Common ELISA kit detection method: double antibody sandwich method and indirect method - Database & Sql Blog Articles

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Kaixin micro test

Test probe P100-M3

According to Shanghai Jinma's analysis, the ELISA kit combines the specific reaction between antigens and antibodies with the high-efficiency catalytic action of enzymes on substrates. The antigen-antibody reaction takes place in a solid-phase carrier, specifically a polystyrene microtiter plate. After each reagent is added, any unbound excess reactants can be removed through washing, ensuring the accuracy and reliability of the results. In practical applications, there are several commonly used methods based on different design approaches. These include: the indirect method for detecting antibodies (Panel A), the double antibody sandwich method for detecting antigens (Panel B), and the competitive method for detecting small molecule antigens or haptens.

Based on years of research experience from Shanghai Jinma Biological, we recommend using the double antibody sandwich method and the indirect method for ELISA kits. Below are six key steps in the testing process, which you can follow for accurate and reliable results:

1. Capture Antibody Coating: Dilute the primary antibody to a concentration of 1–10 μg/ml in 0.05 M carbonate coating buffer (pH 9.6). Add 0.1 ml per well in a polystyrene plate and incubate overnight at 4°C. The next day, discard the solution and wash the wells three times with washing buffer (3 minutes each time).
2. Sample Addition: Add 0.1 ml of the diluted sample to the coated wells and incubate at 37°C for 1 hour. Include blank, negative, and positive control wells during this step.
3. Add Enzyme-Labeled Antibody: Add 0.1 ml of freshly prepared enzyme-labeled antibody (diluted according to titration) to each well. Incubate at 37°C for 0.5 to 1 hour, then wash again.
4. Substrate Addition: Add 0.1 ml of TMB substrate solution to each well and incubate at 37°C for 10–30 minutes to develop color.
5. Stop the Reaction: Add 0.05 ml of 2M sulfuric acid to each well to terminate the reaction.
6. Result Interpretation: Observe the color change visually against a white background. A darker color indicates a stronger positive result, while a light or no color suggests a negative result. You can also measure the OD value at 450 nm (or 410 nm if using ABTS) using an ELISA reader. If the OD value exceeds 2.1 times that of the negative control, it is considered positive.

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