Rockland Immunochemicals produces a wide variety of buffers and substrates for use in ELISAs. Antigen was diluted in ELISA Microwell Coating Stabalizer (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 S

The Rockland Immunochemical family of ELISA buffers includes coating buffers, blocking buffers, stabilization buffers, and substrates. Rockland TMBE ELISA Peroxidase Substrate is specially formulated for stability and sensitivity in HRP based ELISA systems.

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.

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.