AFFINITY PURIFICATION OF A MANNOSE-BINDING PROTEIN, A SENSITIVE TOOL IN THE DIAGNOSTICS OF IGM, VIA SITE-DIRECTED PHOSPHORYLATED MANNAN BOUND TO ALUMINA

Ca2+-dependent mannose-binding proteins (MPBs) belong to the family of animal lectins. They perform in vivo as defence molecules that act as opsonins by enhancing the clearance of mannose rich pathogens and hav e been used in vitro for the purification of IgM. MBPs have been previ ously isolated by methods based on binding the protein moiety of vario us mannan species to different matrices. However, the mannan-protein c omplexes did not have a constant protein content and the yield of the isolated MBPs was variable. In the present study we describe a new app roach for the affinity purification of MBPs based on the main polysacc haride moiety of the complex. After removal of residual phosphate grou ps naturally occurring at the C-3 position of the sugar, which interfe re with MBP recognition, the mannan was phosphorylated enzymatically a t C-6, at which position the OH group is not required for lectin bindi ng. The enzymatically phosphorylated mannan bound to an alumina column was used successfully for MBP separation from rabbit serum. The manno se-binding protein obtained was used in our study for diagnostic purpo ses in the identification and determination of very low concentrations of IgM.