This article was downloaded by: George Mason University On: 01 January 2015, At: 12:00 Publisher: Taylor Methanol CCV; Enzyme immunoassay; Coating antigen preparation; INTRODUCTION Canine coronavirus (CCV) has been identified as one of the causative agents of viral enteritis in dogs around the world. Clinical symptoms range from inapparent 11 to rapidly fatal 2, 31 gastroenteritis. Seroconversion to CCV has 1 Copyright 0 1998 by Marcel Dekker, Inc. Downloaded by George Mason University at 12:00 01 January 2015 2 PALMER-DENSMORE, JOHNSON, AND SABARA been used to determine virus exposure and evaluate vaccine efficacy. A variety of assays, including indirect fluorescent (FA) 4, 5, 61, enzyme linked immunosorbent (ELISA) 7, 8, 91 and serum neutralization (SN) 10, 111 have been developed in order to measure CCV-specific antibody responses. In many cases, the results of these assays are compared even though they measure different type of antibodies due to the varied composition of the antigens used for detection. The antibodies measured by an SN assay differ from those detected by the IFA and ELISA in that they are biologically active (i.e. neutralizing). Several laboratories have further determined that the cell substrate can play a major role in the sensitivity of this assay 121. Detection of antibody by the IFA uses CCV- infected cell monolayers, which are usually fixed with acetone, as the antigen substrate. Most of the variability in this assay arises from the fixation method and the stage at which virus replication is arrested, both of which can determine the conformation, type and amount of individual proteins available for antibody detection. Traditional ELISA assays for CCV are variable in their sensitivity and specificity due to the nature of the antigen preparation used to coat 96-well plates; ranging from purified virus 9, 131 to supernatants from infected cells disrupted by either deoxycholate detergent (DOC) 8 or sonication 7. In general, these three antibody assay systems also differ from each other from the standpoint of time and reagents necessary to perform them, with the SN being Downloaded by George Mason University at 12:00 01 January 2015 NEW ELISA TO MEASURE ANTIBODY RESPONSES 3 the most labor intensive followed by the IFA. For this reason ELISA formats are highly desirable since they lend themselves to validation and automation. Two desirable features for a CCV-specific ELISA is that it be sensitive enough to detect low antibody levels and that the level of antibody detected reflect the magnitude of the SN antibody response. In other words, it is important that the ELISA accurately reflect the time course of the IgG response resulting from infection or vaccination. It is fairly well established in the literature that the major neutralizing antigen of CCV is the spike or S-protein and that nucleocapsid or NC-protein, besides being immunogenic, is one of the more abundant proteins in the virus 14, 151. In one report, an ELISA, using whole virus as the coating antigen, was able to detect CCV-specific IgG in blood samples from 10 week old puppies 4-7 days after oronasal administration of CCV 13. However, the IgG titers did not reflect the increasing SN titers observed between 10-14 days after infection. An ELISA described by Tuchiya et al.8, using DOC disrupted infected cell supernatants as the coating antigen, was even less sensitive in that CCV-specific IgG was first detected three days after the detection of SN antibodies in 2 1 day old puppies experimentally infected with CCV. Based on these reports we hypothesized that one reason for the lack of sensitivity and correlation with the SN response was due to the composition of the ELISA coating antigen, with the S-protein being present in a lower molar quantity than the NC protein as has been reported in structural analyses of Downloaded by George Mason University at 12:00 01 January 2015 4 PALMER-DENSMORE, JOHNSON, AND SABARA coronaviruses 16. In order to test our hypothesis we developed and evaluated the sensitivity of an ELISA method using a methanol treated, S-protein enriched preparation as the coating antigen. This assay should theoretically measure all the NC and S-specific IgG antibody, therefore including the majority of virus neutralizing as well as non-neutralizing antibody, both of which are elicited as a result of vaccination or viral infection. The specificity and sensitivity of this assay was confirmed by the corresponding appearance of S and NC-protein bands in Western blots and SN antibody levels induced in dogs after experimental infection with CCV. MATERIALS AND METHODS EXPERIMENTAL INFECTION OF DOGS Ten, 13-15 week old specific pathogen-free (SPF) dogs (Liberty Labs, New Jersey, USA) were experimentally infected with a total of 1.2 x lo5 plaque forming units of CCV strain CV-6 administered via the intranasal and oral route 13. The virus was obtaine