Elisa kits are among the most popular products offered by Shanghai Hengyuan Biotechnology Co., Ltd. The company is known for its comprehensive pre- and post-sale services, including full technical support throughout the experimental process and free testing services. While many steps in an Elisa experiment are straightforward, some key stages require careful optimization to ensure accurate and reliable results. In this article, Shanghai Hengyuan shares expert tips on optimizing your Elisa experiment:
1. **Optimization of Blocking, Washing, and Protective Solutions**
- **Blocking Solution**: The blocking step was performed using a standard protocol from published literature. The solution consisted of 10 mM phosphate buffer with 1% bovine serum albumin (BSA). This helps prevent non-specific binding during the assay.
- **Washing Solution**: A commonly used washing buffer was prepared according to standard methods, ensuring efficient removal of unbound components without disrupting the immobilized antigen or antibody.
- **Protective Solution for the Enzyme-Linked Plate**: To enhance the stability of the coated plate, a protective solution was added after blocking. This included sugars, unrelated proteins, gentamicin, and divalent metal ions dissolved in 10 mM phosphate buffer. After incubation at 4°C overnight, the protected plates were compared with unprotected ones under high-temperature conditions (37°C) for 15 days to evaluate their performance.
2. **Optimization of Enzyme-Labeled Antibody Dilution**
The antigen coating concentration was set at 4 µg/ml. The enzyme-labeled antibody was serially diluted (1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:1000) and incubated. After washing and color development, the optimal dilution was determined based on the absorbance value (around 1.5), ensuring both sensitivity and specificity.
3. **Optimization of Substrate Solution**
TMB (3,3',5,5'-tetramethylbenzidine) was chosen as the substrate due to its safety and ease of use compared to OPD. The preparation followed standard protocols: TMB was dissolved in DMSO and diluted with ultrapure water to create Substrate B, while hydrogen peroxide was dissolved in a phosphate-citric acid buffer to make Substrate A. These were mixed just before use. Shanghai Hengyuan also adjusted the concentrations of TMB and hydrogen peroxide, adding an enzyme promoter to improve sensitivity. Different dilutions of horseradish peroxidase (HRP) were tested to compare the performance of two substrate formulations.
4. **Optimization of Antigen-Antibody Reaction Time**
The reaction time between antigen and antibody was systematically tested at intervals of 10, 20, 30, 40, 50, 60, 70, and 90 minutes. This helped identify the optimal time when the signal reached equilibrium without excessive background noise.
5. **Optimization of Substrate Incubation Time**
Under consistent conditions, the substrate incubation time was tested at various intervals (5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80 minutes) to determine the best point for stopping the reaction and reading the results.
Shanghai Hengyuan Biotechnology offers a wide range of high-quality Elisa kits and reagents, trusted by customers worldwide. Whether you're looking for wholesale or retail options, our products are designed for precision and reliability. If you need assistance in choosing the right Elisa kit for your research, feel free to contact our sales team today. We’re here to help you achieve the best results in your experiments.
RYEOGLOBAL Co,.LTD , https://www.ryeolab.com