Download NS1 antigen ELISA

Immunoglobulin M, nonstructural 1 antigen and virus
detection of dengue virus in single serum specimen
to develop diagnostic algorithm that maximizes the
likelihood of a correct laboratory diagnosis
Abstract
Dengue fever caused by dengue virus (DENV), a member of Flaviviridae leads to large global disease burden. Detection of immunoglobulin M (IgM) and
nucleic acid to DENV, and virus isolation have been used for laboratory diagnostic assays for dengue. Nonstructural 1 (NS1) antigen which releases from
DENV infected cells is also a useful diagnostic tool. The measure of duration and intensity of immune response and the first detection time of antibody
and antigen to dengue virus are influenced by sensitivity and specificity of assays and specimen collection time. To diagnose a current DENV infection,
the demonstration of seroconversion in paired sera is required. Specimens are collected at wide time range of infection episode. The paired sera are
usually not available in most of cases. Therefore, it is strongly required to develop dengue diagnostic algorithm to support the likelihood of a correct
laboratory diagnosis. Sera were collected from 604 dengue suspected patients who traveled dengue endemic country. Sera were tested by IgM ELISA,
NS1 antigen ELISA and nested RT-PCR. IgM ELISA, NS1 antigen ELISA and RT-PCR showed 38.7 %, 47.5 % and 18.9 % of positive rate, respectively.
The positive rate was increased by combination of assay, 54.0 % in IgM ELISA and NS1 antigen ELISA, 41.4 % in IgM ELISA and RT-PCR, 49.7 % in NS1
antigen ELISA and RT-PCR, 54.8 in three assays. These results suggest combination of NS1 antigen and IgMs give more accurate diagnostic result. This
study was supported by the intramural grant (#2013-NG53003-00) of the National Institute of Health, Korea.
Materials and Method
 Serum samples
-The serum samples (totally 604 sera) of dengue like symptoms between 2011 and 2012 were obtained from Division of Arboviruses at Korea National Institute of Health for serologic diagnosis.
 RT-PCR
Preparation of RNA
cDNA synthesis and
first PCR reaction
Nested PCR reactions
Gel electrophoresis
 All the 604 specimens were extracted viral RNA by
viral RNA extraction kit (Intron 17151)
 Primer information and PCR condition (Table 1 [1,2])
 One step RT-PCR kit (Takara Prime Script RR055A) was
used for reverse transcribed and amplified of RNA.
 The extracted RNA (5 μl) was mixed with one step RTPCR kit constructs and primer (each 1 μl D1 and D2 were
mixed) (Table 2a).
 Second PCR was performed by Emerald Amp PCR
master mix (Takara Emerald Amp PCR master mix
RR300A) and primer mix for seconds PCR (Table 2b).
Figure 1. protocol of RT-PCR
 IgM ELISA and NS1 antigen ELISA
IgM ELISA [3,4]
-using Dengue IgM capture ELISA kit (InBios)
IgM ELISA kit is detection of IgM antibodies to dengue virus antigens in serum.
60 μl of serum samples added anti-human IgM coated and blocked plate and incubated followed by incubation with dengue recombinant antigens.
After incubating , the wells were treated with dengue specific monoclonal antibody and determined absorbance of labeled IgG with substrate at 450 nanometers.
Between each step included wash steps.
Figure 2. Scheme of ELISA
NS1 antigen ELISA
-using NS1 antigen ELISA (Bio-rad)
NS1 antigen ELISA kit is detection of dengue virus NS1 antigen in serum.
serum was incubated at 37 oC for 90 min then added labeled antibody, finally NS1 antigen made the antibody-NS1 antigen-labeled antibody complex within the plate.
After incubation, substrate was added and optical density read at 450/620 nm. Between each step included wash steps.
Results & Discussion
•604 sera were collected and evaluated dengue positive or negative by RT-PCR, IgM ELISA and NS1 antigen ELISA.
•In Table 3 showed dengue positive and negative rate by three assay (RT-PCR, IgM ELISA and NS1 Antigen ELISA).
• RT-PCR showed 111sera (18.9 %) of dengue positive and the other 490 sera (81.1 %) showed dengue negative
• IgM ELISA showed 234 sera (38.7 %) of dengue positive and 368 sera (60.9 %) dengue negative
•,NS1 antigen ELISA showed 287 sera (47.5 %) dengue positive and 278 (46.0 %) dengue negative.
These results showed that NS1 antigen ELISA has the higher positive rate than IgM ELISA and RT-PCR.
• In Table 4 showed dengue positive rate by combination of three assay (RT-PCR, IgM ELISA and NS1 Antigen ELISA).
•
•
•
•
IgM ELISA or NS1 antigen combination assay showed 54.0 % of positive rate
IgM ELISA or RT-PCR combination assay showed 41.4 % of positive rate
NS1 antigen ELISA or RT-PCR combination assay showed 49.7 % of positive rate
In three combination assay showed of 54.8 % positive rate
Theses result suggested that combination of NS1 antigen and IgMs give more accurate diagnosis result
compared with present diagnosis (RT-PCR with IgM ELISA).
Conclusions
 In this study, the commercially available dengue virus detection assay was
evaluated and compared positive rate of dengue virus.
 The results suggested that the detection sensitivity of dengue between three
methods were statistically different (Table 3).
 The results, new combination assay such as IgM ELISA and NS1 antigen or
combination of three assays for dengue diagnosis were necessary to considered
(Table 4).
 This study was supported by the intramural grant (#2013-NG53003-00) of the
National Institute of Health, Korea.
References
[1] dengue diagnosis, advances and challenges, Maria G, Gustavo K, International Journal of
infectious dieases 2004, 8, 69-80.
[2] Typing of Dengue Viruses in clinical Specimens and Mosquitoes
by Single-Tube
Multiplex Reverse Transcription PCR, Eva harris et al., Journal of Clinical Microbiology,
1998, 2, 2634-2639
[3] Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1 Nonstructural
Protein NS1 Reveals Circulation of the Antigen in the Blood during the Acute Phase of
Disease in Patients Experiencing Primary or Secondary Infections, Sophie A, Antoine T,
Monique D, et al, J. Clin. Microbiol, 2002, 40, 376-381
[4] Identification of Dengue type 1 virus (DENV-1) in Koreans traveling abroad, YoungEui J,
YeonHee K, JungEun C et al, Public health Res Perspect, 2011, 2(1), 34-40