Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on "Membrane-engineered" Fibroblast Cells with Virus-Specific Antibodies and Antigens

Abstract

A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV)-associated antigens and anti-HBV is described. The biosensor is based on "membrane-engineered" Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe) or antigens (HBsAg) in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen) triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA). The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV) virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg). The observed response was rapid (45 sec) and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca(2+)]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed.

Keywords: Bioelectric Recognition Assay; Cell biosensor; Hepatitis viruses; Membrane-engineering; Vero cells.

Figure 1.

Schematic representation of the immobilized cell biosensor. The reference electrode is manually inserted in a cell-free gel bead, while the measuring electrode is inserted in the immobilized cell-loaded gel bead. Neither electrode is in direct contact with the sample solution (i.e. only beads are immersed into sample). The approximate diameter of the beads is two mm. Both measurement and reference electrodes are connected to the PMD 1608-FS converter.

Figure 2.

Biosensor response against HBsAg. The sensor is based on Vero cells membrane-engineered with the anti-HBs antibody. Sensor response is expressed as an associated change in the membrane potential of immobilized cells. Error bars represent standard errors of the average value of all replications with each sample (n = 15 replications for each sample).

Figure 3.

Biosensor response against HBeAg. The sensor is based on Vero cells membrane-engineered with the anti-HBe antibody. Sensor response is expressed as an associated change in the membrane potential of immobilized cells. Error bars represent standard errors of the average value of all replications with each sample (n = 15 replications for each sample).

Figure 4.

Biosensor response against the anti-HBs antibody. The sensor is based on Vero cells membrane-engineered with HBsAg. Sensor response is expressed as an associated change in the membrane potential of immobilized cells. Error bars represent standard errors of the average value of all replications with each sample (n = 15 replications for each sample).

Figure 5.

Changes (expressed as differences in fluorescence intensity) of the cytoplasmic calcium ion concentration in Vero cells, membrane-engineered with anti-HBs (A, B), anti-HBe (C, D) and HBsAg (E, F), before (A, C, E) and after (B, D, F) treatment with the HBsAg, HBeAg and anti-HBs respectively.