Este trabajo fue apoyado por el Instituto de Investigaciones sobre el Virus Hepatitis y.

<ol>
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Los autores no tienen nada que revelar.

1. Objetivo de proteínas y selección de anticuerpos de captura Antes del ensayo de microarrays de anticuerpos, algunos reactivos y materiales son necesarios para ser considerado y preparado. Para diseñar un microarray de anticuerpos para el perfil de glicano o cribado glicano biomarcador, un panel de anticuerpos específicos a los candidatos la glicoproteína debe determinarse de acuerdo a la literatura o de los resultados anteriores. Estos anticuerpos fueron adquiridos por lo general de dif...

<table border="1"><tbody><tr><td> Identificación </td><td> Nombre del reactivo </td><td> Abreviación </td><td> Empresa </td><td> Catálogo # </td></tr><tr><td> L1 </td><td> Biotinilado Concanavalina A </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinilado lectina Sambucus nigra </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinilado Lens culinaris aglutinina </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinylated aglutinina de Ricinus communis que </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinilado Aleuria Aurantia lectina </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinilado Erythrina crista lectina </td><td> ECL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinilado Griffonia (Bandeiraea) Simplicifolia lectina II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinilada aglutinina de germen de trigo </td><td> Ventajas de Windows Original </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Erythroagglutinin biotinilado Phaseolus vulgaris </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Leucoaglutinina biotinilado Phaseolus vulgaris </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Aglutinina de maní biotinilado </td><td> ANP </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinylated Pisum sativum aglutinina </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinilado Dolichos biflorus aglutinina </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Datura stramonium lectina biotinilada </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinilado aglutinina Sophora japónica </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Aglutinina de soja biotinilado </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinilado Solanum tuberosum (papa) lectina </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinilado Griffonia (Bandaeiraea) Simplicifolia me lectina </td><td> GSL I </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinilado Vicia villosa lectina </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinilado Lycopersicon esculentum (tomate) lectina </td><td> LEL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Ulex europaeus biotinilado aglutinina I </td><td> UEA I </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinilado jacalin </td><td> Jacalin </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> F cabra (ab &#39;) 2 Fragmento de IgM anti-humano, el anticuerpo Fc5μ </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Burro F (ab &#39;) 2 Frag anti-IgG humana (H + L) untibody </td><td> AB1 </td><td> Jackson Immuno Research </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> Ratón anti-humano IgG F (ab &#39;) 2 anticuerpo monoclonal </td><td> AB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Cabra anti-humano alfa 2 macroglobulina anticuerpo policlonal </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> Conejo anti-humana de alfa-1-antitripsina anticuerpo policlonal </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> Ratón anti-humano alfa-1-antitripsina anticuerpo monoclonal </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> Conejo anti-humana de alfa-1-antitripsina anticuerpo policlonal </td><td> ACT </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> Conejo anti-humanaalfa-1-antiquimotripsina anticuerpo policlonal </td><td> ACT </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Ratón anti-transferrina humana anticuerpo monoclonal </td><td> La transferrina </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> Conejo anti-transferrina humana anticuerpo policlonal </td><td> La transferrina </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Cabra anti-humano apolipoproteína J anticuerpo policlonal </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Ratón anti-humano GP73 anticuerpo monoclonal </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Ratón anti-humano GP73 anticuerpo monoclonal </td><td> GP73 </td><td> Santa Cruz de Biotecnología INC </td><td> sc-101275 </td></tr><tr><td> A14 </td><td> Conejo anti-humana de alfa-1 fetoprotein anticuerpo policlonal </td><td> AFP </td><td> Genway </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> Ratón anti-humano alfa-1-fetoproteína anticuerpo monoclonal </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Ratón anti-humano hemopexina anticuerpo monoclonal </td><td> Hemopexin </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> Ratón anti-humano glipicano-3 (1G12) del anticuerpo monoclonal </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65443 </td></tr><tr><td> A18 </td><td> Ratón anti-humano Quininógeno (BPM) del anticuerpo monoclonal </td><td> Quininógeno </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> Conejo anti-humano MMP-21 anticuerpo monoclonal </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Ratón anti-humano CEACAM-1 anticuerpos monoclonalesy </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Rata anti-humano DPPIV/CD26 anticuerpo monoclonal </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Ratón anti-humano PIVKA II anticuerpo monoclonal </td><td> PIVICA </td><td> Crystal química </td><td> 8040 </td></tr><tr><td> A23 </td><td> Mouse anti-antígeno carcinoembrionario </td><td> CEA </td><td> EE.UU. biológica </td><td> C1300 </td></tr><tr><td> A24 </td><td> Mouse anti-cáncer de antígeno CA125 </td><td> CA125 </td><td> EE.UU. biológica </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Mouse anti-CA19-9 del cáncer de antígeno </td><td> CA19-9 </td><td> EE.UU. biológica </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> Ratón anti-Lewis x anticuerpo monoclonal </td><td> Lewis X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> bio </td><td> Biotinilada BSA (control positivo) </td><td> Bio </td><td> Hecho en casa </td><td> N / A </td></tr></tbody></table> Tabla 1. Lista de las lectinas y anticuerpos utilizados en este protocolo. <table border="1"><tbody><tr><td> Nombre de los equipos o el reactivo s </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Microarrayer sin contacto </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 microplaca </td><td> Pescador </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultrafino de nitrocelulosa Coate microarrays se desliza </td><td> Gentel </td><td> TRAYECTORIA </td></tr><tr><td> Slide Imprinter (opcional) </td><td> La Compañía de gel </td><td> WSP60-1 </td></tr><tr><td> Shaker </td><td> Pescador </td><td> 15-453-211 </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Deslice el lavabo / Slide tinción Plato con bastidor extraíble </td><td> Pescador </td><td> 08-812 </td></tr><tr><td> La incubación de diapositivas de cámara / caja portaobjetos del microscopio </td><td> Pescador </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30 w / v% en agua </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 al </td><td> Pescador </td><td> P337-100 </td></tr><tr><td> Peryodato de sodio (NaIO 4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> L-glutámico ácido γ-hidrazida </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> Acetato de sodio anhidro (CH3COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Albúmina de Suero Bovino (BSA) </td><td> Lampire Biológica Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> Tampón fosfato salino (PBS) (10 veces) </td><td> Denville Científico </td><td> CP4390-48 </td></tr><tr><td> DyLight 549 neutravidin conjugado </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Inhibidor de la proteasa cóctel comprimidos </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure IgG humana, fragmento Fc </td><td> Jackson Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure IgG humana, toda la molécula </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure IgG de ratón, toda la molécula </td><td> Jackson Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure IgG de ratón, fragmento Fc </td><td> Jackson Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure IgG de conejo, molécula entera </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure burro IgG, molécula entera </td><td> Jackson Immunoresearch </td><td> 017-000-003 </td></tr><tr><td> Microarray Scanner </td><td> Tecan </td><td> LS Reloaded </td></tr></tbody></table> Tabla 2. Lista de equipos y reactivos utilizados en este protocolo.

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.

Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

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

En este estudio, se describe un protocolo mejorado para una micromatriz anticuerpo multiplexado de alto rendimiento con el método de detección de lectina que se puede utilizar en perfiles de glicosilación de proteínas específicas. Este protocolo incluye nuevos reactivos fiables y reduce significativamente el tiempo, el coste y los requisitos de equipos de laboratorio, en comparación con el procedimiento anterior.

Anticuerpos, químicamente bloqueado Microarray de multiplexado de alto rendimiento de perfiles de glicosilación de proteínas específicas en muestras complejas

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

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15.3K vistas.  Institute for Hepatitis and Virus Research. En este estudio, se describe un protocolo mejorado para una micromatriz anticuerpo multiplexado de alto rendimiento con el método de detección de lectina que se puede utilizar en perfiles de glicosilación de proteínas específicas. Este protocolo incluye nuevos reactivos fiables y reduce significativamente el tiempo, el coste y los requisitos de equipos de laboratorio, en comparación con el procedimiento anterior.