Gli autori ringraziano il Thin Film Technology Division, strumento Technology Research Center (ITRC) e laboratori di ricerca applicata nazionale, parco scientifico Hsinchu, Taiwan. Questo lavoro è stato supportato dal National Science Council (grant no. La maggior parte 105-0210-01-13-01) e Academia Sinica.

<ol>
	<li>Kim, P. J., Lee, D. Y., Jeong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centralized+modularity+of+N-linked+glycosylation+pathways+in+mammalian+cells.">Centralized modularity of N-linked glycosylation pathways in mammalian cells.</a> PloS one. 4, e7317 (2009).</li><li>Townsley, S., Li, Y., Kozyrev, Y., Cleveland, B., Hu, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+Role+of+an+N-Linked+Glycan+on+the+Surface+Antigen+of+Human+Immunodeficiency+Virus+Type+1+Modulating+Virus+Sensitivity+to+Broadly+Neutralizing+Antibodies+Against+the+Receptor+and+Coreceptor+Binding+Sites.">Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies Against the Receptor and Coreceptor Binding Sites.</a> J.virol. 90, 829-841 (2015).</li><li>Wang, Z., 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+General+Strategy+for+the+Chemoenzymatic+Synthesis+of+Asymmetrically+Branched+N-Glycans.">A General Strategy for the Chemoenzymatic Synthesis of Asymmetrically Branched N-Glycans.</a> Science. 341, 379-383 (2013).</li><li>Li, 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=Efficient+Chemoenzymatic+Synthesis+of+an+N-glycan+Isomer+Library.">Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library.</a> Chem Sci. 6, 5652-5661 (2015).</li><li>Pritchard, L. K., Harvey, D. J., Bonomelli, C., Crispin, M., Doores, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-+and+Protein-Directed+Glycosylation+of+Native+Cleaved+HIV-1+Envelope.">Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope.</a> J.Virol. 89, 8932-8944 (2015).</li><li>Behrens, A. J., 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=Composition+and+Antigenic+Effects+of+Individual+Glycan+Sites+of+a+Trimeric+HIV-1+Envelope+Glycoprotein.">Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein.</a> Cell Rep. 14, 2695-2706 (2016).</li><li>Barouch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+the+development+of+an+HIV-1+vaccine.">Challenges in the development of an HIV-1 vaccine.</a> Nature. 455, 613-619 (2008).</li><li>Horiya, S., MacPherson, I. S., Krauss, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Strategies+Targeting+HIV+Glycans+in+Vaccine+Design.">Recent Strategies Targeting HIV Glycans in Vaccine Design.</a> Nat Chem Bio. 10, 990-999 (2014).</li><li>Wang, L. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+carbohydrate+Antigens+for+HIV+Vaccine+Design.">Synthetic carbohydrate Antigens for HIV Vaccine Design.</a> Curr Opin Chem Biol. 17, 997-1005 (2013).</li><li>Tian, J., 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=Effect+of+Glycosylation+on+an+Immunodominant+Region+in+the+V1V2+Variable+Domain+of+the+HIV-1+Envelope+gp120+Protein.">Effect of Glycosylation on an Immunodominant Region in the V1V2 Variable Domain of the HIV-1 Envelope gp120 Protein.</a> PLoS Comput Biol. 12, e1005094 (2016).</li><li>Geyer, H., Holschbach, C., Hunsmann, G., Schneider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrates+of+human+Immunodeficiency+Virus.+Structures+of+Oligosaccharides+Linked+to+the+Envelope+Glycoprotein+120.">Carbohydrates of human Immunodeficiency Virus. Structures of Oligosaccharides Linked to the Envelope Glycoprotein 120.</a> The J Bio Chem. 263, 11760-11767 (1988).</li><li>Lee, J. H., Ozorowski, G., Ward, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+Structure+of+A+Native,+Fully+Glycosylated,+Cleaved+HIV-1+Envelope+Trimer.">Cryo-EM Structure of A Native, Fully Glycosylated, Cleaved HIV-1 Envelope Trimer.</a> Science. 351, 1043-1048 (2016).</li><li>Lonardi, E., Balog, C. I., Deelder, A. M., Wuhrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+GlycanMicroarrays.">Natural GlycanMicroarrays.</a> Expert Rev Proteomics. 7, 761-774 (2010).</li><li>Paulson, J. C., Blixt, O., Collins, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweet+Spots+in+Functional+Glycomics.">Sweet Spots in Functional Glycomics.</a> Nat. Chem. Bio. 2, 238-248 (2006).</li><li>Dotsey, E. 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=A+High+Throughput+Protein+Microarray+Approach+to+Classify+HIV+Monoclonal+Antibodies+and+Variant+Antigens.">A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.</a> PLoS One. 10, e0125581 (2015).</li><li>Wu, C. Y., Liang, P. H., Wong, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Development+of+Glycan+Arrays.">New Development of Glycan Arrays.</a> Org Biomol Chem. 7, 2247-2254 (2009).</li><li>Scurr, D. J., 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=Surface+Characterization+of+Carbohydrate+Microarrays.">Surface Characterization of Carbohydrate Microarrays.</a> Langmuir. 26, 17143-17155 (2010).</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, 17033-17038 (2004).</li><li>Chang, S. 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=Glycan+Array+on+Aluminum+Oxide-Coated+Glass+Slides+Through+Phosphonate+Chemistry.">Glycan Array on Aluminum Oxide-Coated Glass Slides Through Phosphonate Chemistry.</a> J. Am. Chem. Soc. 132, 13371-13380 (2010).</li><li>Tseng, S. 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=Preparation+of+Aluminum+Oxide-Coated+Glass+Slides+for+Glycan+Microarrays.">Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.</a> ACS Omega. 1, 773-783 (2016).</li><li>Shivatare, S. 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=Efficient+Convergent+Synthesis+of+Bi-,+Tri-,+and+Tetra-Antennary+Complex+Type+N-Glycans+and+Their+HIV-1+Antigenicity.">Efficient Convergent Synthesis of Bi-, Tri-, and Tetra-Antennary Complex Type N-Glycans and Their HIV-1 Antigenicity.</a> J. Am. Chem. Soc. 135, 15382-15391 (2013).</li><li>Shivatare, S. 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=Modular+synthesis+of+N-Glycans+and+Arrays+for+the+Hetero-Ligand+Binding+Analysis+of+HIV+Antibodies.">Modular synthesis of N-Glycans and Arrays for the Hetero-Ligand Binding Analysis of HIV Antibodies.</a> Nat Chem. 8, 338-346 (2016).</li><li>McLellan, J. 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=Structure+of+Hiv-1+gp120+V1/V2+Domain+with+Broadly+Neutralizing+Antibody+PG9.">Structure of Hiv-1 gp120 V1/V2 Domain with Broadly Neutralizing Antibody PG9.</a> Nature. 480, 336-343 (2011).</li><li>Julien, J. 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=Asymmetric+Recognition+of+the+HIV-1+Trimer+by+Broadly+Neutralizing+Antibody+PG9.">Asymmetric Recognition of the HIV-1 Trimer by Broadly Neutralizing Antibody PG9.</a> Proc Natl Acad Sci U S A. 110, 4351-4356 (2013).</li><li>Willis, J. 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=Long+Antibody+HCDR3s+from+HIV-Native+Donors+Presented+on+a+PG9+Neutralizing+Antibody+Background+Mediate+HIV+Neutralization.">Long Antibody HCDR3s from HIV-Native Donors Presented on a PG9 Neutralizing Antibody Background Mediate HIV Neutralization.</a> Proc Natl Acad Sci U S A. 113, 4446-4451 (2016).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Organic+Chemistry.+Performing+1D+Thin+Layer+Chromatography.">Essentials of Organic Chemistry. Performing 1D Thin Layer Chromatography.</a> JoVE. , (2017).</li><li>de Castro, M. D., 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+useful+approach+to+the+automation+of+analytical+processes?.">A useful approach to the automation of analytical processes?.</a> J Automat Chem. 12, 267-279 (1990).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Organic+Chemistry.+Nuclear+Magnetic+Resonance+(NMR)+Spectroscopy.">Essentials of Organic Chemistry. Nuclear Magnetic Resonance (NMR) Spectroscopy.</a> JoVE. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Analytical+Chemistry.+Introduction+to+Mass+Spectrometry.">Essentials of Analytical Chemistry. Introduction to Mass Spectrometry.</a> JoVE. , (2017).</li><li>Tseng, S. 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=Preparation+of+Aluminum+Oxide-Coated+Glass+Slides+for+Glycan+Microarrays.">Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.</a> ACS Omega. 1 (5), 773-783 (2016).</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, 17033-17038 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GenePix Pro 6.0 Microarray Acquisition and Analysis Software for GenePix Microarray Scanners User’s Guide &amp; Tutorial. , (2017).</li></ol>

Gli autori non hanno nulla a rivelare.

Una classe di HIV-1 bNAbs tra cui PG9, PG16 e PGTs 128, 141-145 sono stati segnalati per essere altamente potente nel neutralizzare il 70-80% del circolante isolati HIV-1. Gli epitopi di questi bNAbs sono altamente conservati tra le varianti del gruppo intero di HIV-1 M, quindi si può guidare la progettazione immunogen efficace per un vaccino contro l'HIV suscitare anticorpi neutralizzanti23,24,25 . Come parte dei nostri sforzi...

N- glicani su glicoproteine sono covalentemente ad l'asparagina (Asn) residuo di sequon di Asn-Xxx-Ser/Thr di consenso, che interessano diversi processi biologici come il riconoscimento di conformazione, antigenicità, solubilità e lectina di proteina 1 , 2. la sintesi chimica di N-collegati oligosaccaridi rappresenta una sfida sintetica significativa a causa della loro enorme eterogeneità strutturale micro e architettura altamente ramificato. Un'attenta selezione di proteggere gruppi per ottimizzare la reattività di blocchi predefiniti, raggiungimento di selettività presso centri anomerici e uso corretto del promotore / activator(s) sono elementi chiave nella sintesi di oligosaccaridi complessi. Per risolvere il problema della complessità, una grande quantità di lavoro per far avanzare di N- glycan sintesi è stata segnalata recentemente3,4. Nonostante questi approcci robusti, trovando un metodo efficace per la preparazione di una vasta gamma di N- glicani (~ 20.000) rimane una grande sfida.
Il tasso di mutazione rapida di HIV-1 per ottenere la vasta diversità genetica e la sua capacità di fuggire da neutralizzare la risposta anticorpale, è tra le più grandi sfide per sviluppare un vaccino sicuro e profilattico contro HIV-15,6 , 7. una tattica efficace che l'HIV utilizza per evitare la risposta immunitaria è la glicosilazione post-traduzionale della busta glicoproteina gp120 con una diversificata N-collegato glicani derivato dal host glicosilazione macchine8, 9. Un recente rapporto per quanto riguarda l'analisi precisa dei ricombinanti monomerica glicosilazione di gp120 di HIV-1 da cellule 293T cellule embrionali umane del rene (HEK) suggerisce l'avvenimento di microheterogeneity strutturale con un modello caratteristico di cellula-specifico10 , 11 , 12. Pertanto, la comprensione delle specificità glycan di HIV-1 bNAbs richiede ben caratterizzati gp120 relative N- glycan strutture in quantità sufficiente per l'analisi.
La scoperta della tecnologia microarray glycan fornito alta basati su velocità effettiva esplorazione delle specificità di una gamma diversificata di carboidrati proteine, virus/batterica adesine, tossine, anticorpi e lectines13,14 . La disposizione sistematica glicani in un formato basato su chip allinearono potrebbe determinare interazioni proteina-glycan problematico bassa affinità attraverso presentazione multivalente15,16,17,18. Questa disposizione basata su chip glycan convenientemente sembra imitare efficacemente interfacce cellula-cellula. Per arricchire la tecnologia e superare il problema irregolare connesso con formati di matrice convenzionale, il nostro gruppo ha recentemente sviluppato una matrice glycan su un vetrino in vetro rivestita di ossido di alluminio (ACG) usando glicani attacco acido fosfonico per aumentare l'intensità del segnale, omogeneità e sensibilità19,20.
Per migliorare la comprensione corrente circa glycan epitopi di recente isolato HIV-1 largamente neutralizzando gli anticorpi (bNAbs), abbiamo sviluppato una strategia modulare altamente efficiente per la preparazione di una vasta gamma di N-collegato glicani21 ,22 per essere stampati su un ACG piastrina (Vedi Figura 1). Specificità di profilatura studi di HIV-1 bNAbs su una matrice ACG offerto l'individuazione insolita del comportamento di associazione di etero-glycan di altamente potente bNAb PG9 che è stato isolato da HIV infettato gli individui23,24,25.

<table><tbody><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>64197</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>75058</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma Aldrich</td><td>108247</td><td></td></tr><tr><td>Anhydrous magnesium sulfate</td><td>Sigma Aldrich</td><td>7487889</td><td></td></tr><tr><td>Boron trifluoride ethyl etherate</td><td>Sigma Aldrich</td><td>109637</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>9048468</td><td></td></tr><tr><td>Bio-Gel P2 polyacrylamide</td><td>Bio-Rad</td><td>1504118</td><td></td></tr><tr><td>Bis(cyclopentadienyl)hafnium(IV) dichloride</td><td>Sigma Aldrich</td><td>12116664</td><td></td></tr><tr><td>&amp;beta;-1, 4 Galactosyl transferases from bovine milk</td><td>Sigma Aldrich</td><td>48279</td><td></td></tr><tr><td>BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td><td>Arrayit</td><td></td><td></td></tr><tr><td>Cerium ammonium molybdate</td><td>TCI</td><td>C1794</td><td></td></tr><tr><td>Cerium ammonium nitrate</td><td>Sigma Aldrich</td><td>16774213</td><td></td></tr><tr><td>Clean glass slide&amp;nbsp;</td><td>Schott&amp;nbsp;</td><td></td><td></td></tr><tr><td>Cytidine-5&amp;prime;-monophospho-N-acetylneuraminic acid</td><td>Sigma Aldrich</td><td>3063716</td><td></td></tr><tr><td>Deuterated chloroform</td><td>Sigma Aldrich</td><td>865496</td><td></td></tr><tr><td>Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td><td>Jackson Immuno Research, USA</td><td>709605098</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>75092</td><td></td></tr><tr><td>Diethylaminosulfur trifluoride</td><td>Sigma Aldrich</td><td>38078090</td><td></td></tr><tr><td>Dimethylformamide</td><td>Sigma Aldrich</td><td>68122</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma Aldrich</td><td>141786</td><td></td></tr><tr><td>Ethylene glycol</td><td>Acros Organic</td><td>107211</td><td></td></tr><tr><td>FAST frame slide incubation chambers</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Guanosine 5&#039;-diphospho-b-L-fucose disodium salt&amp;nbsp;</td><td>Sigma Aldrich</td><td>15839700</td><td></td></tr><tr><td>Lab tracer 2.0 software&amp;nbsp;</td><td></td><td></td><td>Section 4 of the Protocol</td></tr><tr><td>GenePix Pro 4300A reader (microarray image analysis)</td><td>moleculardevices</td><td></td><td>www.moleculardevices.com</td></tr><tr><td>GraphPad Prism Software (Image processing )</td><td>GraphPad Software, Inc</td><td></td><td>http://www.graphpad.com/guides/prism/6/user-guide/</td></tr><tr><td>Lithium hydroxide</td><td>Sigma Aldrich</td><td>1310652</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma Aldrich</td><td>7773015</td><td></td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>67561</td><td></td></tr><tr><td>N-butanol</td><td>Sigma Aldrich</td><td>71363</td><td></td></tr><tr><td>Oxalic acid</td><td>Acros Organic</td><td>144627</td><td></td></tr><tr><td>Palladium hydroxide</td><td>Sigma Aldrich</td><td>12135227</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Pyridine</td><td>Sigma Aldrich</td><td>110861</td><td></td></tr><tr><td>P-Toluene sulfonic acid monohydrate</td><td>Sigma Aldrich</td><td>773476</td><td></td></tr><tr><td>Silver triflate</td><td>Sigma Aldrich</td><td>2923286</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>144558</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>7647145</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma Aldrich</td><td>144558</td><td></td></tr><tr><td>Sodium methoxide&amp;nbsp;</td><td>Sigma Aldrich</td><td>124414</td><td></td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>7757826</td><td></td></tr><tr><td>Toluene&amp;nbsp;</td><td>Sigma Aldrich</td><td>108883</td><td></td></tr><tr><td>Tris buffer&amp;nbsp;</td><td>Amresco</td><td>N/A</td><td>Ultra-pure grade</td></tr><tr><td>Tween-20</td><td>Amresco</td><td>9005645</td><td></td></tr><tr><td>Uridine diphosphate galactose (UDP-galactose)</td><td>Sigma Aldrich</td><td>137868521</td><td></td></tr></tbody></table>

chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling

Vi presentiamo un modo altamente efficiente per la preparazione rapida di una vasta gamma di N-collegato oligosaccaridi (stimati a superare i 20.000 strutture) che si trovano comunemente sulle glicoproteine umane. Per ottenere il desiderato diversità strutturale, la strategia ha cominciato con il chemio-enzimatico di sintesi dei tre tipi di moduli di fluoruro di oligosaccharyl, seguite da loro graduale glycosylations α-selettivo presso la 3-O e 6-O posizioni della residui di mannosio del trisaccaride nucleo comune avendo un legame β-mannoside cruciale. Abbiamo attaccato ulteriormente gli N- glicani alla superficie di una lastra di vetro rivestita di ossido di alluminio (ACG) per creare un array misto covalente per l'analisi dell'interazione ligando-etero con un anticorpo HIV. In particolare, il comportamento di associazione di un nuovo isolato HIV-1 largamente anticorpo neutralizzante (bNAb), PG9, alla miscela di ravvicinate Man5GlcNAc2 (uomo5) e 2,6-di-sialilato bi-antennary complesso tipo N- glycan (SCT ) su una matrice ACG, apre una nuova strada per guidare la progettazione immunogen efficace per lo sviluppo del vaccino HIV. Inoltre, la nostra matrice ACG incarna un potente strumento per studiare altri anticorpi anti-HIV per il comportamento di etero-ligando.

Una strategia modulare chemo-enzimatica per la sintesi di una vasta gamma di N -glicani è presentata nella Figura 1. La strategia è basata sul fatto che la diversità può essere creato all'inizio da chemio-enzimatico di sintesi dei tre moduli importanti, seguita dalla mannosilazione α-specifiche con il 3-O e/o 6-O posizione del residuo mannosio del comune trisaccaride nucleo di N- glicani. Considerando la diversità str...

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Un approccio modulare alla sintesi di N- glicani per il collegamento ad un vetro rivestita di ossido di alluminio diapositiva (ACG) come è stato sviluppato un microarray glycan e suo utilizzo per la profilazione di un HIV anticorpo di neutralizzazione ampiamente è stata dimostrata.

Chemio-enzimatico di sintesi della N- glicani matrice dello sviluppo e dell'anticorpo HIV profilatura

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

chemo-enzymatic-synthesis-n-glycans-for-array-development-hiv

Research

JoVE Journal

Chemistry

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

8.6K Views.  Academia Sinica. Un approccio modulare alla sintesi di N- glicani per il collegamento ad un vetro rivestita di ossido di alluminio diapositiva (ACG) come è stato sviluppato un microarray glycan e suo utilizzo per la profilazione di un HIV anticorpo di neutralizzazione ampiamente è stata dimostrata.

Video: Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Profilare IgG anti-Neu5Gc in Sera umana con un assaggio microarray di Sialoglycan

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

Ricerca

Comportamento

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

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

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.

Un miniaturizzato Glycan microarray test per la valutazione avidità e Specificità del virus influenzale A emoagglutinine

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

Immunologia e infezioni

<meta charset="utf8"></head><body>Profilazione dei tessuti tumorali del colon-retto mediante glicoproteomica guidata dai glifumetti

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

<meta charset="utf8"></head><body>La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Ricerca sul cancro

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purificazione e analisi di un anticorpo monoclonale da cellule ovaio Cesster cinese utilizzando un sistema microbioreante automatizzato

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Bioingegneria

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

Stampato Glycan Array: Una tecnica sensibile per l'analisi del repertorio di anticorpi anti-carboidrati nei piccoli animali

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biologia

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Glicoingegneria anticorpale rapida nelle cellule ovariche di criceto cinese

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Chemio-enzimatico di sintesi della N- glicani matrice dello sviluppo e dell'anticorpo HIV profilatura

Riepilogo

Esplora Altri Video

Capitoli in questo video

Profilare IgG anti-Neu5Gc in Sera umana con un assaggio microarray di Sialoglycan

Un miniaturizzato Glycan microarray test per la valutazione avidità e Specificità del virus influenzale A emoagglutinine

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

Purificazione e analisi di un anticorpo monoclonale da cellule ovaio Cesster cinese utilizzando un sistema microbioreante automatizzato

Stampato Glycan Array: Una tecnica sensibile per l'analisi del repertorio di anticorpi anti-carboidrati nei piccoli animali

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine specifico in campioni complessi

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Glicoingegneria anticorpale rapida nelle cellule ovariche di criceto cinese

Chemio-enzimatico di sintesi della N- glicani matrice dello sviluppo e dell'anticorpo HIV profilatura

Riepilogo

Esplora Altri Video

Capitoli in questo video

Profilare IgG anti-Neu5Gc in Sera umana con un assaggio microarray di Sialoglycan

Un miniaturizzato Glycan microarray test per la valutazione avidità e Specificità del virus influenzale A emoagglutinine

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

Purificazione e analisi di un anticorpo monoclonale da cellule ovaio Cesster cinese utilizzando un sistema microbioreante automatizzato

Stampato Glycan Array: Una tecnica sensibile per l'analisi del repertorio di anticorpi anti-carboidrati nei piccoli animali

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Glicoingegneria anticorpale rapida nelle cellule ovariche di criceto cinese

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine specifico in campioni complessi