2 Determination of the optimal dilution of MAbs. An unexpected Rabbit Polyclonal to MMP-19 but useful obtaining was the ability of this CDV- and PDV-specific cELISA to also detect antibodies against the related pair dolphin morbillivirus and porpoise morbillivirus. Based on a subpopulation of 625 sera used in statistical analyses, the overall sensitivity and specificity of cELISA relative to those of the VNT were 94.9 and 97.7%, respectively. Because the cELISA proved to be nearly as sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate screening test for suspect CDV or PDV cases and would also be useful for epidemiological surveillance of morbilliviral infections in marine mammal populations. The last 13 years have witnessed the emergence of newly recognized members of the genus as significant causes of disease and mortality in marine mammals belonging in the and orders. Four morbilliviruses are now known to infect various species of marine mammals: canine distemper virus (CDV) in seals (10) and polar bears (8, 9), phocine distemper virus (PDV) in seals (18), dolphin morbillivirus (DMV) in dolphins and whales, and porpoise morbillivirus (PMV) in porpoises (13). In 1987 to 1988, more than half of the population of bottlenose dolphins (for 1 h. Viral particles were then purified by gradient centrifugation as previously described for other morbilliviruses (22). Briefly, concentrated virus was layered onto a 20 to 60% step sucrose gradient and centrifuged at 125,000 g for 1 h at 4C. The virus band at the interface of the two sucrose layers was removed, pelleted at 125,000 for 1 h at 4C, and layered onto a continuous 15 to 40% potassium tartrate gradient. After being centrifuged for 4 h at 4C, the virus band was collected, diluted 1:15 in sterile phosphate-buffered saline (PBS), and centrifuged at 125,000 for 1 h. The resulting pellet was resuspended in sterile PBS by sonication and used as the antigen for ELISA and for MAb production. MAbs. Gradient purified whole viral antigens of CDV, DMV, PDV, and PMV antigens were submitted to the Hybridoma Center, Oklahoma State University, for MAb production on a contract basis. The resulting four panels of MAbs were screened in our laboratory for their reactivities against all four viruses by ELISA and VNT (see below). Indirect ELISA was used to determine their specificities, while cELISA was used to measure the ability of selected MAbs to compete with specific antisera for binding to solid-phase-gradient-purified whole viral antigen. Two CDV-induced MAbs, designated 1-1E12 and 2-5F8, were selected for development of a diagnostic cELISA for CDV and PDV on the basis of their strong indirect ELISA signals and their ability to compete with specific anti-CDV and anti-PDV sera for binding to CDV antigen. The MAb 1-1E12 was specific for CDV, while the MAb 2-5F8 reacted with both CDV and PDV by ELISA, but none of them neutralized either virus. For simplicity and didactic reasons, these MAbs will be referred to in the rest of this paper as MAb1 (1-1E12) and MAb2 (2-5F8). VNT. The morbilliviruses are antigenically so closely related that they cross-neutralize one another. Morin hydrate However, serum raised against one morbillivirus will neutralize the homologous Morin hydrate virus at a higher titer than it will other (heterologous) morbilliviruses (24). The VNT was therefore used in this study as the gold standard to determine the antibody specificities of diagnostic serum samples. The test was performed by following a modification of the microtiter method (21). Briefly, serial twofold dilutions of heat-inactivated sera were made in eight columns of 96-well plates using Eagle’s minimum essential medium, starting at a 1:2 dilution. Equal volumes (25 l) of the viruses containing about 100 50% tissue culture infective doses were added to duplicate columns. The virus-serum mixtures were incubated at 37C for 1 h in 5% CO2, Morin hydrate and a Vero cell suspension (150 l containing 104 cells/well) was added. The plates were incubated at 37C in 5% CO2 for 4 days. The test was read by examining cell monolayers under an inverted microscope.