Mapping of structure-function relationships in proteins with a panel of monoclonal antibodies. A study on human alpha...

Monoclonal antibody (Mab) F2B2, directed to the receptor-recognition site of human alpha 2 macroglobulin (alpha 2M), has been instrumental in the characterization of that site and in the isolation of the receptor-binding domain. We have now prepared a panel of Mab to study the structure-function relationships in alpha 2 M, and in particular the expression of the receptor-recognition site. Reversed dot-blotting was very effective to screen hybridoma supernatant for specificities to either the native or complex form of alpha 2M. Reaction with the isolated 20 kDa receptor-binding domain of alpha 2M and cross-reaction with pregnancy zone protein was detected by the same technique. Eventually, a panel of 45 Mab was constructed consisting of essentially five types of specificities, although in fact no two Mab reacted with complete identity in all assays. In addition to the assays already mentioned, the Mab were tested for interference with binding of alpha 2M-trypsin to the cellular receptor, for competition with F2B2 for alpha 2M-trypsin and for inhibition of trypsin by alpha 2M. Finally, Western blotting was used as a first approximate mapping of the epitope relative to the internal thiolesters by exploiting the heat-induced fragmentation of alpha 2M at this site. The five categories of Mab thus detected were: (i) five Mab that react with native alpha 2M and not with alpha 2M trypsin; (ii) 18 Mab that react with both native alpha 2M and with alpha 2M-trypsin; (iii) 12 Mab, including F2B2 and F12A3, that react with the receptor-binding domain, neo-antigenically expressed on alpha 2M-trypsin, (iv)O six Mab that are also specific for alpha 2M-trypsin but map outside the receptor-binding domain; (v) three Mab that define hidden determinants, not expressed on undenatured alpha 2M. For completeness, the panel includes the Mab obtained against pregnancy zone protein.