An Enzyme‐Linked Immunosorbent Assay Using a Chaotropic Agent (Sodium Thiocyanate) for Serotype Specific Reaction between Crude Dengue Viral Antigen and Anti‐Dengue Mouse Antibody

Abstract

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti-dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement-fixing antigen (SCF) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted serotype specifically to anti-dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti-type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti-type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.Dengue virus, family Flaviviridae, genus Flavivirus, consists of four closely related serotypes, and serotyping is difficult because of their cross-reaction. When antidengue antibody was prepared in mice, it was difficult to prepare serotype specific antibody as measured by the ELISA. And the use of serotype specific monoclonal antibodies is not simple and is hampered by the circumstances of virus laboratories, e.g., the lack of equipment and skilled personnel, and high cost and long periods to establish specific hybridoma cell clones. These situations are particularly seen for the laboratories in the developing countries where dengue viruses are prevalent. In this regard, the development of a practicable method to distinguish serotype specific reaction from cross-reaction will not only be useful for the identification of dengue isolates but also for the titration of serotype specific antibody in antisera. However, there are no adequate methods to detect only serotype specific reaction by using anti-dengue antibody and crude viral antigens.In 1984, Inouye et al (9) demonstrated a new technique for the differentiation of antibody avidity after virus infection, for example, rubella, rota and Japanese 721

Use of Recombinant Envelope Proteins for Serological Diagnosis of Dengue Virus Infection in an Immunochromatographic Assay

“…Reported methods for diagnosis of dengue virus infection commonly use whole virus (3,26) or crude extracts (1,19,35) for the target antigen. The use of such materials presents a potential health hazard through exposure to infectious virus particles.…”

Chronic viral infection promotes early germinal center exit of B cells and impaired antibody development

“…We next sought to determine whether LCMV-specific serum antibody avidity was impacted by chronic infection. Thus, we measured LCMV-specific serum antibody avidity using an ELISA combined with a chaotropic sodium thiocyanate (NaSCN) wash to remove low avidity antibody (Nawa, 1992;Pullen et al, 1986). In this assay, antibody avidity (Avidity Index) was measured by comparing the fraction of serum antibody sensitive to removal by NaSCN treatment to total antibody levels ( Figure S2A).…”

“…Analysis of GC area was done in using the FIJI distribution (Schindelin et al, 2012) of ImageJ (Rueden et al, 2017) through manual annotation of GC boundaries followed by area determination. Antibody avidity was measured by ELISA with modifications (Nawa, 1992;Pullen et al, 1986). For serum antibody avidity measurements, the anti-LCMV-NP ELISA was modified to include a wash step with either PBS, 1.5M sodium thiocyanate (NaSCN), 3M NaSCN, or 4.5M NaSCN for 15 minutes at room temperature after incubation of serum samples but before application of secondary goat-antimouse IgG(H+L) antibody.…”