European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants.

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut-off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers.

Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry.

During recent years, phytoestrogens have been receiving an increasing amount of interest, as several lines of evidence suggest a possible role in preventing a range of diseases, including the hormonally dependent cancers. In this context, various parts of the pomegranate fruit (Punica granatum; Punicaceae), e.g. seed oil, juice, fermented juice and peel extract, have been shown to exert suppressive effects on human breast cancer cells in vitro. On-line biochemical detection coupled to mass spectrometry (LC-BCD-MS) was applied to rapidly profile the estrogenic activity in the pomegranate peel extract. The crude mixture was separated by HPLC, after which the presence of biologically active compounds, known or unknown, was detected by means of an on-line beta-estrogen receptor (ER) bioassay. Chemical information, such as molecular weight and MS/MS fingerprint, was obtained in real time by directing part of the HPLC effluent towards a mass spectrometer. Using this approach in total three estrogenic compounds, i.e. luteolin, quercetin and kaempferol, were detected and identified by comparing the obtained molecular weights and negative ion APCI MS/MS spectra with the data of an estrogenic compound library. Although well known in literature and widely distributed in nature, the presence of these phytoestrogenic compounds in pomegranate peel extract was not reported previously. Compared to traditional screening approaches of complex mixtures, often characterized by a repeating cycle of HPLC fractionation and biological screening, LC-BCD-MS was shown to profoundly accelerate the time required for compound description and identification.