A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

<p>Abstract</p> <p>Background</p> <p>We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.</p> <p>Methods</p> <p>The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.</p> <p>Results</p> <p>By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.</p> <p>Conclusion</p> <p>We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.</p

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors.

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections

Native human autoantibodies targeting GIPC1 identify differential expression in malignant tumors of the breast and ovary

<p>Abstract</p> <p>Background</p> <p>We have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue.</p> <p>Methods</p> <p>The current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples.</p> <p>Results</p> <p>By screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line). To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors.</p> <p>Conclusion</p> <p>The present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein plays in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools.</p

The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9–97.2), and 97.67 % (95 % CI: 94.15–99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa

The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9-97.2), and 97.67 % (95 % CI: 94.15-99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa

Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment-4

<p><b>Copyright information:</b></p><p>Taken from "Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment"</p><p>http://www.biomedcentral.com/1472-6750/8/3</p><p>BMC Biotechnology 2008;8():3-3.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2254390.</p><p></p>m powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (

Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment-2

<p><b>Copyright information:</b></p><p>Taken from "Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment"</p><p>http://www.biomedcentral.com/1472-6750/8/3</p><p>BMC Biotechnology 2008;8():3-3.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2254390.</p><p></p>in NPD and the other in DI medium and both were kept in standard culture conditions. After a week of growth the supernatants were collected, and the antibody concentrations were measured by a standard sandwich ELISA. Each column represents the mean antibody concentration that was measured in NPD and DI cultures. The error bars denote the standard error of the means. We have observed increased secretion of monoclonal antibody with a series of stable hybridoma clones and presented a detailed analysis with one of them in this manuscript. Panel A: Total antibody concentration measured in the culture supernatants; Panel B: Antibody concentration normalized per cell

Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment-3

<p><b>Copyright information:</b></p><p>Taken from "Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment"</p><p>http://www.biomedcentral.com/1472-6750/8/3</p><p>BMC Biotechnology 2008;8():3-3.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2254390.</p><p></p>ased medium supplemented with 3% FCS. Before seeding the cells were washed in serum-free media to verify the removal of any residual serum. During a period of two weeks the supernatants were collected as indicated and the cells were counted on the same day. The cultures were fed on the 4and 10day and medium was placed in the cultures on day 6. Although the cells in DI culture proliferated normally under these conditions, they failed to produce measurable quantities of antibody. Panel A: IgM production per hybridoma cell in 3% FCS; Panel B: Number of live cells at each antibody titration

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen-4

S analyzed by confocal microscopy and indicates that the target antigen is present in the membrane and cytoplasm. Staining of human breast cancer tissue was analyzed by standard fluorescent microscopy.<p><b>Copyright information:</b></p><p>Taken from "A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen"</p><p>http://www.biomedcentral.com/1471-2407/8/248</p><p>BMC Cancer 2008;8():248-248.</p><p>Published online 24 Aug 2008</p><p>PMCID:PMC2529336.</p><p></p