Preparation of conjugates based on colloidal gold nanoparticles for application in rapid detection of antibodies to hepatitis E virus

Relevance. Hepatitis E virus (HEV) is a common cause of viral hepatitis not only in areas with low levels of water supply and hygiene, but also in industrialized countries. Rapid tests development for the infection seromarkers detection in the absence of special equipment and trained staff remains the most important problem in improving the diagnosis of hepatitis E. Aim. To produce conjugates of recombinant ORF2 antigen of HEV genotype 3 with gold nanoparticles (GNP) of varied sizes and to evaluate their applicability in the immunoassay for the detection of antibodies to HEV. Materials and methods. Specific polyclonal and monoclonal antibodies, recombinant antigen ORF2 of hepatitis E virus genotype 3, blood serum samples of people diagnosed with acute hepatitis. Synthesis of GNPs and their conjugates with recombinant antigen, enzyme immunoassay, dot immunoassay, immunochromatographic analysis, transmission electron microscopy. Results. Three samples of colloidal GNP were synthesized using citrate method with varied concentrations of reducing agent and were subsequently used for preparation of conjugates with recombinant antigen ORF2 of HEV genotype 3. Immunoreactivity of these conjugates was confirmed by dot-immunoassay with blood serum samples containing specific IgG. A conjugate based on a 41 nm GNP was chosen for use in immunochromatographic analysis (ICA). Optimal conditions for preparation of a multi-membrane composite, including formation of analytical and control lines and the conjugate area were identified, and test strips were developed. The obtained conjugate was tested by ICA using blood serum samples which had been subjected to preliminarily characterization by the content of the IgG antibody to HEV. High immunoreactivity of the conjugate was demonstrated. Antibodies to the virus were identified in 100% of the examined (n = 17) IgG-positive serum samples, while in negative samples (n = 17) they were absent. Conclusion. The results demonstrated effectiveness of the obtained immunoreagents (recombinant antigen, antibodies, conjugate) for use in test-systems for rapid diagnosis of hepatitis E.