Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) co njugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG, The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CM D conjugates maintained the retinol-binding activity of native beta-LG. Int rinsic fluorescence study indicated that shielding of the surface of beta-L G by CMD occurred in each conjugate, which was eminent in Conj. 10B. A loca l conformational change around (125)Thr-(135)Lys (alpha-helix) in each conj ugate was detected by ELISA with monoclonal antibodies. The denaturation te mperature of beta-LG evaluated by differential scanning calorimetry was gre atly enhanced in each conjugate. The anti-beta-LG antibody response was mar kedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6,and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope p rofiles of the beta-LG-CMD conjugates were similar to those of beta-LG, whi le the antibody response for each epitope was dramatically reduced. The red uced immunogenicity of beta-LG was most marked in the case of Conj. 10B, wh ich contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decrea sed immunogenicity of the beta-LG in these conjugates.