Single step, direct fluorescence immunoassays based on metal enhanced fluorescence (MEF-FIA) applicable as micro plate-, array-, multiplexing- or point of care-format.

Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lower sub-picomolar concentrations. We hereby present a new assay platform based on metal enhanced fluorescence (MEF), that remedies these problems since it eliminates the need for washing steps, for using enzyme labels and allows detection of analytes down to sub-picomolar concentrations. In addition this technology is fully compatible to standard fluorescence reader equipment as it is found in many laboratories nowadays. Since our present work is focused on single biomarker evaluation, we chose a 96 well plate format for convenience, but any other formate like antibody arrays, strip-like point of care devices etc. is feasible too.

Consumer preferences regarding the introduction of new organic products. The case of the Mediterranean sea bass (Dicentrarchus labrax) in Italy.

The introduction of new products on the market poses several challenges; in particular, whether the characteristics of the proposed product will be judged positively by potential consumers. This paper analyses the preferences of consumers regarding the introduction on the Italian market of a new product: organic Mediterranean sea bass. The aim of this study is to assess the importance given by consumers to four main characteristics of sea bass (country of origin, size, production method - organic or conventional - and price) so as to be able to formulate marketing strategies. We applied a choice experiment (CE) in order to define not only the ordinal ranking of preferences but also the willingness to pay (WTP) for the key characteristics of the newly-introduced product. We found that consumers show a higher WTP for the sea bass country of origin than for the breeding method used. Our results suggest that while organic aquaculture might be a new and important strategy for diversification, if suitable communication, either from a public policy or commercial perspective, and labelling/certification are not taken into consideration, the added value of the production method might not be perceived by the final consumers.

Assembly of rubella virus structural proteins into virus-like particles in transfected cells.

We have developed a stably transfected CHO cell line (CHO24S) that expresses the three structural proteins of rubella virus (RV). RV proteins C (capsid), E2, and E1 are secreted from CHO24S cells in the form of RV-like particles (RLPs) which form by budding into the cisterna of the Golgi complex. RLPs resemble RV virions in their size and morphology and have an identical buoyant density when purified on sucrose gradients. Release of RLPs into the medium was found to be dependent upon the E1 cytoplasmic tail since deletion or substitution of this domain with the same region from vesicular stomatitis virus G protein abrogated release of RV proteins from transfected cells. These results indicate that the RV 40S genomic RNA is not required for efficient particle assembly. Therefore, RLPs may serve as a convenient source of RV antigen for use in diagnostic assays and as an alternative to live attenuated vaccine strains.

Selective tolerance to the E1 protein of rubella virus in congenital rubella syndrome.

Rubella virus (RV) infection of the fetus in the first trimester of pregnancy usually results in severe birth defects collectively termed Congenital Rubella Syndrome (CRS) and is frequently associated with prolonged RV persistence in the infant. Immunological tolerance to RV is believed to contribute to viral persistence, but the mechanism for this is unknown. In this study, RV-specific antibody responses in CRS patients and healthy controls who had experienced Rubella infection postnatally were compared to determine if there were differences that might account for RV persistence in the former group. Levels and functional affinities of IgG specific for individual RV proteins (E1, E2, and C) were measured by enzyme immunoassay (EIA). Relative amounts of RV protein-specific IgG directed to linear and topographic epitopes were compared by immunoblots run under reducing or nonreducing conditions, respectively, and biological activity was determined by hemagglutination inhibition (HAI) assay. Results showed that both CRS patients and control subjects had comparably high levels of IgG directed to whole RV and to RV E2 and C proteins as measured by EIA. However, in contrast to the controls, CRS patients were found to have significantly reduced levels of antibodies directed to RV E1 protein and its linear (but not topographic) epitopes. Also, functional affinities of specific IgG directed to whole RV and E1 protein, as well as hemagglutination inhibition titers, were found to be significantly lower in CRS patients than in controls. The data suggest that intrauterine exposure to RV may result in selective immunological tolerance to the RV E1 protein. A model is presented that accommodates the serological findings of this investigation within a proposed mechanism of RV persistence resulting from selective immunological tolerance to RV E1 protein.

Cellular and humoral immune responses to rubella virus structural proteins E1, E2, and C.

Better understanding of cell-mediated immune responses to rubella virus would provide the basis for the development of safe and effective vaccines against rubella and would aid in analysis of the pathophysiology of congenital rubella syndrome. We have expressed individual rubella virus structural proteins, E1, E2 and C, via vaccinia virus recombinants. Using the expressed recombinant proteins as antigens, we were able to demonstrate antigen-specific lymphocyte proliferative responses in control individuals and individuals with congenital rubella syndrome. Among the two human groups studied, E1 glycoprotein proved to be a better immunogen than E2 or C. For the control individuals, significant differences in proliferative responses to the structural proteins E1, E2, and C were observed. These differences were not significant in individuals with congenital rubella syndrome. In parallel to the lymphoproliferative responses, immunoglobulin G responses were also found directed mainly to the E1 glycoprotein. These results suggest that E1 may be the most important rubella virus antigen to study in determining the domains required for constructing subunit vaccines against rubella.

Detection of rubella virus-specific immunoglobulin G (IgG), IgM, and IgA antibodies by immunoblot assays.

Immunoblot (IB) assays were developed for detection of rubella virus (RV)-specific immunoglobulin G (IgG), IgM, and IgA antibodies in human serum following natural infection or immunization. IB assays performed under nonreducing conditions were compared with those performed under reducing conditions and with immunoprecipitation assays. Significant loss of antigenicity (greater than 90%) of RV E1 and E2 proteins was observed when IB assays were performed in the presence of 2-mercaptoethanol as compared with assays under nonreducing conditions. In contrast, the antigenicity of RV capsid protein was not influenced by reducing agents. Sensitivity of IB for RV-specific IgG antibodies was determined to be 0.01 IU/ml under nonreducing conditions. In the determination of RV-specific IgM and IgA antibodies by IB, pretreatment of serum with protein G to remove competing high-affinity RV-specific IgG or rheumatoid factor significantly improved assay sensitivity. IB assays were observed to be superior to immunoprecipitation assays in their ability to better define the specificities of RV-specific antibodies and to detect antibodies of all immunoglobulin classes. However, the conformational sensitivity of RV protein antigenicity should be an important consideration in the interpretation of RV-specific antibodies by IB assays.

Differential IgG avidity to rubella virus structural proteins.

Avidity maturation of IgG antibody responses directed against the structural proteins of rubella virus (E1, E2, and C) as well as whole rubella virus (RV) was assessed at sequential time intervals in 7 individuals following serologically confirmed wild rubella infection. Individual structural proteins were purified from tissue culture supernatants by differential centrifugation, followed by preparative SDS-PAGE under non-reducing conditions. Avidity of IgG anti-rubella responses was measured by using the 8 M urea elution technique and results expressed as an elution ratio [ER(%)]. A low mean ER(%) of 23% was determined for E1-specific IgG responses during the 10-20 day period following onset of clinical rubella, with subsequent maturation of avidity ER(%) values to 52%, 75%, and 84% at 3 months, 1 year, and 2 years, respectively, post-rubella. In contrast, IgG anti-E2 responses showed minimal avidity maturation with ER(%) values of 20%, 29%, 30%, and 31% over the same time intervals. Similarly, responses to the capsid protein (C) remained at low avidity ER(%) values of 21%, 29%, 36%, and 35% over the 2 year follow-up period. The avidity maturation values for IgG directed against whole RV preparations paralleled observations for E1-specific responses with ER(%) values of 23%, 52%, 85%, and 87%, respectively. These data support the need to assess individual protein-specific antibody avidities in order to more fully understand viral-specific immune responses.

Reduction of rubella ELISA background using heat denatured sample buffer.

During the analysis of rubella-specific antibodies in sera from adult vaccinees, it was observed that the inclusion of heat-denatured blocking proteins in the sample dilution buffer substantially reduced non-specific binding of serum IgG to microtitre plates. Results suggest that this modification to standard ELISA technique may reduce the incidence of false positive results in these assays.

Characterization of carbohydrates linked to rubella virus glycoprotein E2.

Rubella virus contains two envelope glycoproteins, E1 and E2. The amino acid sequence for both glycoproteins is known, as is the number of N-glycosylation sites. This study has demonstrated the presence of O-linked carbohydrates bound to E2 and determined structural characteristics of the N-linked oligosaccharide chains. O-linked sugars were found to be resistant to digestion with N-glycanase but sensitive to beta-elimination with alkaline borohydride. After treatment with neuraminidase, O-linked sugars bound to peanut agglutinin, suggesting the presence of the disaccharide galactose-N-acetylgalactosamine, masked by sialic acid. The N-linked oligosaccharides were large, probably four-branched, and showed a lectin binding pattern suggesting the complex type, with terminal Gal, GlcNAc and sialic acid. No Endo H-sensitive carbohydrates were detected.

pH-dependent solubility shift of rubella virus capsid protein.

The mechanism of capsid uncoating in rubella virus and other togaviridae is not well understood. This study presents data which suggest that rubella virus capsid undergoes a structural change from having hydrophilic to hydrophobic properties, between pH 5 and 5.5. Such a conformational change would allow capsid uncoating to occur within the lysosome, allowing RNA penetration to occur upon fusion of the viral envelope with the limiting membrane of the lysosome.

Amino acid sequence of a deltalike toxin from Staphylococcus epidermidis.

A deltalike toxin produced by a clinical isolate of Staphylococcus epidermidis was purified, and the amino acid sequence was determined. The toxin molecule consisted of 25 amino acid residues and shared a high degree of molecular homology with delta toxin purified from a Staphylococcus aureus human isolate.