The immunogenicity of hyaluronic acid was investigated. Rabbits were immunized with encapsulated group A and C streptococci. Intact long-chain hyaluronate was conjugated to BSA for use as antigen in an ELISA. Antibodies to the hyaluronate-BSA conjugate were detected in peak immune sera. The specificity of the antibodies for both mammalian and streptococcal hyaluronate was shown by inhibition studies. To further confirm the presence of antihyaluronate antibodies, hyaluronidase-digested streptococcal hyaluronate was conjugated to biotin and used as an antigen in the ELISA. A clear immunization effect was shown for each rabbit by the study of preimmune and postimmunization bleedings. Titers for each rabbit increased by greater than 32 - 256 - fold. Inhibition studies using hyaluronidase-digested hyaluronate and periodate-treated hyaluronate showed that the immunodominant site of antibody reactivity was a terminal glucuronic acid residue. Further studies showed that the carboxyl group of the terminal glucuronide was the major immunoreactive site. Both mammalian and streptococcal hyaluronate inhibited the immune rabbit sera reaction to streptococcal hyaluronate, demonstrating crossreactivity of these molecules. Thus, hyaluronate was shown to be immunogenic in rabbits.

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