Goat anti human igm hrp

ZIKV-NS1-specific antibodies in serum were detected by ELISA. To begin, the plate was coated with ZIKV-NS1 antigen (MyBioSource, MBS568704) diluted to 10 μg ml⁻¹ in carbonate binding buffer and incubated overnight at 4°C. The following day, the plate was washed 5-times with PBS-Tween20 and all wells were blocked with 5% nonfat dry milk in PBS for 1 h at 37°C. After the block, the plate was washed, and serum samples diluted 1:100 were added to corresponding wells and incubated for 1 h at 37°C. Subsequently, the plate was washed 5-times and IgG or IgM detection was carried out. ZIKV-NS1-specific rhesus IgG and IgM were detected using the Mouse Anti-Monkey IgG-HRP (Southern Biotech, 4700-05) diluted 1:4,000 and the Goat Anti-Human IgM-HRP diluted 1:2,000 (Southern Biotech, 2023-05), respectively. The diluted detection antibodies were added to corresponding wells and incubated for 1 h at 37°C. The plate was then washed 8-times and developed with TMB substrate at room temperature for 2–3 min. The reaction was then stopped with TMB Stop Solution, and the absorbance was read at 450nm.

Antibody binding to whole virus was determined in a side-by-side DENV1, DENV2, DENV3, DENV4, and ZIKV VCA ELISA. The ELISA plate was coated with the mouse-anti-flavivirus monoclonal antibody 4G2 diluted 1:1,000 in carbonate binding buffer and incubated overnight at 4°C. The next day, the plate was washed 5 times with PBS-Tween20 and the wells were blocked with 5% nonfat dry milk in PBS for 1 h at 37°C. Following the block, the plate was washed and each virus was added to the corresponding VCA wells, respectively, and incubated for 1 h at room temperature. Subsequently, the plate was washed with PBS and mAbs from different time points diluted in 5% nonfat dry milk with PBS were added to designated wells and incubated for 1 h at 37°C. Following sample addition, plates were washed and detection was carried out using one of the following antibodies: goat anti-human IgG HRP (SouthernBiotech, 2045-05) diluted 1:10,000, Mouse Anti-Monkey IgG-HRP (SouthernBiotech, 4700–05) diluted 1:4,000, or the Goat Anti-Human IgM-HRP diluted 1:2,000 (SouthernBiotech, 2023-05). The diluted detection antibody was added to all wells and incubated for 1 h at 37°C. The plate was then washed and the wells were developed with the tetramethylbenzidine (TMB) substrate at room temperature for 3-4 min. The reaction was then stopped with the TMB solution, and absorbance was read at 450 nm.