Immune response in rabbits to dengue viral proteins.

Antisera to all types of dengue and Japanese encephalitis (JE) viruses were raised in rabbits. The first set of rabbits was immunized with crude antigens prepared by a sucrose acetone extraction. The first generation of antisera demonstrated antibody activity towards the group specific flaviviruses, without unwanted antibody activity towards the mouse brain material. Antibody activity towards E, NS3, NS5 and NS1 could be observed by western blot/immunoenzymatic assay. Another set of rabbits was immunized with the precipitin complexes formed between antisera raised against each type of dengue, or JE viruses, and their homologous antigens. Each rabbit serum was screened again by the western blot/immunoenzymatic method, and was absorbed with other types of dengue viruses until the specific activity towards the immunized viral antigen was obtained. The last 2 bands detected on the viral antigen strip were the doublet of protein bands at Mr 67 and 71 kDa, which are the NS3 protein. The specific polyclonal antisera obtained can be used with other tests as well as the monoclonal antibody. Since absorption can lead to type specific antisera, this NS3 protein must be quite unique, it processes a type specific determinant (s) and deserves further study as a target molecule at the polypeptide level as well as at the RNA level.

Antibodies to dengue viral polypeptides. I. Sensitivity and specificity of the viral-antigen-strips/enzymeimmunoassay (VAS/EIA).

The authors applied polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), western blot, and enzymeimmunoassay (EIA) for the detection of antibodies toward individual dengue viral proteins or polypeptides. SDS-PAGE procedure as described by Laemmli et al., was applied and modified. The results can be observed by visualization. The scores, from 0 to 4, can be assigned by comparing the intensity of the color development. Besides being sensitive and rapid, this technique yields information of the polypeptides or molecular level. An increasing of intensities of positive reactions indicated rising in antibodies titers and all serotypes of dengue viruses (from type 1 to type 4) can be tested together allowing reliable comparison among serotypes. With hyperimmune human sera, at least 13 polypeptides reacted with sera while negative non-immune subject showed no reaction. It is highly possible to use this technique as a rapid quantitative and for qualitative analysis of antibodies to individual viral proteins as well.