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

Dana Cavill, Peter J. Macardle, Dimitra Beroukas, Gentaro Kinoshita, Jurgen Stahl, James McCluskey, Tom P. Gordon

    Research output: Contribution to journalArticlepeer-review

    2 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)150-155
    Number of pages6
    JournalApplied Immunohistochemistry
    Volume7
    Issue number2
    Publication statusPublished - 1 Jun 1999

    Keywords

    • Calreticulin
    • Human tissues
    • Immunolocalization

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