| Abstract
| - We prepared two β-lactoglobulin (β-LG)−carboxymethyl dextran (CMD) conjugates (Conj. 10A andConj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of β-LG. The molarratios of β-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The β-LG−CMDconjugates maintained the retinol-binding activity of native β-LG. Intrinsic fluorescence study indicatedthat shielding of the surface of β-LG by CMD occurred in each conjugate, which was eminent in Conj.10B. A local conformational change around 125Thr−135Lys (α-helix) in each conjugate was detected byELISA with monoclonal antibodies. The denaturation temperature of β-LG evaluated by differentialscanning calorimetry was greatly enhanced in each conjugate. The anti-β-LG antibody response wasmarkedly reduced after immunization with the β-LG−CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of β-LG and each conjugate recognized in these mice andfound that the linear epitope profiles of the β-LG−CMD conjugates were similar to those of β-LG,while the antibody response for each epitope was dramatically reduced. The reduced immunogenicityof β-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, andwas effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible forthe decreased immunogenicity of the β-LG in these conjugates.
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