What are the most commonly used enzyme systems in ELISA assays? The Shanghai Hengyuan Technology Department provides a detailed explanation:
1. Alkaline Phosphatase (AP) AP is one of the most frequently used enzymes in ELISA and other immunoassays. Its reaction substrate typically includes nitroblue tetrazolium (NBT), often combined with BCIP, which forms an insoluble blue product when catalyzed by AP. This color development is widely used in immunohistochemistry (IHC), Western blot (WB), immunocytochemistry (ICC), and SOD activity assays. AP is favored for its high sensitivity and stability in certain applications.
2. Horseradish Peroxidase (HRP) HRP is another key enzyme system in ELISA due to its high specificity and versatility. It primarily acts on hydrogen peroxide (Hâ‚‚Oâ‚‚) and small molecule alcohols or urea derivatives. In ELISA, common substrates include o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). These substrates produce different colored products that can be measured spectrophotometrically.
1. OPD (o-phenylenediamine) OPD is one of the most widely used substrates for HRP in ELISA. It offers high sensitivity and ease of detection through colorimetric analysis. However, it has some drawbacks, such as instability after formulation and potential carcinogenicity, which limits its use in some settings.
2. TMB (Tetramethylbenzidine) TMB is gaining popularity due to its excellent performance and safety profile. When acted upon by HRP, it changes from colorless to blue, and adding an acidic stop solution turns it yellow. This clear visual contrast makes it ideal for quantitative measurements, and it's becoming the preferred choice in many laboratories.
3. ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) Although ABTS has lower sensitivity compared to OPD and TMB, it offers a very low background signal, making it suitable for assays where minimal interference is critical. It’s often used in situations requiring high specificity and low noise levels.
Each enzyme-substrate system has its own advantages and limitations, and the choice depends on the specific requirements of the assay, including sensitivity, stability, and detection method. Understanding these differences helps in selecting the most appropriate system for your ELISA experiments.
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