This research was supported by a Hermes Fellowship to Ola Negm and Tanya Monaghan and through separate funding from the Nottingham Digestive Diseases Centre and the NIHR Nottingham Digestive Diseases Biomedical Research Centre.

1. Preparing a Microarray Plate
<ol>
	<li>Dilute C. difficile antigens with a printing buffer [PBS-Tween-trehalose (50 mM)] at the optimum concentration (which was predefined before running the patient sera): toxin A (200 &#181;g/mL) and toxin B (100 &#181;g/mL), pCDTb (200 &#181;g/mL), and purified native whole SLPs derived from ribotypes 001, 002, and 027 (200 &#181;g/mL). 
	NOTE: Toxin B was obtained from a toxin B-expressing strain CCUG 20309 (90 &#181;g/mL).
	<ol>
		<li>Dilute the positive controls, lysates of C. albicans, and tetanus toxoid with the printing buffer at 100 &#181;g/mL. Finally, dilute the purified human immunoglobulin matching the tested antibody isotype serially, starting at 50 &#181;g/mL across 10 dilutions in the printing buffer to create a calibration curve.</li>
	</ol>
	</li>
	<li>Transfer 10 &#181;L of the dilutions in the printing buffer into a 384-well plate.</li>
	<li>Cover the plate with a plate seal and centrifuge at 300 x g for 5 min.</li>
</ol>
2. Printing Arrays with a Contact Robot
<ol>
	<li>Heat the silicon pin using the hand-held gas burner 3x 2 s each and rinse in clean water 3x 3 s each.</li>
	<li>Place the microarray plate into the loading cartridge of the arrayer.</li>
	<li>Place the aminosilane slides on the slide tray. 
	NOTE: The slide tray can hold 27 slides: three rows of nine slides. The slides are arranged in portrait orientation starting from the left side of the bottom row and the rest of the spaces are covered with blank slides to ensure that all the holes on the slide tray are covered.
	<ol>
		<li>Press OK and check that a vacuum has been turned on&#160;and all the slides are secure. Leave the slides in the humid environment for at least 1 h before starting the print. 
		NOTE: The aminosilane slides are provided in a sealed pouch with desiccant. Once opened, any unused slides should be returned immediately to the desiccator for storage up to the expiration date.</li>
	</ol>
	</li>
	<li>Set up the suitable printing programme to print Clostridium difficile antigens. Launch the TAS Application Suite and open the required print run parameters file from the &#39;My Gridding Runs&#39; directory. Select Clostridium difficile file for printing all the Clostridium difficile antigens in quadruplicate.</li>
	<li>Upon double-clicking on the MicroArray icon on the main window a three-tabbed window will appear called &#8216;MicroArraying parameters&#8217;.&#160; The three tabs are &#8220;Source,&#8221;&#160;&#8220;Target,&#8221; and &#8220;Options.&#8221;&#160;The &#8220;Source&#8221; tab shows details the number of samples used in the microarray plate. The &#8220;Target&#8221; tab shows details of how the slides are to be loaded, where the array should be printed on the slide and edit the slide layout (16 subarray formats). The &#8220;Options&#8221; tab shows details of the wash protocol and tool to be used e.g. wash after every completed sample. Clean the silicon pin between samples to avoid any cross contamination between different samples.&#160;&#160;</li>
	<li>The programming options are located under Options&#62;Run Preferences on the TAS Application Suite toolbar. There are 9 tabs to work through in this section and as you complete one tab move to the next by clicking on it. Clicking the &#8220;OK&#8221; the button will take you out of &#8220;Run Preferences.&#8221; In the &#8220;General&#8221; tab of the &#8220;Run preferences,&#8221; there are a number of check boxes available under this section relating to how the instrument will behave when a program is started. Check the &#8220;Prime bath before start of run&#8221; box, all three wash stations will undergo a short priming sequence prior to the run beginning. Check the &#8220;Wash at start of run&#8221; box, the pin tool will be washed before the first source visit. Check &#8220;Wash at end of run&#8221; box, the pin tool will be washed after the last source visit. The user can set the number of wash cycles. In the &#8220;Climate&#8221; tab of the &#8220;Run preferences,&#8221; start the humidification. The setting we use are as follows:&#160; start rate 55%, run rate 55%, minimum humidity 55%, target humidity 60%. Do not start the run until the target humidity has been reached, otherwise the spots will be the wrong size and the library will rapidly evaporate.</li>
	<li>In the menu bar of TAS, click the green go button.&#160; Start the run and periodically observe the arrayer during the print run to be certain everything is OK.&#160; 
	Note: This enables the analysis of 15 samples in parallel on each slide as one subarray is used as a negative control. The design of subarrays is determined by the number of the printed proteins (antigens) and the number of the printed biological replicates.&#160;&#160; 
	Note: After printing each sample, the head moves toward the washing baths to allow multiple dipping of the pin into two wash baths containing distilled water and 0.002% Tween 20 for 1.5 s in each bath, followed by rinsing the pin with ultra-pure water and drying the pin in the main wash station for 4 s.</li>
</ol>
3. Storage of the Arrays
<ol>
	<li>Keep the printed slides stored overnight at room temperature in the desiccator. 
	NOTE: The printed slides can be stored for up to one week.</li>
</ol>
4. Array Probing
<ol>
	<li>Carefully place printed slides in a slide holder of 16 multi-well chambers format. 
	NOTE: The chambers, having a depth of approximately 7.5 mm, provide a generous surface to volume ratio to facilitate mixing and washing steps.</li>
	<li>Allow all reagents to warm to room temperature before use. Unless specified otherwise, perform all the following steps at room temperature.</li>
	<li>Add 100 &#181;L of filtered 5% bovine serum albumin tween (BSAT; use a 0.2 &#181;m syringe filter) to each block, cover the slides, and incubate them with shaking for 1 h.</li>
	<li>Wash the slides 5x 1 min each in 120 &#181;L of phosphate buffered saline with Tween 20 (PBST; 0.1% Tween) per well with shaking. Do not let the slides dry out.</li>
	<li>Thaw the serum samples on ice for 20 min and dilute each sample with a commercially available antibody diluent with background reducing component (see Table of Materials).
	<ol>
		<li>Optimize the dilution of the serum samples according to the tested immunoglobulin as shown in Table 2.</li>
	</ol>
	</li>
	<li>Transfer 100 &#181;L of the diluted serum samples to all blocks except one, which will be used as a negative control; to this block, add 100 &#181;L of antibody diluent only. Incubate the slides with gentle agitation for 1 h.</li>
	<li>Wash the slides 5x 3 min each in 120 &#181;L of PBST (0.1% Tween) per well with shaking.</li>
	<li>Add 100 &#181;L of a biotinylated goat anti-human antibody diluted with antibody diluent.
	<ol>
		<li>Optimize the dilution of the secondary antibody according to the tested immunoglobulin as described in Table 2. Cover the slides and incubate them with gentle shaking for 1 h.</li>
	</ol>
	</li>
	<li>Wash the slides 5x 3 min each in 120 &#181;L of PBST per well with shaking.</li>
	<li>Add 100 &#181;L of streptavidin Cy5 to each block diluted at 1:2000 in 5% BSA. Cover the slides with foil to protect them from light whilst shaking for 15 min with gentle agitation.</li>
	<li>Wash the slides 5x 1 min each in 120 &#181;L of PBST per well with shaking, followed by 2 washes of 1 min each in PBS only with shaking.</li>
	<li>Spin the slides for 5 min at 300 x g to dry them.</li>
	<li>Keep the slides in a dark box for transport and storage at room temperature.</li>
	<li>Proceed to scan the arrays immediately to ensure signal consistency after probing. 
	NOTE: However, probed slides can be stored in the dark and scanned within one week from probing.</li>
</ol>
5. Scanning the Arrays
<ol>
	<li>Switch on the laser scanner (see Table of Materials) 30 min before scanning to warm up the laser. Upload the scanner software and connect the computer to the scanner.</li>
	<li>Place a slide with the printed side face up into the scanner until the play light turns solid green.</li>
	<li>Press the Settings button and adjust the following settings when scanning the slides as follows: Scan Mode, Median; Acquisition, 635 Only; Acquisition Mode, Manual; Gain, 20; Power, Low; Slide Type, Unlabeled Slide; Focus, Auto Focus. Turn Automatic Scrolling off and set Barcode Reading to Pixel Size 10 and Scan 35.</li>
	<li>Save the resultant images as 16-bit grayscale multiple image TIFF format. Press the Import Grid button and select the appropriate array list. 
	NOTE: The array list describes the size and position of subarrays and the names of the printed substances associated with each feature-indicator.
	<ol>
		<li>Align the grid to the spots on the slide.</li>
	</ol>
	</li>
	<li>Analyze the images by pressing the Quantification process&#160;button to measure the fluorescence signal intensities of each spot and save the results in a text format called GenePix Results (GPR). 
	NOTE: The GPR file contains the localization and identification variables of the antigen targets on the array and also the median fluorescence intensity and the local background that represents the antibody binding signal values of each spot.</li>
</ol>
6. Data Analysis
<ol>
	<li>Calculate the median for the replicates of each antigen after background subtraction using a free microarray analysis software called reverse phase protein array (RPPA) analyzer, a module within the R statistical language on CRAN36.</li>
	<li>Plot the standard curve and interpolate the signals of each antigen relative to the immunoglobulin standard curve using commercial scientific graphing and statistics software (see Table of Materials) to calculate the antibody levels.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+microarray+surface+chemistries.">Current microarray surface chemistries.</a> Methods in Molecular Biology. 381, 37-75 (2007).</li><li>Wellhausen, R., Seitz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facing+current+quantification+challenges+in+protein+microarrays.">Facing current quantification challenges in protein microarrays.</a> Journal of Biomedicine and Biotechnology. 2012, (2012).</li><li>Guo, A., Zhu, X. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+role+of+surface+chemistry+in+protein+microarrays.">The critical role of surface chemistry in protein microarrays.</a> Functional Protein Microarrays in Drug Discovery. , (2007).</li></ol>

In this protocol, we have shown that microarray is a suitable platform for defining humoral immune responses to C. difficile protein antigens in patient sera (Figures 3 and 6) and commercial preparations of IVIg (Figure 5). We have also demonstrated that the microarray technique performs well when compared to conventional ELISA (Figure 2) and shows excellent reproducibility, with intra- and inter-assay variabilities fal...

This protocol describes the development and validation of a novel and customized protein microarray assay for the detection and semi-quantification of bacterial strain and isotype-specific antibody responses to C. difficile antigens. We successfully use our C. difficile-specific microarray assay as a promising new tool for the compositional bioanalysis of specific antibody content in patient sera1,2, preparations of IVIg3, and identification of antibody specificities that correlate with poor outcomes in CDI4. We demonstrate how biobanked serum samples and commercial preparations of IVIg can be analyzed on microarray slides, allowing high-quality reproducible profiling of C. difficile pathogen-specific antibody responses in this assay.
Many healthy children and adults have detectable serum IgG and IgA antibodies to C. difficile toxins A and B5,6. These are thought to arise following transient exposure during infancy and following exposure to C. difficile in adulthood. For this reason, polyclonal IVIg has been used off-label to treat both recurrent and fulminant CDI7,8,9. However, its definitive role and mode of action remains unclear. Several studies have shown that the humoral immune response to C. difficile toxins plays a role in disease presentation and outcome. Specifically, asymptomatic patients show an increased serum anti-toxin A IgG concentration compared to patients who develop symptomatic disease10. A demonstrable association has been reported for median anti-toxin A IgG titers and 30-day all-cause mortality11. Several reports have also revealed an association with a protection against recurrence and antibody responses to toxin A, B, and several non-toxin antigens (Cwp66, Cwp84, FliC, FliD, and surface layer proteins (SLPs))12,13,14,15. These observations have spurred the development of the first passive immunotherapy drug targeting C. difficile toxin B (bezlotuxumab), which has recently been approved by the US Food and Drug Administration and the European Medicines Agency for the prevention of recurrent CDI16. Vaccination strategies using inactivated toxins or recombinant toxin fragments are also currently under development17,18,19. These new therapeutic approaches will undoubtedly stimulate the requirement for evaluating humoral immune responses to multiple antigens in large sample sizes.
Today, there is a notable lack of commercially available high-throughput assays capable of simultaneously assessing bacterial strain and isotype-specific antibody responses to C. difficile antigens. There is an unmet need to develop such assays to facilitate future research efforts and clinical applications. Protein microarrays are a method to immobilize large numbers of individually-purified proteins as a spatially organized array of spots onto a microscopic slide-based surface by using a robotic system, which can be either a contact20 or a non-contact printing tool21. The spots may represent complex mixtures such as cell lysates, antibodies, tissue homogenates, endogenous or recombinant proteins or peptides, body fluids or drugs22,23.
Protein microarray technology offers distinct advantages over standard in-house ELISA techniques, which have traditionally been used to assess anti-C. difficile antibody responses. These include an increased capacity for detecting a range of multi-isotype-specific antibodies against a more extensive panel of protein targets, reduced volume requirements for antigens, samples, and reagents, and an enhanced ability to incorporate a larger number of technical replicates, in addition to multiple internal quality control (QC) measures1. Microarrays are therefore more sensitive, accurate, and reproducible and have a greater dynamic range. These factors make microarrays a cheaper and potentially favorable alternative to ELISAs for the large-scale detection of known proteins. However, disadvantages of microarray technology result mainly from the large up-front costs associated with establishing a panel of highly purified antigens and setting up the technological platform.
Protein microarrays have been extensively used over the past two decades as a diagnostic and basic research tool in clinical applications. Specific applications include protein expression profiling, the study of enzyme-substrate relationships, biomarker screening, analysis of host-microbe interactions, and profiling antibody specificity23,24,25,26,27,28. Many new pathogen protein/antigen microarrays have been established, including malaria (Plasmodium)29, HIV-130, influenza31, severe acute respiratory syndrome (SARS)32, viral hemorrhagic fever33, herpesviruses34, and tuberculosis35.
The present protocol relates to the establishment of an easy operating C. difficile reactive antigen microarray assay, which enables accurate, precise, and specific quantification of multi-isotype and strain-specific antibody responses to C. difficile antigens in sera and polyclonal IVIg. Herein, we include representative results pertaining to an acceptable microarray assay performance when compared to ELISA as well as assay precision and reproducibility profiles. This assay could be further developed to profile other clinical samples and sets a new standard for research into the molecular basis of CDI.

<table><tbody><tr><td>BioRobotics MicroGrid II arrayer</td><td>Digilab, Malborough, MA, USA</td><td>N/A</td><td>Contact arrayer used to automated spotting of the antigens onto the slides.</td></tr><tr><td>Scanner InnoScan 710.</td><td>Innopsys</td><td>N/A</td><td>A fluorescent microarray slide scanner with a red (Cy5) laser to read the reaction.</td></tr><tr><td>MAPIX software version 7.2.0</td><td>Innopsys</td><td>N/A</td><td>Measure signal intensities of the spots.</td></tr><tr><td>Silicon contact pin</td><td>Parallel Synthesis Technologies</td><td>SMT-P75</td><td>Print the samples onto the slides.</td></tr><tr><td>Thermo Scientific Nalgene Desiccator</td><td>Thermo Scientific</td><td>41102426</td><td>To store the new and printed slides.</td></tr><tr><td>384-well plate</td><td>Genetix</td><td>X7022UN</td><td>To prepare the antigens.</td></tr><tr><td>Plate cover</td><td>Sigma Aldrich, UK</td><td>CLS6570-100EA</td><td>To reduce evaporation of the samples.</td></tr><tr><td>Aminosilane slides</td><td>Schott,&amp;nbsp; Germany</td><td>1064875</td><td>The slide of choice for printing the antigens.</td></tr><tr><td>Slide holders</td><td>GraceBio Labs, USA</td><td>204862</td><td>Divide the slides into identical 16 subarrays. These holders are re-usable, removable, leak-proof wells .&amp;nbsp;</td></tr><tr><td>Candida albicans lysate</td><td>NIBSC</td><td>PR-BA117-S</td><td>Positive control</td></tr><tr><td>Tetanus Toxoid&amp;nbsp;</td><td>Athens Research and Technology</td><td>04/150</td><td>Positive control</td></tr><tr><td>Immunoglobulin G (IgG), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707</td><td>Purified Native Human Immunoglobulin G IgG, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G1 (IgG1), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-1</td><td>Purified Native Human Immunoglobulin G1 IgG1, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G2 (IgG2), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-2</td><td>Purified Native Human Immunoglobulin G2 IgG2, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G3 (IgG3), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-3</td><td>Purified Native Human Immunoglobulin G3 IgG3, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin G4 (IgG4), Normal Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090707-4</td><td>Purified Native Human Immunoglobulin G4 IgG4, Human Plasma.&amp;nbsp;</td></tr><tr><td>Immunoglobulin A (IgA), Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090701</td><td>Purified Native Human Immunoglobulin A (IgA), Human Plasma.</td></tr><tr><td>Immunoglobulin A1 (IgA1), Human Myeloma Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090701-1M</td><td>Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma.</td></tr><tr><td>Immunoglobulin A2 (IgA2), Human Myeloma Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090701-2M</td><td>Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma.</td></tr><tr><td>Immunoglobulin M (IgM), Human Plasma</td><td>&amp;nbsp;Athens research and&amp;nbsp; technology&amp;nbsp;&amp;nbsp;</td><td>16-16-090713</td><td>Purified Native Human Immunoglobulin M (IgM), Human Plasma.</td></tr><tr><td>Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific</td><td>Vector Labs</td><td>BA-3080</td><td>Goat anti- human IgG (&amp;gamma;-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG1 Hinge-BIOT</td><td>Southern Biotec</td><td>9052-08&amp;nbsp;</td><td>Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG2 Fc-BIOT</td><td>Southern Biotec</td><td>9060-08&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG3 Hinge-BIOT</td><td>Southern Biotec</td><td>9210-08&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgG4 pFc&#039;-BIOT</td><td>Southern Biotec</td><td>9190-08&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Anti-Human IgA, alpha chain specific, made in goat - Biotinylated</td><td>Vector Labs</td><td>BA-3030</td><td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgA1-BIOT</td><td>Southern Biotec</td><td>9130-08</td><td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgA2-BIOT</td><td>Southern Biotec</td><td>9140-08&amp;nbsp;&amp;nbsp;</td><td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>Mouse Anti-Human IgM-BIOT</td><td>Southern Biotec</td><td>9020-08&amp;nbsp;</td><td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td></tr><tr><td>0.2 mm syringe filter</td><td>Thermo scientific</td><td>723-2520</td><td>Filter the 5% BSA.</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich, UK</td><td>A7284</td><td>Use 5% BSA for blocking the slides.</td></tr><tr><td>Antibody diluent</td><td>Dako, UK</td><td>S3022</td><td>To dilute the serum and the secondary antibody.</td></tr><tr><td>Streptavidin Cy5</td><td>eBioscience</td><td>SA1011</td><td>Detection of the immune reaction.</td></tr><tr><td>Purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087)</td><td>Toxins Group, Public Health England</td><td>NA</td><td></td></tr><tr><td>Purified whole C. difficile toxin B (CCUG 20309 toxin B only expressing strain)</td><td>Toxins Group, Public Health England</td><td>NA</td><td></td></tr><tr><td>Precursor form of B fragment of binary toxin, pCDTb</td><td>University of Bath&amp;nbsp;</td><td>NA</td><td>Produced in E. Coli from wholly synthetic recombinant gene construct. Amino acid sequence based on published sequence from 027 ribotype (http:www.uniprot.org/uniprot/A8DS70)</td></tr><tr><td>Purified native whole ribotype-specific (001, 002, 027) surface layer proteins</td><td>Dublin City University&amp;nbsp;</td><td>NA</td><td></td></tr><tr><td>Vigam (IVIg preparation 1)</td><td>Nottingham University Hospitals NHS Trust</td><td>N/A</td><td></td></tr><tr><td>Privigen (IVIg preparation 2)</td><td>Nottingham University Hospitals NHS Trust</td><td>N/A</td><td></td></tr><tr><td>Intratect (IVIg preparation 3)</td><td>Nottingham University Hospitals NHS Trust</td><td>N/A</td><td></td></tr></tbody></table>

a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

Figure 1 illustrates a flowchart describing the major steps in the described protocol. Figure 2 shows Spearman correlation tests demonstrating significant agreement between microarray and ELISA for IgG and IgA anti-toxin A and B levels in the patient test sera. Figure 3 shows differential IgG and IgA antibody-class specific antibody responses to toxin A, toxin B, and binary toxin (pCDTb) in patients ...

Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

a-protein-microarray-assay-for-serological-determination-antigen

Research

JoVE Journal

Immunology and Infection

10.1K Views.  University of Nottingham. Dit artikel bevat een beschrijving van een eenvoudige eiwit microarray methode voor profilering humorale immuunreacties aan een 7-plex panel van sterk gezuiverd Clostridium difficile antigenen in menselijke sera. Het protocol kan worden uitgebreid voor de bepaling van specifieke antilichaam reacties in preparaten van polyklonale intraveneuze immunoglobuline.

Video: A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Environment

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

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 ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Bioengineering

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Biology

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

Medicine

Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen

Source: Khan, S., et al., <a href="https://app.jove.com/v/59973">Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the process for detecting antibody isotypes in multiple human serum samples using a microarray slide printed with antigenic subtypes of influenza virus strains.

Source: Khan, S., et al., Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes. J. Vis. Exp. (2019)
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A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

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