A.C. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada and the Training Program in Regenerative Medicine (Canadian Institutes of Health Research). F.Y.Y.H. was supported by the Training Program in Regenerative Medicine. This work was funded by a grant from Astellas Pharma Canada. We also would like to thank Dr. Mark Menenghini (University of Toronto) for use of his Axon microarray scanner.

1. Diluting Antigens and Generating Antigen Microarrays
<ol>
	<li>Dilute antigens in PBS to a final concentration of 0.2 mg/ml. Print up to 162 unique antigens in duplicate with a microarrayer configuration with 9 pins. Include IgG and IgM antigens in the antigen library as positive controls. Include PBS only as a negative control.</li>
	<li>Add 20 &#181;l of each antigen to the 384 well source plate. Add antigens to the source plate in groups that mirror the setup of the printhead (e.g., arrange antigens in groups of 9 when 9 pins are used for printing).</li>
	<li>Cover source plate with foil and freeze at -80 &#176;C until ready to print arrays.</li>
	<li>Clean solid microarrayer pins by incubating them in a sonicating bath with deionized water for 3 x 1 min. Place pins in rack to dry and then arrange pins in microarray printhead. For 9 pins, use a 3 x 3 configuration.</li>
	<li>Program microarrayer for print run by setting number pins on printhead, number of slides to print, number of pads on each slide, and number of replicate spots for each antigen. Typically, use 9 pins, 70 slides, 2 pads/slide, and 2 replicate spots for each antigen. Program microarrayer to sonicate pins in water between different groups of antigens.</li>
	<li>Thaw source plate and then centrifuge plate for 1 min at 100 x g. Place source plate in designated spot in microarrayer. Remove the section of foil that is covering the first group of antigens to be printed.</li>
	<li>Arrange unprinted slides on arrayer surface and run print program. Print slides at RT with humidity set on the arrayer at 55 - 60% with the build in humidifier of the array machine.</li>
	<li>After each group of antigens is printed on all the slides, pause the microarrayer. Cover the antigens that were just printed with foil (to prevent evaporation) and uncover the next group of antigens to be printed. Continue print program.</li>
	<li>After all antigens have been printed, cover source plate with new piece of foil and freeze at -80 &#176;C. Place printed slides in slide box and vacuum seal. Slides may be used the next day or up to one month later.</li>
</ol>
2. Probing Antigen Microarrays with Diluted Sera
<ol>
	<li>Place slides into frames using incubation chambers. Add 700 &#181;l of blocking buffer (2.5% [vol/vol] Fetal calf serum (FCS), 0.1% [vol/vol] Tween 20 in PBS) to each array surface.</li>
	<li>Place adhesive film over frame and place in a sealed container with a piece of wet tissue. Incubate O/N at 4 &#176;C on a rocker.</li>
	<li>Dilute serum samples 1:100 in blocking buffer. Aspirate blocking solution from arrays and add 500 &#181;l of diluted sample to each array surface.</li>
	<li>Cover with adhesive film and incubate for 1 hr at 4&#176; C with rocking.</li>
	<li>Dilute secondary antibodies in blocking buffer. For human studies, dilute a Cy3 labeled goat anti-human IgG antibody to 0.33 &#181;g/ml and a Cy5 labeled goat anti-human IgM antibody to 0.25 &#181;g/ml. For mouse studies, dilute a Cy3 labeled goat anti-mouse IgG antibody to 0.38 &#181;g/ml and a Cy5 labeled goat anti-mouse IgM antibody to 0.7 &#181;g/ml.</li>
	<li>Aspirate samples from arrays and rinse array surfaces 4 times with rinse buffer (0.1% Tween 20 in PBS). Pour buffer onto to slides and flick off quickly.</li>
	<li>Add 700 &#181;l of blocking buffer to wash each array surface and incubate 10 min at RT with rocking. Repeat wash step two more times.</li>
	<li>Add 500 &#181;l of diluted secondary antibody to each slide surface and cover with adhesive film. Incubate 45 min at 4&#176; C with rocking.</li>
	<li>Aspirate secondary antibody mixture from slides and rinse 4 times as above. Wash slides 3 times with 700 &#181;l of blocking/dilution buffer as above.</li>
	<li>Remove slides from frames and place in metal slide rack that is immersed in PBS. Incubate 20 min at RT with orbital shaking. Place in new container of PBS and incubate another 20 min with shaking.</li>
	<li>Place slide rack in container of deionized water 15 sec. Place rack in a new container of water and incubate another 15 sec.</li>
	<li>To dry slides, place slide rack on an ELISA plate adapter in centrifuge and spin at 220 x g for 5 min at RT.</li>
	<li>Place slides in light-tight box until ready for scanning.</li>
</ol>
3. Scanning Antigen Microarrays and Exporting Data
<ol>
	<li>Scan slides using a microarray scanner that can detect Cy3 and Cy5 fluorescent signals. In order to adjust the photomultiplier tube (PMT) levels of the scanner, pre-scan a slide that was only probed with secondary antibodies. 
	Note: This slide should have the full antigen library printed on it including positive (IgG and IgM) as well as negative (PBS) controls. The pre-scan is a low resolution scan that allows the user quickly to find the optimal PMT settings.</li>
	<li>Set the PMT values so that the human IgG features (on the Cy3 channel) and the human IgM features (on the Cy5 channel) have a similar median fluorescence intensity minus background (MFI-B) (typically 40,000). The PMT levels are set by pressing the &#34;hardware&#34; button. Once set, keep these PMT settings constant.</li>
	<li>Scan the experimental slides on the two channels by pressing the &#34;scan&#34; button. Save the slide images (both channels) after each scan.</li>
	<li>Load the slide to be analyzed into the microarray software by using the &#34;file&#34; button.</li>
	<li>Load the gene array list (GAL) file by using the &#34;file&#34; button. The GAL file has the layout of the array with the identities of the array features.</li>
	<li>Place the array template over the scanned image so that the arrays features match the template as closely as possible.</li>
	<li>Align features in all blocks with the template by pressing the &#34;align&#34; button. The program will generally find the outline of the circular features but some manual adjustments may be necessary. These adjustments can be made by entering the feature mode. The software will then calculate a MFI-B for each of the antigens.</li>
	<li>Use the &#34;file&#34; button to export these results as a text file.</li>
</ol>
4. Analyzing Array Data with Significance Analysis of Microarrays (SAM)
<ol>
	<li>Load individual text files into excel and identify the columns that have MFI-B for the Cy3 and Cy5 channels.</li>
	<li>Calculate the average for the duplicate features.</li>
	<li>For any negative MFI-B, replace the negative number with 10. Transform data by dividing raw MFI-B by 100 and then by calculating log base 2.</li>
	<li>Analyze log transformed data with Significance Analysis of Microarrays (SAM) as described previously7.</li>
	<li>For a study using two groups (e.g., healthy controls vs. patients), label one group &#34;1&#34; and the group &#34;2.&#34; Use a two-class, unpaired analysis in SAM to identify antigens reactivities that are significantly different between the two groups (q value &#60; 0.05).</li>
	<li>Use clustering and heat-map generating software to make images for presentation8.</li>
</ol>

<ol>
	<li>Naparstek, Y., Plotz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+autoantibodies+in+autoimmune+disease.">The role of autoantibodies in autoimmune disease.</a> Annu Rev Immunol. 11, 79-104 (1993).</li><li>Damoiseaux, J., Andrade, L. E., Fritzler, M. J., Shoenfeld, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+2015:+From+diagnostic+biomarkers+toward+prediction,+prognosis+and+prevention.">Autoantibodies 2015: From diagnostic biomarkers toward prediction, prognosis and prevention.</a> Autoimmun Rev. 14, 555-563 (2015).</li><li>Robinson, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantigen+microarrays+for+multiplex+characterization+of+autoantibody+responses.">Autoantigen microarrays for multiplex characterization of autoantibody responses.</a> Nature. medicine. 8, 295-301 (2002).</li><li>Quintana, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+microarrays+identify+unique+serum+autoantibody+signatures+in+clinical+and+pathologic+subtypes+of+multiple+sclerosis.">Antigen microarrays identify unique serum autoantibody signatures in clinical and pathologic subtypes of multiple sclerosis.</a> Proc. Natl. Acad. Sci. USA. 105, 18889-18894 (2008).</li><li>Hueber, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+microarray+profiling+of+autoantibodies+in+rheumatoid+arthritis.">Antigen microarray profiling of autoantibodies in rheumatoid arthritis.</a> Arthritis Rheum. 52, 2645-2655 (2005).</li><li>Price, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarray+analysis+reveals+BAFF-binding+autoantibodies+in+systemic+lupus+erythematosus.">Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus.</a> J. Clin. Invest. 123, 5135-5145 (2013).</li><li>Tusher, V. G., Tibshirani, R., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+analysis+of+microarrays+applied+to+the+ionizing+radiation+response.">Significance analysis of microarrays applied to the ionizing radiation response.</a> Proc. Natl. Acad. Sci. USA. 98, 5116-5121 (2001).</li><li>Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cluster+analysis+and+display+of+genome-wide+expression+patterns.">Cluster analysis and display of genome-wide expression patterns.</a> Proc. Natl. Acad. Sci. USA. 95, 14863-14868 (1998).</li><li>Chruscinski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Antigen+Microarrays+to+Screen+for+Autoantibodies+in+Heart+Failure+and+Heart+Transplantation.">Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.</a> PloS one. 11, e0151224 (2016).</li><li>Price, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+influenza+vaccine+immunogenicity+using+influenza+antigen+microarrays.">Characterization of influenza vaccine immunogenicity using influenza antigen microarrays.</a> PloS one. 8, e64555 (2013).</li><li>Singh, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivity+profiles+of+broadly+neutralizing+anti-HIV-1+antibodies+are+distinct+from+those+of+pathogenic+autoantibodies.">Reactivity profiles of broadly neutralizing anti-HIV-1 antibodies are distinct from those of pathogenic autoantibodies.</a> Aids. 25, 1247-1257 (2011).</li><li>Porcheray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+humoral+rejection+of+human+kidney+allografts+associates+with+broad+autoantibody+responses.">Chronic humoral rejection of human kidney allografts associates with broad autoantibody responses.</a> Transplantation. 89, 1239-1246 (2010).</li><li>Haddon, D. 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The authors have nothing to disclose.

The protocol described here allows for the quantification of autoantibodies using the antigen microarray technique. Antigen microarrays offer several advantages over conventional ELISA in screening for autoantibodies. First of all, a variety of antigens including nucleic acids, proteins, peptides, and cell lysates can be arrayed onto the nitrocellulose-coated slides, thus allowing for multiplexed screening of autoantibodies. In addition, only micrograms of antigen are necessary to generate the arrays since nanoliters of ...

Autoantibodies are present in many disease states and can often have direct pathogenic activity1. Identification of autoantibodies is important for diagnosis of certain diseases, for prognosis of disease outcome, and for the classification of patients who may benefit from specific therapies2. Autoantibodies are typically identified in patient serum using the ELISA technique; however, screening for multiple antigens with this technique is laborious and consumes a large quantity of patient sample. New technologies are therefore needed to profile autoantibodies on a larger scale.
The antigen microarray technique is a proteomic technology that allows autoantibodies to be profiled in a multiplex fashion3. In the first step of this process, an antigen library is arrayed onto a slide surface using a robotic microarrayer. The slides are probed with diluted serum and then fluorescent-labeled secondary antibodies are added. Antibody reactivities are visualized by scanning the slides with a microarray scanner and quantified by fluorescent intensities. Antigen microarrays offer multiple advantages over the ELISA technique in screening for autoantibodies: 1) they require only microliters of serum to profile autoantibodies to multiple antigens simultaneously, 2) they use antigen sparingly, as only nanoliters of antigen are spotted onto the arrays, 3) they have enhanced sensitivity3 compared to ELISA and 4) they allow for the simultaneous yet, separate detection of more than one antibody isotype. Antigen microarrays have been used to profile autoantibodies in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus4-6. In all three of these diseases, new insight into disease pathogenesis was obtained from profiling autoantibodies on a large scale with the arrays. 
Here we describe a protocol to generate antigen microarrays using nitrocellulose-coated slides. A variety of antigens including proteins, peptides, and cell lysates can be arrayed onto the slides using this technique. The arrays can be easily customized by including antigens of interest in the source plate that holds the antigen library. In addition, we show how a pair of secondary antibodies can be used to separate IgG and IgM reactivities on the same slide surface. We have now optimized this technique to measure autoantibodies in both humans and mice.

<table border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Ribosomal P0</td>
			<td style="width:225px;">Diarect</td>
			<td style="width:304px;">14100</td>
			<td style="width:240px;">dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IgG</td>
			<td>Jackson Immuno</td>
			<td>009-000-003</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IgM</td>
			<td>Jackson Immuno</td>
			<td>009-000-012</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse IgG</td>
			<td>Sigma-Aldrich</td>
			<td>I5381</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse IgM</td>
			<td>Biolegend</td>
			<td>401601</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double-stranded DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D1626</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">single-stranded DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D8899</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microarrayer</td>
			<td>Virtek</td>
			<td>VersArray Chipwriter Pro</td>
			<td>many types of arrayers are suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solid printing pins</td>
			<td>Arrayit Corporation</td>
			<td>SSP015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">software for robotic microarrayer</td>
			<td>Virtek</td>
			<td>Chipwriter Pro&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST slides (2 Pad)</td>
			<td>GVS Northa America</td>
			<td>10485317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST frame</td>
			<td>GVS Northa America</td>
			<td>10486001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST incubation chambers (2 Pad)</td>
			<td>GVS Northa America</td>
			<td>10486242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 well plates</td>
			<td>Whatman</td>
			<td>7701-5101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plate sealers</td>
			<td>VWR</td>
			<td>60941-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">foil plate covers</td>
			<td>VWR</td>
			<td>60941-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Invitrogen</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 goat anti-human IgG</td>
			<td>Jackson Immuno</td>
			<td>109-165-096</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5 goat anti-human IgM</td>
			<td>Jackson Immuno</td>
			<td>109-175-129</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 goat anti-mouse IgG</td>
			<td>Jackson Immuno</td>
			<td>115-165-071</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5 goat anti-mouse IgM</td>
			<td>Jackson Immuno</td>
			<td>115-175-075</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray Scanner</td>
			<td>Molecular Devices</td>
			<td>Axon 4200A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray software</td>
			<td>Molecular Devices</td>
			<td>Genepix 6.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clustering software</td>
			<td>eisenlab.org</td>
			<td>Cluster 3.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heatmap software</td>
			<td>eisenlab.org</td>
			<td>Treeview 1.60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray statistical software</td>
			<td>Stanford University</td>
			<td colspan="2">SAM 4.0 (Significance Analysis of Microarrays)</td>
		</tr>
	</tbody>
</table>

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.

Antigens are arranged in a 384 well plate and printed onto slides by a robotic microarrayer as shown in Figure 1. Figure 2 shows slides placed in a frame with incubation chambers and a scanned slide after processing. Figure 3 shows positive and negative control slides. The negative slide is only probed with secondary antibodies, and the positive control slide is probed with serum from a patient with systemic lupus erythematosus. By using ...

Watch this Scientific Journal Video about Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies at JoVE.com

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

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Immunology and Infection

12.7K Views.  University Health Network. 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. 

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

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

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Chapters in this video

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Electrochemiluminescence Assays for Human Islet Autoantibodies

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues