Mass spectrometric proteome profiling of tumor pleural effusion (
TPE
) liquid fraction from ovarian cancer patients was performed to identify the potential biomarkers of the disease. The methodology ...of analysis of the TPE protein composition included the removal of high-abundant proteins by affinity chromatography, additional fractionation of the low-abundant proteins based on their lipophilicity, and high-resolution mass spectrometric analysis. As a result, 190 proteins were indentified, 49% of them belonging to the groups of extracellular and membrane proteins. The application of several criteria to data analysis allowed us to generate a group of 26 proteins that are promising candidates for testing as ovarian cancer biomarkers.
MAb against the antigen CD117 - stem marker of human tumor cells. Strain 406 PPI prepared by cell fusion of mouse myeloma NS-1 cells with spleen mice BALB/ C, pre-immunized three times at an interval ...of two weeks, the cells of the cell line of human melanoma melKor. Merging conducted using a solution of PEG/DMSO. For screening received mAb 406 used human melanoma cell lines which differed in the expression of CD117 antigen FSBSI "N.N. Blokhin RCRC" collection. N.N Blokhin. Antigen expression was studied in immunofluorescence and evaluated on a flow cytometer BD FACS CantoTMII. ICA IC0-406 was compared with commercial ICA against antigen CD 117 (Germany). The results indicated the identity of the frequency of antigen-positive cases and the percentage of antigen cells. ICA IC0-406 block binding to cells melKor ICA anti-CD117. Linking ICA IC0-406 antigen-positive cells causes modulation of antigen CD 117.
High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just ...after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.
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