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Competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of a flavivirus specific antibody

A flavivirus-specific technology, applied in the field of exposure of valuable substances and differential serum evaluation, can solve the problems of difficult diagnosis and unreliable serotyping

Inactive Publication Date: 2009-06-03
NATIONAL ENVIRONMENT AGENCY
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Problems solved by technology

Although some cross-reactivity with heterologous serotypes was observed, the results showed a specificity of 93.94%, but the method was not reliable for serotyping secondary infections
[0019] The problem faced in dengue virus is also encountered in other flaviviruses, both share the same antigenic properties, making the diagnosis difficult

Method used

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  • Competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of a flavivirus specific antibody
  • Competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of a flavivirus specific antibody
  • Competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of a flavivirus specific antibody

Examples

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Embodiment 1

[0194] Example 1 - C-ELISA for detection of dengue specific IgG

[0195] 1. Materials and methods

[0196] 1.1 Preparation of virus lysate antigens for C-ELISA: According to the method described in Cardosa et al., 2002, dengue virus antigen lysates were prepared for all four dengue virus serotypes. Briefly, dengue virus (5m.o.i.) was cultured in C6 / 36 cells, depending on the development of the cytopathic effect and the serotype of the virus, in virus maintenance medium containing 2% fetal bovine serum and incubated for 4- 5 days. The medium was decanted, the culture flask containing infected cells was washed 4 times with PBS, treated with 1 ml hypotonic buffer containing 1% trix100 for 1 hour, and finally centrifuged at 14000 rpm for 10 minutes. The supernatant was collected for detection of dengue population-specific and serotype-specific monoclonal antibodies by indirect ELISA, and 500 μl was dispensed into eppendorf tubes, and stored at -70°C for future use.

[0197] 1.2...

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Abstract

A competitive enzyme-linked immunosorbent assay (C-ELISA), using flavivirus member specific immunological agents was developed to detect antibody specific to members of the flaviviruses indicative of exposure to flavivirus. The test is based on a competition for epitope binding on the envelope protein of the flavivirus antigen captured using anti-flavivirus IgA in the presence of flavivirus positive serum. This test has comparable sensitivity specificity and speed to the virus neutralization assay (VNT). C-ELISA is a versatile technique, which could have various applications. Slight modifications of this protocol could lead to a C-ELISA-based detection method of secondary infection or one that could be used for serotype specific sero-epidemiological studies and / or vaccine evaluation. The protocol developed for C-ELISA was demonstrated using dengue lysate antigen and dengue specific monoclonal antibody. This can be used against other flaviviruses and the results for Japanese encephalitis illustrates this.

Description

technical field [0001] The invention relates in particular to techniques for detecting exposure of humans and / or animals to flaviviruses or equivalents thereof. More particularly, the present invention relates to the rapid and simple analysis of a biological sample taken from a subject (animal or human) to determine whether the subject was previously exposed to a member of the Flaviviridae family or an equivalent thereof. The present invention also provides serotyping of flavivirus exposure based on serological analysis, and provides medical diagnostic kits and differential serum evaluations for infection by flavivirus members or equivalents thereof. The invention is particularly important for detection of flavivirus secondary infection, determination of serotyping of previous infection, seroepidemiology and vaccine evaluation. Background technique [0002] The Flavividae family includes many viruses that cause disease in humans and are usually transmitted by mosquitoes and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C07K1/14C07K1/22C07K2/00C12N7/02
CPCC12N2770/24111G01N2333/18G01N33/56983C07K1/14C07K1/22C12N7/02G01N33/569Y02A50/30
Inventor 比容·库马尔西切尼·诗·祯·李
Owner NATIONAL ENVIRONMENT AGENCY
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