Accepted Manuscript Title: Development and clinical evaluation of a new gold-immunochromatographic assay for the detection of antibodies against field strains of pseudorabies virus Author: Dian-lei Guo Qi-wei Pan Kun-peng Li Jun-qing Li Han-wei Shen Xiang-ling Wang Xun-yun Zhang Xue-song Li Fang Fu Li Feng Xi Li PII: DOI: Reference:
S0166-0934(15)00232-3 http://dx.doi.org/doi:10.1016/j.jviromet.2015.06.016 VIRMET 12830
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
22-1-2015 25-6-2015 28-6-2015
Please cite this article as: Guo, D.-l., Pan, Q.-w., Li, K.-p., Li, J.-q., Shen, H.-w., Wang, X.-l., Zhang, X.-y., Li, X.-s., Fu, F., Feng, L., Li, X.,Development and clinical evaluation of a new gold-immunochromatographic assay for the detection of antibodies against field strains of pseudorabies virus, Journal of Virological Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.06.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manuscript
Development and clinical evaluation of a new
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gold-immunochromatographic assay for the detection of
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antibodies against field strains of pseudorabies virus
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Dian-lei Guoa, Qi-wei Pana, Kun-peng Lia, Jun-qing Lia, Han-wei Shena, Xiang-ling
5
Wanga, Xun-yun Zhanga, Xue-song Lia, Fang Fua, Li Fenga, Xi Lia,*
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a
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Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of
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Agricultural Sciences, No.427 Maduan St., Nangang
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China.
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Division of Swine Infectious Diseases, State Key Laboratory of Veterinary
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District, Harbin 150001,
* Corresponding author. Tel.: +86 18946066147; fax: +86 451 82733132
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E-mail address: [email protected]
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ABSTRACT
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An immunochromatographic strip (ICS) was developed for the detection of
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swine antibodies against glycoprotein E (gE) in Pseudorabies Virus (PRV). In this test,
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Staphylococcal Protein A (SPA) labeled with colloidal gold was dispensed on a
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conjugate pad as the detector. Purified PRV-gE and pig-IgG were blotted on a
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nitrocellulose membrane for the test (T) and control lines (C), respectively. If the
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tested serum contains IgG antibodies against PRV-gE, the IgG will interact with the
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colloidal gold-SPA to form a complex (gold-SPA-swine IgG). The complex will react
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with the immobilized PRV-gE on the T line and the Pig-IgG in the C line of the ICS to
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form two visible red bands. If there is no IgG antibody against PRV-gE in the sample
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serum, only the C line will be visible. The ICS was capable of specifically detecting
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PRV-gE antibody within 5 min, and its stability and reproducibility were quite good
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after storage at 4°C and use within 4 months. Using an IDEXX Pseudorabies Virus gE
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Antibody Test Kit (IDEXX PRV gE Ab Test) as a reference, the relative specificity
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and sensitivity of the ICS were determined to be 81.6% and 90.7%, respectively.
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Furthermore, there was a good agreement between the results obtained by the
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commercial product and the ICS (kappa = 0.7289).
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Keywords: PRV；Antibody detection；ICS
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1. Inroducion
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Pseudorabies virus (PRV), a member of the alphaherpesviridae cause Aujezsky's
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disease (AD), is a pathogen resulting in swine devastating disease and economic
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losses worldwide. PRV usually is asymptomatic, but it can produce fatal encephalitis
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in newborn pigs, respiratory disorders in fattening pigs and reproductive failure in
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sows (Nauwynck et al., 2007; Mettenleiter 2000). Significant eradication efforts,
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including selective culling of PRV-positive herds and widespread vaccination
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programs with marker viruses such as gE-null vaccine strains, make contributions to
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the prevention and control of the Pseudorabies (PR) happening (Pomeranz et al.,
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2005). Although vaccination is effective in reducing the circulation of wild PRV and
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preventing the illness, it generally does not prevent infection and establishment of
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latency (Serena et al., 2013).
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As an important virulence factor, the gE was required for efficient
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anterograde-directed spread of PRV infection in neurons (Kratchmarov et al., 2013).
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Therefore, most PRV vaccines are attenuated modified live virus vaccines by deletion
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of gE (Mettenleiter et al., 1989). Deletion of gE from vaccine strains also makes it
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possible to serologically distinguish gE-null vaccine strain-infected animals from
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virulent field strain-infected animals by using serologic assays that detect antibodies
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to gE (Kimman et al., 1996).
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Some methods had been established previously to detect the antibody or the virus
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in clinical samples (OIE, 2012). Traditionally, PRV detection is based on direct virus
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isolation followed by confirmation using immunofluorescence, immunoperoxidase or
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neutralization tests with specific antiserum. However, this
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consuming and false negative results may occur in submissions from latently infected
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animals (Perez et al., 2009). Serological test for PRV antibody, such as enzyme-linked
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immunosorbent assays testing (ELISA) for a humoral response to gE, is the most
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common method used for herd diagnosis (Zanella et al., 2012). In 1977, indirect
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ELISAs (I-ELISAs) for detection of antibodies to PRV were developed in the USA
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(Snyder et al., 1977). Since then, a number of reports on the application of ELISA as a
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serodiagnostic test for PRV have been published (Banks et al., 1983; Echeverria et al.,
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2000). ELISA specifically identifying the presence of gE in an animal’s blood are
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particularly useful because these distinguish an immune response to a field strain
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infection from an immune response to a vaccine strain, but the method require
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laboratory operation, special equipment and have a prolonged time span, and can not
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used to rapid diagnosis of PRV antibody in the field.
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Gold-immunochromatographic assay (GICA) is a diagnostic technique in which
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a colloidal gold-labeled antigen or antibody is used as tracer to detect antibody or
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antigen respectively (Heeschen et al., 1998). This method has been widely used for
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the diagnosis of many pathogens (virus, bacteria, parasite etc.) due to its simple
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procedure, rapid and low-cost detection (Chen et al., 2012; Jung et al., 2005; Saidin et
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al., 2014). The technology has also been adapted for the surveillance and diagnosis of
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animal infectious diseases by detection of antibodies in serum, such as Porcine
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circovirus disease , Avian Influenza (AI) and Porcine epidemic diarrhea (PED) (Peng
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et al., 2007; Jin et al., 2012).
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In this study, an immunochromatographic strip (ICS) was developed for the
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detection of PRV antibodies against gE protein, which show whether swine are
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infected with field virus.
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2. Materials and Methods
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2.1 Special reagents and materials
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The Ni-NTA His·Bind Resin was purchased from EMD Chemicals Inc.
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Recombinant staphylococcal Protein A (Recombinant SPA) was purchased from
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Prospect Biosystems Inc. Purified pig IgG was purchased from BIO-RAD Inc.
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Tetrachloroauric (Ⅲ) acid hydrate (HAuCl4•xH2O) was purchased from Sinopharm
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Chemical Reagent Beijing Co. Bovine serum albumin (BSA) was purchased from
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Sigma–Aldrich Inc. Pseudorabies vaccine (strain: Bartha-K61; gE/gI deletion) was
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purchased from Harbin Weike Biotechnology Development Company, and the vaccine
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contained at least 5000 50% tissue culture infective doses (TCID50) in 1 ml. An
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IDEXX pseudorabies virus gB Antibody Test Kit (IDEXX PRV gB Ab) and IDEXX
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pseudorabies virus gE Antibody Test Kit (IDEXX PRV gE Ab) were purchased from
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IDEXX Laboratories Inc.
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2.2 Virus and Serum samples The PRV strain used to challenge and PCR was the same, which named HN
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strain and was isolated from He-Nan province of China. Standard virus sera were
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obtained from different sections of the Harbin Veterinary Research Institute (HVRI),
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Which located in the city of Harbin of China, including sera against PRV, porcine
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circovirus type2 (PCV2), pleuropneumonia-like organism (PPLO), porcine epidemic
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diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine
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reproductive and respiratory syndrome virus (PRRSV). The negative control serum
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was obtained from pigs reared under a specific pathogen-free (SPF) environment and
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was certified to be non-reactive with PRV by an IDEXX PRV gE Ab test. A total of
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184 swine serum samples from China were collected.
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2.3 Preparation of the GICA
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2.3.1 Preparation of recombinant gE protein of PRV
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DNA fragments (945bp) encoding the PRV gE five important epitopes were
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amplified by polymerase chain reaction (PCR) from the PRV genome. The following
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primer
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-CGGAATTCATGCCGAGTCTCTCCGCCGAG-
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5’-GACGTCGACTCAGAAGGCGTCGAAGGGGCAGG-3’.
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settings were 94℃ 5min; 98℃ 10s，55℃ 30s，72℃ 1min, 35cycle; 72℃ 10min.
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The purified PCR product was digested with EcoRI and SalI and cloned into the
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PET32a vector.
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sequences
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primer, reverse The
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The recombinant plasmid pET-32a-gE was transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells (obtained from TIANGEN Biotech
Co., Ltd.
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(Beijing)). The transformants were grown at 37°C in Luria-Bertani broth (LB)
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containing 100 μg/mL ampicillin to an optical density of 0.4 at 600 nm, and isopropyl
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thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM. After
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4 hours of induction at 37°C, the cells were harvested by centrifugation (8,000 rpm
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for 10 min) at 4°C. The cells were resuspended in PBS and disrupted by sonication on
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ice and then subjected to centrifugation at 10,000 rpm for 30 min; the supernatant was
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collected. The recombinant protein containing the His-tag was purified using
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nickel–nitrilotriacetic acid (Ni-NTA) resin (Novagen, Madison, USA), following the
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manufacturer’s protocol. The final protein product was examined by sodium
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dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) before being divided
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into small aliquots of 2 mg/ml prior to storage at −80°C. The gE protein, purified
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from the supernatant using a Ni-NTA His·Bind Resin, was analyzed by western blot
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for the presence of gE, which the PRV positive and negative serums respectively used
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as the first antibody, and the HRP-labeled rabbit anti-swine IgG used as the second
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antibody.
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2.3.2 Preparation of colloidal gold-SPA
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Colloidal gold particles with a mean diameter of 20 nm were prepared according
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to the method of Frens (Frens , 1973). Briefly, an aqueous solution of chloroauric acid
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(100 ml 0.01% (W/V) AuCl3•HCl4•H2O) was heated to boiling and, with rapid
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stirring, 1.4 ml of a 1.0% (W/V) sodium citrate solution was added. The reaction
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solution was stirred simultaneously and gently boiled for 14 min until the color of the
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solution turned from light yellow into black and eventually red. The obtained colloidal
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gold solution was cooled to room temperature, then stored at 4°C for several months
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in a dark-colored glass bottle until use.
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A preliminary titration was performed to determine the optimal conditions for
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conjugation between gold and SPA. Briefly, 10μl of purified SPA (10 mg/184μl) was
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added to 20 ml of a colloidal gold solution with the pH value adjusted to 6.0 using 1%
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(W/V) potassium carbonate (K2 CO3). After the mixture was stirred vigorously for 30
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min and rested at 4°C for 30 min, 1 ml of a 10% (w/v) BSA aqueous solution was
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added to the stirred solution and then stabilized for another 60 min at 4°C. Finally,
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The colloidal gold-SPA solution was centrifuged (12,000 rpm) at 4°C for 30 min, and
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the precipitate was resuspended with 2 ml of working buffer (10 mM borax buffer, pH
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8.0) containing 2% (w/v) BSA and 0.05% (w/v) sodium azide.
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2.3.3 Preparation of the ICS
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The immunochromatographic test strip was composed of the sample/conjugate
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pad (glass fiber membrane), the analytical membrane (nitrocellulose filter membrane)
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and the absorbent pad (absorbent paper), which was placed on a backing card (a
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non-porous polyester support having a higher tensile strength, also known as a PVC
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card). The recombinant gE protein (2 mg/ml, containing 0.1% (w/v) sodium azide)
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and the pig IgG (1 mg/ml) were dispensed onto the nitrocellulose (NC) membrane as
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the test (T line) and control lines (C line), respectively, at a rate of 2 μl/cm using a
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XYZ3050 Dispense Workstation (Bio-Dot, Irvine, CA, USA). The two lines were
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applied approximately 0.5 cm apart. Gold-labeled SPA (without dilution) was jetted
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onto the glass fiber and dried at 37°C for approximately 4 hours. Finally, the sample
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application pad, SPA-gold conjugated pad, nitrocellulose membrane and absorption
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pad were laminated and pasted onto the PVC plate successively to form the strip and
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attached to the plastic scale-board, which was cut into 4 mm wide pieces using the
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programmable strip cutter (Beckman, USA) (Fig. 1).
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2.4 Specificity of the ICS
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To evaluate the specificity of the immunochromatographic strips, sera positive
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for PRV and other viruses, including PRRSV, PPLO, PCV, TGEV and PEDV, were
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simultaneously tested using the test strips and the IDEXX PRV gE Ab Kit.
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2.5 Reproducibility and stability of the ICS
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To determine the reproducibility and stability of the strip, 20 unknown pig serum
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samples were randomly selected for the detection of PRV antibody using 4 different
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batches of strips (batch numbers: 140405, 140413, 140416, 140426). All of the strips
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from each batch were stored at 4°C and were used for the repeated testing of the 20
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serum samples 4 times, once a month.
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2.6 Agreement between the ICS and reference methods
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Eight PRV control sera were diluted to 1:10, 1:30, 1:50, 1:100, 1:150; 1:200,
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1:250, and 1:300 for the IDEXX Kit test, and the same positive sera were
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simultaneously tested using the ICS with the same dilution to evaluate the strips’
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detection limit.
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A total of 184 unknown pig serum samples, collected from 10 swine herds in the
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northeast of China, were detected by the prepared strips and the IDEXX PRV gE Ab
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Kit at the same time. The consistency of the percentages of PRV-gE positive sera
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were analyzed using McNemar's test, and the kappa statistic was used to measure the
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strength of agreement among the results between the ICS and the IDEXX PRV gE Ab
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Kit with Statistic Analysis System (SAS) software.
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2.7 Clinical evaluation of the ICS
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2.7.1 Animals and feeding
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A total of 28 colostrum-fed piglets, similar in weight and cross-bred, were
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weaned and purchased at 35 days of age from a PRV-free commercial farm. They
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were all negative for PRV, according to testing for the gB and gE antibody by using
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the IDEXX Pseudorabies Virus Antibody Test Kit. All of the pigs were housed in an
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environmentally controlled building with a pen over completely slatted floors
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throughout the experiment. Based on the experimental requirements, the building was
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completely emptied, cleaned and disinfected with a 3% Sodium hypochlorite solution
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for 3 days and then emptied for an additional 5 days before the pigs were introduced.
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The pigs contained in the raised pen were equipped with four nippled rinkers and
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eight self-feeders and were fed ad libitum with a balanced, pelleted feed ration free of
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antibiotics.
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2.7.2 Experimental design
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A total of 28 pigs were randomly divided into 4 groups (7 pigs per group)
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numbered separately, “01,” “02,” “03,” and “04,” which represent the four
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experimental treatment groups: non-vaccinated and challenged (NVC) group (01),
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non-vaccinated and non-challenged (NVNC) group (02), vaccinated and challenged
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(VC) group (03), and vaccinated and non-challenged (VNC) group (04) (Table 1). In
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order to simulate the conditions of disease free spreading in pigs in the field, the four
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group pigs with different treatment were housed together. At 40 days of age (-25 dpc),
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the pigs of group numbers “03” and “04” were immunized with the PRV vaccine
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according to the product manual. Blood samples from each pig were collected by
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jugular venipuncture at -25 dpc, -18 dpc, -11 dpc and -4 dpc, and the sera were
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stored at -20°C. At 65 days of age (0 days postchallenge [0 dpc]), the pigs of group
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numbers “01” and “03” were each challenged with approximately 3×106.75 TCID50 of
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the PRV by intranasal (2 ml) and intramuscular (1 ml) injection (20-gauge needle,
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right neck area). Serum samples from all of the groups were collected with the same
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method every two days until 84 days of age (19 dpc), and the last blood collection was
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taken at 110 days of age (45 dpc).
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All of the serum samples were tested for PRV gB and gE antibodies using the
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IDEXX Pseudorabies Virus Antibody Test Kit according to the manufacturers’
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instructions to evaluate the immune effect of the PRV vaccine and gE antibody
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changes. The same sera were simultaneously detected by ICS to determine the date of
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a positive result.
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3. Results
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3.1 Expression and purification of the PRV gE protein in E. coli
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The recombinant gE of approximately 42 kDa, containing an inserted
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hexahistidine (His6) tag, was expressed and purified as a monomeric subunit from E.
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coli (BL21). SDS-PAGE (Fig. 2) showed that the target protein existed mainly as a
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soluble protein, and the concentration of gE detected by ultramicrospectrophotometer
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(IMPLEN) was over 2 mg/ml. The target protein, purified from the supernatant using
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a Ni-NTA His·Bind Resin, was analyzed by western blot for the presence of gE (Fig.
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3).
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3.2 Specificity of the immunochromatographic strip
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To determine the specificity of the ICS, as shown in Figure 4, two red bands
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clearly appeared in both the test and control lines when the PRV reference positive
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sera were assayed by the ICS. In contrast, only a red-purple band appeared in the
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control line of the test strip in healthy Bama suckling pig (negative) serum and sera
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containing other viruses, which was similar to the blank control.
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3.3 Reproducibility and stability of the immunochromatographic strip
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The results (Table 2) show that 7 out of the 20 tested serum samples were
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detected as positive, with identical results in both strips from the same batch on
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different detection days as well as in strips from different batches.
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3.4 Coincidence test of the immunochromatographic strip
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The sera with the three highest titers against PRV gE were equal, and five other
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sera with the next highest titers tested with the ICS were slightly higher than those
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tested with the IDEXX PRV gE Ab Kit (Table 3).
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Among the 184 serum samples, 112 samples (60.9%) were determined to be
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positive by ICS. The percentage of positive sera was comparable to the rate of 58.7%
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obtained with the IDEXX PRV gE Ab Kit (P = 0.414; McNemar's test), indicating
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that the positivity rate of these two methods was not significantly different (P > 0.05)
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(Table 4). Using the IDEXX PRV gE Ab Kit as a reference test, the relative specificity and
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sensitivity of the immunochromatographic strip were determined to be 81.6% and
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90.7%, respectively. There was good agreement between the ICS and the commercial
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kit (Kappa = 0.7289; P < 0.0001), indicating that the two methods are comparable
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(Table 4).
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3.5 Clinical application of the immunochromatographic strip
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In all sera analyzed by the IDEXX antibody kit, the results showed that pigs
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unvaccinated (groups 01 and 02) against PRV-gB were seronegative from -25 dpc to
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-4 dpc. In contrast, pigs vaccinated (groups 03 and 04) with pseudorabies vaccine
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seroconverted at seven days post-vaccination, indicating that robust immune
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protection is generated within 7 days of vaccination. As expected, both vaccinated and
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unvaccinated pigs were negative for anti-PRV-gE antibodies prior to and at trial day
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21. Meanwhile, all of the sera from -25 dpc to -4 dpc were negative by the ICS test.
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During the PRV challenge and the flock infection, antibodies against the gB
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protein were found in non-vaccinated group pigs (“01” and “02”), and all of the pigs
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had significantly elevated anti-PRV-gB antibody levels after 9 dpc. The
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PRV-gE-specific antibody, as indicated by a positive result for anti-PRV-gE IgG using
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the IDEXX gE antibody kit, emerged in groups “01,” “02,” “03,” and “04” at 9, 13, 9
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and 13 dpc, respectively (Table 5). Meanwhile, all of the sera from 0 dpc to 19 dpc
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evaluated by the ICS test showed positive results emerging at 9, 13, 9 and 13 dpc,
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which demonstrates consistent results between these two methods (Table 5).
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4. Discussion The immunochromatographic strip has been widely applied for the detection of
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various infectious diseases (Cui et al., 2008; Arai et al., 1999). Several
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immunochromatographic strips for the detection of anti-virus antibodies, such as
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PRRSV (Cui et al., 2008), PCV-2 (Jin et al., 2012) and FMDV (Yang et al., 2010)
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have been developed and applied to the evaluation of antibody titers. In the study,
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the ICS was developed for the detection of gE antibody, which was very useful for
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rapid diagnosis of PRV field strain and helpful to the treatment and control of the PR
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in developing countries.
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Staphylococcal Protein A (SPA), a cell-wall protein of Staphylococcus aureus,
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has been developed as a universal ligand for immunoglobulin G (IgG) purification
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because it binds specifically to the Fc portion of the IgG molecule of many mammals
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(Jungbauer et al., 2004). Protein A contains five highly similar domains (from the
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N-terminus: E, D, A, B, and C), each with specificity for Fc (Ljungberg et al., 1993),
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and the binding capacity for IgG depends on the source species of the particular
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immunoglobulin. Normally, when comparing the relative binding strengths for protein
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A in several species, from strong to medium and finally to weak or no binding, the
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order is Human/Pig > Mouse/Rabbit/Dog/Cow/Horse > Rat/Sheep/Goat. Based on
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this, pig IgG was used on the NC membrane as the C line due to its strong binding
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ability for SPA.
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At the end of the PRV challenge experiment, all of the pigs were killed at the age
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of 110 days (45 dpc) and the sera were evaluated by the IDEXX PRV gE Antibody
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Test Kit and ICS. The results demonstrated that all of the sera were strongly positive
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for PRV gE antibody. In addition, main organs, including the liver, heart, spleen, lung,
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kidney and blood, were tested for the gE gene using a conventional PCR method that
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was previously established in our laboratory (Perez et al., 2009) and showed negative
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results in all of the pigs. The results indicated that compared with the PCR method, an
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antibody against PRV-gE can be easily detected with an ICS over a long period of
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time.
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In conclusion, we have developed an ICS for the detection of antibodies against
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gE of PRV with high specificity, reproducibility and stability. Compared with IDEXX
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PRV gE Ab Test, the relative specificity and sensitivity of the ICS were determined to
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be 81.6% and 90.7%, respectively. Furthermore, there was a good agreement between
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the two method (kappa = 0.7289), and the detection of PRV-gE-specific antibodies by
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the kit only takes approximately 5 min, which is much faster than the time required
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for ELISA or PCR assays. In summary, the results of this study suggest that there is
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potential for the application of our method in the clinical diagnosis of PRV wild
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strain.
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Acknowledgments
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We would like to express our sincere gratitude to Mr. Tian Zhijun at the PRRS
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Virus Section in Harbin Veterinary Research Institute (HVRI) for kindly providing a
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large number of serum samples. This research was supported by grants from Major
308
International Scientific Cooperation Projects in the HVRI (1610302014008).
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Echeverria, M. G., Nosetto, E. O., Etcheverrigaray, M. E., 2000. Evaluation of a
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blocking ELISA using a urease conjugate for the detection of antibodies to
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pseudorabies virus. J Vet Diagn Invest. 12, 266–268.
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Frens, G., 1973. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Physical Science. 241, 20–22. 15
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performance and clinical application of a new rapid bedside assay for the
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Zhi, Y., Wang, L., Yang, S., Zhang. G., 2012. Development of an
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circovirus-2. J Vet Diagn Invest. 24, 1151-1157.
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Jin., Q. Y., Yang, J. F., Lu Q. X., Guo, J. Q., Deng, R. G., Wang, Y. B et al., 2012.
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against Porcine circovirus-2. J VET Diagn Invest. 24, 1151.
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Jung, B. Y., Jung, S. C., Kweon, C. H., 2005. Development of a rapid
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Jungbauer, A., Hahn, R., 2004. Engineering protein A affinity chromatography. Curr Opin Drug Discov Devel. 7, 248-256. Kimman, T. G., Leeuw, O., Kochan, G., Szewczyk, B., Rooij, E., Jacobs, L., Kramps, J.
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A., Peeters, B., 1996. An Indirect Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) using Baculovirus-Expressed Antigen for the
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Detection of Antibodies to Glycoprotein E of Pseudorabies Virus and
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Comparison of the Method with Blocking ELISAs. Clin Diagn Lab Immunol. 3,
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167–174.
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Kratchmarov, R., Kramer, T., Greco, T., Taylor, M. P., Ch’ng, T. H., Cristea, I. M.,
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Enquist, L., 2013. Glycoproteins gE and gI Are Required for Efficient
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KIF1A-Dependent Anterograde Axonal Transport of Alphaherpesvirus Particles
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in Neurons. Journal of Virology. 87, 9431–9440. Ljungberg, U. K., Jansson, B., Niss, U., Nilsson, R., Sandberg, B. E., Nilsson, B.,
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1993. The interaction between different domains of Staphylococcal Protein A and
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human polyclonal IgG, IgA, IgM and F(ab’)2: separation of affinity from
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specificity. Mol Immunol. 30, 1279 –1285.
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Mettenleiter, T. C., 2000. Aujeszky’s disease (pseudorabies) virus: the virus and molecular pathogenesis–state of the art, June 1999. Vet. Res. 31, 99–115.
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Mettenleiter, T. C., Lomniczi, B., Zsak, L., Medveczky, I., Ben-Porat, T., Kaplan, A.
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S., 1989. Analysis of the factors that affect virulence of pseudo-rabies virus. p.
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Nauwynck, H., Glorieux, S., Favoreel, H., Pensaert, M., 2007. Cell biological and
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OIE, 2012. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Chapter 2.1.2. Aujeszky’s disease.
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Peng, D. P., Hu, S. S., Hua, Y., Xiao, Y. C., Li, Z. L., Wang, X. L., Bi, D. R., 2007.
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Comparison of a new gold-immunochromatographic assay for the detection of
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antibodies against avian influenza virus with hemagglutination inhibition and
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Perez, L., Arce, H. D., 2009. Development of a Polymerase Chain Reaction Assay for
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the Detection of Pseudorabies Virus in Clinical Samples. Brazilian journal of
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microbiology. 40, 433-438. Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J., 2005. Molecular biology of
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pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol
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Noordin, R., 2014. Production of recombinant Entamoeba histolytica pyruvate
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phosphate dikinase and its application in a lateral flow dipstick test for amoebic
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liver abscess. BMC Infectious Diseases. 14, 182.
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Serena, M. S., Geisler, C., Metz, G. E., Corva, S. G., Mórtola, E. C., Larsen, A., Jarvis,
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D. L., Echeverria, M. G., 2013. Expression and purification of Suid
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Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose
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Aujeszky’s disease in infected pigs. Protein Expr Purif. 90, 1–8.
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Snyder, M. L., Stewart, W. C., 1977. Application of an enzyme labelled antibody test in pseudorabies. Proc Annu Meet Am Assoc Vet Lab Diagn. 20,17–32.
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385
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Yang, S., Yang, J., Zhang, G., Wang, X., Qiao, S., Zhao, D., Zhi, Y., Li, X., Xing, G.,
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Luo, J., Fan, J., Bao, D., 2010. Development of an immunochromatographic strip
393
for the detection of antibodies against foot-and-mouth disease virus serotype O. J
394
Virol Methods. 165, 139-44.
395
Zanella, E. L., Miller, L. C., Lager, K. M., Bigelow, T. T., 2012. Evaluation of a
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real-time polymerase chain reaction assay for Pseudorabies virus surveillance
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purposes. J VET Diagn Invest. 24, 739.
398
Figure Captions
400
Fig. 1 Schematic diagram of the immunochromatographic strip. For positive samples,
401
IgG against PRV gE protein, combined with gold-SPA, is captured by the gE protein
402
through percolation in the nitrocellulose membrane, while in the test line, the
403
formation of a colloidal gold-SPA – gE IgG – gE protein complex causes the
404
appearance of a red line. For negative samples, the test line zone does not have
405
gold-SPA remaining, and therefore, no red line is evident.
406
Fig. 2 Analysis of the purified recombinant gE protein. M: PageRuler Prest Protein
407
Ladder. 1: The precipitate harvested by centrifugation (8,000 rpm for 10 min) at 4°C
408
after sonication on ice. 2: The supernatant collected after sonication. 3: The target
409
protein purified from the supernatant using Ni-NTA His·Bind Resin.
410
Fig. 3 The result of western blots of gE. M: PageRuler Prest Protein Ladder. 1: The
411
purified gE Igs on the blots were detected by swine negative sera. 2: The purified gE
412
Igs on the blots were detected by PRV positive sera.
413
Fig. 4 Specificity of the immunochromatographic strip. Strip 1: PRV control serum.
414
Strip 2 to 5: Positive sera for PCV, PPLO, PRRSV, PEDV and TGEV, Strip 6:
415
Negative serum. Strip 7: Blank control (1% (V/V) PBST).
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416
19
Page 19 of 25
*Highlights (for review)
1. An ICS was developed with high specificity, reproducibility and stability. 2. This method can be used for detecting PRV wild-type viruse.
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3. The challenge of PRV in swine used to evaluate the result of the ICS.
Page 20 of 25
Table(s)
Table 1 Study design with vaccination and challenge statuses for PRV. No. of
Vaccination Challenge
Group no.
name
pigs
(－25 dpc)
(0 dpc)
01
NVC
7
-
+
02
NVNC
7
-
-
03
VC
7
+
04
VNC
7
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Group
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+ -
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+
Table 2
140405
no. Positive
140426
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Negative no.
140416
1
Ac
no.
140413
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Batches
ed
Reproducibility and stability of the ICS
13
Notes: 1 to 4 defined as after one to four months later, the same 20 serums were detected by the ICS strip each batches.
Page 21 of 25
Table 3 The detection limit of the ICS compared with the IDEXX PRV gE Ab Kit Serums no.
1
2
3
4
5
6
7
8
1:200 1:150 1:250 1:200 1:150 1:250 1:200 1:200
The IDEXX PRV gE
1:150 1:150 1:200 1:200 1:150 1:200 1:150 1:150
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The ICS
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Ab Kit
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Table4
Agreement between the immunochromatographic strip (ICS) and reference methods.
Positive
Positive
ed
PRV gE Ab Kit
The ICS
ce pt
Negative Total
M
The IDEXX
Total
Negative
98
10
108
14
62
76
112
72
184
Notes: The positive rate of the ICS: 112/184 =60.9%, the positive rate of the IDEXX
Ac
PRV gE Ab Kit: 108/184 =58.7%, P (The value of significant test for McNemar's test) = 0.414 >0.05. Relative to The IDEXX PRV gE Ab Kit, specificity of ICS: 62/76 × 100% = 81.6%, sensitivity of ICS: 98/108 × 100% = 90.7%; Kappa = 0.7289, P (The value of significant test for kappa)
S0166-0934(15)00232-3 http://dx.doi.org/doi:10.1016/j.jviromet.2015.06.016 VIRMET 12830
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
22-1-2015 25-6-2015 28-6-2015
Please cite this article as: Guo, D.-l., Pan, Q.-w., Li, K.-p., Li, J.-q., Shen, H.-w., Wang, X.-l., Zhang, X.-y., Li, X.-s., Fu, F., Feng, L., Li, X.,Development and clinical evaluation of a new gold-immunochromatographic assay for the detection of antibodies against field strains of pseudorabies virus, Journal of Virological Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.06.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manuscript
Development and clinical evaluation of a new
2
gold-immunochromatographic assay for the detection of
3
antibodies against field strains of pseudorabies virus
4
Dian-lei Guoa, Qi-wei Pana, Kun-peng Lia, Jun-qing Lia, Han-wei Shena, Xiang-ling
5
Wanga, Xun-yun Zhanga, Xue-song Lia, Fang Fua, Li Fenga, Xi Lia,*
6
a
7
Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of
8
Agricultural Sciences, No.427 Maduan St., Nangang
9
China.
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Division of Swine Infectious Diseases, State Key Laboratory of Veterinary
an
District, Harbin 150001,
* Corresponding author. Tel.: +86 18946066147; fax: +86 451 82733132
11
E-mail address: [email protected]
12
ABSTRACT
ed
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An immunochromatographic strip (ICS) was developed for the detection of
14
swine antibodies against glycoprotein E (gE) in Pseudorabies Virus (PRV). In this test,
15
Staphylococcal Protein A (SPA) labeled with colloidal gold was dispensed on a
16
conjugate pad as the detector. Purified PRV-gE and pig-IgG were blotted on a
17
nitrocellulose membrane for the test (T) and control lines (C), respectively. If the
18
tested serum contains IgG antibodies against PRV-gE, the IgG will interact with the
19
colloidal gold-SPA to form a complex (gold-SPA-swine IgG). The complex will react
20
with the immobilized PRV-gE on the T line and the Pig-IgG in the C line of the ICS to
21
form two visible red bands. If there is no IgG antibody against PRV-gE in the sample
22
serum, only the C line will be visible. The ICS was capable of specifically detecting
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Page 1 of 25
PRV-gE antibody within 5 min, and its stability and reproducibility were quite good
24
after storage at 4°C and use within 4 months. Using an IDEXX Pseudorabies Virus gE
25
Antibody Test Kit (IDEXX PRV gE Ab Test) as a reference, the relative specificity
26
and sensitivity of the ICS were determined to be 81.6% and 90.7%, respectively.
27
Furthermore, there was a good agreement between the results obtained by the
28
commercial product and the ICS (kappa = 0.7289).
29
Keywords: PRV；Antibody detection；ICS
30
1. Inroducion
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Pseudorabies virus (PRV), a member of the alphaherpesviridae cause Aujezsky's
32
disease (AD), is a pathogen resulting in swine devastating disease and economic
33
losses worldwide. PRV usually is asymptomatic, but it can produce fatal encephalitis
34
in newborn pigs, respiratory disorders in fattening pigs and reproductive failure in
35
sows (Nauwynck et al., 2007; Mettenleiter 2000). Significant eradication efforts,
36
including selective culling of PRV-positive herds and widespread vaccination
37
programs with marker viruses such as gE-null vaccine strains, make contributions to
38
the prevention and control of the Pseudorabies (PR) happening (Pomeranz et al.,
39
2005). Although vaccination is effective in reducing the circulation of wild PRV and
40
preventing the illness, it generally does not prevent infection and establishment of
41
latency (Serena et al., 2013).
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As an important virulence factor, the gE was required for efficient
43
anterograde-directed spread of PRV infection in neurons (Kratchmarov et al., 2013).
44
Therefore, most PRV vaccines are attenuated modified live virus vaccines by deletion
2
Page 2 of 25
of gE (Mettenleiter et al., 1989). Deletion of gE from vaccine strains also makes it
46
possible to serologically distinguish gE-null vaccine strain-infected animals from
47
virulent field strain-infected animals by using serologic assays that detect antibodies
48
to gE (Kimman et al., 1996).
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Some methods had been established previously to detect the antibody or the virus
50
in clinical samples (OIE, 2012). Traditionally, PRV detection is based on direct virus
51
isolation followed by confirmation using immunofluorescence, immunoperoxidase or
52
neutralization tests with specific antiserum. However, this
53
consuming and false negative results may occur in submissions from latently infected
54
animals (Perez et al., 2009). Serological test for PRV antibody, such as enzyme-linked
55
immunosorbent assays testing (ELISA) for a humoral response to gE, is the most
56
common method used for herd diagnosis (Zanella et al., 2012). In 1977, indirect
57
ELISAs (I-ELISAs) for detection of antibodies to PRV were developed in the USA
58
(Snyder et al., 1977). Since then, a number of reports on the application of ELISA as a
59
serodiagnostic test for PRV have been published (Banks et al., 1983; Echeverria et al.,
60
2000). ELISA specifically identifying the presence of gE in an animal’s blood are
61
particularly useful because these distinguish an immune response to a field strain
62
infection from an immune response to a vaccine strain, but the method require
63
laboratory operation, special equipment and have a prolonged time span, and can not
64
used to rapid diagnosis of PRV antibody in the field.
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Gold-immunochromatographic assay (GICA) is a diagnostic technique in which
66
a colloidal gold-labeled antigen or antibody is used as tracer to detect antibody or
3
Page 3 of 25
antigen respectively (Heeschen et al., 1998). This method has been widely used for
68
the diagnosis of many pathogens (virus, bacteria, parasite etc.) due to its simple
69
procedure, rapid and low-cost detection (Chen et al., 2012; Jung et al., 2005; Saidin et
70
al., 2014). The technology has also been adapted for the surveillance and diagnosis of
71
animal infectious diseases by detection of antibodies in serum, such as Porcine
72
circovirus disease , Avian Influenza (AI) and Porcine epidemic diarrhea (PED) (Peng
73
et al., 2007; Jin et al., 2012).
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In this study, an immunochromatographic strip (ICS) was developed for the
75
detection of PRV antibodies against gE protein, which show whether swine are
76
infected with field virus.
77
2. Materials and Methods
78
2.1 Special reagents and materials
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The Ni-NTA His·Bind Resin was purchased from EMD Chemicals Inc.
80
Recombinant staphylococcal Protein A (Recombinant SPA) was purchased from
81
Prospect Biosystems Inc. Purified pig IgG was purchased from BIO-RAD Inc.
82
Tetrachloroauric (Ⅲ) acid hydrate (HAuCl4•xH2O) was purchased from Sinopharm
83
Chemical Reagent Beijing Co. Bovine serum albumin (BSA) was purchased from
84
Sigma–Aldrich Inc. Pseudorabies vaccine (strain: Bartha-K61; gE/gI deletion) was
85
purchased from Harbin Weike Biotechnology Development Company, and the vaccine
86
contained at least 5000 50% tissue culture infective doses (TCID50) in 1 ml. An
87
IDEXX pseudorabies virus gB Antibody Test Kit (IDEXX PRV gB Ab) and IDEXX
88
pseudorabies virus gE Antibody Test Kit (IDEXX PRV gE Ab) were purchased from
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89
IDEXX Laboratories Inc.
90
2.2 Virus and Serum samples The PRV strain used to challenge and PCR was the same, which named HN
92
strain and was isolated from He-Nan province of China. Standard virus sera were
93
obtained from different sections of the Harbin Veterinary Research Institute (HVRI),
94
Which located in the city of Harbin of China, including sera against PRV, porcine
95
circovirus type2 (PCV2), pleuropneumonia-like organism (PPLO), porcine epidemic
96
diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine
97
reproductive and respiratory syndrome virus (PRRSV). The negative control serum
98
was obtained from pigs reared under a specific pathogen-free (SPF) environment and
99
was certified to be non-reactive with PRV by an IDEXX PRV gE Ab test. A total of
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184 swine serum samples from China were collected.
101
2.3 Preparation of the GICA
102
2.3.1 Preparation of recombinant gE protein of PRV
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DNA fragments (945bp) encoding the PRV gE five important epitopes were
104
amplified by polymerase chain reaction (PCR) from the PRV genome. The following
105
primer
106
-CGGAATTCATGCCGAGTCTCTCCGCCGAG-
107
5’-GACGTCGACTCAGAAGGCGTCGAAGGGGCAGG-3’.
108
settings were 94℃ 5min; 98℃ 10s，55℃ 30s，72℃ 1min, 35cycle; 72℃ 10min.
109
The purified PCR product was digested with EcoRI and SalI and cloned into the
110
PET32a vector.
Ac
103
sequences
were
used:
forward 3’,
and
primer, reverse The
5’ primer,
temperature
5
Page 5 of 25
111
The recombinant plasmid pET-32a-gE was transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells (obtained from TIANGEN Biotech
Co., Ltd.
113
(Beijing)). The transformants were grown at 37°C in Luria-Bertani broth (LB)
114
containing 100 μg/mL ampicillin to an optical density of 0.4 at 600 nm, and isopropyl
115
thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM. After
116
4 hours of induction at 37°C, the cells were harvested by centrifugation (8,000 rpm
117
for 10 min) at 4°C. The cells were resuspended in PBS and disrupted by sonication on
118
ice and then subjected to centrifugation at 10,000 rpm for 30 min; the supernatant was
119
collected. The recombinant protein containing the His-tag was purified using
120
nickel–nitrilotriacetic acid (Ni-NTA) resin (Novagen, Madison, USA), following the
121
manufacturer’s protocol. The final protein product was examined by sodium
122
dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) before being divided
123
into small aliquots of 2 mg/ml prior to storage at −80°C. The gE protein, purified
124
from the supernatant using a Ni-NTA His·Bind Resin, was analyzed by western blot
125
for the presence of gE, which the PRV positive and negative serums respectively used
126
as the first antibody, and the HRP-labeled rabbit anti-swine IgG used as the second
127
antibody.
128
2.3.2 Preparation of colloidal gold-SPA
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Colloidal gold particles with a mean diameter of 20 nm were prepared according
130
to the method of Frens (Frens , 1973). Briefly, an aqueous solution of chloroauric acid
131
(100 ml 0.01% (W/V) AuCl3•HCl4•H2O) was heated to boiling and, with rapid
132
stirring, 1.4 ml of a 1.0% (W/V) sodium citrate solution was added. The reaction
6
Page 6 of 25
solution was stirred simultaneously and gently boiled for 14 min until the color of the
134
solution turned from light yellow into black and eventually red. The obtained colloidal
135
gold solution was cooled to room temperature, then stored at 4°C for several months
136
in a dark-colored glass bottle until use.
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A preliminary titration was performed to determine the optimal conditions for
138
conjugation between gold and SPA. Briefly, 10μl of purified SPA (10 mg/184μl) was
139
added to 20 ml of a colloidal gold solution with the pH value adjusted to 6.0 using 1%
140
(W/V) potassium carbonate (K2 CO3). After the mixture was stirred vigorously for 30
141
min and rested at 4°C for 30 min, 1 ml of a 10% (w/v) BSA aqueous solution was
142
added to the stirred solution and then stabilized for another 60 min at 4°C. Finally,
143
The colloidal gold-SPA solution was centrifuged (12,000 rpm) at 4°C for 30 min, and
144
the precipitate was resuspended with 2 ml of working buffer (10 mM borax buffer, pH
145
8.0) containing 2% (w/v) BSA and 0.05% (w/v) sodium azide.
146
2.3.3 Preparation of the ICS
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The immunochromatographic test strip was composed of the sample/conjugate
148
pad (glass fiber membrane), the analytical membrane (nitrocellulose filter membrane)
149
and the absorbent pad (absorbent paper), which was placed on a backing card (a
150
non-porous polyester support having a higher tensile strength, also known as a PVC
151
card). The recombinant gE protein (2 mg/ml, containing 0.1% (w/v) sodium azide)
152
and the pig IgG (1 mg/ml) were dispensed onto the nitrocellulose (NC) membrane as
153
the test (T line) and control lines (C line), respectively, at a rate of 2 μl/cm using a
154
XYZ3050 Dispense Workstation (Bio-Dot, Irvine, CA, USA). The two lines were
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Page 7 of 25
applied approximately 0.5 cm apart. Gold-labeled SPA (without dilution) was jetted
156
onto the glass fiber and dried at 37°C for approximately 4 hours. Finally, the sample
157
application pad, SPA-gold conjugated pad, nitrocellulose membrane and absorption
158
pad were laminated and pasted onto the PVC plate successively to form the strip and
159
attached to the plastic scale-board, which was cut into 4 mm wide pieces using the
160
programmable strip cutter (Beckman, USA) (Fig. 1).
161
2.4 Specificity of the ICS
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To evaluate the specificity of the immunochromatographic strips, sera positive
163
for PRV and other viruses, including PRRSV, PPLO, PCV, TGEV and PEDV, were
164
simultaneously tested using the test strips and the IDEXX PRV gE Ab Kit.
165
2.5 Reproducibility and stability of the ICS
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To determine the reproducibility and stability of the strip, 20 unknown pig serum
167
samples were randomly selected for the detection of PRV antibody using 4 different
168
batches of strips (batch numbers: 140405, 140413, 140416, 140426). All of the strips
169
from each batch were stored at 4°C and were used for the repeated testing of the 20
170
serum samples 4 times, once a month.
171
2.6 Agreement between the ICS and reference methods
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166
Eight PRV control sera were diluted to 1:10, 1:30, 1:50, 1:100, 1:150; 1:200,
173
1:250, and 1:300 for the IDEXX Kit test, and the same positive sera were
174
simultaneously tested using the ICS with the same dilution to evaluate the strips’
175
detection limit.
176
A total of 184 unknown pig serum samples, collected from 10 swine herds in the
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Page 8 of 25
northeast of China, were detected by the prepared strips and the IDEXX PRV gE Ab
178
Kit at the same time. The consistency of the percentages of PRV-gE positive sera
179
were analyzed using McNemar's test, and the kappa statistic was used to measure the
180
strength of agreement among the results between the ICS and the IDEXX PRV gE Ab
181
Kit with Statistic Analysis System (SAS) software.
182
2.7 Clinical evaluation of the ICS
183
2.7.1 Animals and feeding
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A total of 28 colostrum-fed piglets, similar in weight and cross-bred, were
185
weaned and purchased at 35 days of age from a PRV-free commercial farm. They
186
were all negative for PRV, according to testing for the gB and gE antibody by using
187
the IDEXX Pseudorabies Virus Antibody Test Kit. All of the pigs were housed in an
188
environmentally controlled building with a pen over completely slatted floors
189
throughout the experiment. Based on the experimental requirements, the building was
190
completely emptied, cleaned and disinfected with a 3% Sodium hypochlorite solution
191
for 3 days and then emptied for an additional 5 days before the pigs were introduced.
192
The pigs contained in the raised pen were equipped with four nippled rinkers and
193
eight self-feeders and were fed ad libitum with a balanced, pelleted feed ration free of
194
antibiotics.
195
2.7.2 Experimental design
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A total of 28 pigs were randomly divided into 4 groups (7 pigs per group)
197
numbered separately, “01,” “02,” “03,” and “04,” which represent the four
198
experimental treatment groups: non-vaccinated and challenged (NVC) group (01),
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Page 9 of 25
non-vaccinated and non-challenged (NVNC) group (02), vaccinated and challenged
200
(VC) group (03), and vaccinated and non-challenged (VNC) group (04) (Table 1). In
201
order to simulate the conditions of disease free spreading in pigs in the field, the four
202
group pigs with different treatment were housed together. At 40 days of age (-25 dpc),
203
the pigs of group numbers “03” and “04” were immunized with the PRV vaccine
204
according to the product manual. Blood samples from each pig were collected by
205
jugular venipuncture at -25 dpc, -18 dpc, -11 dpc and -4 dpc, and the sera were
206
stored at -20°C. At 65 days of age (0 days postchallenge [0 dpc]), the pigs of group
207
numbers “01” and “03” were each challenged with approximately 3×106.75 TCID50 of
208
the PRV by intranasal (2 ml) and intramuscular (1 ml) injection (20-gauge needle,
209
right neck area). Serum samples from all of the groups were collected with the same
210
method every two days until 84 days of age (19 dpc), and the last blood collection was
211
taken at 110 days of age (45 dpc).
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All of the serum samples were tested for PRV gB and gE antibodies using the
213
IDEXX Pseudorabies Virus Antibody Test Kit according to the manufacturers’
214
instructions to evaluate the immune effect of the PRV vaccine and gE antibody
215
changes. The same sera were simultaneously detected by ICS to determine the date of
216
a positive result.
217
3. Results
218
3.1 Expression and purification of the PRV gE protein in E. coli
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219
The recombinant gE of approximately 42 kDa, containing an inserted
220
hexahistidine (His6) tag, was expressed and purified as a monomeric subunit from E.
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Page 10 of 25
coli (BL21). SDS-PAGE (Fig. 2) showed that the target protein existed mainly as a
222
soluble protein, and the concentration of gE detected by ultramicrospectrophotometer
223
(IMPLEN) was over 2 mg/ml. The target protein, purified from the supernatant using
224
a Ni-NTA His·Bind Resin, was analyzed by western blot for the presence of gE (Fig.
225
3).
226
3.2 Specificity of the immunochromatographic strip
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To determine the specificity of the ICS, as shown in Figure 4, two red bands
228
clearly appeared in both the test and control lines when the PRV reference positive
229
sera were assayed by the ICS. In contrast, only a red-purple band appeared in the
230
control line of the test strip in healthy Bama suckling pig (negative) serum and sera
231
containing other viruses, which was similar to the blank control.
232
3.3 Reproducibility and stability of the immunochromatographic strip
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The results (Table 2) show that 7 out of the 20 tested serum samples were
234
detected as positive, with identical results in both strips from the same batch on
235
different detection days as well as in strips from different batches.
236
3.4 Coincidence test of the immunochromatographic strip
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The sera with the three highest titers against PRV gE were equal, and five other
238
sera with the next highest titers tested with the ICS were slightly higher than those
239
tested with the IDEXX PRV gE Ab Kit (Table 3).
240
Among the 184 serum samples, 112 samples (60.9%) were determined to be
241
positive by ICS. The percentage of positive sera was comparable to the rate of 58.7%
242
obtained with the IDEXX PRV gE Ab Kit (P = 0.414; McNemar's test), indicating
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243
that the positivity rate of these two methods was not significantly different (P > 0.05)
244
(Table 4). Using the IDEXX PRV gE Ab Kit as a reference test, the relative specificity and
246
sensitivity of the immunochromatographic strip were determined to be 81.6% and
247
90.7%, respectively. There was good agreement between the ICS and the commercial
248
kit (Kappa = 0.7289; P < 0.0001), indicating that the two methods are comparable
249
(Table 4).
250
3.5 Clinical application of the immunochromatographic strip
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In all sera analyzed by the IDEXX antibody kit, the results showed that pigs
252
unvaccinated (groups 01 and 02) against PRV-gB were seronegative from -25 dpc to
253
-4 dpc. In contrast, pigs vaccinated (groups 03 and 04) with pseudorabies vaccine
254
seroconverted at seven days post-vaccination, indicating that robust immune
255
protection is generated within 7 days of vaccination. As expected, both vaccinated and
256
unvaccinated pigs were negative for anti-PRV-gE antibodies prior to and at trial day
257
21. Meanwhile, all of the sera from -25 dpc to -4 dpc were negative by the ICS test.
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During the PRV challenge and the flock infection, antibodies against the gB
259
protein were found in non-vaccinated group pigs (“01” and “02”), and all of the pigs
260
had significantly elevated anti-PRV-gB antibody levels after 9 dpc. The
261
PRV-gE-specific antibody, as indicated by a positive result for anti-PRV-gE IgG using
262
the IDEXX gE antibody kit, emerged in groups “01,” “02,” “03,” and “04” at 9, 13, 9
263
and 13 dpc, respectively (Table 5). Meanwhile, all of the sera from 0 dpc to 19 dpc
264
evaluated by the ICS test showed positive results emerging at 9, 13, 9 and 13 dpc,
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265
which demonstrates consistent results between these two methods (Table 5).
266
4. Discussion The immunochromatographic strip has been widely applied for the detection of
268
various infectious diseases (Cui et al., 2008; Arai et al., 1999). Several
269
immunochromatographic strips for the detection of anti-virus antibodies, such as
270
PRRSV (Cui et al., 2008), PCV-2 (Jin et al., 2012) and FMDV (Yang et al., 2010)
271
have been developed and applied to the evaluation of antibody titers. In the study,
272
the ICS was developed for the detection of gE antibody, which was very useful for
273
rapid diagnosis of PRV field strain and helpful to the treatment and control of the PR
274
in developing countries.
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Staphylococcal Protein A (SPA), a cell-wall protein of Staphylococcus aureus,
276
has been developed as a universal ligand for immunoglobulin G (IgG) purification
277
because it binds specifically to the Fc portion of the IgG molecule of many mammals
278
(Jungbauer et al., 2004). Protein A contains five highly similar domains (from the
279
N-terminus: E, D, A, B, and C), each with specificity for Fc (Ljungberg et al., 1993),
280
and the binding capacity for IgG depends on the source species of the particular
281
immunoglobulin. Normally, when comparing the relative binding strengths for protein
282
A in several species, from strong to medium and finally to weak or no binding, the
283
order is Human/Pig > Mouse/Rabbit/Dog/Cow/Horse > Rat/Sheep/Goat. Based on
284
this, pig IgG was used on the NC membrane as the C line due to its strong binding
285
ability for SPA.
286
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At the end of the PRV challenge experiment, all of the pigs were killed at the age
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Page 13 of 25
of 110 days (45 dpc) and the sera were evaluated by the IDEXX PRV gE Antibody
288
Test Kit and ICS. The results demonstrated that all of the sera were strongly positive
289
for PRV gE antibody. In addition, main organs, including the liver, heart, spleen, lung,
290
kidney and blood, were tested for the gE gene using a conventional PCR method that
291
was previously established in our laboratory (Perez et al., 2009) and showed negative
292
results in all of the pigs. The results indicated that compared with the PCR method, an
293
antibody against PRV-gE can be easily detected with an ICS over a long period of
294
time.
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In conclusion, we have developed an ICS for the detection of antibodies against
296
gE of PRV with high specificity, reproducibility and stability. Compared with IDEXX
297
PRV gE Ab Test, the relative specificity and sensitivity of the ICS were determined to
298
be 81.6% and 90.7%, respectively. Furthermore, there was a good agreement between
299
the two method (kappa = 0.7289), and the detection of PRV-gE-specific antibodies by
300
the kit only takes approximately 5 min, which is much faster than the time required
301
for ELISA or PCR assays. In summary, the results of this study suggest that there is
302
potential for the application of our method in the clinical diagnosis of PRV wild
303
strain.
304
Acknowledgments
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We would like to express our sincere gratitude to Mr. Tian Zhijun at the PRRS
306
Virus Section in Harbin Veterinary Research Institute (HVRI) for kindly providing a
307
large number of serum samples. This research was supported by grants from Major
308
International Scientific Cooperation Projects in the HVRI (1610302014008).
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309
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Luo, J., Fan, J., Bao, D., 2010. Development of an immunochromatographic strip
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for the detection of antibodies against foot-and-mouth disease virus serotype O. J
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Zanella, E. L., Miller, L. C., Lager, K. M., Bigelow, T. T., 2012. Evaluation of a
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Figure Captions
400
Fig. 1 Schematic diagram of the immunochromatographic strip. For positive samples,
401
IgG against PRV gE protein, combined with gold-SPA, is captured by the gE protein
402
through percolation in the nitrocellulose membrane, while in the test line, the
403
formation of a colloidal gold-SPA – gE IgG – gE protein complex causes the
404
appearance of a red line. For negative samples, the test line zone does not have
405
gold-SPA remaining, and therefore, no red line is evident.
406
Fig. 2 Analysis of the purified recombinant gE protein. M: PageRuler Prest Protein
407
Ladder. 1: The precipitate harvested by centrifugation (8,000 rpm for 10 min) at 4°C
408
after sonication on ice. 2: The supernatant collected after sonication. 3: The target
409
protein purified from the supernatant using Ni-NTA His·Bind Resin.
410
Fig. 3 The result of western blots of gE. M: PageRuler Prest Protein Ladder. 1: The
411
purified gE Igs on the blots were detected by swine negative sera. 2: The purified gE
412
Igs on the blots were detected by PRV positive sera.
413
Fig. 4 Specificity of the immunochromatographic strip. Strip 1: PRV control serum.
414
Strip 2 to 5: Positive sera for PCV, PPLO, PRRSV, PEDV and TGEV, Strip 6:
415
Negative serum. Strip 7: Blank control (1% (V/V) PBST).
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416
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Page 19 of 25
*Highlights (for review)
1. An ICS was developed with high specificity, reproducibility and stability. 2. This method can be used for detecting PRV wild-type viruse.
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3. The challenge of PRV in swine used to evaluate the result of the ICS.
Page 20 of 25
Table(s)
Table 1 Study design with vaccination and challenge statuses for PRV. No. of
Vaccination Challenge
Group no.
name
pigs
(－25 dpc)
(0 dpc)
01
NVC
7
-
+
02
NVNC
7
-
-
03
VC
7
+
04
VNC
7
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Group
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+ -
M
an
+
Table 2
140405
no. Positive
140426
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Negative no.
140416
1
Ac
no.
140413
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Batches
ed
Reproducibility and stability of the ICS
13
Notes: 1 to 4 defined as after one to four months later, the same 20 serums were detected by the ICS strip each batches.
Page 21 of 25
Table 3 The detection limit of the ICS compared with the IDEXX PRV gE Ab Kit Serums no.
1
2
3
4
5
6
7
8
1:200 1:150 1:250 1:200 1:150 1:250 1:200 1:200
The IDEXX PRV gE
1:150 1:150 1:200 1:200 1:150 1:200 1:150 1:150
ip t
The ICS
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cr
Ab Kit
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Table4
Agreement between the immunochromatographic strip (ICS) and reference methods.
Positive
Positive
ed
PRV gE Ab Kit
The ICS
ce pt
Negative Total
M
The IDEXX
Total
Negative
98
10
108
14
62
76
112
72
184
Notes: The positive rate of the ICS: 112/184 =60.9%, the positive rate of the IDEXX
Ac
PRV gE Ab Kit: 108/184 =58.7%, P (The value of significant test for McNemar's test) = 0.414 >0.05. Relative to The IDEXX PRV gE Ab Kit, specificity of ICS: 62/76 × 100% = 81.6%, sensitivity of ICS: 98/108 × 100% = 90.7%; Kappa = 0.7289, P (The value of significant test for kappa)
