Antibodies to calnexin and mutated calreticulin are common in human sera.

PURPOSE OF THE STUDY: Calreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. PATIENTS AND METHODS: We analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. RESULTS: Antibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. CONCLUSION: Collectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.

Virus-specific antibody indices may supplement the total IgG index in diagnostics of multiple sclerosis.

Intrathecal antibody synthesis to viruses is associated with multiple sclerosis (MS). Here, IgG levels to Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1), EBV nuclear antigen 1 (EBNA1), mumps virus (MuV) nucleoprotein (NuP), measles virus (MeV) NuP and rubella virus (RuV) capsid protein (CaP) were found to be elevated in serum and cerebrospinal fluid (CSF) of MS patients compared to healthy controls (HCs), whereas the opposite was found for cytomegalovirus (CMV) pp52. Strong correlations between serum and CSF IgG were seen for MeV, CMV and RuV in both MS patients and HCs. The antigen panel obtained high sensitivity (81%) and specificity (86%), demonstrating that antigen panels may supplement the total IgG index used in MS diagnosis.

Production and characterization of peptide antibodies.

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies.