TechnicalGeneration of a Monoclonal Antibody Against Human Calreticulin by Immunization with a Recombinant Calreticulin Fusion Protein: Application in Paraffin-Embedded SectionsCavill, Dana B.Sc. Hons; Macardle, Peter J. Ph.D.; Beroukas, Dimitra B.Sc.; Kinoshita, Gentaro M.D.; Stahl, Jurgen F.R.C.P.A.; McCluskey, James M.D.; Gordon, Tom P. M.D.Author Information From the Departments of Immunology, Allergy & Arthritis (D.C., P.J.M., D.B., G.K., T.P.G.) and Anatomical Pathology (J.S.), Flinders Medical Centre and Flinders University of South Australia, SA; and the Department of Microbiology and Immunology (J.McC.), University of Melbourne, Victoria, Australia. Manuscript received November 2, 1998; accepted March 4, 1999. Address correspondence and reprint requests to Dr. T.P. Gordon, Department of Immunology, Allergy & Arthritis, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. E-mail: [email protected] Applied Immunohistochemistry & Molecular Morphology: June 1999 - Volume 7 - Issue 2 - p 150-155 Buy Abstract Calreticulin (CR) is a highly conserved, calcium-binding protein with a diverse 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 in 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. Immunohistochemical analysis of a range of normal tissues confirmed the widespread expression of CR, notably in parenchymal epithelial cells, neurons, endothelial cells, and lymphocytes, predominantly of B-cell origin. The pattern of staining was cytoplasmic, not nuclear. Only weak staining was found in stromal cells. This first mAb to be produced against human CR will be a valuable reagent for studying the expression of CR and its putative role in autoimmune disease and malignancy. Recombinant fusion proteins in which the target protein is fused with a foreign moiety may be useful immunogens for breaking tolerance and generating mAbs against extremely conserved proteins such as CR. © 1999 Lippincott Williams & Wilkins, Inc.