Automated type specific ELISA probe detection of amplified NS3 gene products of dengue viruses.

Aim: To apply an automated system of nucleic acid hybridisation coupled with the enzyme linked immunosorbent assay (ELISA) for the type specific detection of amplification products of dengue viruses.

Methods: Non-structural 3 (NS3) gene targets of reference strains of all four dengue and other flaviviruses, as well as dengue patient viraemic sera, were subjected to reverse transcription and polymerase chain reaction using consensus and dengue type specific primers and digoxigenin-11-dUTP label incorporation. The amplification products were detected by biotinylated type specific primers which served as ELISA capture probes bound to streptavidin coated tubes.

A shortened dengue IgM capture ELISA using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody.

A shortened IgM capture ELISA for the detection of dengue IgM antibodies using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody was described. The shortened two-step assay was compared with the four-step IgM capture ELISA on sera from dengue patients confirmed by the hemagglutination inhibition (HI) test. When paired acute and convalescent sera were tested, the shortened ELISA showed 100% agreement with HI results.