Generation of a monoclonal antibody against human calreticulin by immunization with a recombinant calreticulin fusion protein: Application in paraffin-embedded sections

Calreticulin (CR) is a highly conserved, calcium-binding protein with a div erse functional repertoire located primarily in the endoplasmic reticulum ( ER). A murine monoclonal antibody (mAb) reactive with human CR was produced by immunizing with a maltose-binding protein-CR fusion protein expressed i n Escherichia coli. This mAb (FMC75) bound recombinant and native human 60- kDa CR on Western blots but, unlike a polyclonal anti-CR antibody, did not cross-react with mouse CR. FMC75 gave a staining pattern identical to that of the polyclonal antibody on confocal microscopy of cultured cells and was positive on microwave-treated tissue sections embedded in paraffin. Immuno histochemical analysis of a range of normal tissues confirmed the widesprea d expression of CR, notably in parenchymal epithelial cells, neurons, endot helial cells, and lymphocytes, predominantly of B-cell origin. The pattern of staining was cytoplasmic, not nuclear. Only weak staining was found in s tromal cells. This first mAb to be produced against human CR will be a valu able reagent for studying the expression of CR and its putative role in aut oimmune disease and malignancy. Recombinant fusion proteins in which the ta rget protein is fused with a foreign moiety may be useful immunogens for br eaking tolerance and generating mAbs against extremely conserved proteins s uch as CR.