A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity.In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity.     

微孔板弓形虫染色试验

以细胞培养的方法传代弓形虫，建立微孔板弓形虫染色试验。血清与弓形虫虫体经孵育后，用碱性美蓝染色，计数100个虫体，记录染色与未染色虫体的比例，标本中50%虫体着色的稀释度为终点稀释度。弓形虫悬液浓度为10⁹/ml，辅助因子中含1%枸橼酸钠时，弓形虫染色效果最好，微孔板染色试验的稳定性良好。     

FAME24/20中两步法ELISA试剂工作表的优化设计

[目的]为了缩短FAME24／20全自动酶联免疫分析仪的工作时间，提高工作效率。[方法]在FAME用户软件中编制好实验步骤，指定洗液的洗板单元和试剂的分配单元，改进酶标板进板方式和顺序，对ELISA两步法试剂的工作表进行优化。[结果]编制出2个能在53min内完成15个酶标板进板的工作表。[结论]优化设计两步法ELISA试剂的工作表，可以充分利用FAME24／20的仪器资源，并有效提高其使用效率。     

广东地区TTV感染情况及基因分型

目的 调查广东地区ＴＴ病毒（ＴＴＶ）感染流行情况和基因分型。方法 选取ＴＴＶＯＲＦ１的保守序列作内外引物,Ⅰ型、Ⅱ型同源性极高的序列ｎｔ２４６４～ｎｔ２５２０作包被探针,异源性大于５０％的序列ｎｔ２１２５～ｎｔ２１５９作显色探针Ⅰ和显色探针Ⅱ,采用微板核酸杂交－ＥＬＩＳＡ技术对２８０例肝功能损害的肝炎患者血清进行ＴＴＶ检测及ＴＴＶ基因分型研究。结果 检测出４４例ＴＴＶ阳性患者,检出率为１５．７１％     

单双波长在生物活性测定中的比较研究

目的：通过比较研究,确定酶标仪单双波长哪个更适于生物制品活性测定实验。方法：样品通过多次实验单双波长扫描,比较得到的实验结果,进行统计分析。结果：选用双波长可以排除其他因素对结果的影响,实验结果的标准差和离散系数均较低。结论：单双波长法更适于生物活性测定实验,提高结果的准确度。     

Antibacterial activity of selected Cameroonian dietary spices ethno-medically used against strains of Mycobacterium tuberculosis

Ethnopharmacological relevance

Tuberculosis (TB) is considered as a re-emerging disease and one of the most important public health problems worldwide. The use or (in most cases) misuse of existint anti-tuberculosis drugs over the years has led to an increasing prevalence of resistant strains, establishing an urgent need to search for new effective agents. Spices are largely used ethno-medically across Africa.

Aim of the study

The present study aimed to evaluate the in vitro antimycobacterial activities of a total of 20 methanol crude extracts prepared from 20 Cameroonian dietary spices for their ability to inhibit the growth of or kill Mycobacterium tuberculosis strains H₃₇Rv (ATCC 27294) and H₃₇Ra (ATCC 25177).

Materials and methods

The antituberculosis screening was performed using the Microplate Alamar Blue Assay (MABA) method to determine the minimum inhibitory concentration (MIC) and the minimum mycobactericidal concentration (MBC).

Results

Fifteen (15) plant extracts out of 20 showed varied levels of antimycobacterial activity against the strains M. tuberculosis H₃₇Rv and H₃₇Ra, with MICs in the range of 2.048–0.016 mg/ml. The extract of Echinops giganteus exhibited the most significant activity with a MIC value of 32 μg/ml and 16 μg/ml, respectively against H₃₇Ra and H₃₇Rv. To the best of our knowledge, the antimycobacterial activity of the tested spices has not been reported before and therefore our results can be evaluated as the first report about the antimycobacterial properties.

Conclusions

The results of this study suggest that Echinops giganteus and Piper guineense could be important sources of bactericidal compounds against M. tuberculosis and could probably be promising candidates that can be further investigated.     

TTV基因分型的研究

目的调查华南地区 ＴＴＶ感染流行情况和基因分型。方法选取 ＴＴＶ开放读码枢１的保守序列作内外引物, Ｇ１、 Ｇ２、 Ｇ３、 Ｇ４、 Ｇ５、 Ｇ６同源性极高的系列 ２ １６０— ２ １９６ ｎｔ作包被探针,异源性大于 ２０％的序列作显色探针Ｇ１、Ｇ２、Ｇ３、Ｇ４、Ｇ５、Ｇ６,采用ＰＣＲ微板核酸杂交ＥＬＩＳＡ技术对３８０例肝功能损害的肝炎患者血清进行ＴＴＶ检测及ＴＴＶ基因分型研究。结果检测出６１例ＴＴＶ阳性患者,检出率为１６．０％。对６１例ＴＴＶ进行基因分型,Ｇ１型４４例,Ｇ２型５例,Ｇ１、Ｇ２混合型感染１０例,Ｇ３、Ｇ４各１例,尚未发现Ｇ５、Ｇ６型。慢性肝炎中ＴＴＶ感染者与急性肝炎中ＴＴＶ感染者比例两者相近。结论在华南地区的肝炎疾病中, ＴＴＶ有较高的感染率; ＴＴＶ存在四个基因型,主要为Ｇ１型,其次为Ｇ２型,尚未发现Ｇ５、Ｇ６型。     

面部多发展折的手术整复

目的：探讨面部多发骨折复位治疗的手术方法。方法：对53例不同类型的面部多发展折行发开复位坚固内固定。结果：患者的面部外观及功能均有较大改善。结论：面部多发骨折行切开复位坚固内固定技术治疗能效恢复面部的形态和功能。     

全自动化学发光免疫分析仪在梅毒螺旋体抗体检测中的临床应用

目的探讨CHEMCLIN(600型全自动化学发光免疫分析仪(下称CLIA法)在梅毒螺旋体抗体检测中的临床应用价值。方法通过测定CLIA法在梅毒螺旋体抗体检测中的精密度、携带污染现象、灵敏度、干扰试验及对比试验。并进行统计分析1484份临床血清标本用CLIA法、甲苯胺红不加热血清试验(TRUST)和梅毒螺旋体明胶凝集试验(TPPA)三种方法的平行检测的结果。结果 CHEMCLIN(600型系统对高、中、低度3个浓度梅毒螺旋体抗体阳性标本检测的重复性结果显示,其批内、批间的变异系数均低于10%。通过比较强阳性混合血清混合前后及阴性混合血清,系统吸样针无携带污染,差异无显著性(P0.05)。用CLIA法和TPPA法同时检测弱阳性标本,CLIA法1∶16稀释检测结果为阴性,TPPA法1∶4稀释检测为阴性。三酰甘油浓度高达20.0mmol/L、血红蛋白浓度高达30.0g/L、胆红素高达95mol/L时对测定结果的发光值影响度3.5%,且前后判断结果不变。比较CLIA法与进口仪器I2000两种方法 ,差异无统计学意义P0.05。TPPA法与CLIA法两种方法检测结果比较,差异也无显著性(P0.05)。CLIA的敏感度为98.4%,特异度为99.8%。结论 CHEMCLIN(600型全自动化学发光免疫分析仪系统作为国内新开发的梅毒血清学特异性抗体检测方法 ,其检测精密度较好,无携带污染现象,检测灵敏度高于TPPA法,抗干扰能力强。其敏感性和特异性都很高,自动化程度高,值得在临床推广应用。