SHORT RESEARCH COMMUNICATION Year : 2022  |  Volume : 59  |  Issue : 1  |  Page : 98-101

Detection of recombinant dengue virus 2 NS1 protein in Aedes aegypti mosquitoes using commercial Dengue NS1 ELISA kit

Philip Raj Abraham1, T Sankari1, N Pradeep Kumar2, Ashwani Kumar1
1 ICMR-Vector Control Research Centre, Indira Nagar, Puducherry, India
2 ICMR-Vector Control Research Centre Field Station, Kottayam, Kerala, India

Date of Submission26-Feb-2021Date of Acceptance03-Sep-2021Date of Web Publication07-Jun-2022

Correspondence Address:
Ashwani Kumar
ICMR-Medical Complex, VCRC Road, Indra Nagar, Puducherry 605006
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0972-9062.328975

Dengue, a vector-borne disease remains as one of the most serious public health problems globally. Incidence of this disease is on an increasing trend and currently over a billion people in tropical and subtropical regions are at risk. In the absence of an operational vaccine, prevention of dengue virus (DENV) is primarily focused upon controlling mosquito vectors. Mosquito vector surveillance programmes require simple and rapid tools to detect mosquitoes infected with DENV. Here, we tested the commercially available DENV Detect™ NS1 ELISA kit (InBios International, Inc.) for detection of recombinant DENV-NS1 protein in Aedes mosquito samples. The kit was evaluated to find out the minimum detection limit of recombinant DENV-2 NS1 protein following the manufacturer’s instructions. Initially, the NS1 protein detection threshold of the kit was determined and later the assay was standardized for detection of NS1 protein in Aedes aegypti mosquito pools containing 5, 10 and 25 mosquitoes. The ELISA kit displayed high sensitivity towards detection of recombinant dengue virus-2 NS1 protein in mosquito pools (up to 25 mosquitoes per pool) at 25 pico gram concentration. Since the commercial NS1 ELISA is highly sensitive and follows a very simple procedure, it could be employed for DENV surveillance in Aedes aegypti mosquitoes, after carrying out laboratory and field bioassays with DENV infected specimens.

Keywords: Aedes aegypti, commercial kits, Dengue virus, ELISA, NS1 antigen

How to cite this article:
Abraham PR, Sankari T, Kumar N P, Kumar A. Detection of recombinant dengue virus 2 NS1 protein in Aedes aegypti mosquitoes using commercial Dengue NS1 ELISA kit. J Vector Borne Dis 2022;59:98-101
How to cite this URL:
Abraham PR, Sankari T, Kumar N P, Kumar A. Detection of recombinant dengue virus 2 NS1 protein in Aedes aegypti mosquitoes using commercial Dengue NS1 ELISA kit. J Vector Borne Dis [serial online] 2022 [cited 2022 Jun 7];59:98-101. Available from: https://www.jvbd.org/text.asp?2022/59/1/98/328975
Dengue, a mosquito-borne viral infection is one of the most important public health problems in many tropical and subtropical regions of the world. It has been estimated that dengue infection range between 100 to 400 million worldwide annually and 70% of the disease burden occurs mainly in Asia[1],[2]. Prevention of dengue virus (DENV) is mainly focused upon controlling mosquito vectors due to non-availability of specific treatment and vaccine. For effective management of vector, surveillance of DENV prevalence in natural mosquito populations is important. It offers timely implementation of mosquito control measures thereby limiting impending possible outbreak[3]. However, with the limitations of existing tools, it remains difficult to undertake efficient surveillance programmes. Therefore, simple and rapid tests that support the surveillance programmes for detection of DENV infected mosquitoes are urgently required.

The RNA genome of DENV encodes for 3 structural and 7 non-structural (NS) proteins. The NS1 protein antigen of DENV is a 352-amino acid non-structural protein and based on glycosylation, the molecular weight ranges from 46 to 55 kDa[4],[5]. The protein antigen is found at different cellular locations as membrane-associated, cell surface bound or secreted form (sNS1). It involves in viral replication and known to correlate with viremia[6],[7],[8]. The sNS1 is widely used as a biomarker for serological diagnosis of DENV infection. It is present in the patients’ sera from the onset of illness at a concentration of 50 μg/ ml sera[9],[10] and several NS1- protein based RDT and ELISA formats are commercially available for diagnosis of DENV infection. These tests are simple, rapid and also less expensive. These readily available and easy to use NS1 protein-based dengue diagnostic kits can be explored as a tool for DENV surveillance. In this study, we have examined the potential application of commercial DENV Detect™ NS1 ELISA kit (InBios International Inc) for detection of recombinant dengue virus 2 NS1 protein (R & D Systems; Catalog No. 9439-DG) in pools of Aedes aegypti mosquito samples.

Initially, for determination of minimum detection limit of recombinant dengue virus 2 NS1 protein (rDENV-2 NS1), 25 nano gram (ng) to 2.5 pico gram (pg) rDENV-2 NS1 antigen were prepared in 0.5M phosphate-buffered saline (PBS) and the assay was carried out as per the instructions of the manufacturer. Briefly, 50 μl sample dilution buffer containing secondary antibody was added to individual wells of the plate followed by addition of 50 μl different concentrations of rDENV-2 NS1 or mosquito homogenate containing rDENV-2 NS1 and control samples. The plate was incubated at 37° C for 1 hour. Conjugate solution (100 μl) supplied with the kit was added to the wells after washing the plate. Later, adding the substrate and stopping the reaction, Optical Density (OD) was measured at 450 nm in a microplate ELISA reader (Thermo scientific).

For detection of rDENV-2 NS1 in Ae. aegypti mosquito samples, five days old adult female Ae. aegypti mosquitoes (unfed/blood fed) were obtained from the institutional Rearing and Colonization facility. The spiking experiment for detection of rDENV-NS1 in Ae. aegypti mosquito was carried out following two different procedures. In the first procedure, unfed mosquitoes (5/pool) were homogenized in different concentrations (750 pg, 250 pg, 75 pg and 25 pg) of rDENV-2 NS1 in tissue lyser II (Qiagen, Germany) at 24 frequencies for 5 minutes and centrifuged at 5000 rpm for 5 minutes and afterwards ELISA was carried out. In the second procedure, different concentrations of NS1 protein was added to the mosquito homogenate and then ELISA was performed. Based on the results, we followed first procedure for carrying out subsequent experiments for detection of NS1 protein in Ae. aegypti mosquito pools.

ELISA result was interpreted as per the manufacturer’s instructions. In brief, after ensuring the test passes quality control measures (negative control OD < 0.2, positive control OD ≥ 0.5, cut-off control OD > negative control and ratio of positive to negative control ≥ 8.00), the positivity or immune status ratio (ISR) was calculated using the formula: Positivity or ISR = absorbance of rDENV-2 NS1 / absorbance of conjugate control

The ratio of absorbance of rDENV-2 NS1 concentration and conjugate control was plotted and the minimum detection limit was determined. The mosquito pool is considered positive only if the ISR is ≥ 1. The OD value of rDENV-2 NS1 from 25 ng to 250 pg concentration by DENV Detect NS1 ELISA, was > 6 and could not be read by the microplate ELISA reader. However, the kit was found sensitive to detect rDENV-2 NS1 as low as 2.5 pg (ISR = 1.03) and the ISR values remains same from 250 pg onwards [Figure 1].

Figure 1: Determination of rDENV-2 NS1 detection limit of DENV Detect NS1 ELISA kit. Different concentrations of dengue NS1 protein was added to the wells of ELISA plate and the assay was performed as per the manufacturers’ instructions. Absorbance was read at 450 nm in an ELISA reader. Dotted horizontal line indicates cut-off value. N – negative control; P – positive control; C – cut-off control

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Based on the rDENV-2 NS1 detection limit of the ELISA kit, we used 25 to 750 pg protein in further experiments in Ae. aegypti mosquito samples. The assay was standardized for detection of NS1 protein either in the Ae. aegypti mosquito homogenate or the protein homogenized along with the mosquitoes. On comparison of both the procedure, it was observed that the ISR values in the second procedure were comparatively reduced [Figure 2]a. Following the initial procedure, the spiking experiment was carried out for the mosquitoes of pool size 10 and 25. It was observed that the kit is sensitive to detect the rDENV-2 NS1 even at 25 pg level in a pool of 25 mosquitoes [Figure 2]b.

We further tested rDENV-2 NS1 protein in blood fed mosquitoes of pool size 5, 10 and 25. We observed that the ELISA kit was sensitive to detect the rDENV-2 NS1 protein in all the pools [Figure 3]. On comparison of ISR values of the cut-off control and mosquito pools, the kit was sensitive to detect rDENV-2 NS1 when there is minimum 25 pg protein in pool size 5 and 10 and 75 pg in the 25 blood fed mosquitoes per pool.

Figure 3: Determination of sensitivity of DENV-NS1 ELISA kit in Blood fed mosquito pools. Pools of Ae. aegypti mosquitoes (5, 10 and 25 /pool) were homogenized in different concentrations of rDENV-2 NS1 and ELISA was performed. Dotted horizontal line indicates cut-off value. Mq. – mosquitoes; N – negative control; P – positive control; C – cut-off control; M – mosquito homogenate only

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Dengue NS1 protein is strongly immunogenic and elicits antibodies in infected individuals. Since this protein antigen can be detected in circulating blood during the acute dengue infection[6], an array of commercial kits has been developed to test DENV infection in humans. Nevertheless, attempts were also made for testing the NS1 antigen in DENV infected mosquito samples using these kits. Since 2011, several studies were under taken to utilize the application of commercial NS1 antigen-based dengue diagnostic tests[11],[12],[13],[14].

Commercial In Bios DENV Detect NS1 kits were evaluated for detection of DENV infection in clinical samples[15],[16],[17]. However, this kit is not explored for detection of rDENV-2 NS1 or NS1 protein antigen in DENV infected Ae. aegypti mosquito. In the current study, we attempted for the first time to test this kit for detection of rDENV-2 NS1 in pools of Ae. aegypti mosquitoes. We observed that the rDENV-2 NS1 detection limit of this kit is very high as it detected the rDENV-2 NS1 at 2.5 pg. We have also observed that the ISR value of the assay was reduced when the rDENV-2 NS1 protein was added to the mosquito homogenate. However, the pool having five mosquitoes was found to be positive at 25 pg rDENV-2 NS1 when the mosquitoes were homogenized along with the protein. Since, detection of rDENV-2 NS1 protein in a single mosquito will be very expensive, the mosquitoes could be pooled and tested for the DENV infection. We tested the protein in pools of 10 and 25 mosquitoes and found that the kit was sensitive to detect the rDENV-2 NS1 protein at 25 pg in unfed and 75 pg in blood fed Ae. aegypti mosquitoes with a pool size of 25 mosquitoes. This will reduce the cost of DENV infection detection in wild-caught Ae. aegypti mosquitoes when surveillance programmes are planned. Since the NS1 protein is a secretory protein, it was necessary to test the sensitivity of the ELISA kit to test the NS1 protein in blood fed mosquitoes.

The major limitations of the study are, the kit has not been tested in dengue virus infected mosquitoes in the laboratory as well as field-caught mosquitoes. However, this pilot study shows that the commercial InBios DENV Detect NS1 kit is highly sensitive for detection of recombinant dengue NS1 protein in Ae. aegypti mosquito. However, as highlighted, it needs further validation with Dengue infected field-caught mosquitoes to use the methodology as a surveillance tool for DENV infection.

Conflict of interest: None

 

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  [Figure 1], [Figure 2], [Figure 3]