Anti-anti-idiotypic (Ab3) antibodies that bind progesterone-11 alpha-bovine serum albumin differ in their combining sites from antibodies raised directly against the antigen

Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antipr ogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone respon se in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-5 0-times lower than that of DB3, their steroid-binding specificity showed co nsiderable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal ant ibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progestero ne conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound fr ee progesterone, although with low affinity and very broad cross-reactivity . Like DB3, 1A4 is encoded by a heavy-chain variable region (V-H) gene segm ent from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11 alpha-BSA. In contrast, 3B11 bind s progesterone-11 alpha-BSA but not free progesterone and is encoded by an unrelated V-H gene from the J558 family. The light chain variable region (V L) of 1A4 lacks the intradomain disulphide bridge owing to replacement of C ysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short compl ementarity determining region (CDR) H3 loops, comprising three and four ami no acids, respectively. Modelling of the combining site of 1A4 from the X-r ay crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.