Diese Arbeit wurde durch das Institut für Hepatitis-Virus und Forschung gefördert.

<ol>
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1. Zielprotein und Fängerantikörper Auswahl Vor der Antikörper-Mikroarray-Test werden einige Reagenzien und Materialien benötigt, um in Betracht gezogen werden und vorbereitet. Um einen Antikörper Microarray-Profiling für Glykan oder Glykan Biomarker-Screening, ein Gremium von spezifischen Antikörpern gegen Glykoprotein Kandidaten entwerfen sollte nach der Literatur oder von früheren Ergebnissen festgestellt werden. Diese Antikörper wurden in der Regel von verschiedenen Anbietern wie R...

<table border="1"><tbody><tr><td> Identifikation </td><td> Name des Reagenzes </td><td> Abkürzung </td><td> Firma </td><td> Katalog # </td></tr><tr><td> L1 </td><td> Biotinyliertes Concanavalin A </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinyliertes Sambucus nigra-Lectin </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinyliertes Lens culinaris Agglutinin </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinylierte Ricinus communis-Agglutinin I </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinyliertes Aleuria aurantia-Lectin </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinyliertes Erythrina cristagalli Lektin </td><td> ECL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinyliertes Griffonia (Bandeiraea) simplicifolia Lektin II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinyliertes Weizenkeim-Agglutinin </td><td> WGA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Biotinyliertes Phaseolus vulgaris Erythroagglutinin </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Biotinyliertes Phaseolus vulgaris Leucoagglutinin </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Biotinyliertes Erdnußagglutinin </td><td> PNA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinylierte Pisum sativum Agglutinin </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinyliertes Dolichos biflorus Agglutinin </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Biotinyliertes Datura Stramonium Lektin </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinyliertes Sophora Japonica Agglutinin </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Biotinyliertes Sojabohnen Agglutinin </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinyliertes Solanum tuberosum (Kartoffel) Lektin </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinyliertes Griffonia (Bandeiraea) simplicifolia Lektin I </td><td> GSL ich </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinylierte Vicia villosa-Lectin </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinyliertes Lycopersicon esculentum (Tomate) Lektin </td><td> UEG </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Biotinyliertes Ulex europaeus Agglutinin I </td><td> UEA I </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinyliertes Jacalin </td><td> Jacalin </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> Ziegen-F (ab &#39;) 2 Fragment Anti-Human-IgM, Antikörper Fc5μ </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Esel F (ab &#39;) 2 Frag Anti-Human-IgG (H + L) einkörper- </td><td> AB1 </td><td> Jackson Immuno Research </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> Maus Anti-Human-IgG F (ab &#39;) 2-Antikörpers </td><td> AB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Anti-Human-Alpha-2-Makroglobulin polyklonalen Antikörper </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> Kaninchen anti-Human-alpha-1-Antitrypsin polyklonaler Antikörper </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> Maus Anti-Human-alpha-1-Antitrypsin monoklonalen Antikörper </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> Kaninchen anti-Human-alpha-1-Antitrypsin polyklonaler Antikörper </td><td> HANDELN </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> Kaninchen anti-humanAlpha-1-Antichymotrypsin polyklonaler Antikörper </td><td> HANDELN </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Maus Anti-Human-Transferrin monoklonalen Antikörper </td><td> Transferrin </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> Kaninchen anti-Human-Transferrin polyklonaler Antikörper </td><td> Transferrin </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Anti-Human-Apolipoprotein J polyklonaler Antikörper </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Maus Anti-Human-GP73 monoklonalen Antikörper </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Maus Anti-Human-GP73 monoklonalen Antikörper </td><td> GP73 </td><td> Santa Cruz Biotechnology INC </td><td> sc-101275 </td></tr><tr><td> A14 </td><td> Kaninchen anti-human alpha-1 fetoprotein polyklonaler Antikörper </td><td> AFP </td><td> Genway </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> Maus Anti-Human-alpha-1 Fetoprotein monoklonalen Antikörper </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Maus Anti-Human-Hämopexin monoklonalen Antikörper </td><td> Hämopexin </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> Maus Anti-Human-Glypican-3 (1G12) monoklonaler Antikörper </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65443 </td></tr><tr><td> A18 </td><td> Maus Anti-Human-Kininogen (LMW) monoklonaler Antikörper </td><td> Kininogen </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> Kaninchen anti-Human-MMP-21 monoklonalen Antikörpers </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Maus Anti-Human-CEACAM-1-monoklonalen Antiköry </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Ratten-Anti-Human-DPPIV/CD26 monoklonalen Antikörper </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Maus Anti-Human-PIVKA-II-monoklonalen Antikörpers </td><td> PIVICA </td><td> Kristall-chem </td><td> 8040 </td></tr><tr><td> A23 </td><td> Maus anti-carcinoembryonales Antigen </td><td> CEA </td><td> US biologischen </td><td> C1300 </td></tr><tr><td> A24 </td><td> Maus anti-CA125 Cancer Antigen </td><td> CA125 </td><td> US biologischen </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Maus anti-CA19-9 Cancer Antigen </td><td> CA19-9 </td><td> US biologischen </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> Maus Anti-Lewis-x monoklonalen Antikörper </td><td> Lewis-X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> Bio </td><td> Biotinyliertes BSA (positive Kontrolle) </td><td> Bio </td><td> Hausgemachte </td><td> N / A </td></tr></tbody></table> Tabelle 1. Liste der Lektine und Antikörper in diesem Protokoll verwendet. <table border="1"><tbody><tr><td> Name des Reagenzes s / Ausrüstungen </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Berührungslose Microarrayer </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 Mikroplatte </td><td> Fischer </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultradünne Nitrocellulose Coate Microarray-Slides </td><td> Gentel </td><td> PATH </td></tr><tr><td> Slide Imprinter (optional) </td><td> Das Gel Unternehmen </td><td> WSP60-1 </td></tr><tr><td> Shaker </td><td> Fischer </td><td> 15-453-211 </td></tr><tr><td> Zentrifugieren </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Slide Waschbecken / Glaszylinder, Schale mit Wechselrahmen </td><td> Fischer </td><td> 08-812 </td></tr><tr><td> Slide Inkubationskammer / Objektträger-Box </td><td> Fischer </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30 w / v% igen Lösung in Wasser </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> Fischer </td><td> P337-100 </td></tr><tr><td> Natriumperiodat (NaIO 4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> L-Glutaminsäure-γ-hydrazid </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> Natriumacetat wasserfrei (CH 3 COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Rinderserumalbumin (BSA) </td><td> Lampire Biologische Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> Phosphatgepufferte Kochsalzlösung (PBS) (10X) </td><td> Scientific Denville </td><td> CP4390-48 </td></tr><tr><td> DyLight 549 konjugierten NeutrAvidin </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Protease Inhibitor Cocktail Tablets </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure Human-IgG, Fc-Fragment </td><td> Jackson Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure Human-IgG, ganze Molekül </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure Maus-IgG, ganze Molekül </td><td> Jackson Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure Mouse IgG, Fc-Fragment </td><td> Jackson Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure Rabbit IgG, ganze Molekül </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure Donkey IgG, ganze Molekül </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> Tabelle 2. Liste der Geräte und Reagenzien in diesem Protokoll verwendet.

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

In dieser Studie beschreiben wir ein verbessertes Protokoll für eine Multiplex-Hochdurchsatz-Antikörper Mikroarray mit Lektin Detektionsverfahren, das in der Glykosylierung Profilierung spezifischer Proteine ​​verwendet werden können. Dieses Protokoll bietet zuverlässige neue Reagenzien und reduziert den Zeitaufwand, Kosten und Laborgeräte Anforderungen an das vorherige Verfahren verglichen.

Chemisch-Antikörper blockiert Microarray für Multiplex-High-Throughput-Profiling von spezifischen Protein-Glykosylierung bei 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 ...

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Biology

15.3K Aufrufe.  Institute for Hepatitis and Virus Research. In dieser Studie beschreiben wir ein verbessertes Protokoll für eine Multiplex-Hochdurchsatz-Antikörper Mikroarray mit Lektin Detektionsverfahren, das in der Glykosylierung Profilierung spezifischer Proteine ​​verwendet werden können. Dieses Protokoll bietet zuverlässige neue Reagenzien und reduziert den Zeitaufwand, Kosten und Laborgeräte Anforderungen an das vorherige Verfahren verglichen.