Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling

Abstract

Dengue fever, a mosquito-borne disease in tropical and subtropical climates caused by the dengue virus (DENV), has become a major social and economic burden in recent years. However, current primary detection methods are inadequate for early diagnosis of DENV because they are either time-consuming, expensive, or require training. Non-structural protein 1 (NS1) is secreted during DENV infection and is thus considered a suitable biomarker for the development of an early detection method. In the present study, we developed a detection method for the NS1 protein based on a previously reported thio-NAD cycling ELISA (i.e., ultrasensitive ELISA) and successfully achieved a LOD of 1.152 pg/mL. The clinical diagnosis potential of the detection system was also evaluated by using 85 patient specimens, inclusive of 60 DENV-positive and 25 DENV-negative specimens confirmed by the NAAT method. The results revealed 98.3% (59/60) sensitivity and 100% (25/25) specificity, which was in almost perfect agreement with the NAAT data with a kappa coefficient of 0.972. The present study demonstrates the diagnostic potential of using an ultrasensitive ELISA as a low-cost, easy-to-use method for the detection of DENV compared with NAAT and could be of great benefit in low-income countries.

Quantification of HPV16 E7 Oncoproteins in Urine Specimens from Women with Cervical Intraepithelial Neoplasia

“…We consider that such a discrepancy could be explained by a variation in the HPV life cycle or in the oncogenic activity. We previously developed an ultrasensitive ELISA antigen test for use in dengue fever [26], and these ) was detected between the absorbance of the blank and that of HeLa cells (n = 3 each, p > 0.05 by a t-test). That is, our ultrasensitive ELISA specifically detects HPV16 and its related type but not HPV18.…”

“…We consider that such a discrepancy could be explained by a variation in the HPV life cycle or in the oncogenic activity. We previously developed an ultrasensitive ELISA antigen test for use in dengue fever [ 26 ], and these studies have produced excellent results comparable to those of PCR-based tests, demonstrating that our ultrasensitive ELISA can be used to detect precise amounts of trace proteins. Therefore, it is reasonable that some specimens in the present study containing no or very low levels of E7 oncoproteins, contrary to the uniplex E6/E7 PCR-based HPV typing, represent inactive infections.…”

“…The resultant precipitates were resolved with 2 mL of ThinPrep solution (Hologic, Marlborough, MA, USA) and stored at –80 °C. Our ultrasensitive ELISA with thio-NAD cycling was based on a sandwich ELISA and developed by Watabe and Ito [ 15 ]; the details are described elsewhere [ 25 , 26 ]. Briefly, 2 μg/mL of primary antibody (NM2) against HPV16 E7 oncoprotein, provided by an author (M.M.…”

“…Specimens were processed for measurements using the ultrasensitive ELISA with thio-NAD cycling as follows: 45 mL of urine was collected from each patient and centrifuged at 3000 rpm for 5 min. The resultant precipitates were resolved with 2 mL of ThinPrep solution (Hologic, Marlborough, MA, USA) and stored at -80 • C. Our ultrasensitive ELISA with thio-NAD cycling was based on a sandwich ELISA and developed by Watabe and Ito [15]; the details are described elsewhere [25,26]. Briefly, 2 µg/mL of primary antibody (NM2) against HPV16 E7 oncoprotein, provided by an author (M.M.…”