Anti-anti-idiotypic (Ab3) antibodies that bind progesterone-11alpha-bovine serum albumin differ in their combining sites from antibodies raised directly against the antigen.
Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antiprogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone response in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-50-times lower than that of DB3, their steroid-binding specificity showed considerable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal antibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progesterone conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound free progesterone, although with low affinity and very broad cross-reactivity. Like DB3, 1A4 is encoded by a heavy-chain variable region (VH) gene segment from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11alpha-BSA. In contrast, 3B11 binds progesterone-11alpha-BSA but not free progesterone and is encoded by an unrelated VH gene from the J558 family. The light chain variable region (VL) of 1A4 lacks the intradomain disulphide bridge owing to replacement of CysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short complementarity determining region (CDR) H3 loops, comprising three and four amino acids, respectively. Modelling of the combining site of 1A4 from the X-ray crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.
Kirsch
R D
RD
Laboratory of Molecular Recognition, The Babraham Institute, Babraham, Cambridge, UK.
Beale
D
D
He
M
M
Corper
A L
AL
Krawinkel-Brenig
U
U
Taussig
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003429:Cross Reactions; D005260:Female; D005803:Genes, Immunoglobulin; D007114:Immunization; D007074:Immunoglobulin G / Q000096:biosynthesis; D007075:Immunoglobulin M / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011374:Progesterone / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000276:immunology / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; C086103:progesterone-11-hemisuccinyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D011374:Progesterone