Study: NCT04520880

Intervention type: DIAGNOSTIC_TEST
Intervention: Testing procedure for Binding antibodies
Description: Levels of S-specific and N-specific binding antibodies will be measured by enzyme immune-sorbent assay (ELISA). Briefly, a purified, recombinant S or N proteins will be coated into 96-well plates and incubated for 1 hour at room temperature. The plates will be then washed with 1X PBS five times. The unbound regions of the coated S proteins are blocked by 5% non-fat dry milk. After overnight incubation at 4 C. The plates are washed several times with 5X PBS. Patients sera samples are added in duplicate and incubated for 1 hour at room temperature. After several washes. anti-human IgG HRP conjugate is added. After 1-hour incubation at room temperature, the plates are washed with 1X PBS five times 3,3', 5,5' tetramethylbenzidine (TMB) substrate is added to each wells and incubated for 5 minutes. The reaction will be then stopped with 0.2 M sulfuric acid and the absorbance is measured at 450nm.

Intervention type: DIAGNOSTIC_TEST
Intervention: Testing procedure for Binding antibodies
Description: Levels of S-specific and N-specific binding antibodies will be measured by enzyme immune-sorbent assay (ELISA). Briefly, a purified, recombinant S or N proteins will be coated into 96-well plates and incubated for 1 hour at room temperature. The plates will be then washed with 1X PBS five times. The unbound regions of the coated S proteins are blocked by 5% non-fat dry milk. After overnight incubation at 4 C. The plates are washed several times with 5X PBS. Patients sera samples are added in duplicate and incubated for 1 hour at room temperature. After several washes. anti-human IgG HRP conjugate is added. After 1-hour incubation at room temperature, the plates are washed with 1X PBS five times 3,3', 5,5' tetramethylbenzidine (TMB) substrate is added to each wells and incubated for 5 minutes. The reaction will be then stopped with 0.2 M sulfuric acid and the absorbance is measured at 450nm.

Intervention type: DIAGNOSTIC_TEST
Intervention: Neutralizing antibodies
Description: Neutralizing antibodies against the S protein of SARS-CoV-2 are measured by PV microneutralization assay. Briefly, individual plasmids expressing S.fl gene, luciferase genes, and reporter genes are co-transfected into 293T cells to produce luciferase-expressing pseudo-viruses. Then, pseudo-viruses are added at equal volume into 96 well plates and incubated at 37 for 1 hour. Then, the ACE2 expressing 293T cells are added into the 96well pates with 100 ul of patients' sera in duplicates. After, a single round of replication the cells are lysed with lysis buffer. Luciferase activity is measured by the luciferase assay kit. The activity of luciferases is proportional to the number of cells infected. Luciferase activity is then measured by the luminometer device and the neutralization rate is calculated.

Intervention type: DIAGNOSTIC_TEST
Intervention: Neutralizing antibodies
Description: Neutralizing antibodies against the S protein of SARS-CoV-2 are measured by PV microneutralization assay. Briefly, individual plasmids expressing S.fl gene, luciferase genes, and reporter genes are co-transfected into 293T cells to produce luciferase-expressing pseudo-viruses. Then, pseudo-viruses are added at equal volume into 96 well plates and incubated at 37 for 1 hour. Then, the ACE2 expressing 293T cells are added into the 96well pates with 100 ul of patients' sera in duplicates. After, a single round of replication the cells are lysed with lysis buffer. Luciferase activity is measured by the luciferase assay kit. The activity of luciferases is proportional to the number of cells infected. Luciferase activity is then measured by the luminometer device and the neutralization rate is calculated.