Development of a Rapid Diagnostic Test Kit to Detect IgG/IgM Antibody against Zika Virus Using Monoclonal Antibodies to the Envelope and Non-structural Protein 1 of the Virus

Kim, Yeong Hoon; Lee, Jihoo; Kim, Young-Eun; Chong, Chom-Kyu; Pinchemel, Yanaihara; Reisdörfer, Francis; Coelho, Joyce Brito; Dias, Ronaldo Ferreira; Bae, Pan Kee; Gusmão, Zuinara Pereira Maia; Ahn, Hye-Jin; Nam, Ho-Woo

The mAb-containing supernatant and ascitic fluid of BALB/c mice were collected and screened using ELISA to determine the antibody properties. The mAbs were purified on a protein G agarose resin (Amicogen, Inc, Jinju, Korea) and identified using SDS-polyacrylamide gel electrophoresis and western blotting. The mAb isotypes were determined using goat anti-mouse immunoglobulins (Sigma-Aldrich), according to the manufacturer's instructions. An indirect competitive ELISA was conducted to measure the affinity of mAbs, as previously described [32] . Various concentrations of the recombinant NS1 or E antigen were incubated with mAbs for 1 hr at room temperature (RT). This antigen-antibody mixture was then transferred to wells where recombinant antigens are coated. After 1-hr incubation at RT, wells were rinsed with PBS containing 0.1% Tween-20. A 1-hr incubation was followed with anti-mouse IgG-horseradish peroxidase (HRP) conjugate at RT and 3,3´,5,5´-tetramethylbenzidine solution was added after final washing. Finally, the absorbance was measured by a microplate reader (Benchmark Plus; Bio-Rad, Hercules, California, USA) at 450 nm after the termination of coloring reaction by H2SO4. Dissociation constant (Kd) was calculated by generating Klotz plot.