Immunochemical characterisation of species-specific antigens in bovine crude heparin

In order to develop immunoassays for the control of the species origin of crude heparins, polyclonal antisera were produced in rabbits against samples obtained from the last purification steps of bovine intestinal crude heparin. The reactivity of the antisera was analysed by agar gel double immunodiffusion, immunoelectrophoresis, crossed and line immunoelectrophoresis. Up to 13 antigenic components were detected in the effluents of the ion-exchange chromatographic step, and three in the final crude heparin. The major and most anodic antigen (Ag1) was recovered in bovine crude heparin purified by the two different industrial processes. This bovine specific antigen was found in high concentrations in lung, liver, small intestine, spleen and kidney. It displayed an apparent molecular weight of 45 kDa by size-exclusion chromatography. Though the identification of Ag1 is not yet fully elucidated, a single radial immunodiffusion assay has been developed for the quantification of this antigen, allowing the detection of 6 p 1000 bovine crude heparin in porcine heparin (50 mg/ml) after 2 h diffusion.