A rapid, sensitive chemifluorescent ELISA substrate for peroxidase enzymes.

Thermo Scientific QuantaBlu Fluorogenic Peroxidase Substrate allows rapid stopped and kinetic ELISA assays to be performed with greater sensitivity than TMB and OPD substrates.

Highlights of QuantaBlu Fluorogenic ELISA Substrate:

• More sensitive than TMB, OPD or ABTS substrates
• Flexible - stopped, non-stopped or kinetic assays possible
• Large dynamic range (4 log peroxidase concentration range)
• Excellent stability: working solution is stable for 24 hours
• Large stokes shift; excitation/emission maxima of 325/420; range of 315-345/370-460
• Does not photobleach

About QuantaBlu Fluorogenic ELISA Substrate:

A variety of substrates are available for detecting peroxidase activity in ELISA-based assays. Colorimetric substrates (e.g., TMB, OPD and ABTS) have been widely used for years. Each of these substrates varies greatly with respect to its performance characteristics such as detection sensitivity, working range and attainable signal-to-noise ratios. Substrate flexibility is also a key issue that affects an assay. Stability, development time requirements and the capacity to perform stopped and/or kinetic assays vary significantly among these substrates. Ideally, a substrate is stable, very sensitive, has high attainable signal-to-noise ratios, possesses a broad dynamic range, and allows the user to perform stopped, non-stopped and kinetic assays.

QuantaBlu Fluorogenic Peroxidase Substrate meets all the requirements of an ideal substrate for use in peroxidase detection. QuantaBlu Substrate generates a blue fluorescent product upon reaction with peroxidase. The fluorescent product does not photobleach. Fluorometric-based detection overcomes the limitations of colorimetric substrate detection, which does not allow for quantitation of greater than four optical density units. QuantaBlu Substrate allows for stopped, non-stopped and kinetic assays to be performed. Incubation times with QuantaBlu Substrate for stopped and non-stopped assays may be varied between one to 90 minutes at either room temperature or 37°C. QuantaBlu Substrate exhibits a flat baseline in assays, which facilitates low-level detection sensitivity and allows for high signal-to-noise ratios.

Comparison of Thermo Scientific QuantaBlu Fluoregenic Peroxidase Substrate to TMB, ABTS and OPD ELISA substrates. QuantaBlu fluorescent substrate and other colorimetric substrates were incubated for 30 minutes at room temperature with increasing amounts of horseradish peroxidase enzyme followed by addition of a stop solution. QuantaBlu Fluorogenic Peroxidase Substrate gave the greatest signal-to-noise ratios and exhibited the lowest detection limit.

Thermo Scientific QuantaBlu Fluorogenic Peroxidase Substrate provides the ability to rapidly detect peroxidase at very low concentrations in short periods of time. QuantaBlu fluorescent substrate was read in a non-stopped mode using a 1-minute instrument cycle time between reads. The graph below illustrates the detection of horseradish peroxidase enzyme amounts ranging from 0-10pg per well from 1.5-6.5 minutes of substrate incubation time. At 1.5 minute, as little as 2.5pg of peroxidase was detected. At 6.5 minutes, 0.625pg of peroxidase was detected.

References:

1. Savage, M.D.,
et al. (1998). QuantaBlu Fluorogenic Peroxidase Substrate: the ideal peroxidase substrate. Previews 2(1), 6-9.
2. Savage, M.D.,
et al. (1998). QuantaBlu Fluorogenic Peroxidase Substrate offers rapid data acquisition and high sensitivity. Previews 2(2), 18-19.
3. Ayala, P., et al. (2002). Infect. Immun. 70, 5965-5971.
4. Atamna, H., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 686-691.
5. Jefcoat, A.M., et al. (2001). Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L704-712.