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Development of two polyclonal antibodies against the human periostin (PN) and analysis of their specificities in serum by Western blot (WB) and in bone by immunohistochemistry (IHC)

Vergnaud P, Pagnon A, Bertholon C, Lhoste Y, Chassaing E, Borel O, Gineyts E, Schubert T, Clezardin P, Chapurlat R, Rousseau JCh

PURPOSE

Periostin belongs to the matricellular family of proteins and is mainly expressed in the periosteum. However, several authors have suggested roles for PN inside the cells: regulation of Notch1 expression (Tanabe, 2010), regulation of cell invasiveness (Isono, 2009) and activation of lysyl-oxydase (Kudo, 2011). The aims of this study were to develop two polyclonal antibodies against the Fas-1 region of human PN and investigate what they recognize in human serum and bone.

METHODS

By in silico study of the human PN we have detected two sequences of amino acids suitable for generate antibodies (Patent n° 13/375,870) in the second Fasciclin-like domain (ETLEGNTIEIGCDGDSI, Ab-34) and in the fourth Fasciclin-like domain (CKGFEPGVTNILKTTQGSK, Ab-12). These two peptides were injected in rabbits for immunization following a standard protocol of 12 weeks. Specific polyclonal antibodies Ab12 and Ab34 were purified by immunoaffinity. We assessed the specificity of these antibodies for PN by IHC in human bone tissue and by WB analysis in human sera previously depleted for albumin, IgG and transferrin.

RESULTS

In bone as in serum Ab-12 and Ab-34 antibodies did not have the same pattern of recognition. WB analysis showed that in non-reducing conditions Ab-12 recognized two bands, a high molecular band around 200 kDa and a second one at 75 kDa. After serum reduction by dithiothreitol, these bands disappeared and 3 bands were detected at 70, 50 and 25 kDa. On the other hand, Ab-34 recognized in non-reducing conditions only one band at 75 kDa. After reduction, the band at 75 kDa migrates slightly higher at 100 kDa suggesting that the reduction of intra-chain disulfide bridges leads to a more extended conformation of this PN form and makes it run more slowly on gel. By IHC, we showed that Ab-12 stained the cytoplasm of osteoblasts located at the interface between cortical bone and bone marrow and the cytoplasm of osteoblasts present in the Havers canals. On the contrary, Ab-34 has a matrix staining in the periosteum and in the Havers canals.

CONCLUSIONS

We have generated two polyclonal antibodies against the Fas-like domains of human PN. Our preliminary results suggest that they highlighted in serum and in bone at least two forms of PN molecules.

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