Molecular and biochemical analyses of combining sites of monoclonal anti-morphine antibodies.
V region nucleotide sequences were determined by mRNA sequencing for 11 monoclonal anti-morphine antibodies with slightly different specificities for morphine-related opiates. The VH region nucleotide sequences of the antibodies MOR8, MOR33, MOR35, MOR44, MOR83, and MOR76 were classified into the VH-5 (7183) family, while the antibodies MOR39, MOR115, MOR131, MOR158 and MOR180 used VH-1 (J558) family genes. MOR39, MOR115 and MOR131 used the V lambda-1 gene for their L chain V region. MOR158 and MOR180 used the Vk-10 gene. MOR8, MOR33, OR35, MOR44, MOR76 and MOR83 used VK-21D. The antibody sets MOR158 and MOR180; MOR39 and MOR131; and MOR8, MOR33, MOR35, MOR44, MOR76 and MOR83 appeared to be somatic mutants derived from the same clones since they showed the same VH/VL usage and V(D)J recombination pattern. The pH-reactivity profiles for these antibodies revealed that the binding of morphine to the antibodies is highly dependent on the pH value of the assay solution, suggesting the importance of the electrostatic interaction between the positive charge of morphine and the negative charges at or near the combining sites. Direct UV-photoaffinity labeling with 3H-morphine was carried out in order to estimate the orientation of morphine in the combining sites. The H chains were preferentially labeled in MOR8, MOR33, MOR35, MOR76, and MOR83, whereas most of the crosslinked hapten was found in the L chains in MOR39, MOR115, MOR131, MOR158 and MOR180. Thus, these 11 antibodies were classified into two types in terms of reactivity in the photoaffinity labeling.
Sawada
J
J
Division of Biochemistry and Immunochemistry, National Institute of Hygienic Sciences, Tokyo, Japan.
Yamazaki
T
T
Terao
T
T
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics*; D003061:Codeine / Q000276:immunology; D003429:Cross Reactions; D004090:Dihydromorphine / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009020:Morphine / Q000276:immunology*; D009270:Naloxone / Q000276:immunology; D012333:RNA, Messenger / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D009270:Naloxone; D009020:Morphine; D004090:Dihydromorphine; D003061:Codeine