Breast carcinoma specific antibody selection combining phage display and immunomagnetic cell sorting.

To discover new specific antibodies directed against disseminated carcinoma cells in breast cancer patients, a strategy combining single-chain variable fragment (scFv) phage display and immunomagnetic cell sorting was developed. A selection model, in which ErbB2-expressing breast carcinoma SKBR3 cells are spiked into a 50-fold excess of lymphocytes, was setup. Selection conditions, optimized using the previously characterized ErbB2-specific F5 phage scFv, led to an outstanding phage enrichment yield of 25,000 after only one round. This protocol applied to human nai ve and synthetic phage display antibody libraries led to the selection, in only two rounds, of individual scFv clones (43 out of 46 tested) specific for non-epithelial carcinoma antigens expressed on SKBR3 cells. This strategy is fully applicable to metastatic cells in effusions from breast carcinoma patients and shall lead to the discovery of immunotools crucial for novel diagnostic and therapeutic approaches.

Expression, purification, crystallization and preliminary X-ray studies of the outer membrane efflux proteins OprM and OprN from Pseudomonas aeruginosa.

OprM and OprN belong to the outer membrane factor family proteins. These approximately 52 kDa proteins are part of the tripartite efflux pumps found in Pseudomonas aeruginosa and are responsible in part for the antibiotic resistance observed in these bacteria. Both proteins have been expressed in Escherichia coli as His-tag proteins and purified accordingly by affinity chromatography in the presence of n-octyl-beta-D-glucopyranoside detergent. OprM and OprN were crystallized using PEG 20 000/ammonium citrate and ammonium sulfate as precipitating agents, respectively. Crystals belong to space group C2, with unit-cell parameters a = 152.6, b = 87.9, c = 355.9 A, beta = 98.9 degrees and a = 151.3, b = 87.6, c = 356.5 A, beta = 98.1 degrees for OprM and OprN, respectively. Using the ESRF synchrotron-radiation source, OprM diffraction data extended to 3.4 A.