Monoclonal antipeptide antibodies as tools to dissect closely related gene products. A model using peptides encoded by the calcitonin gene.
We have explored the possibility of using mAb as tools for distinguishing between closely related gene products. We utilized calcitonin (CT) gene products as a model, because this 32-amino-acid-amidated hormone is biosynthesized by post-translational processing of a larger precursor. By using CT as a hapten, we had previously identified a mAb (CT07) with restricted specificity to mature CT, and had shown that another mAb (CT08) directed to a different epitope bound to both CT and the CT precursor. In this study, we used synthetic peptides analogous to various regions of biosynthetic intermediates of CT as haptens, and generated a library of mAb which define distinct epitopes. First, we identified two separate epitopes located in either the 1-11 or the 12-21 region of the C-terminal flanking peptide of CT (katacalcin, KC), and which were recognized by mAb KC01 and KC04, respectively. Second, we identified a conformational epitope in the C-terminal region of the putative glycine-extended form of CT (CT-Gly). This epitope was recognized by mAb CT19 and was shared by mature CT but not by CT precursors. Third, we identified an epitope restricted to CT-Gly and recognized by mAb CT20. For dissecting between related products of the CT gene, we designed different monoclonal immunoradiometric assays (m-IRMA) based on CT08 as the radiolabeled indicator antibody. A first m-IRMA based on CT07 as the capture antibody specifically recognized mature CT and did not cross-react with CT precursors. Conversely, another m-IRMA with KC01 as the capture antibody was specific for CT precursors and did not cross-react with either mature CT or CT-Gly. A third assay based on CT20 as the capture mAb was specific for CT-Gly and was not affected by the presence of either CT precursors or mature CT. We also used these antibodies to demonstrate that neoplastic C cells incompletely released processed CT precursors in serum, in addition to mature CT. This study demonstrates that mAb can be used as tools to selectively recognize closely related gene products. These findings might be applied to the study of other molecules biosynthesized by enzymatic modifications of a larger precursor.
Ghillani
P
P
Unité d'Immunochimie, Institut Gustave-Roussy, Villejuif, France.
Motté
P
P
Bohuon
C
C
Bellet
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D002116:Calcitonin / Q000097:blood / Q000235:genetics* / Q000276:immunology; D005998:Glycine; D007202:Indicators and Reagents; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D002116:Calcitonin; D005998:Glycine