Kai 1 and Kai 2: Characterization of these dog erythrocyte antigens by monoclonal antibodies

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Abstract

Dog Erythrocyte Antigens (DEA) have thus far been found by sensitizing dogs with canine allogeneic blood and are clinically important regarding blood transfusion incompatibilities, but remain poorly defined. The goals of this study were to discover and characterize two DEAs, named as Kai 1 and Kai 2. The monoclonal antibodies were produced by mouse hybridoma techniques and examined by ELISA isotyping, immunoblotting, and affinity chromatography. Canine blood samples were typed and the development of alloantibodies was examined in transfused dogs. The monoclonal Kai 1 and Kai 2 antibodies were isotyped as IgM kappa and IgG3 lamda, respectively, and identified two different erythrocyte membrane proteins of 200 kDa and 80 kDa in molecular weights, respectively. Either Kai 1 or Kai 2 can be expressed but not both in an individual dog. There were no naturally occurring anti-Kai 1 or Kai 2 alloantibodies. In addition, Kai 1-and/or Kai 2-dogs developed Kai 1 and Kai 2 alloantibodies, respectively, when transfused with mismatched blood. This is the first discovery of canine blood types by screening monoclonal antibodies. Kai 1 and Kai 2 are novel blood types which can induce anti-Kai 1 or anti-Kai 2 alloantibodies when Kai 1-and/or Kai 2-dogs are transfused with Kai 1+ or Kai 2+ blood. These canine blood types may explain some of the blood incompatibilities and transfusion reactions observed in dogs in clinical practice.

Figures

  • Fig 1. SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) of purified (A) anti-Kai 1 and (B) anti-Kai 2 monoclonal antibodies. M: Marker (kDa); Lane 1: Normal mouse IgG, Lane 2 and 3: Purified mAb.
  • Fig 2. Isotyping of (A) anti-Kai 1 and (B) anti-Kai 2 antibodies by ELISA. Results were expressed as optical density (O.D.) values in ELISA, and values >0.2 (horizontal dashed line) were considered positive.
  • Table 1. Comparison of Kai and DEA 1 in 50 dogs.
  • Fig 3. Immunoblot of canine erythrocyte membrane proteins with with (A) anti-Kai 1 and (B) anti-Kai 2 monoclonal antibodies. M: Marker (kDa), Lane 1: Kai 1+/Kai 2- RBCs, Lane 2: Kai 1-/Kai 2+ RBCs, Lane 3: Kai 1-/Kai2- RBCs.
  • Fig 4. Affinity purification and chromatography of Kai 1 and Kai 2 antigens from erythrocyte membrane extracts. (A) Schematic diagram of affinity purification procedure. (B) Membrane proteins from Kai 1+ RBCs bound with anti-Kai 1 mAbs and IgM heavy & light chains of anti-Kai 1 antibody. M: marker (kDa), Lane 1: purified membrane protein (10 μg) from Kai 1+ RBCs. (C) Membrane proteins Kai 2+ from RBCs bound with anti-Kai 2 antibody and IgG heavy & light chains of Kai 2 antibody. M: marker, Lane 1: bovine serum albumin (2 μg), Lane 2: purified membrane protein (10 μg) from Kai 2+ blood. Molecular weights are indicated on the left side in kDa.
  • Table 2. Major cross-match results in the transfused dogs related to DEA 1 and Kai.
  • Fig 5. Major crossmatch test incompatibility in two dogs 21 days after receiving Kai mismatched transfusions. (A) Microscopic agglutination in case of a Kai 2-/DEA 1+ dog receiving Kai 2+/DEA 1+ blood. (B) Microscopic agglutination in case of a Kai 1-/DEA 1+ dog receiving Kai 1+/DEA 1+ blood. Bar = 200 μm.

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Canine blood groups and their importance in veterinary transfusion medicine.

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APA

Lee, J. H., Giger, U., & Kim, H. Y. (2017). Kai 1 and Kai 2: Characterization of these dog erythrocyte antigens by monoclonal antibodies. PLoS ONE, 12(6). https://doi.org/10.1371/journal.pone.0179932

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