A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

Abstract

Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm 3 . Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.INDEX TERMS: Enzyme-linked immunosorbent assay, isopycnic centrifugation, porcine circovirus type 2, sucrose cushion, antibody.

Colorimetric Kit for Rapid Porcine Circovirus 2 (PCV-2) Diagnosis

“…Antibodies used in the experiments were produced by our research group, which used a female rabbit immunized with 50 µg/mL ( v/v ) of purified PCV-2 [ 8 ]. The purified IgG protein concentration of 8 mg/mL was herein adopted by using bovine serum albumin (BSA) as protein standards based on the bicinchoninic acid (BCA) methods.…”

“…PCV-2 is often diagnosed based on the assessment of clinical signs in association with antigen or nucleic acid identification through tests-based immunofluorescence, hybridization in situ, immunoperoxidase, and PCR or quantitative PCR (qPCR), which are the gold-standard methods used for porcine circovirus 2 detection purposes [ 6 , 7 , 8 , 9 , 10 ]. A TaqMan rtPCR assay was recently developed to help detecting porcine circovirus 4.…”

Development of a Gold Nanoparticle-Based ELISA for Detection of PCV2

“…Afterward, the serum obtained from the animals was purified using a HiTrap ® Protein G 5 mL column (GE Healthcare, Buckinghamshire, UK). The protein concentration was determined (8 mg/mL) using the bicinchoninic acid (BCA) method and bovine serum albumin (BSA) as a protein standard (protocol number 103/2010-CEUA-Ethics Committee Animal Use of FMVZ, São Paulo State University, Botucatu Campus) [ 13 ]. In all the experiments, water was sourced from a Millipore unit (Merck, Burlington, MA, USA).…”

“…PCV2 can infect wild and domestic pigs, which may have varying health statuses [5]. The diagnosis of porcine circovirus disease (PCVD) is frequently performed by evaluating clinical signs [6] and identifying antigens or nucleic acids via PCR [7] or quantitative PCR (qPCR) [8], immunofluorescence [9], immunohistochemistry [10], in situ hybridization [11], immunoperoxidase [12], or conventional enzyme-linked immunosorbent assay (ELISA) [13]. The main limitation of all these techniques is that they require specific equipment to be operated by specialized individuals.…”

A Methodology for Porcine Circovirus 2 (PCV-2) Quantification Based on Gold Nanoparticles

“…The purified IgG protein concentration was 8 mg/mL, which was based on the BCA methods, using BSA as protein standards, as described by Smith et al, 1985. (Protocol No. 103/2010-CEUA, FMVZ Animal Use Ethics Committee, São Paulo State University-Botucatu [14].…”