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 support, including full technical guidance throughout the experimental process and free testing services. While many steps in an Elisa experiment are straightforward, certain key procedures 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**: A standard blocking solution was used, consisting of 10 mM phosphate buffer with 1% bovine serum albumin (BSA), as described in previous studies. This helps prevent non-specific binding during the assay.
- **Washing Solution**: The washing buffer was prepared according to standard protocols, ensuring thorough removal of unbound reagents.
- **Protective Solution for Enzyme-Linked Plates**: To enhance the stability of the coated plates, a protective solution containing sugar, unrelated protein, gentamicin, and divalent metal ions was added after blocking. The plates were then stored at 4°C overnight. Afterward, both protected and unprotected plates were incubated at 37°C for 15 days to evaluate the effectiveness of the protective solution.
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 an absorbance value of approximately 1.5, as measured by an automated microplate reader.
3. **Optimization of Substrate Solution**
TMB (3,3',5,5'-tetramethylbenzidine) was selected as the substrate due to its safety and ease of use compared to OPD. The preparation involved dissolving TMB in DMSO and diluting it with ultrapure water to create Substrate B. Substrate A was made by dissolving urea hydrogen peroxide in a 100 mM phosphate-citric acid buffer. Before use, equal volumes of A and B were mixed. Shanghai Hengyuan also adjusted the concentrations of TMB and hydrogen peroxide, adding an enzyme promoter to improve sensitivity. Different dilutions of horseradish peroxidase (1:1000, 1:10,000, 1:100,000, etc.) were tested alongside two versions of the substrate to compare performance.
4. **Optimization of Antigen-Antibody Reaction Time**
The reaction time between antigen and antibody was tested at various intervals (10, 20, 30, 40, 50, 60, 70, 90 minutes). This helped identify the optimal time when the signal reached equilibrium without over-saturation.
5. **Optimization of Substrate Incubation Time**
Under consistent conditions, the substrate incubation time was tested at 11 different intervals (5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80 minutes) to determine the best time for maximum signal detection.
Shanghai Hengyuan Biotechnology offers high-quality Elisa kits and a wide range of reagents, trusted by customers worldwide. Whether you're looking for wholesale or retail options, our team is here to assist you. If you need help choosing the right Elisa kit, feel free to contact our sales representatives today!
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