Active Spike Protein (SP)

The coronavirus spike (S) glycoprotein is a class I viral fusion protein on the outer envelope of the virion that plays a critical role in viral infection by recognizing host cell receptors and mediating fusion of the viral and cellular membranes. The S proteins consist of two subunits, S1 and S2.Be-
-sides,S1 binding to ACE2 and CLEC4M/DC-SIGNR receptors and internalizationof thevirus into t-
-he endosomes of the host cell induces conformational changes in the S glycoprotein. Thus a binding ELISA assay was conducted to detect the interaction of recombinant human ACE2 and recombinant Spike glycoprotein(S1). Briefly, S1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to ACE2-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-S1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant Spike glycoprotein(S1) and recombinant human ACE2 was shown in Figure 1, the EC50 for this effect is 0.034µg/mL.