Simple and Rapid Method for Production of Whole-Virus Antigen for Serodiagnosis of Caprine Arthritis-Encephalitis Virus by Enzyme-Linked Immunosorbent Assay

Abstract

Polyethylene glycol (PEG) was used to produce whole-virus antigen derived from tissue culture cells infected with a Canadian strain of caprine arthritis-encephalitis virus. PEG antigen batches were obtained after precipitation and concentration of infected tissue culture material with PEG 8000 and final treatment with sodium dodecyl sulfate. The optimum time of harvest of tissue culture extracted material to produce the maximum amount of viral proteins was determined in roller bottles, after cocultivation of infected and noninfected fetal lamb corneal cells. Samples from day 9 to day 25 postculture were collected and processed. By Western blotting, the optimum time of harvest was found to be day 25 following the coculture. Two large batches of PEG antigen were prepared at the optimum time of harvest. Both batches gave similar results when tested by Western blotting and enzyme-linked immunosorbent assay (ELISA), using reference control sera from infected and noninfected goats. For further testing in ELISA, cutoff values and ratios were determined for PEG batch 1, using 200 known serum samples from goats free of the disease. The PEG antigen batch was compared with an in-house ELISA antigen in a kinetic mode, using 498 serum samples from field goats. The in-house ELISA antigen was produced following two rounds of ultracentrifugation and treatment with sodium dodecyl sulfate (R. A. Heckert, W. B. McNab, S. M. Richardson, and M. R. Briscoe, Can. J. Vet. Res. 56:237-241, 1992). The PEG antigen batch was found suitable for ELISA, with a relative specificity of 100% and a relative sensitivity of 99.4% compared to the in-house ELISA antigen. This method of antigen production for ELISA was found to be rapid, inexpensive, and reliable for the diagnosis of caprine-arthritis encephalitis, without requiring the use of sophisticated laboratory equipment.Caprine arthritis-encephalitis virus (CAEV) belongs to the Retroviridae family and the subfamily Lentivirinae. The virus is distributed worldwide and causes leuko-encephalomyelitis in goat kids (13), chronic progressive arthritis-synovitis, indurative mastitis, and chronic interstitial pneumonia in adult goats (6,9,12). The development of clinical disease takes a few months to a few years, with infections in most animals remaining subclinical (7,15). For both the clinical and asymptomatic forms, the CAEV causes a lifetime infection despite humoral and cellular immune responses (8), with no known effective treatment; the infected animals remain reservoirs of the virus for their entire lives. Transmission between infected and susceptible animals occurs mainly through colostrum and milk consumption (1,15). Contact transmission between goats of all ages has also been demonstrated (15). The early detection of the infected animals and their segregation and/or eradication from the flock form an efficient practice to limit the spread of the virus (17). The infection is primarily detected by the demonstration of specific CAEV antibodies in the body fluids of the infected goats (15). The...

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

“…The pelleted cell debris were dissolved in 1/100 of the starting volume in PBS and stored in −80°C until used. The supernatants were precipitated and concentrated with 10% polyethylene glycol (PEG) as described by Simard et al [ 16 ] with minor modifications. Briefly, the supernatants were mixed with 30% (w/v) PEG 8000 (Fisher Scientific) in 0.4 M NaCl at a ratio of 2:1 and stirred for 1 h at 4°C.…”

Presence of Antibodies against Sindbis Virus in the Israeli Population: A Nationwide Cross-Sectional Study

“…al. [14] Briefly, the SINV antigen was prepared from Vero cells infected with a SINV strain isolated in Israel, while the mock antigen was obtained from non-infected Vero cells. Vero cell lysates were incubated overnight with polyethylene glycol (PEG) 6000 buffer and centrifuged at 4 °C at 10,000 g for 30 min.…”

Diagnostic performance of ID Screen® MVV-CAEV Indirect Screening ELISA in identifying small ruminant lentiviruses-infected goats

“…This, in turn, depends on quality of gold standard used. Procedures applied so far in investigations regarding accuracy of ELISAs for SRLV infection have mostly consisted in comparison with another imperfect test such as agar gel immunodiffusion (Castro et al 1999), radioimmunoprecipitation (Vander Schalie et al 1994, Herrmann et al 2003, western blotting (Clavijo and Thorsen 1995) or another ELISA (Simard et al 2001). Rarely, has it been based on comparison with combination of two or three of the aforementioned tests (Hecker et al 1992, Rimstad et al 1994, Kwang et al 1995.…”