This work was supported by &#34;Fondo de Investigaciones Sanitarias&#34; (FIS) grant PI13/01098 from Carlos III Health Institute, Spanish Ministry of Health. DB-G was benefited from a post-doctoral research position funded by the European Union Seventh Framework Programme (FP7/2007-2013) under the Grant Agreement 603049 (TRANSLINK). Work of NK, NS, and NB was supported by grant #14-50-00131 of Russian Science Foundation. DB-G wants to express his gratitude to Marta Broto, J. Pablo Salvador and Ana Sanchis for excellent technical assistance, and Alexander Rakitko for assistance in statistical analysis.&#160;With the support of the &#34;Pla de Doctorats Industrials de la Secretaria d&#39;Universitats i Recerca del Departament d&#39;Empresa i Coneixement de la Generalitat de Catalunya (grant number 2018 DI 021). We thank CERCA Programme / Generalitat de Catalunya for institutional support.

1. Glycochips Production
<ol>
	<li>Microarray preparation
	<ol>
		<li>Print the glycans (50 mM) and polysaccharides (10 &#181;g/mL) in 300 mM phosphate buffered saline (PBS, pH 8.5) at 6 replicates onto N-hydroxysuccinimide-derivatized glass slides, using non-contact robotic arrayer (drop volume ~900 pL). Each slide contains 4 different blocks of sub-arrays (Figure 1A, in colors) repeated 6 times. Every single sub-array is formed by 112 different glycan spots, including controls (8 rows &#215; 14 columns) (Figure 1B). 
		NOTE: Glycan-related information is provided in the Supplementary Table 1. The glycan library used for printing microchips is the result of a long-term synthetic effort of the IBCh team; examples of synthesis are described in related publications29,30,31,32,33,34,35,36,37,38. The glycan library included blood group antigens and some of the most frequently occurring terminal oligosaccharides, as well as core motifs of mammalian N- and O-linked glycoproteins and glycolipids, tumor-associated carbohydrate antigens, and polysaccharides from pathogenic bacteria.</li>
		<li>Incubate the slides in moisture box (relative humidity ~70%) at room temperature (25 &#176;C) for 1 h.</li>
		<li>Blocking microarrays: incubate the slides for 1.5 h with blocking buffer at room temperature (100 mM boric acid, 25 mM ethanolamine, 0.2% (v/v) Tween-20 in ultrapure water).</li>
		<li>Wash the glycochip with ultrapure water and dry it by air.</li>
	</ol>
	</li>
	<li>Glycochip quality control
	<ol>
		<li>Analyze two microarrays from each batch using 1mg/mL solution of complex immunoglobulin preparation (CIP, containing IgG, IgM and IgA), 10 &#181;g/mL solution of biotinylated goat anti-human immunoglobulins as a secondary antibody (IgM + IgG + IgA), followed by 1 &#181;g/mL solution of the corresponding fluorescent streptavidin conjugate (via protocol described below, see step 2).</li>
		<li>Scan and analyze the glycochips (see step 3, analysis of glycan array).</li>
		<li>Use microarray batches with intra- and inter-chip correlation higher than 0.9.</li>
	</ol>
	</li>
</ol>
2. Glycan Array Technique
<ol>
	<li>Prepare the following aqueous solutions (in ultrapure water) and store them at room temperature (25 &#176;C):
	<ul>
		<li>Buffer-1: 1% (w/v) bovine serum albumin (BSA) in PBS, 1% (v/v) Tween-20 and 0.01% (w/v) NaN3</li>
		<li>Buffer-2: 1% (w/v) BSA in PBS, 0.1% (v/v) Tween-20 and 0.01% (w/v) NaN3.</li>
		<li>Buffer-3: 0.1% (v/v) Tween-20 in PBS.</li>
		<li>Buffer-4: 0.001% (v/v) Tween-20 in PBS.</li>
	</ul>
	</li>
	<li>Glycochip and sample preparation
	<ol>
		<li>Put the storage box with the slides on the table until they reach room temperature (25 &#176;C). 
		NOTE: Use powder-free latex gloves. The glycochip must be manipulated by the bottom part of the glass slide, where the barcode is located. The barcode will help you to identify the right side, avoiding the contact with the surface where the glycans are printed.</li>
		<li>Open the box, take the glycochip and place it in the incubation chamber (25 &#176;C), already conditioned with wet filter paper to keep humidity constant inside the chamber.</li>
		<li>Meanwhile, dilute the mice serum with Buffer-1 (1:20) in 1.5 mL tubes. Homogenize the serum solution (5 s) with a vortex mixer. 
		NOTE: The volume needed to totally cover a single glycochip surface is approximately 1 mL.</li>
		<li>After the homogenization, incubate the diluted serum at 37 &#176;C for 10 min in a water bath to avoid immunoglobulin aggregation. Centrifuge the tubes for 3 min at 10,000 x g and 25 &#176;C, collect the supernatant and discard any precipitated material.</li>
		<li>Place the glycochip carefully in the incubation chamber. Incubate it for 15 min at 25 &#176;C with 1 mL of Buffer-3 to eliminate any residual material on the surface of the glycochip.</li>
		<li>Hold the glycochip in a vertical position and rewash it with some drops of Buffer-3 using a plastic Pasteur pipette. Carefully remove the buffer from the glycochip surface using filter paper.</li>
	</ol>
	</li>
	<li>Reaction: antibodies binding
	<ol>
		<li>Place the glycochip in the incubation chamber. Spread the diluted serum sample over the glycochip surface using a micropipette. Incubate with orbital agitation (30 rpm) at 37 &#176;C for 1.5 h. Ensure that all dry area of the glycochip surface is covered by the diluted serum sample using the tip of the pipette.</li>
		<li>Remove any excess sample and immerse the glycochip for 5 min in Buffer-3 at 25 &#176;C. Then, move the glycochip to a container with Buffer-4 (5 min) and finally wash (5 min) the glycochip with ultrapure water. Centrifuge the glycochip for 1 min at 175 x g and 25 &#176;C to remove the liquid.</li>
	</ol>
	</li>
	<li>Detection: secondary antibody
	<ol>
		<li>Place the glycochip in the incubation chamber. Spread over the glycochip surface a solution (5 &#181;g/mL) of goat anti-mouse (IgG + IgM) conjugated to biotin in Buffer-2. Incubate with orbital agitation (30 rpm) at 37 &#176;C for 1 h.</li>
		<li>Remove the unbound fraction and repeat the washing steps.</li>
		<li>After the centrifugation, incubate the glycochip in darkness at 25 &#176;C for 45 min (30 rpm) with 2 &#181;g/mL of the corresponding fluorochrome-labeled streptavidin solution (in Buffer-2).</li>
		<li>In darkness, remove the unbound fraction and repeat the washing steps.</li>
		<li>Dry the glycochip by air. 
		Note: Glycochip should be scanned as soon as possible. But if it&#39;s impossible to do scan immediately after staining, glycochips can be stored in a cool and dry place in darkness.</li>
	</ol>
	</li>
</ol>
3. Analysis of Glycan Array
<ol>
	<li>Scan the array
	<ol>
		<li>Leave the glycochip on the table until it reaches room temperature in dark. At the same time, turn on the slide scanner and the laser (excitation wavelength of 633 nm).</li>
		<li>Holding the microarray, slide the glycochip into the slot until it touches the back.</li>
		<li>Scan the glycochip (&#34;run easy scan&#34;), and save the scan as a &#34;.TIFF&#34; file.</li>
	</ol>
	</li>
	<li>Array quantification
	<ol>
		<li>Quantify the array using a ScanArray Analysis System. Open previously scanned images, by clicking &#34;File&#34; in the &#34;Configure &#38; File group&#34; on the Main Window (Figure 1B-D)</li>
		<li>Load the corresponding array file template in GAL format (disposition of printed glycans on the glass slide) (Figure 1C).</li>
		<li>Adjust GAL template by carefully aligning the array (grids) with the spots in the image and initiate quantification (Figure 1D).</li>
		<li>Select the quantification parameters:
		<ul>
			<li>Quantification type: Run Easy Quant.</li>
			<li>Quantification method: Fixed Circle</li>
			<li>Auto find Spots: unclick all options</li>
			<li>Normalization Method: LOWESS (Locally Weighted Scatter Plot Smoothing).</li>
		</ul>
		</li>
		<li>Save the quantified data as &#34;.CSV&#34; file (Figure 1D). Transfer this data into a common spreadsheet file using Microsoft Excel or another appropriate application.</li>
		<li>Use the interquartile range (IQR) as the main statistical method: calculation of the median (Quartile 2) of all signals for each ligand and the interquartile deviation (75th and 25th percentiles, or upper and lower quartiles Q3 and Q1, respectively).</li>
		<li>Perform interactive exploration of data by using a Hierarchical Clustering Explorer application.</li>
		<li>Use clustering parameters: Average Linkage (UPGMA) and Euclidean distance as similarity distance measure. Perform hierarchical clustering by rows without normalization.</li>
	</ol>
	</li>
</ol>

Nailya Khasbiullina and Alexey Nokel are employees of Semiotik LLC, which is the supplier of the glycochips used in this study.

Glycan microarrays have become indispensable tools for studying protein-glycan interactions40. The present work describes a protocol based on PGA technology to study the repertoire of circulating of anti-carbohydrate antibodies in BALB/c mice. Since PGA offers the possibility to screen large numbers of biologically unknown glycans, it is an exceptionally convenient discovery tool13,15,28. The proposed met...

Antibodies play a central role in our defense against invading pathogens by directly neutralizing viruses1,2 and bacteria2,3, by activating the complement system4,5 and the enhancement of phagocytosis6. Additionally, they are essential elements in cancer targeting and elimination of malignant cells7, and homeostasis maintenance8,9.
Disorders of the immune system can result in autoimmune and inflammatory diseases10 and cancer11. All these pathological conditions ideally demand a prompt diagnosis for an efficient treatment. In the case of autoimmune disorders, the serological presence of autoantibodies in most of the cases is a predictor for diagnostic of autoimmunity10,12. These antibodies react with the cell surface and extracellular autoantigens and, they are often present for many years before the presentation of autoimmune disease10,12. Immune deficiencies and cancer are also diagnosed with blood tests that either measure the level of immune elements such as antibodies, or their functional activity11.
The identification of the repertoire of circulating antibodies and their serological levels are paramount to set a prognosis and evaluate the progression of all of the mentioned pathological conditions. We have previously demonstrated the potential of PGA technique for the analysis of circulating antibodies in different animal species13-16, minimizing the use of large volumes of serological samples, avoiding the problem associated with antibodies cross-reactivity17 and allowing high-throughput profiling of an extensive repertoire of antibodies15.
Glycan-based immunoassays are mainly conditioned, among other factors, by the origin and production of carbohydrates, which determine the affinity and binding of ligands15,18,19,20,21. Glycan-based immunoassays can be developed in suspension (microspheres)15,21,22 or in flat-activated surfaces15,21,22,23,24. The last include ELISA (the most conventional of these methods) and PGA. There is not much data comparing these methodologies in the same experimental setting15,25,26,27. We have previously compared the efficacy and selectivity of these immunoassays to profile anti-glycan antibodies in individual human plasma samples15. For some antibodies such as those targeting anti-A/B blood group, all the immunoassays could detect them with statistical significance and they positively correlated with each other15,18,21. Meanwhile, anti-P1 antibodies were primarily detected by PGA with the highest discriminative power, and there was no correlation in the determinations made by the different glycan-based immunoassays15,18,21. These differences between methods were mainly related to the antibody/antigen ratio and glycan orientation15. ELISA and suspension arrays are more susceptible to unspecific binding than PGA because there is an excess of antigen over antibodies in these methods15. Additionally, the orientation of glycans in the PGA is more restricted than in ELISA and suspension arrays15. ELISA is convenient when the study includes a limited panel of glycans. Along with suspension arrays, ELISA offers broader flexibility regarding assay reconfiguration. PGA is exceptionally convenient for discovery approaches15,18,21,28. Despite these clear advantages and disadvantages, the three mentioned immunoassays could be used to study different aspects of glycan-antibody interactions. The final goal of the study is the one will guide the selection of the more suitable methodology.
The present work aims to extend the use of PGA technology for the analysis of the repertoire of circulating anti-glycan antibodies in small animals. As a representative result, we present here a detailed protocol to assess the repertoire of natural anti-carbohydrate antibodies in adult BALB/c mice by PGA.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>biotinylated goat anti-human Igs</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>Ref. #: 31782</td><td></td></tr><tr><td>biotinylated goat anti-mouse IgM + IgG</td><td>Thermo Fisher Scientific</td><td>Ref. #: 31807</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Robotic Arrayer sciFLEXARRAYER S5&amp;nbsp;</td><td>Scienion AG, Berlin, Germany</td><td>http://www.scienion.com/products/sciflexarrayer/</td><td></td></tr><tr><td>Stain Tray (slide incubation chamber)</td><td>Simport, Beloeil, QC, Canada</td><td>Ref. #: M920-2</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hamburg, Germany&amp;nbsp;</td><td>Ref. #: 5810 R</td><td></td></tr><tr><td>Pipettes</td><td>Gilson, Middleton, WI, USA</td><td>http://www.gilson.com/en/Pipette/</td><td></td></tr><tr><td>Slide Scanner&amp;nbsp;</td><td>PerkinElmer, Waltham, MA, USA</td><td>ScanArray G&lt;sub&gt;X&lt;/sub&gt; Plus&amp;nbsp;</td><td></td></tr><tr><td>Shaking incubator</td><td>Cole-Parmer, Staffordshire, UK</td><td>Ref. #: SI50</td><td></td></tr><tr><td>&lt;strong&gt;Biological samples&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BALB/c mice sera</td><td>This paper</td><td>N/ A</td><td></td></tr><tr><td>Complex Immunoglobulin Preparation (CIP)</td><td>Immuno-Gem, Moscow, Russia</td><td>http://www.biomedservice.ru/price/goods/1/17531</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals, Reagents and Glycans&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glycan library</td><td>Institute of Bioorganic Chemistry (IBCh), Moscow, Russia</td><td>N/ A</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich, St. Louis, MO,&amp;nbsp;</td><td>Ref. #: A9418</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>Ref. #: 411000</td><td></td></tr><tr><td>Tween-20</td><td>Merck Chemicals &amp;amp; Life Science S.A., Madrid, Spain</td><td>Ref. #: 655204</td><td></td></tr><tr><td>Phospahte buffered saline (PBS)</td><td>VWR International Eurolab S.L, Barcelona, Spain</td><td>Ref. #: E404</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>Ref. #: S2002</td><td></td></tr><tr><td>Streptavidin Alexa Fluor 555 conjugate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>Ref. #: S21381</td><td></td></tr><tr><td>Streptavidin Cy5 conjugate</td><td>GE Healthcare, Little Chalfont, Buckinghamshire, UK</td><td>Ref. #: PA45001</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N-hydroxysuccinimide-derivatized glass slides H&amp;nbsp;</td><td>Schott-Nexterion, Jena, Germany</td><td>Ref. #: 1070936</td><td></td></tr><tr><td>Whatman filter paper&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Ref. #: WHA10347509</td><td></td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf&amp;nbsp;</td><td>Ref. #: 0030120086</td><td></td></tr><tr><td>&lt;strong&gt;Software and algorithms&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ScanArray Express Microarray Analysis System</td><td>PerkinElmer</td><td>http://www.per</td><td></td></tr><tr><td>kinelmer.com/microarray</td><td></td><td></td><td></td></tr><tr><td>Hierarchical Clustering Explorer application</td><td>University of Maryland, MD, USA</td><td>http://www.cs.umd.edu/hcil/hce/</td><td></td></tr></tbody></table>

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.

Here, we present a summary of representative results obtained from the quantification of the repertoire of natural anti-glycan antibodies in a population of 20 BALB/c mice. The glycochips used in this study contained 419 different glycan structures. Most glycans were synthesized as -CH2CH2CH2NH2 spacer-armed O-glycosides, in several cases as -CH2CH2NH2 or -NHCOCH2NH2 glycosides. All...

Watch this Scientific Journal Video about Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals at JoVE.com

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

This work shows the potential of printed glycan array (PGA) technology for the analysis 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 ...

printed-glycan-array-sensitive-technique-for-analysis-repertoire

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Immunology and Infection

6.8K Views.  Bellvitge Biomedical Research Institute (IDIBELL). Het algemene doel van dit protocol is het bestuderen van het profiel van circulerende anti-glycan antilichamen in het serum van kleine dieren met behulp van een gedrukte glycan array gebaseerde technologie. Deze aanpak heeft een potentieel in de vroege diagnose en de juiste behandeling in sommige pathologische aandoeningen waar antilichamen tegen glycanstructuren een belangrijke rol spelen. Het belangrijkste voordeel van deze techniek is de mogelijkheid om in dezelfde experimentele setting ho...

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

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

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.

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

Behavior

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

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 commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

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

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

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 ...

Chemistry

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.

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

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

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.

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

Bioengineering

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.

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

Biology

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

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

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

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

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app.jove.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

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

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