Antibodies In Cell Lines Research Articles

Expression of DOCK9 and DOCK11 Analyzed with Commercial Antibodies: Focus on Regulation of Mutually Exclusive First Exon Isoforms.

Dedicators of cytokinesis 9 and 11 (DOCK9 and DOCK11) are members of the dedicator of cytokinesis protein family encoding the guanosine nucleotide exchange factors for Rho GTPases. Together with DOCK10, they constitute the DOCK-D or Zizimin subfamily. Two alternative full-length amino terminal isoforms of DOCK9 are known, which we will call DOCK9.1 and DOCK9.2. In order to investigate the relevance of the presence of the alternative first exon isoforms within this family, and to lay the groundwork for future studies that seek to investigate their potential role as biomarkers of disease, the expression levels of DOCK9 and DOCK11 were measured by qRT-PCR in 26 human tissues and 23 human cell lines, and by Western blot analysis, using commercial antibodies in cell lines. DOCK9.1 and DOCK9.2 were widely distributed. High levels of expression of both isoforms were found in the lungs, placenta, uterus, and thyroid gland. However, only DOCK9.1 was significantly expressed in the neural and hematopoietic tissues. The unique first exon form of DOCK11 was highly expressed in hematopoietic tissues, such as the peripheral blood leukocytes, spleen, thymus, or bone marrow, and in others such as the lungs, placenta, uterus, or thyroid gland. In contrast to tissues, the expression of DOCK9.1 and DOCK9.2 differed from one another and also from total DOCK9 in cell lines, suggesting that the amino terminal isoforms of DOCK9 may be differentially regulated. This study demonstrates the usefulness of antibodies in investigating the regulation of the expression of DOCK9.1, total DOCK9, and DOCK11.

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

Characterization of anti-BRCA2 antibodies in cell lines by Western blot analysis

BRCA2 is a tumor suppressor gene associated with familial predisposition to breast and ovarian cancer. BRCA2 has been implicated in response to DNA damage, cell cycle control and transcription. However, the mechanisms by which the BRCA2 protein suppresses tumor cell growth are largely unknown. To begin to understand the contribution of BRCA2 protein to tumorigenesis, we evaluated the specificity of 4 anti-BRCA2 antibodies directed against several different epitopes using immunoblotting techniques. The two monoclonal antibodies (3E6 and 5F6) detected a specific 384-kDa protein in human breast cancer cell lines (MCF7 and MDA-MB 231) and in a human colon carcinoma cell line (CCL 221). The two polyclonal antibodies (9433 and 9434) recognized the 384-kDa BRCA2 protein respectively in MCF7 and in CCL 221 cells, but both BRCA2 polyclonal antibodies also cross-reacted with smaller proteins.

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients. © 2000 Cancer Research Campaign

Neutralization of Aggregated Strains of Enterovirus 71 and Echovirus Type 4 in RD and Vero or GMK‐AH1 Cells

Strains of enterovirus 71 and echovirus type 4 containing aggregates which were poorly neutralized by antibody in cell lines of green monkey kidney origin such as Vero and GMK-AH1, were as easily neutralized as non-aggregated strains when grown in human RD cells, The explanation for this may be a lesser degree of aggregation of viral materials originating from RD cells and/or the use of lower doses of virus for neutralization tests in such cells. The latter was a consequence of a higher sensitivity of RD cells to minimal amounts of virus than that shown by Vero and GMK-AH1 cells. Thus, virus grown in RD, Vero or GMK-AH1 cells reached a titre 10 to 100 times higher in RD cells than in the other cell lines.