Biotin ELISA Kit (UNFI0098)

From Assay Genie, this Competitive ELISA is designed for the quantitative measurement of Biotin with universal reactivity. The kit uses a double-antigen sandwich detection method in which the microplate is pre-coated with a specific antigen; standards and appropriately diluted samples are added to the wells and incubated, allowing target antibodies in the sample to bind to the immobilised antigen. After washing to remove unbound components, a biotin-labelled antigen is added to form a sandwich complex, followed by HRP-conjugated Streptavidin and TMB substrate to generate a colour reaction that is read at 450 nm on a microplate reader.

The assay has a stated sensitivity of 10 pg/mL and a working range of 100-2000 pg/mL, and accepts serum, plasma, cell culture supernatant, cell or tissue lysate, and other liquid samples. Reported linearity for 1:2, 1:4 and 1:8 dilutions (n=10) spans 88-102% / 82-101% / 80-96% for serum, 86-99% / 82-97% / 94-99% for EDTA plasma, and 87-103% / 87-98% / 85-98% for heparin plasma. Recovery averages 95% for serum (range 86-104%), 93% for EDTA plasma (range 90-98%), and 99% for heparin plasma (range 95-105%). Kits should be stored sealed at 2-8°C and not cryopreserved.

The workflow begins by equilibrating TMB substrate for 30 minutes at room temperature and pre-washing the plate twice; 50 µL of standard or sample is then added per well together with 50 µL of biotin-labelled antibody and incubated at 37°C for 45 minutes. The plate is washed five times with a 1 minute soak, TMB substrate is added and incubated in the dark at 37°C for 10-20 minutes, and the stop solution is used to terminate the reaction, turning the colour from blue to yellow before absorbance is read at 450 nm.