Figure 1.
Reactivity of the18 Neutralizing HmAbs with SARS CoV 12-510-S1 proteins.
Medisorp ELISA plates were coated with 100 ng/well of Urbani and RBD mutant 12-510S1-IgG proteins and 2.5 µg/ml of each HmAb was used as the primary antibody. Anti-human IgG2 HRP mouse monoclonal antibody was used as secondary antibody. OD was measured at 450 nm. Error bars represent SD of a representative experiment performed in triplicates. (A) Urbani versus Sin845 mutant. (B) Urbani versus GD01 mutant. (C) Urbani versus GZ0402 mutant. (D) Urbani versus GZ-C mutant.
Figure 2.
Reactivity of Urbani SARS-CoV-S protein antibodies with Urbani S1 protein and mutant S1 proteins.
(A) Different dilutions of a rabbit anti-Urbani SARS-CoV-S protein immune serum were tested in an ELISA against Urbani as well as mutant S1-IgG proteins. Anti-rabbit donkey polyclonal HRP antibody was used as the secondary antibody. (B) Competitive ELISA assay: Different protein concentrations of Urbani or GZ-C proteins were pre-incubated with 5A7 antibody then the protein/Ab mixtures were tested for binding to the other protein by ELISA. OD was measured at 450 nm.
Figure 3.
In vitro pseudovirus neutralization assay.
Eighteen neutralizing HmAbs were tested against different mutant as well as Urbani pseudoviruses. Pseudoviruses equivalent to 10 ng of HIVp24 were incubated for 1 hr with 25 µg/ml of each of the HmAbs at 37°C. The virus/Ab mixtures were then added to 293/ACE2 stable cell line. Seventy two hours later, the virus entry was determined by luciferase expression. The percentage entry inhibitions obtained with Abs were calculated and normalized to HIV/Urbani-S inhibitions (A) HIV/GZ-C and HIV/Sin845 inhibitions (B) HIV/GZ0402 and HIV/GD01 inhibitions. Polyclonal rabbit immune serum (PolyAb) was used as a positive control. Error bars represent SD of a representative experiment performed in triplicates.
Table 1.
HmAbs to HR1 and HR2 can efficiently neutralize surrogate clinical isolates.
Figure 4.
Combinations of HmAbs more efficiently inhibit the entry of SARS-CoV RBD surrogate clinical isolates.
Neutralizing HmAbs binding to different regions of S protein 4D4 (S1), 1F8 (HR1), 5E9 (HR2)) were tested for their ability to neutralize pseudoviruses in different combinations as well as individually at a concentration of 6.25 µg/ml each. The virus/Ab mixture was incubated for 1 hr at 37°C then added to 293/ACE2 stable cell line. Seventy two hours later, the virus entry was determined by luciferase expression. The percentage entry inhibitions by individual antibodies as well as combinations of antibodies were calculated. Error bars represent SD of representative experiment performed in triplicates. Statistical analysis was done using Student-t test, significant differences are indicated by asterisks,* p<0.05.