Name: a miniaturized glycan microarray assay for assessing avidity and specificity of influenza a virus hemagglutinins
Uploaded: 2016-05-29T14:00:00
Description: Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

这项工作由斯克里普斯芯片核心设施，并从疾病控制中心（JCP）合同的一部分得到了支持。 RPdV是来自荷兰科学研究组织（NWO）一个人Rubicon和VENI授权的收件人。由NIGMS授予GM62116（JCP）资助的财团为功能糖组学（http://www.functionalglycomics.org/）提供在这项研究中使用的几个聚糖。这是来自斯克里普斯研究所的出版物29113。

注：所有步骤都在室温下进行，除非另有说明。 1.阵列建设<ol><li>多糖制备及板设置<ol><li>准备打印缓冲区的股票。首先使500毫升磷酸氢钠的150mM的储备液。也使50毫升磷酸二氢钠溶液的150毫原液。在烧瓶中，使用磁力搅拌棒和pH计，直到达到pH为8.5缓慢滴定与一元溶液的磷酸氢钠溶液中。添加吐温-20〜0.005％。 </li><li>准备聚糖样品。稀释PAA结合聚糖（diLacNAc（Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA）?...

<ol>
	<li>Tumpey, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-amino+acid+change+in+the+hemagglutinin+of+the+1918+influenza+virus+abolishes+transmission.">A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission.</a> Science. 315 (5812), 655-659 (2007).</li><li>Xu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+balance+of+the+hemagglutinin+and+neuraminidase+activities+accompanies+the+emergence+of+the+2009+H1N1+influenza+pandemic.">Functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 H1N1 influenza pandemic.</a> J Virol. 86 (17), 9221-9232 (2012).</li><li>Bouvier, N. M., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+influenza+viruses.">The biology of influenza viruses.</a> Vaccine. 26, D49-D53 (2008).</li><li>Freidl, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+at+the+animal-human+interface:+a+review+of+the+literature+for+virological+evidence+of+human+infection+with+swine+or+avian+influenza+viruses+other+than+A(H5N1).">Influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1).</a> Euro Surveill. 19 (18), (2014).</li><li>Xu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+recognition+of+avian-like+receptors+in+human+influenza+A+H7N9+viruses.">Preferential recognition of avian-like receptors in human influenza A H7N9 viruses.</a> Science. 342 (6163), 1230-1235 (2013).</li><li>Paulson, J. C., de Vries, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H5N1+receptor+specificity+as+a+factor+in+pandemic+risk.">H5N1 receptor specificity as a factor in pandemic risk.</a> Virus Res. 178 (1), 99-113 (2013).</li><li>Tzarum, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+receptor+binding+of+the+hemagglutinin+from+a+human+H6N1+influenza+virus.">Structure and receptor binding of the hemagglutinin from a human H6N1 influenza virus.</a> Cell Host Microbe. 17 (3), 369-376 (2015).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human-Infecting+H10N8+Influenza+Virus+Retains+a+Strong+Preference+for+Avian-type+Receptors.">A Human-Infecting H10N8 Influenza Virus Retains a Strong Preference for Avian-type Receptors.</a> Cell Host Microbe. 17 (3), 377-384 (2015).</li><li>Carroll, S. M., Higa, H. H., Paulson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+cell-surface+receptor+determinants+of+antigenically+similar+influenza+virus+hemagglutinins.">Different cell-surface receptor determinants of antigenically similar influenza virus hemagglutinins.</a> J Biol Chem. 256 (16), 8357-8363 (1981).</li><li>Gambaryan, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+receptor-binding+phenotypes+of+influenza+virus+isolates+from+different+hosts+using+synthetic+sialylglycopolymers:+non-egg-adapted+human+H1+and+H3+influenza+A+and+influenza+B+viruses+share+a+common+high+binding+affinity+for+6'-sialyl(N-acetyllactosamine).">Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine).</a> Virology. 232 (2), 345-350 (1997).</li><li>Rogers, G. N., D'Souza, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+properties+of+human+and+animal+H1+influenza+virus+isolates.">Receptor binding properties of human and animal H1 influenza virus isolates.</a> Virology. 173 (1), 317-322 (1989).</li><li>Rogers, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+amino+acid+substitutions+in+influenza+haemagglutinin+change+receptor+binding+specificity.">Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity.</a> Nature. 304 (5921), 76-78 (1983).</li><li>Paulson, J. C., Rogers, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resialylated+erythrocytes+for+assessment+of+the+specificity+of+sialyloligosaccharide+binding+proteins.">Resialylated erythrocytes for assessment of the specificity of sialyloligosaccharide binding proteins.</a> Methods Enzymol. 138, 162-168 (1987).</li><li>Paulson, J. C., Sadler, J. E., Hill, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+specific+myxovirus+receptors+to+asialoerythrocytes+by+incorporation+of+sialic+acid+with+pure+sialyltransferases.">Restoration of specific myxovirus receptors to asialoerythrocytes by incorporation of sialic acid with pure sialyltransferases.</a> J Biol Chem. 254 (6), 2120-2124 (1979).</li><li>Chutinimitkul, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+attachment+pattern+and+replication+efficiency+of+H5N1+influenza+A+viruses+with+altered+receptor+specificity.">In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity.</a> J Virol. 84 (13), 6825-6833 (2010).</li><li>Glaser, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+amino+acid+substitution+in+1918+influenza+virus+hemagglutinin+changes+receptor+binding+specificity.">A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity.</a> J Virol. 79 (17), 11533-11536 (2005).</li><li>Herfst, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+transmission+of+influenza+A/H5N1+virus+between+ferrets.">Airborne transmission of influenza A/H5N1 virus between ferrets.</a> Science. 336 (6088), 1534-1541 (2012).</li><li>Nobusawa, E., Ishihara, H., Morishita, T., Sato, K., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+in+receptor-binding+specificity+of+recent+human+influenza+A+viruses+(H3N2):+a+single+amino+acid+change+in+hemagglutinin+altered+its+recognition+of+sialyloligosaccharides.">Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides.</a> Virology. 278 (2), 587-596 (2000).</li><li>Gambaryan, A. S., Matrosovich, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+solid-phase+enzyme-linked+assay+for+influenza+virus+receptor-binding+activity.">A solid-phase enzyme-linked assay for influenza virus receptor-binding activity.</a> J Virol Methods. 39 (1-2), 111-123 (1992).</li><li>Totani, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoenzymatic+synthesis+and+application+of+glycopolymers+containing+multivalent+sialyloligosaccharides+with+a+poly(L-glutamic+acid)+backbone+for+inhibition+of+infection+by+influenza+viruses.">Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses.</a> Glycobiology. 13 (5), 315-326 (2003).</li><li>Gambaryan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+specificity+of+influenza+viruses+from+birds+and+mammals:+new+data+on+involvement+of+the+inner+fragments+of+the+carbohydrate+chain.">Receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain.</a> Virology. 334 (2), 276-283 (2005).</li><li>Ito, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+specificity+of+influenza+A+viruses+correlates+with+the+agglutination+of+erythrocytes+from+different+animal+species.">Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species.</a> Virology. 227 (2), 493-499 (1997).</li><li>Watanabe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+human-type+receptor+binding+specificity+by+new+H5N1+influenza+virus+sublineages+during+their+emergence+in+birds+in+Egypt.">Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.</a> PLoS Pathog. 7 (5), e1002068 (2011).</li><li>Chandrasekaran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+topology+determines+human+adaptation+of+avian+H5N1+virus+hemagglutinin.">Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin.</a> Nat Biotechnol. 26 (1), 107-113 (2008).</li><li>Blixt, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printed+covalent+glycan+array+for+ligand+profiling+of+diverse+glycan+binding+proteins.">Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.</a> Proc Natl Acad Sci U S A. 101 (49), 17033-17038 (2004).</li><li>Childs, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-binding+specificity+of+pandemic+influenza+A+(H1N1)+2009+virus+determined+by+carbohydrate+microarray.">Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray.</a> Nat Biotechnol. 27 (9), 797-799 (2009).</li><li>Nycholat, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+Sialylated+Poly-N-acetyllactosamine+Chains+on+N-+and+O-Linked+Glycans+by+Human+and+Avian+Influenza+A+Virus+Hemagglutinins.">Recognition of Sialylated Poly-N-acetyllactosamine Chains on N- and O-Linked Glycans by Human and Avian Influenza A Virus Hemagglutinins.</a> Angew Chem Int Ed Engl. , (2012).</li><li>Rillahan, C. D., Paulson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+microarrays+for+decoding+the+glycome.">Glycan microarrays for decoding the glycome.</a> Annu Rev Biochem. 80, 797-823 (2011).</li><li>Song, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sialylated+glycan+microarray+reveals+novel+interactions+of+modified+sialic+acids+with+proteins+and+viruses.">A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses.</a> J Biol Chem. 286 (36), 31610-31622 (2011).</li><li>Gulati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+H3N2+Influenza+Viruses+Isolated+from+1968+To+2012+Show+Varying+Preference+for+Receptor+Substructures+with+No+Apparent+Consequences+for+Disease+or+Spread.">Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread.</a> PLoS One. 8 (6), (2013).</li><li>Stevens, J., Blixt, O., Paulson, J. C., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+microarray+technologies:+tools+to+survey+host+specificity+of+influenza+viruses.">Glycan microarray technologies: tools to survey host specificity of influenza viruses.</a> Nat Rev Microbiol. 4 (11), 857-864 (2006).</li><li>de Vries, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+hemagglutinin+protein+of+the+new+pandemic+H1N1+influenza+virus:+maintaining+optimal+receptor+binding+by+compensatory+substitutions.">Evolution of the hemagglutinin protein of the new pandemic H1N1 influenza virus: maintaining optimal receptor binding by compensatory substitutions.</a> J Virol. 87 (24), 13868-13877 (2013).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+receptor+binding+preferences+of+recombinant+human+A(H3N2)+virus+hemagglutinins.">Structure and receptor binding preferences of recombinant human A(H3N2) virus hemagglutinins.</a> Virology. 477, 18-31 (2015).</li><li>de Vries, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influenza+A+virus+hemagglutinin+glycosylation+state+affects+receptor-binding+specificity.">The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity.</a> Virology. 403 (1), 17-25 (2010).</li></ol>

评估IAV的受体特异性在分析禽流感病毒大流行潜力的重要一步。由病毒唾液酸识别被链接到几个生物性能如从细胞结合和释放。其中知识氨基酸的突变是必要的禽流感病毒实现α2-6结合和跨越物种屏障使大流行的准备。数测定法用于确定受体特异性;但是，所有有其缺点，包括尺寸仅为亲和力，而不是特异性， 反之亦然 。 这里，我们描述基于广泛使用和接受的ELISA，一?...

野生水禽是IAV的自然宿主，但是IAV能够跨越物种屏障，禽类和哺乳动物，包括人类。禽流IAVs认识α2-3连接的唾液酸（禽型受体），而人类病毒结合α2-6连接的唾液酸（人型受体）。为了能够有效复制和人类禽类IAV需要绑定到人型受体1之间传递。 IAVs是基于血清学表征其血凝素（HA）和神经氨酸酶（NA）包膜糖蛋白的抗原性划分。 HA结合到唾液酸，而NA是在病毒的生命周期并切割唾液酸2的另一端的受体破坏酶。所有的人感染病毒，包括H1N1，H2N2和H3N2，有禽源3。在过去的二十年中几个禽类到人类交叉的发生，用H5N1，H7N7，和H7N9是最公知的;豪版本，其他亚型传染人类更零星（H6N1，H7N1，H7N2，H9N2，H10N7，H10N8）4。幸运的是，似乎没有这些病毒已经能够完全适应人型α2-6连接的唾液酸受体5-8。禽流感或其他人畜共患的病毒适应，以适应人类宿主复制和传播可能对人体健康产生破坏性的影响。因此，这些病毒是如何发展结合人型受体将有助于新兴流感病毒全球范围内监视的先验知识。 受体偏好的测定采用不同物种的红细胞首次阐明并保持流感研究者9-12之间的青睐检测。即禽流感病毒识别α2-3唾液酸α2-6连接的唾液酸人类病毒的示威最初是基于使用酶设计包含每个李红细胞血凝化验nkages 13,14。虽然读出是血细胞凝集，一个标准测定为病毒学家，底层聚糖结构没有定义，只有终端联系​​。此外，唾液酸转移酶，用于重新sialylate细胞的有限的可用性，已经限制了使用该测定15-18中。接着，使用连接于聚丙烯酰胺（PAA）或聚谷氨酸（PGA）的结构在基于板的测定法19,20唾液酸化聚糖结构中引入确定受体结合偏好的其他方法。几种变化是在涂覆或聚糖或病毒微量滴定板，其中每一个导致一个健壮的，可靠的和非常敏感的ELISA型测定21-23可能。可替代地，生物素连接的聚糖可以取代的PAA / PGA和可以缀合到链霉抗涂层板2,24。虽然可能需要一些特定的血清，酶联免疫吸附都与PAA标准和几个聚糖容易commercially和非商用（财团为功能糖组学（http://www.functionalglycomics.org））。 聚糖微阵列技术已成为一个宝贵的工具，以确定受体特异性，为多个不同的聚糖发现，并结合到多种不同结构可以在单一实验中25-29评估。 IAV的这些结构的结合提供了更好的理解，即IAV优先识别30-33的聚糖结构。聚糖微阵列需要少量的样本量来进行结合分析，只有使用每点（2 NL）聚糖微量。然而，这些阵列通常仅用于评估聚糖受体特异性。的多种病毒，或血凝素蛋白，在多个浓度范围分析可以望而却步由于所需的幻灯片的数量。此外，迄今为止，没有相对亲合力测定已使用聚糖芳开发射线技术。 结合由聚糖微阵列技术和基于ELISA的测定法的PAA联聚糖的灵敏度，得到低样品要求，我们试图开发一个多井聚糖阵列将允许对具有相似或更好的分辨率高通量分析相比传统的基于ELISA的测定法。同时，我们希望最小化消耗的生物制品和报道的化学品的量。最终的结果是一个小型化的亲合力测定中，专门用于监测IAV特异性开发的，同样适用于评估其他聚糖结合蛋白。使用由聚四氟乙烯面具分为48微孔载玻片，6种不同的聚糖看准每孔6个重复。该芯片平台能提供受体趋势相同绑定看到在宏观ELISA格式几个优点。这些包括（I）中6次重复化合物的印刷，使用最少的样品，与若干行的涂布我呐板，使用每孔100μl; （二）多种不同的化合物在单井，包括控制同时进行分析; （Ⅲ）在培养体积和大规模减少; （IV）使用荧光读出一个较大的动态范围。单个幻灯片可以被计算为等于18倍的96孔板。 用下面的协议，任何实验室能够制造和分析的斑点的微阵列应该能够制造这种小型化的ELISA形式。

<table>
	<tbody>
		<tr>
			<td>NEXTERION&#174; Slide H MPX-48&#160;</td>
			<td>Schott</td>
			<td>1091525</td>
			<td>Microwell slides</td>
		</tr>
		<tr>
			<td>ProScanArray Plus&#160;</td>
			<td>PerkinElmer</td>
			<td>discontinued</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>Innoscan 1100AL Scanner/Mapix Software</td>
			<td>Innopsys</td>
			<td>-</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>MicroGrid II&#160;</td>
			<td>Digilab</td>
			<td>-</td>
			<td>microaray printer</td>
		</tr>
		<tr>
			<td>SNA</td>
			<td>Vector Labs</td>
			<td>B-1305&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>ECA</td>
			<td>Vector Labs</td>
			<td>B-1145&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>Anti-Strep Antibody</td>
			<td>IBA</td>
			<td>2-1509-001&#160;</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Anti-Mouse Alexa-647</td>
			<td>Life</td>
			<td>A-21235</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287&#160;</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>di-basic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>255793</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>mono-basic Sodium phosphate</td>
			<td>Sigma</td>
			<td>229903&#160;</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>poly-l-lysine solution</td>
			<td>Sigma</td>
			<td>P8920&#160;</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>221465</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma</td>
			<td>493546</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>incubation/washing buffer</td>
		</tr>
		<tr>
			<td>SMP4B pins</td>
			<td>Telechem</td>
			<td>SMP4B</td>
			<td>printing pin</td>
		</tr>
		<tr>
			<td>Compressed Nitrogen (Grade5)</td>
			<td>Praxair</td>
			<td>UN1066</td>
			<td>general dusting/drying tool</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma</td>
			<td>B6768-500G</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>ethanolamine</td>
			<td>Sigma</td>
			<td>E9508-500ML</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>Atto 488</td>
			<td>AttoTec</td>
			<td>AD 488-91</td>
			<td>Gridmarker on array</td>
		</tr>
		<tr>
			<td>PAA-LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA368</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA362</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA343</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te98</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te175</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te176</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>384-well microtiter plate</td>
			<td>Matrix TechCorp</td>
			<td>4361</td>
			<td>Printing plate</td>
		</tr>
		<tr>
			<td>VWR lab marker</td>
			<td>VWR</td>
			<td>52877-150</td>
			<td>Slide Numbering</td>
		</tr>
		<tr>
			<td>Wheaton slide staining dish</td>
			<td>Sigma</td>
			<td>Z103969-1EA</td>
			<td>Blocking and Drying</td>
		</tr>
	</tbody>
</table>

a miniaturized glycan microarray assay for assessing avidity and specificity of influenza a virus hemagglutinins

A型流感病毒（IAV）血细胞凝集素识别在细胞表面上的唾液酸作为功能​​性受体来获得进入细胞。野生水禽是IAV的自然宿主，但是可以IAV跨越物种屏障家禽，猪，马和人。禽流感病毒识别由α2-3键（禽型受体）附连到倒数第二个半乳糖，而人类病毒优先识别唾液酸与α2-6键（人型受体）唾液酸。并监控如果禽流感病毒正在适应人型受体，可使用几种方法。用合成唾液酸糖苷的多样库聚糖微阵列越来越多地用于评估受体特异性。然而，这种技术不用于测量亲合力。亲合力的测量一般是通过评估系列稀释血凝素或病毒的结合以吸附到常规聚丙烯96孔板聚糖实现。在该测定中，聚糖与α2-3或α2-6唾液酸连接到生物素并吸附到链霉板，或连接到聚丙烯酰胺（PAA），其直接吸附到塑料。我们通过直接印刷的PAA联唾液酸糖苷及其非PAA联的对应于微井载玻片已显著小型化该测定。这种设置，与在单个幻灯片48阵列，使得能够6聚糖8稀释结合蛋白，询问6个不同的聚糖，包括两个非唾液酸化的控制的同时测定。这相当于在传统的平板测定18倍的96孔板。该聚糖阵列格式降低化合物和生物制品的消费，从而大大提高了工作效率。

打印，扫描和数据分析为确保正确打印，至关重要的是有聚四氟乙烯面罩内的斑点网格，它描绘了在幻灯片上的每个阵列的正确对准。在印刷中，由于聚四氟乙烯涂层的性质，点不能由上MPX幻灯片肉眼看到。注意的是然后在聚-L-赖氨酸包被的玻片预斑点的外观。印刷后直接，各滑动应当由眼对每个斑点化合物是可见的存在，由...

Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...