Follow the step-by-step procedure below to carry out your ELISA assay efficiently.
| Step | Protocol |
| Number | Number the samples and controls in order across multiple wells and keep a record of control wells and sample wells. Set 1 well for blank control, 3 wells for negative control, and 1 well for positive control. Test samples in duplicate. (Blank well is not necessary for dual-wavelength detection.) |
| Add Sample | a) Add 100 μL of negative/positive control respectively to 3 negative control wells and 1 positive control well. Keep the blank control well empty. b) Dilute the serum being tested with Sample Diluent at a 1:10 ratio (add 100 μL of Sample Diluent to the reaction well, then add 10 μL of serum sample), and mix thoroughly. |
| Incubate | Gently tap the plate to mix thoroughly. Cover the ELISA plate with a sealer and incubate for 30 minutes at 37℃ in the dark. |
| Wash | Remove the plate sealer and aspirate the liquid from each well. Repeat the washing procedure 5 times with Wash Buffer, allowing the buffer to immerse for 30-60 seconds each time. Invert the plate and tap it against thick clean absorbent paper. (If bubbles form in the wells, use clean tips to prick them.) |
| HRP Conjugate | Add 100 μL of HRP Conjugate to each well except the blank control well. |
| Incubate | Cover the ELISA plate with a sealer and incubate for 30 minutes at 37℃ in the dark. |
| Wash | Repeat the washing procedure as described in step 4. |
| Add Substrate | Add 50 μL of Substrate Reagent A and 50 μL of Substrate Reagent B to each well. Cover the plate with a sealer, mix thoroughly, and incubate for 10 minutes at 37℃ in the dark. |
| Stop Reaction | Add 50 μL of Stop Solution to each well and gently tap the plate to mix thoroughly. |
| OD Measurement | Set the Micro-plate Reader to a wavelength of 450 nm (dual-wavelength setting of 450 nm/630 nm is recommended) to measure the A value of each well. Blank wells are not essential when using dual-wavelength detection. Note: Read the results within 30 minutes. |
