Overview of ELISA assays and NADH/NADPH conversion detection
Enzyme Linked Immunosorbent Assay (ELISA) is a biochemical assay that detects the presence of an antigen in a sample. A sample is incubated with a secondary antibody that recognizes an antigen and that is bioconjugated to an enzyme. This enzyme reacts with a substrate producing a solution whose change in absorbance can be measured.
The two most commonly used bioconjugated enzymes are horse radish peroxidase (HRP) and alkaline phosphatase (AP). AP's substrate, PNPP absorbs at 405 nm. HRP's substrate is hydrogen peroxide which is coupled with the following chromogens that can be measured with absorption spectroscopy: ABTS (405-420 nm), TMB (650 nm or 450 nm), and OPD (492 nm).
Microplate readers from BMG LABTECH have a UV/Vis spectrometer that measures any absorbance range from 220-1000 nm in less 1 second per well. Here, we have shown the power of the spectrometer in measuring ELISA assays or assays that need the cofactors NAD+, NADH, NADP+ and NADPH.