Production and characterization of a murine anti-dal monoclonal antibody for blood typing in dogs

Considering the strong immunogenicity of the Dal antigen, and that > 98% of dogs, including blood donors, are Dal-positive, finding compatible blood for a previously transfused Dal-negative patient may be challenging. This is exacerbated by limited access to typing reagents, which currently rely on polyclonal antibodies (PAb) produced following sensitization of dogs. Therefore, the objective of this study was to produce and characterize an anti-Dal murine monoclonal antibody (MAb). Conventional hybridoma technology was used to produce MAb directed against canine red blood cells (cRBC). Briefly, female BALB/c mice were immunized via repeated intraperitoneal injections of washed Dal-positive cRBC (DEA 1,3,7 negative; DEA 4,5 positive) until serologic titers were sufficient (>1:1000). Following fusion with myeloma cells, 573 hybridoma cell culture supernatants were obtained and screened for MAb of interest using a gel column agglutination technique and known Dal-negative and Dal-positive cRBC. Fifteen supernatants led to cRBC agglutination, but only one had the desired pattern (i.e. anti-Dal). To assess its specificity and sensitivity, Dal blood typing of 62 canine EDTA-blood samples was performed using the anti-Dal MAb and two canine PAb: 45 Dal-positive and 17 Dal-negative were identified with 100% agreement between reagents (kappa =1). The anti-Dal MAb produced was further determined to be an IgG1. Conventional hybridoma technology, aided by a gel column technique, has enabled the production of a murine MAb specific against the canine Dal antigen. This will ensure long-term perennity of Dal blood typing, facilitate clinical management and research, as well as avoid resorting to repeat dog sensitization.