TMB ELISA Peroxidase Substrate  

TMB ELISA Peroxidase Substrate

ELISA results of Mouse Anti-SARS CoV-2 Spike Protein (S2) Antibody using TMBE-1000. Each well was coated in duplicate with 0.5μg of rec SARS CoV-2 Whole Spike protein [RED line], rec SARS CoV-2 Spike (S1) [GREEN line], rec SARS CoV-2 Spike (S1) RBD protein [BLUE line], and rec SARS CoV-2 Spike (S2) glycoprotein [PURPLE line]. The working dilution for SARS-CoV-2 whole spike is 1:190,000 and SARS CoV-2 Spike (S2) glycoprotein is 1:120,000. The starting dilution of antibody was 0.55μg/ml and the X-axis represents the Log10 of a 3-fold dilution. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using Rabbit Anti-Mouse IgG HRP conjugated (p/n 610-403-C46) at 1:8000 and TMB substrate (p/n TMBE-1000). SARS CoV-2 Spike (S2) EC50: 8ng/mL; SARS CoV-2 Whole Spike EC50: 5ng/mL.

Rockland Immunochemicals produces a wide variety of buffers and substrates for use in ELISAs. Antigen was diluted in ELISA Microwell Coating Stabilizer (p/n MB-063-0100) added to the microwell plate and incubated overnight at 4°C. The plate was then blocked with ELISA Microwell Blocking Buffer with Stabilizer (p/n MB-064-1000) for 2 hours. The primary antibody was diluted in PBS Fish Gel Concentrate (1:10)(p/n MB-066-0100), added to the plate, and allowed to incubate 1 hour at room temperature. HRP conjugated secondary antibody was diluted in HRP Conjugate Stabilizer (p/n MB-060-0100), added to the plate, and allowed to incubate for 30 minutes at room temperature. TMB ELISA Peroxidase Substrate (p/n TMBE-1000) was added to the plate and allowed to incubate for 30 minutes at room temperature. The reaction was then stopped with 1M HCl and read at 450nm.