Application

The BM Chemiluminescence ELISA Substrate (POD) provides a substrate solution for peroxidase-based (POD, HRP) secondary detection systems with highly sensitive, enhanced chemiluminescence. This reagent has been optimized for ELISA applications to be run on a microplate chemiluminescence reader (96-well format) or tube-format luminometers.

Features and Benefits

Improved sensitivity Large dynamic range Rapid and constant signal ContentsSubstrate Reagent A (buffered solution that contains luminol/4-iodophenol)Starting Reagent B (buffered solution that contains a stabilized form of H₂O₂)

General description

The sensitivity of enzyme-linked immunosorbent assays often depends on the detection limit of the colorimetric substrates used. In addition, the dynamic range of a colorimetric assay is restricted to a maximum of two orders of magnitude due to the physical/chemical limitations of absorbance measurement (Law of Lambert & Beer). With chemiluminescence technology, a nonradioactive method, which combines the convenience of tube or microplate-based immunoassays with the advantages of isotopic assays, is now available. The BM Chemiluminescence ELISA Substrate (POD) has been evaluated with different immunoassays on the LB 96 P microplate reader and the LB 953 tube chemiluminescence analyzer from EG&G;Berthold.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Preparation Note

Working solution: Preparation of working solutionThe BM Chemiluminescence ELISA Substrate is supplied as a set of two stable solutions. Depending on the scale of the assay, the appropriate amount of substrate solution has to be prepared 15 minutes before use: Add to 100 parts of solution A one part of solution B. Stir the mixture for at least 15 minutes at 15 to 25 °C to equilibrate the components.Premixed working solution stored at 2 to 8 °C should be warmed up to 15 to 25 °C to avoid temperature effects during measurement.Storage conditions (working solution): The premixed solution is stable for 1 week, when stored at 2 to 8 °C.

Principle

Horseradish peroxidase (POD, HRP), in the presence of hydrogen peroxide (H₂O₂), catalyzes the oxidation of diacylhydrazides such as luminol. A reaction product in an excited state is thus formed, then decays to the ground state by emitting light. Strong enhancement of the light emission is achieved by the agent 4-iodophenol (contained), which acts as a radical transmitter between the formed oxygen radical and luminol.