Background At present, pig industry in China is faced with the complex situation of mixed infection caused by multiple pathogens. It is urgent to develop some new high-throughput molecular diagnosis assays to simultaneously detect pathogens or antibodies. Biochip array technology has made it possible to screen thousands of samples simultaneously; it has been twice named as one of the top 10 scientific and technological breakthroughs. Studies have reported encouraging results using protein biochips for detecting antibodies against avian infectious bronchitis virus and ruminant bluetongue virus, but the research of this technology for the diagnosis of swine diseases is still sparse. Results In this study, a novel protein chip was developed that can simultaneously detect the antibodies of four important swine viruses as follow, classical swine fever virus (CSFV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine reproductive and respiratory syndrome virus (PRRSV). Four prokaryotic expression plasmids pET-32a-E2 of CSFV, −VP2 of PPV, −EDIII of JEV, and -N of PRRSV were induced by IPTG (Isopropyl β-D-1-Thiogalactopyranoside) and overexpressed in E.coli, respectively. The purified proteins were identified by Western blotting and then printed on epoxy-coated glass slides. The optimized parameters of this diagnostic chip showed that the spotting concentrations of E2、VP2、EDIII、N proteins were 0.2, 0.4, 0.4, and 0.4 mg/mL. The optimal primary and secondary antibody dilutions were 1:50 and 1: 600. Compared with the commercial ELISA (Enzyme-linked immunosorbent assay) kits, the positive and negative coincidence rates of this chip were 95.8% ~ 100 and 86.2% ~ 100%, as well as, no cross-reaction. Conclusion This protein chip provided a fast, specific, and sensitive method for simultaneous detection of antibodies in clinical serum samples. Compared with traditional methods, this protein chip can monitor very small amount of serum.
Background: 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. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complementfixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies. Results: We 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 Cy3labeled 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. Conclusion: Overall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.
Background: 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. 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 Results: We 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.Conclusion: Overall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput. Key words: Protein chip; Porcine parvovirus (PPV); Antibody detection; Clinical serum
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