Name: zika virus specific diagnostic epitope discovery
Uploaded: 2017-12-12T13:00:00
Description: Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

Current support is provided by an SBIR (Small Business Innovation Research) administrative supplement grant from NIDCRR44 DE024456. NIDCR HIV grant that evolved out of NIDCR grant U01 DE017855 for the development of a confirmatory point-of- care diagnostic for HIV. We gratefully acknowledge Silke Weisenburger(PEPperPRINT Heidelberg, Germany) for her technical assistance and kind support. We also thank NYU Langone Medical Center for the LI-COR Odyssey Imaging System.

These data are a part of an ongoing research study conducted at New York University College of Dentistry and were approved by the Institutional Review Board of the New York University School of Medicine, IRB # H10-01894. Clinical samples used in this study were de-identified samples used previously for diagnosis and with permission from the Wadsworth Center of New York State Department of Health, Albany, NY.
1. Installing the ZIKV High-density Peptide Microarray Slide in a specific Incubation Tr...

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We designed a protocol utilizing a high-density peptide microarray containing the entire Zika virus protein sequence (French Polynesian strain). The microarray was manufactured by printing 3,423 different overlapping linear peptides. Each peptide was 15 amino acids and varied by only one residue from its nearest neighbor in the sequence (i.e., 14 residue overlap). Although shorter overlaps can be printed, epitope mapping is more precise with longer overlaps. Each peptide was printed in duplicate to increase reli...

Zika virus (ZIKV) diagnosis based on clinical symptoms is challenging because it shares vectors, geographic distribution, and symptoms with Dengue and Chikungunya virus infection1. Given the risk for adverse pregnancy outcomes in women infected with ZIKV during pregnancy, it is important to distinguish between the 3 viruses. Although the current molecular diagnostic tests are specific, they are only useful in blood or saliva during the relatively short period of acute infection2,3. Serological assays are essential for diagnosis outside this initial period of infection4.
The development of a ZIKV specific serological assay is challenging for two reasons: first, the Zika antigens that the human immune system responds to are not currently known; and second, conserved flaviviruses amino acid sequences induce antibody cross-reactivity. Our objective was to discover unique ZIKV specific peptides to be used in diagnostics. Different approaches have been developed to screen peptide libraries covering entire proteins including phage, bacterial, and yeast surface display5,6,7,8,9,10. Our strategy was to use a high-density peptide microarray that permits rapid and inexpensive high-throughput serological screenings11,12 and subsequently the identified peptides can be used to improve current serological assays for detection of ZIKV infection.
This protocol enables the discovery of Zika virus specific diagnostic peptides using a high-density peptide microarray (Figure 1). The high-density peptide microarray was produced using the peptide laser printing technology. The entire ZIKV protein sequence consisting of 3,423 amino acid residues based on the French Polynesian strain (GenBank: KJ776791.2), was printed on a standard glass slide in blocks of 15 linear residues with an overlap of 14 amino acid residues in duplicate for a total of 6,846 peptide spots. In addition to the entire Zika protein sequence peptides, the microarray utilizes Influenza hemagglutinin (HA) peptides for internal controls.
A Zika validated positive serum sample obtained from Wadsworth Center (Albany, NY), was used to identify specific Immunoglobulin M (IgM) and Immunoglobulin G (IgG) reactive peptides. After incubating with the sample overnight, the microarray was stained with a secondary fluorochrome conjugated antibody (anti-human IgM or anti-human IgG), and analyzed on a microarray scanner. Quantification of spot intensities and peptide annotation was performed with a specific software provided by the same company that manufactured the microarray.

<table>
	<tbody>
		<tr>
			<td>PEPperCHIP Custom Peptide Microarray</td>
			<td>PEPperPRINT</td>
			<td>PPC.001.001</td>
			<td>Custom peptide micarray: our microarray contains the entire Zika virus protein sequence</td>
		</tr>
		<tr>
			<td>PEPperCHIP incubation tray 3/1</td>
			<td>PEPperPRINT</td>
			<td>PPC.004.001</td>
			<td></td>
		</tr>
		<tr>
			<td>PEPperCHIP Staining Kit (680 nm) (anti-HA, DyLight labeling)</td>
			<td>PEPperPRINT</td>
			<td>PPC.037.002</td>
			<td></td>
		</tr>
		<tr>
			<td>PepSLide Analyzer</td>
			<td>PEPperPRINT</td>
			<td>PSA.004.001</td>
			<td>14-days free License for Windows</td>
		</tr>
		<tr>
			<td>Rockland Blocking buffer</td>
			<td>Rockland</td>
			<td>MB-070</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgM (mu chain) DyLight 800</td>
			<td>Rockland</td>
			<td>609-145-007</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgG Fc DyLight 680</td>
			<td>Thermo Scientific</td>
			<td>SA5-10138</td>
			<td></td>
		</tr>
		<tr>
			<td>LI-COR Odyssey Imaging System</td>
			<td>LI-COR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker device</td>
			<td>IKA</td>
			<td>MTS 2/4 digital microtiter shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deltagraph</td>
			<td>Redrocks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

zika virus specific diagnostic epitope discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

The results obtained using the protocol described are shown in Figure 2 and Figure 3. No background interactions were noted when pre-staining the microarray with secondary anti-human IgM (data not shown). Staining with an anti-human IgM conjugate resulted in several areas above background green fluorescence intensities, indicating the binding of these peptides with IgM in the host serum sample (<strong class="xfi...

Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

Zika Virus Specific Diagnostic Epitope Discovery

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

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