Os autores graças a divisão de tecnologia de película fina, centro de pesquisa de tecnologia de instrumento (ITRC) e nacional aplicada Research Laboratories, Hsinchu Science Park, Taiwan. Este trabalho foi financiado pelo Conselho Nacional de ciência (conceder n. A maioria dos 105-0210-01-13-01) e Academia Sinica.

<ol>
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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. 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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. 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Os autores não têm nada para divulgar.

Uma classe de bNAbs de HIV-1, incluindo PG9 e PG16 PGTs 128, 141-145 foram relatados para ser altamente potente para neutralizar a 70-80% de isolados de HIV-1 em circulação. Os epítopos destes bNAbs são altamente conservados entre as variantes de todo o grupo HIV-1 M, portanto eles podem guiar o projeto de immunogen eficaz para uma vacina contra o HIV eliciar neutralizando anticorpos23,24,25 . Como parte dos nossos esforços...

Os N- glicanos em glicoproteínas estão covalentemente ligados à asparagina (Asn) resíduo de sequon de Asn-Xxx-Ser/Thr o consenso, que afetam vários processos biológicos, tais como reconhecimento de conformação, antigenicidade, solubilidade e lectina de proteína 1 , 2. a síntese química de N-oligossacarídeos vinculados representa um desafio sintético significativo devido à sua enorme heterogeneidade micro estrutural e arquitetura altamente ramificada. A seleção cuidadosa de proteger grupos de sintonizar a reatividade dos blocos de construção, alcançar a seletividade em centros anomérico e uso correto de promotor / activator(s) são elementos-chave na síntese de oligossacarídeos complexos. Para resolver este problema da complexidade, uma grande quantidade de trabalho para avançar N- glicano síntese foi relatada recentemente3,4. Apesar dessas abordagens robustas, encontrar um método eficaz para a preparação de uma vasta gama de N- os glicanos (~ 20.000) permanece um grande desafio.
A taxa de mutação rápida do HIV-1 para atingir a extensa diversidade genética e sua capacidade de escapar de neutralizar a resposta imunitária, está entre os maiores desafios para desenvolver uma vacina segura e profilática contra HIV-15,6 , 7. uma tática eficaz que o HIV usa para evitar a resposta imune do hospedeiro é a glicosilação pós-traducional de gp120 de glicoproteína do envelope com um diverso N-ligados os glicanos derivados do anfitrião glycosylation máquinas8, 9. Um relatório recente sobre a análise precisa de recombinação monomérico HIV-1 gp120 glicosilação de células de rim humano embrionário (HEK) 293T sugere a ocorrência de microheterogeneity estrutural com um padrão característico de célula específico10 , 11 , 12. portanto, a compreensão das especificidades de glicano do HIV-1 bNAbs requer gp120 bem caracterizadas relacionados N- glicano estruturas em quantidade suficiente para análise.
A descoberta da tecnologia microarray de glicano fornecido alta exploração baseada na taxa de transferência de especificidades de uma diversa gama de proteínas carboidratos, adesinas vírus/bactérias, toxinas, anticorpos e lectines13,14 . O arranjo os glicanos sistemática em um formato de matriz baseada no chip poderia determinar interações do proteína-glicano problemático baixa afinidade a apresentação multivalentes15,16,17,18. Este arranjo de chip baseada glicano convenientemente aparece efetivamente imitar interfaces de celular. Para enriquecer a tecnologia e a superar a questão desigual associada com formatos de matriz convencional, nosso grupo desenvolveu recentemente um glicano matriz sobre uma lâmina de vidro revestido de óxido de alumínio (ACG) usando os glicanos de terminou de ácido fosfônico para aumentar a intensidade do sinal, homogeneidade e sensibilidade19,20.
Para melhorar o entendimento atual sobre glicano epitopos de HIV-1 recém isoladas amplamente neutralizando anticorpos (bNAbs), temos desenvolvido uma estratégia modular altamente eficiente para a preparação de um vasto leque de N-ligados os glicanos21 ,22 para serem impressos em um ACG deslize (ver Figura 1). Especificidade de estudos de perfil de bNAbs de HIV-1 em uma matriz ACG ofereceram a detecção incomum de comportamento de vinculação de hetero-glicano de bNAb altamente potente PG9 que foi isolado do HIV infectados indivíduos23,24,25.

<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

Apresentamos uma maneira altamente eficiente para a preparação rápida de uma vasta gama de N-ligadas a oligossacarídeos (estima-se que para exceder 20.000 estruturas) que são comumente encontrados em glicoproteínas humanas. Para alcançar a desejada diversidade estrutural, a estratégia começou com a síntese enzimática de quimioterapia de três tipos de módulos de fluoreto de oligosaccharyl, seguidos por sua gradual glycosylations α-seletiva para o 3-Ó e 6-O posiciona do resíduo de manose do trissacarídeo núcleo comum, tendo uma ligação crucial β-mannoside. Ainda mais, nós unimos os N- glicanos à superfície de uma lâmina de vidro revestido de óxido de alumínio (ACG) para criar uma matriz covalente mista para a análise da interação de hetero-ligante com um anticorpo de HIV. Em particular, o comportamento de vinculação de um recém isolados VIH-1 amplamente neutralizante anticorpo (bNAb), PG9, a mistura de espaçadas homem5GlcNAc2 (homem5) e 2,6-di-sialylated bi-antennary complexo tipo N- glicano (SCT ) em uma matriz ACG, abre uma nova avenida para guiar o projeto immunogen eficaz para o desenvolvimento de vacina contra HIV. Além disso, nossa matriz ACG encarna uma poderosa ferramenta para estudar outros anticorpos de HIV para o comportamento de vinculação de hetero-ligante.

Uma estratégia modular de quimio-enzimático para a síntese de um vasto leque de N-os glicanos é apresentada na Figura 1. A estratégia é baseado no fato de que a diversidade pode ser criada no início por quimioterapia-enzimático síntese dos três módulos importantes, seguido pelo mannosylation α-específicos para o 3-O e/ou 6-Ó posição do resíduo manose do comum núcleo trissacarídeo de N- os glicanos. Consid...

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

Uma abordagem modular para a síntese de N- os glicanos para fixação a um vidro revestido de óxido de alumínio slide (slide ACG) como desenvolveu um microarray glicano e demonstrou-se seu uso para a criação de perfil de um HIV amplamente neutralizando anticorpos.

Quimio-enzimático síntese de N- os glicanos para desenvolvimento de matriz e anticorpos de HIV de criação de perfil

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 visualizações.  Academia Sinica. Uma abordagem modular para a síntese de N- os glicanos para fixação a um vidro revestido de óxido de alumínio slide (slide ACG) como desenvolveu um microarray glicano e demonstrou-se seu uso para a criação de perfil de um HIV amplamente neutralizando anticorpos.

Vídeo: 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.

Perfis Anti-Neu5Gc IgG em Sera Humana com um Ensaio de Microarray de Sialoglicano

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

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.

A Miniaturized Glycan Microarray Ensaio para a Avaliação de avidez e especificidade do Hemaglutininas vírus da gripe A

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

Imunologia e Infecção

<meta charset="utf8"></head><body>Perfil de tecidos de câncer colorretal usando glicoproteômica guiada por glicômicos

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>A glicoproteômica guiada por glicômicos facilita o perfil abrangente do glicoproteoma em microambientes tumorais complexos

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.

A glicoproteômica guiada por glicômicos facilita o perfil abrangente do glicoproteoma em microambientes tumorais complexos 

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

Pesquisa do câncer

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.

Purificação e análise de um anticorpo monoclonal de células de ovário de hamster chinês usando um sistema de Microbioreator automatizado

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

Bioengenharia

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.

Matriz de glicano impressa: Uma técnica sensível para a análise do repertório de anticorpos anti-hidrato de carbono em pequenos animais em circulação

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.

Microarray de anticorpos quimicamente bloqueada para Multiplexed perfil de alta capacidade de glicosilação proteína específica em amostras complexas

 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 ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Engenharia de Agentes Antivirais via Ressonância de Plasmon de Superfície

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Quimio-enzimático síntese de N- os glicanos para desenvolvimento de matriz e anticorpos de HIV de criação de perfil

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Purificação e análise de um anticorpo monoclonal de células de ovário de hamster chinês usando um sistema de Microbioreator automatizado

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