以下实验：（1）四氯化碳（CCl₄）诱导小鼠急性肝损伤。对照组和0.5 mL/kg、1.0 mL/kg、2.0 mL/kg CCl₄组。（2）筛选脂多糖（LPS）干预小鼠的剂量。对照组和0.1 mg/kg、0.5 mg/kg、2.5 mg/kg LPS组。（3）LPS干预CCl₄（1.0 mL/kg）诱导急性肝损伤模型小鼠。对照组、CCl₄组、0.1 mg/kg LPS+CCl₄组、0.5 mg/kg LPS+CCl₄组。每组12只。诱导24 h后处死小鼠,采用酶速率法检测血清丙氨酸转氨酶（ALT）,重氮法检测总胆红素（TBil）水平;Western blot法检测肝组织HNF4α、胱天蛋白酶3剪切体（Cleaved caspase-3）蛋白表达;原位末端标记法检测肝细胞凋亡情况。结果 0.5、1.0、2.0 mL/kg CCl₄组的血清ALT、TBil及肝组织HNF4α、Cleaved caspase-3蛋白表达水平高于对照组,且呈剂量依赖性增高。2.5 mg/kg LPS组血清ALT、TBil及肝组织Cleaved caspase-3蛋白表达水平高于对照组和0.1、0.5 mg/kg LPS组,肝组织HNF4α蛋白表达水平低于对照组和0.1、0.5 mg/kg LPS组（P<0.05）。CCl₄组和0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织HNF4α、Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数均高于对照组（P<0.05）;0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数高于CCl₄组,HNF4α蛋白表达水平低于CCl₄组（P<0.05）。结论 内毒素血症通过下调HNF4α表达增加肝细胞凋亡,可能是其介导肝损伤进展的机制之一。     

循环酶法测定血清同型半胱氨酸试剂盒的评价

目的建立直接简便测定血清同型半胱氨酸(Hcy)的酶循环法。方法在日立7170A全自动生化分析仪上,采用两点速率法,通过两点线性校准,直接测定血清同型半胱氨酸。结果批内不精密度为2.21%~4.98%,批间不精密度为2.61%~6.73%;回收率为97.3%~104.9%;线性范围为1.5~50μmol/L;干扰实验说明加入的干扰物无明显干扰。结论在全自动生化分析仪上以酶循环法测定血清同型半胱氨酸操作简便、灵敏度高、能快速得到检测结果,适用于常规生化分析。     

吡非尼酮通过miRNA-425-5p调控TGF-β/Smad通路对大鼠心肌纤维化的影响

目的 探讨吡非尼酮通过调控miRNA-425-5p及转化生长因子（TGF）-β/Smad通路改善心肌梗死大鼠心肌纤维化程度的作用。方法 将60只雄性SD大鼠分为3组：假手术组（不结扎冠状动脉）、模型组、吡非尼酮组（吡非尼酮0.3 g/kg灌胃）。造模4周后采用心脏多普勒超声评价各组大鼠心脏功能; 酶联免疫吸附试验检测血浆中白细胞介素6（IL-6）、Ⅰ型胶原蛋白α2（COL1α2）、Ⅲ型胶原蛋白α1（COL3α1）的水平;采用Masson染色法评价大鼠心肌纤维化程度,使用Image-Pro Plus 6.0分析各组心肌胶原容积分数;Western blot检测心肌组织中TGF-β1、Smad2、Smad3蛋白表达水平;实时荧光定量PCR（qPCR）检测心肌组织中TGF-β1和miRNA-425-5p表达水平。同时在体外分离、培养乳鼠心肌成纤维细胞,分为对照组（不加任何物质）、miRNA-425-5p mimic组（加入转染miRNA-425-5p mimic）、吡非尼酮组（加入1.5 g/L的吡非尼酮）。采用qPCR检测各组miRNA-425-5p和TGF-β1 mRNA表达水平。结果 模型组大鼠与假手术组相比心脏左心室舒张末期内径（LVEDd）、左心室收缩末期内径（LVESd）增大,左心室射血分数（LVEF）、左心室短轴缩短率（FS）降低（P<0.05）;Masson染色及定量分析结果显示心肌纤维化加重;炎性因子IL-6、COL3α1、COL1α2水平明显升高（P<0.05）;心肌组织TGF-β1、Smad2、Smad3蛋白表达升高,miRNA-425-5p表达水平降低（均P<0.05）。吡非尼酮组与模型组相比,LVEDd、LVESd缩小,LVEF、FS升高（P<0.05）,Masson染色及定量分析结果示心肌纤维化程度减轻,血浆炎性因子IL-6、COL3α1、COL1α2水平明显降低（P<0.05）;心肌组织TGF-β1、Smad2、Smad3蛋白表达水平降低,miRNA-425-5p表达水平升高（均P<0.05）。体外细胞实验结果表明,miRNA-425-5p mimic组与对照组相比,miRNA-425-5p表达水平升高,TGF-β1 mRNA表达水平降低（P<0.05）。吡非尼酮组与对照组相比,miRNA-425-5p表达水平升高,TGF-β1 mRNA表达水平降低（P<0.05）。结论 吡非尼酮可通过调节miRNA-425-5p表达水平调控TGF-β/Smad信号通路,减轻心肌纤维化,改善心肌梗死后心力衰竭大鼠心脏功能。     

细菌性肺炎患儿血清降钙素原水平变化及临床意义

目的 观察细菌性肺炎患儿血清降钙素原（PCT）水平变化及临床意义.方法 31例肺炎患儿,在抗生素治疗前后均检测PCT、hs-CRP、WBC水平,以观察其变化情况.结果 31例患儿治疗前PCT均升高.患儿治愈后,PCT正常28例,但其中WBC升高者仍有10例（35.7％）,hs-CRP升高者仍有4例（14.3％）.另外3例PCT分别由入院时的5.69 ng/mL、1.25 ng/mL及0.83 ng/mL,分别下降为0.51 ng/mL、0.55 ng/mL及0.52 ng/mL,此3例WBC及hs-CRP升高各1例,但是WBC由13.1×10^9/L下降为12.1×10^9/L、hs-CRP由20.92 mg/L下降为7.38 mg/L.结论 细菌性肺炎患儿血清急性时相指标中PCT敏感性最高,可指导抗生素的使用.     

胃蛋白酶原光激化学发光免疫测定方法的建立

目的建立一种检测人血清胃蛋白酶原浓度的光激化学发光免疫测定方法。方法采用双抗体夹心法建立检测人血清中胃蛋白酶原Ⅰ和胃蛋白酶原Ⅱ浓度的方法,评估其分析灵敏度、回收率和批内精密度,并与雅培(化学发光法)进行比较。结果胃蛋白酶原Ⅰ/Ⅱ的分析灵敏度分别为0.8ng/mL和0.6ng/mL,回收率分别为100.3%、106.5%,批内变异系数(CV)为0.93%~4.1%、1.2%~4.3%,与化学发光法的相关性较好(r=0.99)。结论该方法测定胃蛋白酶原具有较高灵敏度、精密度和准确性,适用于临床。方法建立后可进一步进行长期稳定性试验。     

血清肌酐可见光匀相速率法检测技术的建立

目的建立快速、灵敏的血清肌酐匀相速率法检测新技术,为新试剂盒的研制奠定基础。方法本项目凭借血清小分子物质捕获技术、液体酶稳定技术等,建立了适用于生化自动分析法的可见光匀相速率法,采用经典的方法学评价体系,对血清肌酐匀相速率法检测体系进行评估,并将建立的检测方法与酶法的检测结果进行比较。结果可见光匀相速率法测定血清肌酐,线性范围达3360μmol/L,平均回收率为100.8%,批内变异系数（CV）和批间变异系数分别为0.0071～0.0204和0.0086～0.0316,与酶法检测结果进行线性回归分析,线性回归方程和相关系数分别为Y=1.01X-0.12、r=0.9945,健康人群的血清肌酐为57～129μmol/L（男性）、41～105μmol/L（女性）。结论本研究所建立的测定血清肌酐含量的可见光匀相速率法,各项方法学指标满足临床实际应用需要,具有选择性好、灵敏度高、测定简便、抗干扰能力强等优点,可满足临床实验室需要,适合临床推广应用。     

血清同型半胱氨酸和高敏C反应蛋白在不稳定型心绞痛危险度分层中的作用

目的探讨同型半胱氨酸（homocysteine,Hcy）和高敏C反应蛋白（hypersensitive C-reactive protein,hs-CRP）在不稳定型心绞痛（unstable angina pectoris,UAP）危险度分层中的作用。方法（1）按照中华医学会心血管病学分会《不稳定型心绞痛和非ST段抬高心肌梗死诊断与治疗指南》中的危险性分层将102例UAP患者分为低、中、高3个危险组;（2）测定102例UAP、20例稳定型心绞痛（stable angina pectoris,SAP）及46例对照者的血清Hcy和hs-CRP的含量,分别比较其在3组中的差异。结果（1）UAP组血清Hcy[（21±9）ng/L]、[hs-CRP（12.2±7.3）mg/L]水平明显高于SAP组及对照组（P〈0.05,P〈0.01）;（2）UAP患者低、中、高危组血清Hcy浓度分别为（17±6）ng/L、（21±8）ng/L、（25±10）ng/L,后2组与前者比较差异有统计学意义（P〈0.05）;（3）UAP患者低、中、高危组血清hs-CRP浓度分别为（9±5）mg/L、（12±7）mg/L、（16±8）mg/L,后2组与前者比较差异有统计学意义（P〈0.05）。结论UAP患者血清Hcy、hs-CRP水平明显升高,且随危险度分层的升高而升高,可作为UAP患者危险度分层的重要指标。     

流式微球分析技术联合检测血清MMP-9、MPO CD40L和t-PA的方法学建立及评价

目的 建立流式微球分析技术联合检测人血清中基质金属蛋白酶-9 (MMP-9)、髓过氧化物酶(MPO)、CD40配体(CD40L)、血浆纤溶酶原激活物(t-PA)的方法并对其进行系统性评价.方法 分别将四种选择好一定浓度的捕获抗体(MMP-9、MPO、CD40L和t-PA鼠抗人单克隆抗体)包被在四种已激活的不同荧光强度编码的羧基化聚苯乙烯微球上,用棋盘法对试验条件进行优化选择,并应用CLSI的有关规则进行方法学评价.结果 反应体系中四种鼠抗人单克隆抗体最佳加入量为10 μg,最佳生物素标记抗体浓度为1∶4000倍稀释,最佳反应时间为2h,亲和素最适孵育时间为1h.MMP-9线性范围是0.20～333.33 ng/mL,批内变异系数为4.60％～8.80％,批间变异系数为8.80％～9.60％,准确度相对偏倚为2.80％～3.18％,回收率是94.8～102.50％,灵敏度为0.20 ng/mL;MPO线性范围是0.26～111.11ng/mL,批内变异系数为5.90％～7.70％,批间变异系数为9.10％～11.40％,准确度相对偏倚为1.44％～4.12％,回收率是97.8～103.2％,灵敏度为0.26 ng/mL;CD40L线性范围是0.32～111.11g/mL,批内变异系数为5.40％～6.50％,批间变异系数为8.90％～12.40％,准确度相对偏倚为2.72％～5.95％,回收率是97.20～105.30％,灵敏度为0.32 ng/mL;t-PA线性范围是1.21～111.11 ng/mL,批内变异系数为2.20％～2.90％,批间变异系数为6.30％～12.20％,准确度相对偏倚为1.23％～4.60％,回收率是97.20～101.30％,灵敏度为1.21 ng/mL.高浓度的甘油三酯、胆固醇和胆红素对四种因子有一定的干扰率,低浓度的甘油三酯、胆固醇和胆红素对三种因子干扰较小.分别与ELISA方法比较无显著差异.结论 自建的MMP-9、MPO、CD40L和t-PA1流式微球联合检测技术,可拓展流式细胞分析技术,值得临床推广使用.     

Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway

Aim:

To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.

Methods:

Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.

Results:

Plumbagin (2.5–40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC₅₀ value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5–20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.

Conclusion:

Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.     

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.     

Quantification of brodifacoum in plasma and liver tissue by HPLC

A simple high-performance liquid chromatographic method has been developed for detection and quantification of brodifacoum in plasma and liver tissue. After adding difenacoum as the internal standard, brodifacoum and difenacoum are extracted from 2 mL of plasma with two sequential 10-mL volumes of acetonitrile-ethyl ether (9:1) and from 2 g of liver tissue by grinding the tissue with 10 mL acetonitrile. The extracts are evaporated to dryness under nitrogen, 2 mL of acetonitrile is added to reconstitute the residues, and the resulting solution is analyzed using reversed-phase chromatography and fluorescence detection. The limits of detection for plasma and tissue are 2 micrograms/L and 5 ng/g, respectively. Using internal standardization, the mean intra-assay recovery from plasma is 92% and the mean inter-assay recoveries is 109%. The mean intra-assay and inter-assay recoveries from tissue are 96%. No interferences were observed with any of the following related compounds: brodifacoum, bromadiolone, coumarin, difenacoum, diphacinone, warfarin, and vitamin K1.     

Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-α (TNF-α) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-α induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-α dose-dependent (5~100 ng/ml). TNF-α-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-α-induced COX-2 expression relative to that seen in the control cells (Adβgal). Pretreatment with 10 µM of SB203580 suppressed TNF-α-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-α, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.     

A sensitive indirect competitive enzyme-linked immunosorbent assay for the detection of sarsasapogenin in rat plasma

Aim:

To generate a polyclonal antibody against sarsasapogenin and to develop an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) method for the pharmacokinetic study of Sarsasapogenin in rats.

Methods:

The antigen of sarsasapogenin was produced using an active ester method and subsequently used for raising polyclonal antibodies in rabbits. The specificity and sensitivity of the antibody were measured by IC-ELISA. Using the ELISA method, sarsasapogenin levels were measured in the serum of rats after an oral dose of 100 mg/kg.

Results:

Polyclonal antibodies raised against sarsasapogenin-bovine serum albumin were generated and showed a high reactivity to sarsasapogenin. The antibodies exhibited minor cross-reactivity to ruscogenin (23%), diosgenin (22%), 25 (R, S) ruscogenin l-O-[β-D-glucopyranosyl (1→2)][β-D-xylopyranosyl (1→3)]-β-D-fucopyranoside (26%) and no cross-reactivity to diammonium glycyrrhizinate and notoginseng R1. The detection range of sarsasapogenin by this ELISA method was approximately 2.4−760 ng/mL. The recovery rates of 10 ng/mL, 100 ng/mL, and 500 ng/mL were in the range of 91.0%−96.2% for intra-assay and 89.0%–92.0% for inter-assay. The coefficients of variation (CV%) for intra- and inter-assays at the three different sarsasapogenin levels were 3.1%–8.3% (n=6) and 6.0%-14.1% (n=6), respectively.

Conclusion:

The IC-ELISA method is a sensitive test for the determination of sarsasapogenin concentration in rat plasma and for pharmacokinetic (PK) studies.     

时间分辨荧光免疫分析法检测游离F-βHCG的方法学评价

目的对时间分辨荧光免疫分析法(TRFIA)定量检测F-βHCG的各种性能指标进行方法学评价,验证其临床适用性。方法通过灵敏度、批内和批间精密度、标准曲线稳定性参数等指标评价TRFIA检测F-βHCG的性能,同时通过回收试验分析其准确度。结果 F-βHCG的最小测定值为0.09ng/ml,低、中、高3种质控品批内和批间变异系数均小于10%,标准曲线稳定性参数各变异系数小于5%,回收试验结果在97%～104.7%之间。结论 TRFIA法检测F-βHCG具有准确性好、灵敏度高、特异性强、能自动化分析等优点,能充分满足临床检测的需求。     

Development and Advanced Validation of an Optimized Method for the Quantitation of Aβ42 in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) biomarkers have been extensively utilized in the diagnosis of Alzheimer’s disease (AD) and characterization of progression. One important CSF biomarker is the amyloid beta 42 (Aβ₄₂) peptide, a key player in AD pathogenesis. The INNOTEST® Aβ₄₂ ELISA kit has been widely used but an advanced level of method development and validation has not been reported. To support a clinical trial in AD, we successfully completed a Good Laboratory Practices (GLP)-level validation of the method to establish the parameters of precision, accuracy, parallelism, selectivity, specificity, and linearity of dilution of the assay in CSF matrix, as well as CSF storage stability. Several modifications were required to optimize the assay and ensure consistent results in a clinical-trial setting. These included the use of additional calibrators, an adjusted standard curve range, a minimum required dilution (MRD) of CSF by 6-fold to avoid matrix interference and mitigation of analyte adsorption to labware by the addition of Tween-20. The optimized method displayed a quantitative range of 375–4,500 pg/mL. The inter-assay precision was ≤12.1 % CV and the inter-assay relative accuracy was ≤10.9 % absolute bias, bringing the total error of the assay to ≤23 %. The intra-assay precision of the assay at the high validation standard and below was ≤5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTEST® Aβ₄₂ ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-012-9360-7) contains supplementary material, which is available to authorized users.Key words: AB42, Alzheimer, biomarker, enzyme-linked immunosorbent assay, method validation