Anti-dengue IgG detection by an indirect ELISA.
Protein-free culture media were originally developed for hybridomas to simplify downstream processing and purification. For the same reasons, we have used these protein-free media for passaging dengue 2 virus in C6/36 cells. This provided us with an infected supernatant (DenPF) which could then be used as coating antigens for an indirect enzyme-linked immunosorbent assay (ELISA) to determine dengue IgG levels. Using this preparation, the main immunogenic band as seen by immunoblot appeared to be viral envelope protein (E). Without the high concentrations of "competing" proteins from fetal calf serum (FCS), the Den2PF could be directly coated onto 96-well ELISA plates. The amount of viral proteins in Den2PF appeared to be sufficient so that there was no need for further purification steps, eg polyethylene glycol (PEG) precipitation, which made this preparation cost effective. It compared favorably with the dengue dot enzyme immunoassay (DEIA; sensitivity of 95.7% and specificity of 95.2%).
Tio
P H
PH
Medical Molecular Biology Center, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Malasit
P
P
D000914:Antibodies, Viral / Q000097:blood*; D016022:Case-Control Studies; D003362:Cost-Benefit Analysis; D016895:Culture Media, Serum-Free / Q000592:standards*; D003715:Dengue / Q000175:diagnosis* / Q000276:immunology; D003716:Dengue Virus / Q000145:classification / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000592:standards; D007074:Immunoglobulin G / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D016895:Culture Media, Serum-Free; D007074:Immunoglobulin G