A saline or antiglobulin gel column system was used instead of a tube assay to type samples with the polyclonal 1.1 and 1.X antibodies. alleles: DEA 1 (0) and DEA 1+ weak (1+), intermediate (2+) and strong (3+ and 4+). Samples from 6 dogs previously typed as DEA 1.2+ were typed as DEA 1+ or DEA 1 using monoclonal antibodies. Human typing reagents produced varied reactions in tube agglutination experiments against DEA 1+ and DEA 1 RBCs. Polypeptide bands were not detected on immunoblots using a monoclonal anti-DEA 1 antibody, therefore the anti-DEA 1 antibody may be specific for conformational epitopes lost during denaturation. == Conclusions == The autosomal dominant inheritance of DEA 1 with 4 alleles indicates a complex blood group system; the antigenicity of each DEA 1+ type will need to be determined. The biochemical nature of the DEA 1 antigen(s) appears different from human blood group systems tested. Keywords:Blood groups, EW-7197 Blood typing, Canine, Polymorphism, Transfusion == Introduction == Dog Erythrocyte Antigens (DEA) refer to canine blood group systems that were originally defined by an international committee for canine immunogenetics, and were based on studies utilizing induced alloantibodies.1,2Since then, a few additional blood groups and high frequency RBC antigens, such asDal3, have been proposed, but the biochemical and molecular characteristics of the DEA system remain undefined. Dogs are either positive or negative for each DEA blood group, e.g. DEA 4+ or DEA 4.4The DEA 1 blood group system represents an exception, as it was originally reported to contain 23 types: DEA 1.1 (A1), 1.2 (A2), and possibly 1.3 (A3) based upon experiments using polyclonal alloantibodies. The DEA EW-7197 1.1+ type appeared to be dominant over DEA 1.2 and DEA 1.2+ seemed dominant over DEA 1.3; thus dogs had to be DEA 1.1 in order to be DEA 1.2+ and only DEA 1.2 dogs could be DEA 1.3+.5,6 However, our recent flow cytometric and immunochromatographic studies utilizing a murine monoclonal anti-DEA 1 antibody (Alvedia, Lyon, France) found that antigenic binding was quantitatively different among dogs, indicating that DEA 1 is a complex blood group system with varied surface expression levels. Consequently, a dog could be DEA 1 or weakly to strongly DEA 1+.7Experimentally, as well as clinically, DEA 1 incompatibilities have been associated with serious blood transfusion reactions. As approximately half of all dogs are DEA 1+8,9, it is important to understand how the weak to strong DEA 1+ phenotypes are inherited and how Rabbit polyclonal to RABAC1 they relate to DEA 1+ antigenicity in order to provide sound clinical transfusion guidelines. Here we determined the mode of inheritance of DEA 1 in several canine families, attempted to characterize the DEA 1 antigen using EW-7197 immunoblotting procedures, and screened canine RBCs with typing reagents against human blood groups to check for cross-reactivity. We also screened one monoclonal anti-DEA 1 antibody against human RBC panels expressing different blood group antigens used in human blood banking and transfusion medicine to determine if potential orthologs exist. Identification of a conserved RBC membrane protein in dogs could improve characterization of the DEA 1 antigen. It can also contribute to the development of more specific blood-typing reagents. == Materials and Methods == == Animals and Samples == Blood samples, anticoagulated with Ethylenediaminetetraacetic acid (EDTA) and kept at 4C, were obtained from research colony dogs or EW-7197 received as left-over samples from canine individuals in the Clinical Pathology Laboratory at the School of Veterinary Medicine of the University or college of Pennsylvania (Penn Vet). For confirmatory DEA 1 typing, blood samples previously tested as DEA 1.2+ using conventional polyclonal antibody typing methods were received from EW-7197 Animal Blood Resources International (ABRI, Dixon, CA) and HemoSolutions (Colorado Springs, CO). Samples from several canine families were provided by commercial study puppy colonies (Covance, Cumberland, VA and Marshall, North Rose, NY) and the Penn Vet study puppy colony for DEA 1 typing. These studies were authorized by the Institutional Animal Care and Use Committee in the University or college of Pennsylvania. == DEA 1 Blood Typing by Immunochromatographic Strip and Circulation Cytometry == Following a preparation of 20% RBC suspensions from each blood sample, blood typing was performed using immunochromatographic strip packages (Alvedia, Lyon, France). The band strength was read on a level from 0 to 4+ by one author (KP) prior to densitometric analysis of the strip using a protocol previously explained by our laboratory.7For circulation cytometry, diluted monoclonal murine anti-DEA 1 antibody identical to the antibody impregnated.