What are the most commonly used enzyme systems for ELISA assays? Let's explore this with insights from the Shanghai Hengyuan Technology Department:
1. **Alkaline Phosphatase (AP)**
AP is a widely used enzyme in immunoassays. Its common substrate is nitroblue tetrazolium (NBT), often combined with BCIP. Under the action of AP, this mixture forms an insoluble blue product, making it ideal for visualization in techniques like immunohistochemistry (IHC), Western blot (WB), and SOD activity assays. It is known for its stability and clarity in detection.
2. **Horseradish Peroxidase (HRP)**
HRP is one of the most popular enzymes in ELISA due to its high specificity and versatility. It primarily acts on hydrogen donors in the presence of hydrogen peroxide (Hâ‚‚Oâ‚‚). Common substrates include o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).
- **OPD** is highly sensitive and easy to measure colorimetrically, but it has poor stability and can be carcinogenic.
- **TMB** is more stable than OPD and produces a clear blue color that turns yellow when acid is added, making it easier to quantify using a spectrophotometer. This makes TMB increasingly popular in modern ELISA protocols.
- **ABTS** offers lower sensitivity compared to OPD and TMB but provides a very low background signal, which is beneficial in certain applications.
These enzyme-substrate systems play a crucial role in determining the accuracy, sensitivity, and reliability of ELISA results. Choosing the right system depends on the specific experimental requirements, such as detection speed, sensitivity, and ease of use. Whether you're working in research, diagnostics, or quality control, understanding these enzyme systems can help optimize your ELISA procedures.
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