Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles.
The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.
National Institute of Hygienic Sciences, Tokyo, Japan.
J
J
Sawada
S
S
Mizusawa
T
T
Terao
M
M
Naito
Y
Y
Kurosawa
D019326:17-alpha-Hydroxyprogesterone; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000915:Antibody Affinity; D000916:Antibody Diversity; D001483:Base Sequence; D003350:Cortodoxone / Q000276:immunology*; D005803:Genes, Immunoglobulin; D006863:Hydrogen-Ion Concentration; D006908:Hydroxyprogesterones / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D006908:Hydroxyprogesterones; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D009841:Oligonucleotides; D012333:RNA, Messenger; D019326:17-alpha-Hydroxyprogesterone; D003350:Cortodoxone