Sars cov 2 spike elisa kit

Spike S1 (40591-V08H; Sino Biological) was conjugated to lentivirus (Cellomics Technology LLC) to create a SARS-CoV-2 mimic. His-tagged spike S1 was linked to Ni nitrilotriacetate (Ni-NTA) through the chemical interaction. NTA with mercapto group (N-[Nα,Nα-Bis(carboxymethyl)-L-lysine]-12-mercaptododecanamide) was first reacted with 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) to give NTA-SMCC and then was added to the lentivirus. The NTA groups were conjugated to the lentivirus through the −NH₂ group on lentivirus and N-hydroxysuccinimide ester on NTA-SMCC. The free NTA-SMCC was removed by centrifugation using an ultrafiltration tube (100 kDa MWCO; Millipore) to give SARS-CoV-2 mimicking virus (spike S1-lentivirus). The successfully conjugated spike S1 on lentivirus was confirmed using TEM. Briefly, SARS-CoV-2 mimics were incubated with anti-Spike S1 antibodies overnight at 4°C. Free antibodies were removed using an ultrafiltration tube (100 kDa MWCO; Millipore) and washed with PBS three times. Spike S1 on the SARS-CoV-2 mimics was labeled with immunogold (10 nm) antibodies and negatively stained for TEM visualization. The conjugation efficiency of spike S1 on lentivirus was determined using ELISA (Sino Biological SARS-COV-2 SPIKE ELISA KIT, Sino Biological) according to manufacturer’s protocol.

Recombinant spike S1 (Sino Biological 40591-V08H, 10 ng/mL, MW = 76.5 kDa) was added to nanodecoys at different concentrations (5×10⁹, 1×10⁹, 2×10⁸, 4×10⁷, 8×10⁶, 1.6×10⁶, and 3.2×10⁵) and incubated for three hours. After that, the unbound spike S1 was removed by ultracentrifugation (100 kDa). Spike S1 before and after binding to nanodecoys was determined using an ELISA kit (Sino Biological SARS-CoV-2 SPIKE ELISA KIT, Sino Biological) according to manufacturer’s protocol. To study the neutralization of spike S1 with nanodecoys in primary lung derived cells (LSCs), spike S1 was first labeled using NHS-Rhodamine (46406, Thermo Fisher Scientific) according to the manufacturer’s instructions. The RhB-spike S1 (100 ng) was first incubated with LSCs (2×10⁴) in 4-well slides for 1 h and washed with PBS for three times. After that, DiD labeled nanodecoys (2×10⁷) were added and incubated for another 4 h. Cells were washed and fixed using 4% PFA prior to stain with Alexa Fluor^™ 488 Phalloidin (Invitrogen^™ A12379). Cells were imaged using an Olympus FLUOVIEW confocal microscope.