Mouse ifn γ elisa kit

The enzyme-linked immunosorbent assay (ELISA) experiment was performed for evaluating the CD4⁺ T-cell response in mice. First, the splenocytes in HC and rSMEV groups were co-cultured with the rSMEV vaccine protein (5 μg/mL). The splenocytes were incubated with RPMI-1640 medium containing 10% FCS for 48 hours in a 37 °C under 5% CO₂ humidified incubator. Then the culture supernatant was collected, and the samples and standards were added into the IFN-γ and IL-4 ELISA plates after plates were washed. The ELISA plates were incubated at 37 °C for 1.5 hours and washed for 5 times. The biotinylated detecting antibody was added to the plates and incubated at 37 °C for 1 hour. After the plates were washed for 5 times, the Streptavidin-ALP was added and incubated at 37 °C for 30 minutes. The BCIP/NBT solution were added for chromogenic reaction at 37 °C away from light for 15 minutes. Finally, the termination solution was added, and the OD450nm value was measured immediately by Spectrophotometer (Thermo Fisher, FI-01620 Vantaa, Finland) for detecting the corresponding level of IFN-γ and IL-4. The Mouse IFN-γ ELISA Kit (SEKM-0031, Solarbio Science & Technology Co., Ltd, Beijing, China) and Mouse IL-4 ELISA Kit (SEKM-0005, Solarbio Science & Technology Co., Ltd, Beijing, China) were adopted for ELISA experiment.

Lymphocytes were separated from spleens and re-stimulated with a synthetic N peptides pool or purified S1 protein as described in Methods. After 60 h, concentrations of IFN-γ, IL-2, and IL-4 in the splenocyte culture supernatants (1 × 10⁶ cells/mL) were determined using commercial Mouse IFN-γ ELISA KIT (Solarbio; SEKM-0031), Mouse IL-4 ELISA KIT (Solarbio; SEKM-0005), Mouse IL-2 ELISA KIT (Solarbio; SEKM-0004) according to the instructions for uses, respectively. Signals were measured at 450 nm and 630 nm wavelengths within 5 min by the multi-functional enzyme standard instrument (Omega, Germany).