Rapid and sensitive HBsAg immunoassay based on fluorescent nanoparticle labels and time-resolved detection.

The detection of hepatitis B virus in blood specimens is carried out commonly by measuring surface antigen (HBsAg) levels with assays designed for various random access immunoanalysers or rapid near-patient testing. These methods leave much to be desired in performance or throughput. The aim of this study was to develop a nanoparticle label-based rapid and sensitive HBsAg immunoassay and evaluate its performance compared to a well-established reference immunoassay (Enzygnost HBsAg 5.0). The assay developed is based on kinetic format and relies on one-step two-site antibody-antigen interaction. Europium(III)-chelate-doped nanoparticles and microtiter wells were coated with anti-HBsAg monoclonal antibodies specific for discrete epitopes. The adaptation of nanoparticle labels for quantitative HBsAg detection showed improved sensitivity (LLD: 0.028 ng/ml) and dynamics (up to 1000 ng/ml) with reasonably low coefficients of variation (concentration-CV%s 2.8-21.9%). Furthermore, concurrent sample runs with the ELISA reference method showed 100% agreement. The time required for the assay was only 10 min facilitating a rapid and convenient method for hepatitis B screening.

A sensitive adenovirus immunoassay as a model for using nanoparticle label technology in virus diagnostics.

BACKGROUND: Currently, PCR-based hybridization assays are widely applied in adenovirus diagnostics. However, the technology requires tedious sample preparation, and the amplification phase is susceptible to various contaminants leading to inconvenient and time-consuming assay procedure. Methods relying on viral antigen detection, e.g. immunofluorometric assays (IFMAs) and enzyme immunoassays (EIAs), are less complicated to carry out, but they provide limited sensitivity. OBJECTIVE: Our aim was to develop a simple and sensitive adenovirus assay based on direct antigen detection via sandwich-forming immunoreaction. The assay employed highly fluorescent europium(III)-chelate-doped nanoparticle labels and selection of high affinity monoclonal antibodies (anti-hexon) coated on label particles and microtitration wells. RESULTS AND CONCLUSIONS: The extremely high specific activity of the nanoparticle labels enabled the detection limit over 5000 virus particles per millilitre with purified virus particles. The sensitivity was improved by three orders of magnitude (800-fold) compared to concurrent time-resolved IFMA. Furthermore, the nanoparticle assay showed reasonably low coefficients of variation (4.0-20%) and excellent linearity of more than four orders of magnitude (from below 10(5) to 10(9) virus particles per millilitre). Analyzed nasopharyngeal patient specimens revealed a minor disturbance of matrix components, which could be avoided by dilution. The average signal difference between negative and positive samples was nearly four orders of magnitude. The developed assay was sensitive and more convenient approach to adenovirus screening compared to available assays. In addition, the study demonstrates the potential of nanoparticles in sensitive screening of viral analytes.

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl.

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.