Haplotype analysis reveals that the recurrent BRCA1 deletion of exons 23 and 24 is a Greek founder mutation.

A recurrent large genomic rearrangement (LGR) encompassing exons 23 and 24 of the BRCA1 gene has been identified in breast-ovarian cancer families of Greek origin. Its breakpoints have been determined as c.5406 + 664_*8273del11052 (RefSeq: NM_007294.3) and a diagnostic polymerase chain reaction (PCR) has been set up for rapid screening. In a series of 2,092 high-risk families completely screened for BRCA1 and BRCA2 germline mutations, we have found the deletion in 35 families (1.68%), representing 7.83% of the mutations identified in both genes and 10.3% of the total BRCA1 mutations. In order to characterize this deletion as a founder mutation, haplotype analysis was conducted in 60 carriers from 35 families, using three BRCA1 intragenic microsatellite markers and four markers surrounding the BRCA1 locus. Our results demonstrate a common shared core disease-associated haplotype of 2.89Mb. Our calculations estimate that the deletion has originated from a common ancestor 1450 years ago, which most probably inhabited the Asia Minor area. The particular (LGR) is the third mutation of such type that is proven to have a Greek founder effect in the Greek population, illustrating the necessity for LGRs testing in individuals of Greek descent.

Development of a sensitive cost-effective capture ELISA for detection of murine monoclonal antibodies: correlation with SPR biosensor technology.

The development of antibodies for diagnostic and therapeutic applications in inflammatory diseases is a major focus for biotechnology and pharmaceutical companies. Production of monoclonal antibodies requires the development of fast, high-throughput methodologies for screening and selecting appropriate candidate antibodies for development. Capture (sandwich) enzyme linked immunosorbent assay (ELISA) provides a quick and reliable method that could be used for hybridoma screening of potential candidates accompanied with surface plasmon resonance (SPR) biosensor technology for identifying high affinity biomolecular interactions. A sensitive, cost-effective, robust and accurate capture ELISA for detection of murine monoclonal antibodies in culture supernatants was developed. This assay was optimized for high sensitivity and specificity with a capture anti-mouse polyclonal antibody. Using serial dilutions of a defined murine IgG antibody, a linear dose-response was observed between 2 and 1200 ng/ml antibody with a coefficient of determination r2 of 0.98. The detection limit of the assay was established as 2ng/ml (12.5pM). A similar concentration-dependent doseresponse was also observed using serial dilutions of antibody-containing supernatants from anti-alpha glycophorinproducing hybridomas (detection limit 1:2000). Specific capture of antibodies from supernatants in a similar setting was also confirmed using SPR biosensor technology and correlated well with the immunoassay results. The latter technology can be performed in order to provide quick screening results and kinetic analysis of antibody binding interactions aiming at identifying candidates with high affinity and specificity.