Two novel protein chips for the detection of antibodies against porcine parvovirus

Yue Wu,Xiaobo Huang,Bin Zhou,Xudan Wu,Jinxiu Hou,Xiongnan Chen

doi:10.1186/s12917-020-02280-z

Abstract

BackgroundPPV is one of the most important pathogens causing porcine reproductive disorder. It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complement-fixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies.ResultsWe establish the visible protein chip and the cyanine dye 3 (Cy3)-labeled protein chip to detect the clinical serum from pigs. In this study, the recombinant protein VP2 of PPV was expressed in E.coli, purified with nickel magnetic beads, and then printed onto epoxy-coated glass slides for preparation of the protein chip. After a series of experiments, the conditions of antigen protein concentration, incubation time of primary antibody or secondary antibody, and optimal serum dilution fold were optimized, resulting in a successful visible protein chip and Cy3-labeled protein chip. The results showed that the positive serum, diluted up to 6000-fold, can be detected by the visible protein chip, and the positive serum, diluted up to 12,800-fold, can be detected by the Cy3-labeled protein chip, suggesting the high sensitivity of these protein chips. Moreover, the positive detection ratio, sensitivity, and specificity of these two kinds of protein chips were higher than those of commercial ELISA antibody detection kits.ConclusionOverall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.

Highlights

Porcine parvovirus (PPV) is one of the most important pathogens causing porcine reproductive disorder
Protein chip preparation PPV VP2 protein was diluted to 0–0.4 mg/mL with 2 × printing buffer to determine the optimal protein concentration
Preparation of protein chips based on the dot sequence are shown in Fig. 2a and b, respectively; the results showed 0.3 mg/mL as the optimal protein concentration of the visible protein chip detection microarray (Fig. 2c, e), and 0.1 mg/mL as the optimal protein concentration of the cyanine dye 3 (Cy3)-labeled protein chip detection microarray (Fig. 2d, f)

Summary

Introduction

PPV is one of the most important pathogens causing porcine reproductive disorder It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. PPV was first detected in China in 1980 [4].PPV is one of the most important pathogens causing porcine reproductive disorder It has been shown in clinical cases to be a commonly mixed infection with Pseudorabies virus (PRV), Wu et al BMC Veterinary Research (2020) 16:57. Previous studies have shown that the major antigenic site regions of VP2 are amino acids 60–68, 81–88, 266–275, 351–357, and 398–404, where amino acids 378, 383, and 436 are critical sites to determine PPV virulence [15,16,17,18]

Methods

Results

Discussion

Conclusion