Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran.
We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (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-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly 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 profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.
Hattori
M
M
Department of Applied Biological Science and Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-City, Tokyo 183-8509, Japan. makoto@cc.tuat.ac.jp
Nagasawa
K
K
Ohgata
K
K
Sone
N
N
Fukuda
A
A
Matsuda
H
H
Takahashi
K
K
D000818:Animals; D003911:Dextrans / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D005260:Female; D018796:Immunoconjugates / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D013050:Spectrometry, Fluorescence; D014801:Vitamin A / Q000378:metabolism
D003911:Dextrans; D000939:Epitopes; D018985:Epitopes, B-Lymphocyte; D018796:Immunoconjugates; D007782:Lactoglobulins; D014801:Vitamin A; C014392:carboxymethyl dextran