Name: profiling anti-neu5gc igg in human sera with a sialoglycan microarray assay
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Dette arbejde blev delvist støttet af en Research Career Development Award fra Israels Kræftforskningsfond, et tilskud fra det israelske nationale nanoteknologiske initiativ og Helmsley Charitable Trust for et fokalt teknologisk område for nanomedicin til personlig terapeutisk medicin (VP-K) og nationalt Institutter for sundhedsbidrag R01GM076360 (til XC).

Human sera prøver blev opnået fra den israelske blodbank og blev brugt i overensstemmelse med Helsinki-erklæringen og Tel Aviv Universitets Institutional Review Board. 1. Array Fabrication Planlægning og Layout <ol><li> Bestem dias layout. BEMÆRK: Hvert dias indeholder 16 sub-arrays opdelt i 16 identiske blokke nummereret B1 til B16 ( Figur 1B , Supplerende Figur 1A ). </li><li> Bestem pin layout. Brug fire stifter, hver udskriver ...

<ol>
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En vellykket glycan microarray-fremstilling kræver omhyggelig planlægning og indeholder flere vigtige trin i protokollen. Disse omfatter: (1) planlægning af blok- og pladelayouterne, der definerer alle efterfølgende parametre ( f.eks. Afstande, afstand, antal prøver og udskrivning); (2) rensning af stifterne og sikring af pin integritet, hvilket er kritisk for styring af spot homogenitet; (3) opretholdelse af høj luftfugtighed under tryk, hvilket er kritisk for at undgå prøveinddampning under lange prin...

Sias er sure sukkerarter, der dækker glycan kæder på celleoverflade glycoproteiner og glycolipider hos hvirveldyr. Sia-ekspression er modificeret i cancerceller 1 og korrelerer med progression og / eller metastase 2 , 3 . To af de store Sia former i pattedyr er Neu5Ac og dens hydroxyleret form Neu5Gc 2. Mennesker kan ikke syntetisere Neu5Gc på grund af en specifik inaktivering af genet kodende for CMAH-enzymet. Denne ikke-humane Sia inkorporerer metabolisk i menneskelige celler som &quot;selv&quot;, der stammer fra kostholdige Neu5Gc-rige fødevarer ( fx rødt kød) 4 , 5 . Neu5Gc er til stede i lave niveauer på celleoverfladerne af human epithelia og endothelia, men det akkumuleres især i carcinomer. Neu5Gc er anerkendt som fremmed af det humane humoral immune system 2 , 6 .Den antigeniske kompleksitet af Neu5Gc-glycaner kan opstå på flere niveauer, herunder Neu5Gc modifikation, binding, underliggende glycaner og stilladser, og deres tæthed, som alle afspejles af kompleksiteten af ​​anti-Neu5Gc-antistofrespons hos mennesker 6 . Nogle af disse antistoffer tjener som carcinombiomarkører og potentielle immunoterapeutika 7 . Fremkomsten af ​​den kemoenzymatiske syntese af forskellige sialoglycaner 8 banede vejen for den mere dybtgående analyse af sådanne antistoffer, lette ved anvendelse af glycan mikroarray teknologi 9 , 10 . Med den lettere forberedelse og manipulation af store biblioteker af naturlige og syntetiske kulhydrater er glycanmikarrayser blevet en stærk høj-gennemstrømningsteknologi til undersøgelse af vekselvirkningerne af kulhydrater med et utal af biomolekyler 10 , 11 , </sup&gt; 12 , 13 . I et arrayformat anvendes der små mængder materialer, og denne multivalente visning af biologisk relevante glycaner muliggør undersøgelse af tusindvis af bindende interaktioner i et enkelt forsøg. Det er vigtigt, at denne teknologi også kan anvendes til biomarkøropdagelse og til overvågning af immunresponser i forskellige prøver 7 , 12 . Succesfuld fremstilling af glykan mikroarray kræver overvejelse af tre vigtige aspekter: printerrobotypen, glykan-konjugationskemi og detektionsoptik. Med hensyn til udskrivningsinstrumentet er der to teknikker til rådighed: kontakt- og ikke-kontaktprintere. Ved kontaktudskrivning dyppes 1-48 stålstifter i en multi-well-kildeplade indeholdende glycanopløsning og ses på funktionaliserede glasskinner ved direkte at kontakte glasskærmens overflade. Opløsningen er deLeveret til diaset er en funktion af den langvarige varighed på diasoverfladen. Normalt er prøverne først forspottet på en glasblok (for at nå homogene punkter), før de trykkes på glidens overflade. I ikke-kontaktprintere ( fx den piezo-elektroniske printer), udskrives glycanerne fra en glaskapillær ved hjælp af kontrollerede elektriske signaler. Det elektriske signal kan finskalibreres for at opnå mere præcis udskrivning i forhold til kontaktudskrivning. Pletternes størrelse og morfologi er også relativt mere homogene. En yderligere fordel er genbrug af prøven tilbage til kildeskiltet efter udskrivning. Ikke desto mindre er den største ulempe ved piezo-elektroniske printere trykbegrænsningen (4 eller 8), hvilket resulterer i en meget lang udskrivningstid, hvilket kræver særlig opmærksomhed på glidestabilitet, temperatur, fugtighed og prøvefordampning. Inkjet-printeren uden kontakt kræver større prøvevolumener 14 . <p clasS = &quot;jove_content&quot;&gt; I modsætning til de begrænsede tilgængelige muligheder for trykmetoder er glykan-konjugationskemi en mere kompleks betragtning, med mange muligheder at vælge imellem. Udvalgt immobiliseringskemi skal tegne sig for både de aktive grupper på glycanerne og glidoverfladereaktiviteten. Glycanerne, der skal immobiliseres på en specifik mikroarrayoverflade, enten syntetisk syntetiseret eller naturligt isoleret, kræver alle en identisk reaktiv gruppe. Derudover skal glycans være rene og homogene. På den anden side bør immobiliseringsoverfladen og kæden tilvejebringe reproducerbarhed og pålidelig fastgørelsestæthed. Flere immobiliseringsmetoder er blevet udviklet med enten kovalent eller ikke-kovalent (fysisk absorption) vedhæftning 10 , 11 , 12 , 13 . For meget detaljerede oplysninger om trykt glycan microarray teknologi til uindviedeD undersøger, henvis til disse gode anmeldelser 13 , 15 . Det er vigtigt, at den seneste minimale information, der er påkrævet for et glykamikforsøg (MIRAGE), beskriver retningslinjer for prøvepræparation 16 og til rapportering af data fra glykanmikarrayanalyser 17 for at forbedre standarderne i dette voksende felt. Her beskriver vi en detaljeret protokol til fremstilling af sialoglycan-mikroarrays ved hjælp af en bestemt kontakt nano-printer i et 16-brøndsformat. Hver af glycans har en primær amin, der formidler deres kovalente forbindelse til epoxyaktiverede glasskinner. Vi beskriver også udviklingen og analysen af ​​et dias ved hjælp af forskellige humane sera prøver, antistoffer og Sia-bindende plante lectins. Sialoglycan microarray analyser involverer flere store trin, der omfatter array fabrikation, behandling, udvikling og analyse. Array fabrikation kræver planlægning af arRay-layout, forberedelse af glycans og kildeplade, programmering af nano-printeren og udskrivning af diasene. Derefter behandles, udvikles og analyseres diasene ( figur 1 ).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:269px;">Primary-amine containing sialoglycans</td>
			<td style="width:235px;">Glycohub, Inc., Davis, CA, USA (http://www.glycohub.com/services)</td>
			<td style="width:227px;">Contact info@glycohubusa.com for compound requests</td>
			<td style="width:476px;">Printed glycans</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Monosodium phosphate monohydrate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">S9638</td>
			<td style="width:476px;">Printing buffer component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Disodium phosphate heptahydrate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">S9390</td>
			<td style="width:476px;">Printing buffer component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Phosphate buffered saline</td>
			<td style="width:235px;">Hy-Labs</td>
			<td style="width:227px;">BP-507/500D</td>
			<td style="width:476px;">Printing buffer/ incubation/washing buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Tris-base</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">T1503</td>
			<td style="width:476px;">Slide blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Glycerol</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">G-7893</td>
			<td style="width:476px;">Printing buffer component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Ethanolamine</td>
			<td style="width:235px;">Thermo-Fisher Scientific</td>
			<td style="width:227px;">0700/08</td>
			<td style="width:476px;">Slide blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Ovalbumin (Grade V)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">A5503</td>
			<td style="width:476px;">Slide Blocking protein</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Tween-20</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">P7949</td>
			<td style="width:476px;">Slide washing detergent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Alexa 555-Hydrazide</td>
			<td style="width:235px;">Thermo-Fisher Scientific</td>
			<td style="width:227px;">A20501MP</td>
			<td style="width:476px;">Marker on array</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">ChromPure Human IgG, whole molecule</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">009-000-003</td>
			<td style="width:476px;">Printing component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Biotinylated- SNA</td>
			<td style="width:235px;">Vector Laboratories</td>
			<td style="width:227px;">B-1305</td>
			<td style="width:476px;">Plant Lectin &#8211; binding Sia-alpha2&#8211;6-linked</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Biotinylated-MALII</td>
			<td style="width:235px;">Vector Laboratories</td>
			<td style="width:227px;">B-1265</td>
			<td style="width:476px;">Plant Lectin &#8211; binding Sia-alpha2&#8211;3-linked</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Chicken-anti Neu5Gc IgY</td>
			<td style="width:235px;">BioLegend</td>
			<td style="width:227px;">146903</td>
			<td style="width:476px;">Primary detection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cy3-Streptavidin</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">016-160-0848</td>
			<td style="width:476px;">Biotin binding</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cy3-anti Human IgG</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">109-165-088</td>
			<td style="width:476px;">Secondary detection against human IgG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cy3-anti Chicken IgY</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">703-165-155</td>
			<td style="width:476px;">Secondary detection against chicken IgY</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Human sera samples</td>
			<td style="width:235px;">Israeli Blood Bank</td>
			<td style="width:227px;"></td>
			<td style="width:476px;">Primary detection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Compressed Nitrogen (Grade 5)</td>
			<td style="width:235px;"></td>
			<td style="width:227px;"></td>
			<td style="width:476px;">General dusting/drying tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Epoxy-coated slides</td>
			<td style="width:235px;">Corning</td>
			<td style="width:227px;">40044</td>
			<td style="width:476px;">Slides</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:269px;">Epoxy-coated slides</td>
			<td style="width:235px;">PolyAn</td>
			<td style="width:227px;">2D 104-00-221</td>
			<td style="width:476px;">Slides. In this type of slides the surface is more hydrophobic (compared to Coring slides) therefore the glycans Print Buffer would need to be supplemented with 0.005% Tween-20 to obtain 100 &#181;m size spots.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">384-well microtiter plate</td>
			<td style="width:235px;">Genetix</td>
			<td style="width:227px;">2070</td>
			<td style="width:476px;">Printing plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">VWR lab marker</td>
			<td style="width:235px;">VWR</td>
			<td style="width:227px;">52877-310</td>
			<td style="width:476px;">Slide labeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Staining Tube</td>
			<td style="width:235px;">ArrayIt</td>
			<td style="width:227px;">MST</td>
			<td style="width:476px;">Slide developing tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Staining bath</td>
			<td style="width:235px;">VWR</td>
			<td style="width:227px;">25608-904</td>
			<td style="width:476px;">Slide developing tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Slides glass holders</td>
			<td style="width:235px;">VWR</td>
			<td style="width:227px;">631-9321</td>
			<td style="width:476px;">Slide developing tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">GenePix Scanner</td>
			<td style="width:235px;">Molecular devices</td>
			<td style="width:227px;">4000B</td>
			<td style="width:476px;">Slide scanner</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LM-60 NanoPrinter</td>
			<td style="width:235px;">ArrayIt</td>
			<td style="width:227px;">LM-60</td>
			<td style="width:476px;">Array printer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Pins</td>
			<td style="width:235px;">ArrayIt</td>
			<td style="width:227px;">946MP3</td>
			<td style="width:476px;">Printing pins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">ProPlate Module</td>
			<td style="width:235px;">Grace Bio-Labs</td>
			<td style="width:227px;">P37004</td>
			<td style="width:476px;">Slide developing module</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Distilled water</td>
			<td style="width:235px;">Bio-Lab</td>
			<td style="width:227px;">2321020500</td>
			<td style="width:476px;">Required for arrayer and humidifier</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:269px;">Electronic Multi Pippete, 8 Channel , volume range 2-125 &#956;L</td>
			<td style="width:235px;">Thermo-Fisher Scientific (Matrix)</td>
			<td style="width:227px;">MA-2131 Impact2 Equalizer 384</td>
			<td style="width:476px;">Multi pippete for sample dispansing into 384-well plate</td>
		</tr>
	</tbody>
</table>

profiling anti-neu5gc igg in human sera with a sialoglycan microarray assay

Celler er dækket af en kappe af kulhydratkæder (glycaner), der almindeligvis ændres i kræft, og som omfatter variationer i sialinsyre (Sia) -ekspression. Disse er sure sukkerarter, der har en 9-carbon backbone, og at hatte hvirveldyr glycaner på celleoverflader. To af de vigtigste Sia-former i pattedyr er N- acetylneuraminsyre (Neu5Ac) og dens hydroxylerede form, N- glycolylneuraminsyre (Neu5Gc). Mennesker kan ikke producere endogent Neu5Gc på grund af inaktiveringen af ​​genet kodende for cytidin 5&#39;monophosphat-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Udenlandske Neu5Gc er erhvervet af humane celler gennem kostforbruget af rødt kød og mejeri og ses efterfølgende på forskellige glykaner på celleoverfladen, der hovedsagelig ophobes på carcinomer. Følgelig har mennesker cirkulerende anti-Neu5Gc-antistoffer, der spiller forskellige roller i kræft og andre kroniske inflammationsmidlede sygdomme, og som bliver potentielle diagnostiske og terapeutiske lægemidlerrgets. Her beskriver vi et high-throughput sialoglycan microarray assay for at vurdere sådanne anti-Neu5Gc antistoffer i humane sera. Neu5Gc-holdige glycaner og deres matchede kontrolpar (Neu5Ac-holdige glycaner), hver med en primær amin, er kovalent bundet til epoxy-coatede glasskinner. Vi eksemplificerer udskrivning af 56 dias i 16-brøndsformat ved hjælp af en specifik nanoprinter, der kan generere op til 896 arrayer pr. Print. Hvert dias kan bruges til at screene 16 forskellige humane sera prøver til evaluering af anti-Neu5Gc antistof specificitet, intensitet og mangfoldighed. Protokollen beskriver kompleksiteten af ​​dette robuste værktøj og giver en grundlæggende retningslinje for dem, der sigter mod at undersøge svaret på Neu5Gc diætcarbohydratantigen i forskellige kliniske prøver i et arrayformat.

Array Udskrivning, udvikling og analyse: Udskrivning af en sialoglycan-mikroarray med flere glycanprøver og humane IgG STD-kurver i 16 forskellige blokke kræver grundig kalibrering for at sikre, at alle prøver udskrives så ensartet som muligt i alle 16 blokke pr. Dias og til alle dias i samme print runde. Derfor kræves der flere kalibreringsforsøg, inden de specifikke trykparametre bestemmes, herunder buffersammensætning f...

Watch this Scientific Journal Video about Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay at JoVE.com

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

Profilering Anti-Neu5Gc IgG i humant serum med en Sialoglycan Microarray Assay

Et sialoglycan-mikroarray-assay kan anvendes til at evaluere anti-Neu5Gc-antistoffer i humane sera, hvilket gør det til et potentielt diagnostisk test med høj gennemløb for kræft og andre kroniske inflammationsmedierede humane sygdomme.

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

The overall goal of the sialoglycan microarray assay is to evaluate Anti-Neu5Gc IgG in human sera in a high-throughput manner against a diverse collection of sialoglycans. This method can help answer key questions in the glycoimmunology field, such as how Anti-Neu5Gc antibodies are involved with various human diseases including cancer, heart diseases, and xenotransplantation. The main advantage of this technique is that it allows high-throughput profiling of Anti-Neu5Gc antibodies against a myriad of sialoglycan antigens.Demonstrating the procedure will be Sharon Yehuda, a master student from my laboratory. Careful and detailed planning of array fabrication and layout is important to ensure success. This process is detailed in the text protocol and the supplementary information.Dilute each glycan to 100 micromolar in a total volume of 100 microliters of glycan printing buffer in microcentrifuge tubes. Next, prepare primary amine containing fluorescent dye to one milligram per millimeter with marker buffer. Then, dilute to one microgram per milliliter in a one milliliter total volume.Prepare human IgG standard curve dilutions as described in the text protocol. Place the 384 well plate on ice. Using an electronic multi-pipette, aliquot seven microliters of each glycan, marker, and stardard curve IgG according to the plate layout found in the text protocol.Cover the plate with parafilm and spin down for two minutes at 250 times g and 12 degrees Celsius. Printing must be carried out in dust-free area with a humidity of 60 to 70%Here, all steps are carried out in a clean room with appropriate gloves and clothing for protection. Turn on the nano printer machine and humidifier.After programming the printing machine as described in the text protocol, position the 384 well plate with aliquoted samples into its location on the arrayer deck and remove the parafilm cover. At this point, start printing. Number the slides with an alcohol solvent-resistant marking pen, and store them in a vacuum-sealed box in the dark.To process the arrays, first remove a slide from the vacuum-sealed box and place it in a chemical hood for five minutes, array facing up, to evaporate the excess liquid. Next, hydrate the slide by placing it it in a staining tube filled with deionized water. Cover the tube with foil and incubate for five minutes with slow and gentle shaking at room temperature.To block the reactive epoxy groups on the slide, dip the slide into the 50 milliliter staining tube filled with pre-warmed ethanolamine blocking solution. Cover the tube with foil and incubate for 30 minutes with slow and gentle shaking at room temperature. Discard the ethanolamine blocking solution into the appropriate biohazard trash receptacle.Then, place the slide in a new, clean staining tube filled with 50 degree celsius deionized water. Cover the tube with foil and incubate for 10 minutes with slow and gentle shaking at room temperature. Discard the water and transfer the slides to a glass staining holder.Place the apparatus into a swinging plate holder and centrifuge the slide dry. Next, place the slide onto a 16-well divider. After pre-wetting the wells, use a multi-pipette to aliquot 200 microliters of PBST into the slide wells.Then, place them in a covered, humid chamber and shake for five minutes. Remove the PBST by flicking and tapping gently on a clean paper towel. Then, aliquot 200 microliters of PBS-Ovalbumine blocking buffer into each well.Place the slides in a humid chamber and incubate for one hour at room temperature with gentle shaking. Next, prepare the primary detection, diluted in PBS-OVA blocking buffer as described in the text protocol. Flick PBS-OVA blocking buffer off the slide and add 200 microliters of primary antibody per well.Incubate the slides in a humid chamber for two hours. Then, flick the primary detection off the slide and wash three times with PBST. Incubate the slide in PBST for five minutes in a humid chamber before washing a final time with PBST.Then, wash once with PBS. Next, prepare the secondary antibody in PBS as described in the text protocol. Flick the PBS off the slide and add 200 microliters of secondary antibody.Flick the secondary antibody off the slide and wash four times with PBST as before. Carefully remove the frame, then place the slides into a 50 milliliter slide staining tube filled with PBS and shake for five minutes. Next, fill two slide staining baths with deionized water.Place the slide into the slide holder and dip the slide into the first bath. Quickly dip the slide ten times. Then, transfer the slide to the second bath and dip ten more times.After dipping, incubate the slide for ten minutes at room temperature with shaking. Following incubation, discard the water and transfer the slide to a glass staining holder. Place the glass staining holder in a swinging plate holder and centrifuge the slide dry.Open the scanner door and place the slide inside, array facing down. At this point, start the scanning. Save the scanned slides as a TIFF file.To prepare a slide for analysis, select Load Array List"and upload the appropriate GAL file that maps the arrays in each block on the slide. Slides were developed with 16 different primary detection moieties, one in each block. The plant Lectin SNA binds a2-6 linked sialic acid.Whereas the plant Lectin MALII binds a2-3 linked sialic acid. Polyclonal Anti-Neu5Gc IgY binds Neu5Gc-containing sialoglycans but does not bind Neu5Ac-containing sialoglycans. To profile Anti-Neu5Gc IgG, 12 sera from healthy human donors were analyzed on the printed sialoglycan microarray and developed using fluorescently labeled anti-human IgG in normalized to nanograms per microliter.All tested human sera contained various levels of Anti-Neu5Gc IgG with almost no recognition of the matched pairs of Neu5Ac-containing sialoglycans. Furthermore, Anti-Neu5Gc IgG recognition patterns are highly diverse between these 12 different sera, both in the level of intensity and diversity. After watching this video, you should have a good understanding of how to profile Anti-Neu5Gc antibodies in various human samples in a high-throughput manner, using a glycan microarray system.

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