key: cord-0804049-obb4r1x3 authors: Kumar, Manoj; Nandi, Sukdeb; Chidri, Sunil title: Development of a polyclonal antibody-based AC-ELISA and its comparison with PCR for diagnosis of canine parvovirus infection date: 2010-10-08 journal: Virol Sin DOI: 10.1007/s12250-010-3132-x sha: ad78394e0ca89b96d143ab58b91ef79a12214394 doc_id: 804049 cord_uid: obb4r1x3 A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 10(2.8) TCID(50)/mL whereas PCR sensitivity was 10(0.8) TCID(50)/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection. . A total of 129 faecal samples were collected or received at Virus Laboratory, CADRAD, for diagnosis from dogs suspected of CPV-2 infection. The rectal swabs were taken from individual dogs suspected of CPV-2 infection and suspended (in the ratio 1:9) in Hank's balanced salt solution (HBSS) containing streptomycin (100 mg/L) and penicillin (100 000 IU/L). It was filtered through a disposable syringe filter (0.45 µm) (Millex, Milipore, USA) and then centrifuged at 10 000 r/min at 4℃ for 5 min in a refrigerated centrifuge. The supernatant was carefully pipetted out and stored at -20℃ till further use [18] . MDCK cell line was obtained from National Centre for Cell Science, Pune. It was maintained at the Virus Laboratory, CADRAD by using Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) with 10% fetal calf serum (FCS) as growth medium. Gentamicin was added in the medium at the rate of 50 mg/L (Life Technologies). The MDCK cell line was sub-cultured and grown to 70% of cell culture flask, 0.5 mL of processed faecal samples were added and incubated for 1 h at 37 ℃ . After incubation, the infected cell monolayer was washed three times with DMEM and 5 mL of DMEM medium with 2% FCS was added. The infected cells were incubated at 37℃ for 3-5 days. Purification of CPV was carried out according to the protocol described by Teramoto et al. with some modifications [30] . Infected cells were disrupted and then centrifuged at 3 000×g for 30 min at 4℃ to obtain cell pellet. The pellet was resuspended in PBS, sonicated at 30 µ amplitude for 1 min to disrupt the cells, centrifuged at 8 000×g for 10 min and supernatant was collected. The supernatant was treated with 8% PEG-6000 and kept on the magnetic stirrer overnight for precipitation of the virus. The PEG-treated supernatant was pelleted by centrifugation at 6 000×g for 30 min and the pellet was re-suspended in PBS for ultracentrifugation. Two mL of the resuspended virus was then carefully layered over discontinuous gradient of 60% and 40% sucrose in 12 mL centrifuge tube (4.5 mL 60% sucrose + 4.5 mL 40% sucrose) and centrifuged at 100 000 ×g for 2 h at 4℃ in an ultracentrifuge (Sorvall, Ultracentrifuge, swinging rotor AH629, USA). At the interface of sucrose gradients thick opaque band was observed. The band containing the virus was collected, diluted with equal amount of PBS and again centrifuged at 100 000×g for 2 h at 4℃ in an ultracentrifuge (Sorvall, Ultracentrifuge, USA) using fixed rotor. Hyperimmune serum was raised in rabbits and guinea pigs by inoculating purified virus for three times over a period for 28 days. One hundred µg of The primer set pCPV-2ab (F) 5'-GAAGAGTGGT for 10 min [18] . At the end of PCR, the amplified products were analyzed on 1.0% agarose gel containing ethidium bromide to a final concentration of 0.5 μg/mL. 10 μL of amplified product was mixed with 2 μL of bromophenol dye (6×) and loaded into the well and run along with 100 bp DNA ladder in 1×TAE electrophoresis buffer at 5 V/cm 2 and the progress of mobility was monitored by migration of dye. Three faecal samples found positive in PCR were subjected to blind passages in MDCK cells. In the first 3 passages, no cytopathic effects (CPE) were seen. However, from 4th passage onwards, MDCK cells exhibited CPE characterized by rounding of cells, granulation and aggregation of cells after 72 hpi which increased subsequently and widely distributed in whole monolayer (Fig. 1) . The results are in accordance with the Joshi et al [11] . Then the virus was harvested and freeze-thawed thrice and stored at -20℃ until further use. The virus titre was determined by Reed and Muench method [25] . The titre of virus was calculated from three different readings and the mean was found to be 10 4.8 TCID 50 /mL. Using this as inoculums, 2 000 mL of CPV-2 infected cell culture fluid was produced for purification of virus. In the sucrose density gradient ultracentrifugation, the pellet obtained was resuspended in 2 mL of PBS. The protein content of the purified virus was estimated by U.V. method in nanodrops spectrophotometer (Thermo-scientific, USA) and found to be 350 µg/mL. After test bleeding, the presence of antibody was evaluated by AGPT and a distinct band was visualized between the raised hyperimmune serum in both species and purified virus (Fig. 2) . Final bleeding was done after 10 days of the final immunization, serum collected, inactivated and kept at -20℃ for further use. coronavirus, canine adenovirus etc. and found to be nonreactive. The CPV-2 variants used in the study were typed previously on the basis of PCR and sequence analysis and maintained in the Virus Laboratory, CADRAD [19] . The analytical sensitivity of the AC-ELISA was determined using serial 10-fold dilutions of CPV-2 infected cell culture supernatant of known titer (10 4.8 TCID 50 /mL) and the detection limit of the AC-ELISA assay was up to 10 -2 dilution, equivalent to 10 2.8 TCID 50 /mL. The analytical sensitivity of the AC-ELISA was compared with the pCPV-2ab primer set based PCR [22] and detection limit of the PCR was up to 10 -4 dilution, equivalent to 10 0.8 TCID 50 /mL (Fig. 3) . So, PCR was found to be about 100 times more sensitive than the AC-ELISA for detection of CPV in cell culture system (Table 1) . At the end of the electrophoresis, the gel was visualized under the UV transilluminator and an amplicon of 681 bp of VP2 genes was seen from positive faecal samples and positive control, but not in fecal sample from healthy dog (Fig. 4) . Table 2 ). The relative sensitivity, specificity and accuracy of the AC-ELISA were found to be 88.4%, 100% and 91.4% respectively ( Table 2) . Status report: canine viral enteritis Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy Detection of canine parvovirus in fecal sample using loop-mediated isothermal amplification Isolation of canine parvovirus from clinical cases of gastroenteritis Haemagglutinating activity of canine parvovirus Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera A canine parvovirus mutant is spreading in Italy Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in fecal specimens Detection of canine parvovirus in faeces, using a parvovirus ELISA test kit A novel antigenic variant of canine parvovirus from a Vietnamese dog Molecular characterization and phylogenetic analysis of a canine parvovirus isolate in India Occurrence of canine parvovirus type 2c in the dogs with haemorrhagic enteritis in India The authors would like to acknowledge the Director, Indian Veterinary Research Institute (IVRI) for providing the facilities to carry out the work.