A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay

<div><p>Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world’s most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe₃O₄ magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10⁻³ ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10⁶ ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.</p></div

IMR (%)–ϕ_ICP11 (spiked-rICP11-concentration in PBS) curve showing concentration-dependent IMR signals for ICP11 with standard deviations (duplicate measurements).

<p>IMR (%)–ϕ_ICP11 (spiked-rICP11-concentration in PBS) curve showing concentration-dependent IMR signals for ICP11 with standard deviations (duplicate measurements).</p

Linear correlation between actual ICP11 concentration (ϕ_ICP11) and ICP11 concentration (ϕ_ICP11-c) as measured by IMR in Fig 5.

<p>Linear correlation between actual ICP11 concentration (ϕ_ICP11) and ICP11 concentration (ϕ_ICP11-c) as measured by IMR in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138207#pone.0138207.g005" target="_blank">Fig 5</a>.</p

IMR detection of ICP11 protein in shrimp.

<p>(A) Measured ICP11 protein concentration (ϕ_ICP11-IMR) in non-challenged shrimp (normal control) and in shrimp with a light or severe WSSV infection. The cut-off value (the dashed line) was based on ROC curve analysis of the IMR results. (B) Sensitivity and specificity of the IMR assay as determined by ROC curve analysis.</p

Correlation between detected ICP11-IMR concentration and WSSV copy number.

<p>Pleopod samples were collected from shrimp and subjected to IMR assay and real-time PCR. Dots indicate shrimp belonging to the non-challenged control group (0.08 ~ 7 WSSV copies/mg tissue); crosses indicate light infection (10 ~ 3100 WSSV copies/mg tissue); triangles indicate severe infection (1.57 x 10⁴ ~ 2.83 x 10⁵ WSSV copies/mg tissue).</p

Western blot analysis of WSSV ICP11 and VP28 in total lysates from shrimp pleopods (PL) collected from healthy (PBS) and WSSV-infected shrimp.

<p>After separation and transfer to PVDF membrane, samples were probed with (A) anti-ICP11 antibody or (B) anti-VP28 antibody. (C) To show the protein loading, the SDS-PAGE was stained with coomassie blue. M: Prestained Protein Ladder (10–170 kDa)</p

Time-dependent AC magnetic susceptibility of reagent reacting with rICP11 protein in PBS solution with various concentrations of ICP11.

<p>Time-dependent AC magnetic susceptibility of reagent reacting with rICP11 protein in PBS solution with various concentrations of ICP11.</p

The magnetic reagent used in this study.

<p>(A) Distribution of the diameter of anti-ICP11 coated Fe₃O₄ nanoparticles. (B) Magnetic hysteresis curve of reagent containing anti-ICP11 coated Fe₃O₄ nanoparticles.</p