Name: the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva
Uploaded: 2012-05-16T13:00:00
Description: Watch this Scientific Journal Video about The Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

研究是由哈利·温斯顿研究基金会的支持。

1。准备新鲜的工作方案<ol><li> TBS电视台：0.05 mol的Tris缓冲液，pH值7.8公司（TBS）。准备60毫升TBS /微孔板。 </li><li> 个人防护装备：猪胰弹性蛋白酶，1型（PPE）的暂停。摇匀，稀释约0.01 U / ml的个人防护装备在TBS。准备6毫升工作液/微孔板）。 </li><li> SA的3 NA：琥珀- L-丙氨酸-L-丙氨酸- L-丙氨酸-P-硝基苯胺（SA的3，NA）。准备在二甲基亚砜（DMSO...

<ol>
	<li>Friedlander, Y., Kark, J. D., Eisenberg, S., Stein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+LDL-cholesterol+from+total+cholesterol,+triglyceride+and+HDL-cholesterol:+a+comparison+of+methods+in+the+Jerusalem+Lipid+Research+Clinic+Prevalence+Study.">Calculation of LDL-cholesterol from total cholesterol, triglyceride and HDL-cholesterol: a comparison of methods in the Jerusalem Lipid Research Clinic Prevalence Study.</a> Isr. J. Med. Sci. 18, 1242-1252 (1982).</li><li>Lapidot, T., Petit, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+stem+cell+mobilization:+The+roles+of+chemokines,+proteolytic+enzymes,+adhesion+molecules,+cytokines,+and+stromal+cells.">Current understanding of stem cell mobilization: The roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells.</a> Exp. Hematol. 30, 973-981 (2002).</li><li>Bristow, C. L., Alfano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;1Antitrypsin+therapy+increases+CD4++lymphocytes+to+normal+values+in+HIV-1+patients.">&#945;1Antitrypsin therapy increases CD4+ lymphocytes to normal values in HIV-1 patients.</a> Soluble factors mediating innate immune responses to HIV infection. , (2010).</li><li>Bristow, C. L., Patel, H., Arnold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+antigen+prognostic+for+human+immunodeficiency+virus+disease+progression.">Self antigen prognostic for human immunodeficiency virus disease progression.</a> Clin. Diagn. Lab. Immunol. 8, 937-942 (2001).</li><li>Bristow, C. L., Babayeva, M. A., LaBrunda, M., Mullen, M. P., Winston, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;1Proteinase+Inhibitor+regulates+CD4++lymphocyte+levels+and+is+rate+limiting+in+HIV-1+disease.">&#945;1Proteinase Inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease.</a> PLoS One. , (2011).</li><li>de Almeida, P. D. V., Gregio, A. M. T., Machado, M. A. N., de Lima, A. A. S., Azevedo, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva+Composition+and+Functions:+A+Comprehensive+Review.">Saliva Composition and Functions: A Comprehensive Review.</a> J. Contemp. Dent. Pract. 9, 72-80 (2008).</li><li>Edgar, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva:+its+secretion,+composition+and+funcitons.">Saliva: its secretion, composition and funcitons.</a> Br. Dent. J. , 172-305 (1992).</li><li>Bristow, C. L., Meo, F. d. i., Arnold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+activity+of+&#945;1proteinase+inhibitor+and+++2macroglobulin+in+human+serum:+Application+to+insulin-dependent+diabetes+mellitus.">Specific activity of &#945;1proteinase inhibitor and 2macroglobulin in human serum: Application to insulin-dependent diabetes mellitus.</a> Clin. Immunol. Immunopathol. 89, 247-259 (1998).</li><li>Pederson, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+levels+of+&#945;2-macroglobulin,+&#945;1-antitrypsin,+C-reactive+protein,+cathepsin+G+and+elastase+in+humans+with+or+without+destructive+periodontal+disease.">Salivary levels of &#945;2-macroglobulin, &#945;1-antitrypsin, C-reactive protein, cathepsin G and elastase in humans with or without destructive periodontal disease.</a> Arch. Oral Biol. 40, 1151-1155 (1995).</li><li>Shak, S., Capon, D. J., Hellmiss, R., Marsters, S. A., Baker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+DNase+I+reduces+the+viscosity+of+cystic+fibrosis+sputum.">Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.</a> PNAS. 87, 9188-9192 (1990).</li><li>LeVeque, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicenter,+randomized,+double-blind,+placebo-controlled,+dose-+titration+study+of+oral+pilocarpine+for+treatment+of+radiation-induced+xerostomia+in+head+and+neck+cancer+patients.">A multicenter, randomized, double-blind, placebo-controlled, dose- titration study of oral pilocarpine for treatment of radiation-induced xerostomia in head and neck cancer patients.</a> J. Clin. Oncol. 11, 1124-1131 (1993).</li><li>Stevens, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+new+CD4+enumeration+technologies+for+resource-constrained+countries.">Evaluating new CD4 enumeration technologies for resource-constrained countries.</a> Nat. Rev. Microbiol. 6, S29-S38 (2008).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2010 ART guidelines for adults and adolescents - evidence map. , (2010).</li></ol>

未稀释的血清中含有的中位数为36μM的α1 PI和3.6微米α2巨球蛋白（α2米 ），10倍的差异8。这两种蛋白质的竞争结合个人防护装备。利用这两个特点，浓度和个人防护装备的亲和力，已发展出一种方法，是专门测量在正常血清中存在竞争α2米 8的α1有价证券。这种方法可以在0.3％血清检测低至50纳米μ1有价证券。在血清稀释度为2.5...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> sialogogue，0.05M柠檬酸</td><td> EDA的无糖糖果，4900后方N.20th街，费城，PA的19144 </td><td>无糖柠檬滴</td></tr><tr><td>猪胰弹性蛋白酶，1型（欧共体3.4.21.36） </td><td> Sigma-Aldrich公司，圣路易斯，密苏里州63103 </td><td> E1250 </td></tr><tr><td>琥珀 - L-丙氨酸-L-丙氨酸 - L-丙氨酸-P-硝基苯胺</td><td> Sigma-Aldrich公司</td><td> S4760 </td></tr><tr><td>考马斯亮蓝R-250 </td><td> Sigma-Aldrich公司</td><td> B7920 </td></tr><tr><td>组织培养处理，96孔板</td><td>碧迪，富兰克林湖，NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td>酶标仪405 nm和595 nm的过滤器</td><td> MTX的实验室系统，8456泰科路，裴lding研发，维也纳，弗吉尼亚州22182 </td><td> Dynex的P97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva

有负担得起的CD4枚举监测HIV疾病的迫切需要。的CD4枚举是在资源有限的地区，由于时间和温度的限制，技术复杂，试剂成本，尤其是单克隆抗体的血细胞，目前唯一可以接受的方法来衡量的CD4遥不可及。节约成本和节省时间的一种常用实验室的策略，而不是衡量某些血液值，是计算。例如，低密度脂蛋白水平计算，总胆固醇，高密度脂蛋白（HDL），甘油三酯1使用的测量水平。因此，确定无细胞的相关因素，直接调节的CD4 + T细胞可以提供一个准确的方法，计算CD4计数由于生理意义的相关因素。 进入血液，并注定要成为循环CD4 + T细胞的干细胞数量确定的趋化因子CXCL12的1ND及其受体CXCR4，由于他们对运动2的影响。此外，进入血液干细胞的运动过程调节细胞表面人类白细胞弹性蛋白酶（HLE CS）和HLE CS-反应活性α1蛋白酶抑制剂（α1有价证券，α1抗胰蛋白酶，SerpinA1）3。在HIV-1病毒病，α1 PI灭活由于疾病的进程4。 α1 PI在HIV-1疾病的早期症状类别，积极发现低于100％未经治疗的HIV-1患者（中位数= 12μM，以及达到正常水平，在对症4大类，5正常。格局已被归因于免疫灭活，不足够的合成，蛋白失活，或充氧。我们观察到，在HIV-1&gt; 220的CD4细胞/μl的科目，CD4细胞计数了积极的α1 PI水平呈正相关（R 2 = 0.93，P＆LT; 0.0001，N = 26）和无效的α1的PI（R 2 = 0.91，P &lt;0.0001，N = 26）5。 α1的PI HIV-1感染和未感染者的管理，导致在α1 PI参与调节的CD4 + T细胞，血液中的3中的CD4计数显着增加。 与刺激，全唾液中含有足够的浆液性渗出物（血浆含有蛋白质的物质通过血管壁进入唾液传递）允许测量活性α1 PI与此测量的相关性的证据表明，它是一种精确的方法计算CD4细胞计数。简言之，如嚼口香糖或柠檬酸sialogogues刺激到整个口腔唾液，血清渗出。刺激血清渗出后，血清α1 PI唾液中的活性是衡量其能力，抑制弹性蛋白酶的活性。猪胰弹性蛋白酶（PPE）的弹性蛋白酶是一个现成的廉价来源。个人防护装备的α1有价证券结合形成一个到一个复杂的，防止切割其特定的底物，其中之一是的比色法肽，琥珀- L-丙氨酸-L-丙氨酸- L-丙氨酸-P-硝基苯胺（SA的个人防护装备3不适用）。孵化器为在室温下10分钟的个人防护装备的饱和浓度的唾液，允许个人防护装备的所有活动α1 PI唾液中的约束力。可以衡量的PPE抑制活性α1有价证券加入的PPE基板SA的3不适用。（图1）。虽然CD4细胞计数测量血容量（CD4细胞/μL），α1 PI唾液中的浓度与唾液中的血清浓度，唾液量，因为体积可以改变白天和人大大6人。然而，几乎所有的唾液中的蛋白质是由于血清含量，唾液中的蛋白质含量是测量surable 7。因此，活性α1 PI唾液中的唾液蛋白质含量的比率计算，被称为α1 PI指数。结果提出此表明，α1 PI指数提供了一个准确和精确的计算CD4细胞计数的生理方法。

Watch this Scientific Journal Video about The Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

被描述的CD4枚举法，α测试，它采用全唾液，以提供快速，准确的CD4细胞计数。 α-测试成本便士和消除技术培训，如昂贵的试剂，单克隆抗体，仪器仪表，制冷，样品运输，以及收集和处理血液的需要。

测试：α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Research

JoVE Journal

Medicine

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

酸化浸渍法评价全肾肾素数

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

使用台式流变仪快速表征气道粘液的粘弹性

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Whole-Kidney Three-Dimensional Staining with CUBIC

用立方体进行全肾三维染色

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood ...