Enzyme-linked immunosorbent assay (ELISA) kits are widely used in research to quantify specific proteins, antibodies, or antigens in biological samples. Here is a step-by-step guide on how to use ELISA kits in research:
1. Sample preparation
Prepare biological samples (e.g., serum, plasma, cell culture supernatant) according to the kit instructions. Depending on the assay type, you may need to dilute the sample in the provided buffer to fit the assay's detection range.
2. Coating the plate (if necessary)
For direct or indirect ELISA, coat the provided microplate wells with the antigen or antibody and incubate overnight at 4°C. Some kits come with pre-coated plates, so you can skip this step.
3. Blocking
To prevent nonspecific binding, add blocking buffer to each well after the coating step. Blocking buffer (such as BSA or skim milk) covers unbound sites on the plate, reducing background noise. Incubate according to the kit instructions, typically for 1 hour at room temperature.
4. Add Primary and Secondary Antibodies
After blocking, wash the plate and add a primary antibody specific for the target protein, then incubate. Wash again and add an enzyme-conjugated secondary antibody (such as HRP or alkaline phosphatase). This antibody binds to the primary antibody, amplifying the signal.
5. Add Substrate and Detection
Add the substrate solution provided in the kit, which reacts with the enzyme to produce a measurable color change. Allow the reaction to proceed for the recommended time, then terminate the reaction with the provided stop solution.
6. Data Collection
Measure the optical density (OD) of each well at the recommended wavelength (usually 450 nm) using a microplate reader. Compare sample readings to a standard curve to quantify the target analyte concentration.
By following these steps, ELISA kits provide a reliable, quantifiable method to detect the target protein in a sample.