Anti hamster igg

Nunc MaxiSorp 96-well flat bottom plates (Sigma-Aldrich, USA) were coated with 100 μL of SARS-CoV-2 RBD (1 μg/mL) (GenScript Laboratories, USA) in carbonate bicarbonate buffer (pH 9.6) and incubated at 4°C overnight. The next day, the wells were washed six times with PBS containing 0.05% (v/v) Tween-20 (PBS-T) and blocked with 3% (w/v) skim milk (BD Biosciences, USA) in PBS-T for 2 hours in agitation at RT. The plates were then washed six times with PBS-T. Then, 100 μL of each collected serum sample diluted 1:100 with 1% (w/v) skim milk was added to each plate for 1 hour at 37°C. The wells were washed six times with PBS-T and incubated with 100 μL (1:10000) of Goat Anti Mouse IgG (Genscript Laboratories, USA) or Anti Hamster IgG (Abcam, USA) conjugated to HRP diluted in skim milk in PBS-T for 1 hour at 37°C. The plates were washed six times and were incubated with 100 μL of TMB for 15 min at RT. Finally, the reaction was stopped by adding 50 μL per well of 2 N H2SO4, and the plates were read at 450 nm using an Epoch 2 microplate reader (Biotek, USA). The negative control was obtained from serum samples of the control group.

Microplates precoated with recombinant antigens of RBD, spike ectodomain, S1, or S2 were provided by Beijing Wantai. For detections, serially diluted (twofold) serum samples (100 μl per well) were added to the wells, and the plates were incubated at 37°C for 30 min, followed by washing with PBST buffer [20 mM PBS (pH7.4), 150 mM NaCl, and 0.05% Tween-20]. Then, horseradish peroxidase–conjugated anti-mouse IgG (Proteintech) for measurements of mouse sera, anti-human IgG (Thermo Fisher Scientific) for measurements of monkey sera, or anti-hamster IgG (Abcam) for measurements of hamster sera (100 μl per well) were added according to the species of samples. After a further 30-min incubation followed by washing with PBST buffer, tetramethylbenzidine (Wantai) chromogen solution (100 μl per well) was added to each well. Ten minutes later, the chromogen reaction was stopped by adding 50 μl of 2 M H₂SO₄, and optical density (OD)_450–630 was measured. The IgG titer of each serum was defined as the dilution limit to achieve a positive result (greater than the mean plus 3 SDs of ODs of negative controls). Each plate contained five tests of negative control sera, and their ODs were used to determine the cutoff value. Representative data from technical replicates were performed at least twice for plotting.