Production and diagnostic evaluation of a murine polyclonal antibody against canine erythrocyte antigen DEA 1 for hemagglutination-based blood typing in dogs

Abstract Accurate typing of dog erythrocyte antigen (DEA) 1 helps prevent sensitization and acute hemolytic transfusion reactions in dogs, yet typing reagents are imported and costly in many countries, including Iran, and the feasibility of polyclonal reagents for canine blood typing has not been well defined. To produce a murine polyclonal anti-DEA 1 antibody adsorbed with DEA 1-negative erythrocytes, and to determine its serological characteristics and its concordance, for hemagglutination-based DEA 1 typing, with a commercial gel-column reference kit. Blood was collected from 120 clinically healthy dogs. DEA 1 status was established with a gel-column reference kit (Bio-Rad/DiaMed ID-Gel), and a membrane antigen extract prepared by hypotonic lysis of DEA 1-positive erythrocytes was used to immunize BALB/c mice (alum adjuvant; primary injection plus three boosters). Immunoglobulin from the best-responding mouse was purified by Protein A/G chromatography and adsorbed with DEA 1-negative canine erythrocytes. Agglutination titer, sensitivity, specificity, predictive values, Cohen’s kappa, reproducibility and storage stability were determined; accuracy measures describe concordance with the reference kit at a single working dilution. Sixty-eight dogs (56.7%; 95% CI 47.7–65.2) were DEA 1-positive by the reference kit. Final titers ranged from 1:2048 to 1:8192 (median 1:4096), and the working dilution was 1:160. At this dilution, concordance with the reference kit was: sensitivity 97.1% (95% CI 89.8–99.6), specificity 98.1% (89.7–100), positive and negative predictive values 98.5% and 96.2%, and kappa 0.95. The antibody did not react with DEA 1-negative erythrocytes; DEA 3, DEA 7, Dal, Kai 1 and Kai 2 were not typed or tested separately. The coefficient of variation was 4.1%. Activity was stable at − 80 °C for 12 months but declined by about 25% after 1 month at + 4 °C. In this sample of 120 healthy dogs, an adsorbed polyclonal antibody derived from a single best-responding mouse showed high concordance with a gel-column reference kit. It is a promising locally producible reagent, but batch-to-batch reproducibility, performance on weakly positive samples, specificity against other blood group systems, and validation in larger, breed-diverse and clinically ill populations remain to be established before clinical use.