Extraordinary Merits of Ultrasensitive ELISA with Thio-NAD Cycling in Determining Dengue NS1 Protein
1. IntroductionDengue fever is an acute infectious disease caused by the Dengue virus through the bite of an infected Aedes species (Ae. aegypti or Ae. albopictus) mosquito, with the main symptoms such as high fever, dizziness, headache, rash and severe pains (eye, muscle, joint, or bone pain), etc. This disease is widely prevalent in tropical and subtropical climates. An estimated 100 to 400 million people are infected each year, where over 80% are usually mild and asymptomatic. Nonetheless, severe Dengue fever can increase the risk of death if not treated properly. Thus, early diagnosis of this disease is significant to its timely treatment.
Noteworthily, non-structural protein 1 (NS1), a highly conserved glycoprotein, is chiefly secreted during the infection of Dengue virus, which is intensively deemed as the main pathogenesis factor of Dengue fever. Hence, NSI is generally used as a biomarker for the early detection of this disease.
In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) in combination with the thio nicotinamide adenine dinucleotide (thio-NAD/S-NAD) cycling method (hereafter termed ultrasensitive ELISA) is utilized to detect NSI, followed by the comparison with NAAT to confirm its detection accuracy.
2. Advantages and disadvantages of traditional detection methods for Dengue feverAt present, there are four main detection methods for Dengue fever.
Viral isolation and identification have high specificity but are time-consuming, taking at least 5 days. The rapid antigen test is the fastest and most cost-efficient among the other methods, but the sensitivity and specificity are relatively low. Serologic test based on IgM and IgG is limited by the number of days of infection, as the test must be delayed until the level of antibodies rises to a detectable level.
In clinic, NAAT is often applied to determine Dengue fever by dirt of its high sensitivity and specificity. However, this method is expensive, laborious, and prone to false positivity, which must be conducted by the trained personnel. To overcome the disadvantages of these methods, a new detection method ultrasensitive ELISA is applied in this study.
3. Workflow of ultrasensitive ELISA with thio-NAD cycling
A pair of antibodies is used for capturing the NS1 protein in the sandwich ELISA, and alkaline phosphatase is labeled on the secondary antibody. Aside from the antibodies, an androsterone derivative, 3α-hydroxysteroid dehydrogenase, thio-NAD, and NADH are used to construct the thio-NAD enzyme cycling system. During the thio-NAD cycling reaction, thio-NADH constantly accumulated in a triangular number fashion and could be directly measured at an absorbance of 405 nm.
4. The comparison of ultrasensitive ELISA and NAAT in the detection of Dengue NS1 ProteinIn NAAT, 60 specimens are dengue-positive, and 25 are dengue-negative. The NAAT cycling threshold (CT) value of those dengue-positive specimens ranged from 12.42 to 31.41. In the ultrasensitive ELISA, 59 specimens are correspondingly positive to the NAAT results, whereas 25 specimens are completely correspondingly negative to the NAAT results.
Compared with NAAT, the sensitivity and specificity of the ultrasensitive ELISA are 98.3% and 100%, respectively (Table 2). Of 60 NAAT-confirmed dengue-positive patient specimens, only 1 specimen is negative in the ultrasensitive ELISA. The NAAT data showed that the specimen is a type 4 DENV infection case with a CT value of 21.59. The results of the ultrasensitive ELISA are in almost perfect agreement with the NAAT results, with a kappa value of 0.972 (95% CI: 0.917-1.0).