Antigen capture anti-dengue IgA ELISA (ACA-ELISA) for the detection of a flavivirus specific antibody

A flavivirus, specific technology, applied in antiviral immunoglobulins, measuring devices, resistance to vector-borne diseases, etc., can solve problems such as low IgM detection sensitivity

Inactive Publication Date: 2009-07-08
NATIONAL ENVIRONMENT AGENCY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of these kits claim to differentiate between primary and secondary dengue virus infection, however this is not always reliable
The results show that these kits generally have higher IgG detection sensitivity and lower IgM detection sensitivity, as well as different specificity compared to E / M specific capture IgM and IgG ELISA

Method used

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  • Antigen capture anti-dengue IgA ELISA (ACA-ELISA) for the detection of a flavivirus specific antibody
  • Antigen capture anti-dengue IgA ELISA (ACA-ELISA) for the detection of a flavivirus specific antibody
  • Antigen capture anti-dengue IgA ELISA (ACA-ELISA) for the detection of a flavivirus specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Example 1: Development of anti-dengue IgA using serum (ACA-ELISA)

[0188] a) Preparation of antigens: Lysate dengue virus antigens against all four dengue virus serotypes were prepared according to the method described by Cardosa et al. (2002). 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 the infected cells was rinsed 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 and aliquoted into eppendorf tubes in 250 μl, and stored at -70°C for future use.

[0189] b) Serum samples: A total of 292 dengue PCR-confirmed serum samples from 3 groups were used as positive samples. 182 dengue PCR-negative patient serum sam...

Embodiment 3

[0199] Example 3: Comparison of Analytical Sensitivity of Two IgA Assays:

[0200] The sensitivity levels of the two anti-dengue IgA assays (AAC-ELISA and ACA-ELISA) were further analyzed using dengue positive IgA serum samples in 2 different diluents (eg dengue negative serum and dilution buffer). Figure 4 showed that in AAC-ELISA, the sensitivity of sera with high levels of anti-dengue IgA when diluted in negative sera was 32-fold lower than in dilution buffer, due to suppression by high levels of non-dengue-specific IgA in the serum diluent 43.71%~79.79% ( Figure 4 ).

[0201] On the other hand, in ACA-ELISA, the detection level of dengue-specific IgA was the same in both diluents (negative serum and diluent buffer), and the suppression of non-dengue-specific IgA present in the serum diluent was negligible is -11.08%~-61.76% ( Figure 4 ).

Embodiment 4

[0202] Example 4: Sensitivity and specificity of ACA-ELISA

[0203] This example was carried out using 296 dengue-confirmed and 182 dengue-negative serum samples collected during the acute and convalescent phases. Among the confirmed dengue samples, 96 samples were collected from days 1-3 of febrile onset, 97 samples were collected from days 3-7, and 102 samples were collected from days 10-37 of febrile onset. Serum samples from 182 patients were collected from dengue PCR-negative patients who developed fever during sample collection. The sensitivity and specificity of ACA- and AAC-ELISA are shown in Table 1 and Table 2.

[0204] Table 1. Sensitivity and specificity of serum ACA-ELISA

[0205]

[0206] Table 2. Sensitivity and specificity showing AAC-ELISA

[0207]

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Abstract

An antigen capture IgA Enzyme Linked Immunosorbent Assay (ACA-ELISA) was developed for the detection of anti-flavivirus IgA. The assay utilizes flavivirus lysate antigen, preferably dengue virus lysate antigen captured by a monoclonal antibody. Captured anti-flavivirus IgA from test sera are preferably detected using rabbit anti-IgA conjugated with a reporter group such as horseradish peroxidase (HRP). The assay was found to be at least 8 times more sensitive than anti-human IgA capture ELISA (AAC-ELISA). The ACA-ELISA, based either on serum or saliva, was found to be more sensitive and rapid compared to the ''gold standard'' anti-dengue IgM detection technique and can be utilized as a diagnostic tool for the confirmation of dengue in the early phase of infection.

Description

technical field [0001] The present invention relates to techniques for the detection of recent exposure of humans or animals to flaviviruses or their equivalents. 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 has been definitively exposed to a flavivirus or its equivalent. The present invention also provides a medical diagnostic kit and serum evaluation by detecting flavivirus-specific antibody (IgA) produced by flavivirus infection or its equivalent. More preferably, the invention relates to dengue virus. Background technique [0002] The Flaviviridae family includes many viruses that cause disease in humans and are usually transmitted by mosquitoes or ticks. The Flavivirus genus includes a variety of viruses including yellow fever virus (YF), dengue fever (DF) virus, West Nile (WN) virus, and Japanese encephalitis (JE) virus, each of which ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N33/569G01N33/563C07K16/08Y02A50/30
Inventor 比容·库马尔西叶秀莲
Owner NATIONAL ENVIRONMENT AGENCY
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