All products have special prices for bulk purchase, please contact for more details if required.

Cat. No.: SEAFH-100 (for 100T)

Cat. No.: SEAFH-500 (for 500T)

Cat. No.: SEAFH-2k (for 2000T)

Description

Super Sensitive ELISA Assay Kit with Fluorescent HRP Substrate is a reagent kit that enables ultra-sensitive fluorescence detection of horseradish peroxidase (HRP) in ELISA reaction systems. It is based on the reaction between the fluorescent HRP substrate ADHP (10-acetyl-3,7-dihydroxyphenoxazine) and H2O2 in the presence of HRP, resulting in the generation of resorufin, which can be detected by fluorescence.

This assay kit offers a detection sensitivity that is typically more than 10 times higher than conventional substrates such as TMB. This allows for a significant reduction in the amount of precious samples required and enables the detection of samples with low concentrations that cannot be detected by the TMB method. When conducting ELISA assays, especially for precious samples or target molecules with low concentrations that are difficult to detect, the use of this assay kit is strongly recommended.

Common substrates used for detecting HRP activity in ELISA assays are typically colorimetric substrates such as TMB, OPD, and ABTS, which require absorbance measurement. These substrates differ in performance, including detection sensitivity and dynamic range. Among them, TMB is one of the most sensitive colorimetric substrates and widely used in ELISA assays. However, absorbance-based detection methods are limited in their ability to accurately quantify optical density (OD) values above four units, which restricts the upper limit of ELISA detection. In contrast, fluorescence detection is not affected by this limitation and can accurately measure high fluorescence intensities, given that the instrument allows it. Moreover, fluorescence detection is more sensitive and has a wider detection range compared to absorbance detection methods.

ADHP, also known as 10-acetyl-3,7-dihydroxyphenoxazine, Amplex Red, Ampliflu™ Red, OxiRed Probe, Amplisyn Red, or A6550, is a fluorescent red dye and a peroxidase substrate that can be used for absorbance or fluorescence detection. It is commonly used in fluorescence analysis. ADHP is one of the most sensitive and stable fluorescent probes known for detecting horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). In the presence of HRP, ADHP reacts with H2O2 in a 1:1 stoichiometric ratio, generating a highly fluorescent resorufin with a maximum excitation wavelength of 571nm and a maximum emission wavelength of 585nm. It also exhibits strong visible light absorption at the excitation wavelength, which can be measured at A570 for absorbance detection. ADHP has been widely used in immunological analysis for detecting HRP. Compared to other commercially available HRP substrates such as luminol and lucigenin, ADHP has lower background fluorescence and is less affected by air oxidation.

Features

• The assay kit has low background detection. The emitted wavelength of the generated resorufin is close to the red light region. At this wavelength, the interference from the spontaneous fluorescence of biological samples (which is located in the shorter wavelength blue-green light region) is minimized, making ADHP an ideal substrate for detecting target molecules in biological samples.
• The assay kit has a wide detection range and high sensitivity. It significantly expands the detection range of regular ELISA experiments and has much higher sensitivity than TMB substrates, with the detection limit increased by more than 10 times.
• The assay kit is complete and easy to use. It provides all the necessary reagents, including the ADHP substrate solution (ADHP Substrate Solution), stable peroxide solution (Stable Peroxide Solution), assay buffer (Assay Buffer), and stop solution (Stop Solution). Users only need to mix the reagents provided and use them without the need for additional buffer preparation or reagent dissolution.
• The assay reaction is controllable. The provided stop solution in the assay kit effectively terminates the generation of fluorescent products catalyzed by HRP, allowing stable signals to be obtained for at least 1 hour.

For a 96-well plate, based on using 100 μl of ADHP working solution per well, the small packaging of this assay kit can be used for the detection of 100 samples, the medium packaging can be used for 500 samples, and the large packaging can be used for 2000 samples.

Precautions

• Each solution should be equilibrated to room temperature (around 25°C) before use, as the temperature of the solution can affect the reaction kinetics. Stop Solution may form crystals at low temperatures, so it can be placed in a 37°C water bath before use to quickly bring it back to room temperature and dissolve the crystals.
• After each use, please tightly close the bottle caps of the solutions to prevent them from becoming ineffective. In particular, ADHP Substrate Solution (100X) and Stable Peroxide Solution (50X) are less stable in the air and may lose effectiveness if exposed for too long.
• ADHP Substrate Solution (100X) can be aliquoted and stored according to your needs.
• ADHP Substrate Solution (100X) and Stable Peroxide Solution (50X) should be protected from light during use.
• During the usage process, it is necessary to change the pipette tips when aspirating each solution to avoid cross-contamination, as this can lead to gradual loss of effectiveness and affect subsequent use.
• To ensure accurate reading results from the instrument, avoid the formation of bubbles during the sample addition process.
• It is recommended to use 96-Well Black Opaque Plates for the assay.
• Stop Solution is irritating to the human body, so please handle it with care and take appropriate precautions to avoid direct contact with the body or inhalation.
• This product is intended for scientific research purposes by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in residential areas.
• For your safety and health, please wear lab attire and disposable gloves when handling.

Storage

Store at -20°C, valid for one year. Store at 4°C, valid for one month. ADHP Substrate Solution (100X) and Stable Peroxide Solution (50X) should be protected from light during storage.

Only for research and not intended for treatment of humans or animals

Journals Using SBS Genetech Products                                       Universities Using SBS Genetech Products

SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory