Enzyme-Linked Immunosorbent Assay (ELISA) Operation Procedure

Before starting the ELISA experiment, all reagents should be equilibrated to room temperature. When diluting reagents or samples, ensure thorough mixing while avoiding foam formation as much as possible.

1. Antibody Coating: According to experimental needs, dilute the coating antibody to a certain dilution using carbonate buffer solution (CBS) or phosphate buffer solution (PBS), coat at 100 μL/well, and incubate at 37°C for 2 h or overnight at 4°C.

2. Plate Washing: Discard the liquid in the wells, spin dry, wash the plate with 1xTBST twice, soaking for 1-2 minutes each time, 350 μL/well, spin dry (you can also tap to dry the liquid in the wells).

3. Blocking: Add 200 ~350 μL blocking solution per well, incubate at 37°C for 1 h.

4. Plate Washing: Same as step 2.

Note: Commercial kits usually have pre-coated antibodies on the microplate, so steps 1-4 are not required. Please verify before use.

5. Sample Addition: Set zero wells, standard wells, and test sample wells respectively. Add 100 μL sample dilution to blank wells, and add 100 μL standard or test sample to the remaining wells. Cover the microplate with a lid or film, and incubate at 37°C for 60-120 minutes. To ensure the validity of experimental results, use a new standard solution for each experiment.

Note: Do not generate bubbles during sample addition. Add the sample to the bottom of the microplate well, try not to touch the well wall, and finish loading a plate within 10 minutes.

6. Plate Washing: Discard the liquid in the wells, spin dry, wash the plate with 1xTBST four times, soaking for 1-2 minutes each time, 350 μL/well, spin dry (you can also tap to dry the liquid in the wells).

7. Detection Antibody Addition: According to experimental needs, dilute the detection antibody to a certain dilution using 1xPBS, add 100 μL/well, and incubate at 37°C for 45 minutes.

8. Plate Washing: Same as step 6.

9. Secondary Antibody Addition: According to experimental needs, dilute the secondary antibody to a certain dilution using 1xPBS, add 100 μL/well, and incubate at 37°C for 30 minutes.

10. Plate Washing: Same as step 6.

11. Color Development: Mix TMB color development solution A and B at a ratio of 1:1 thoroughly, add 100 μL/well, and incubate at 37°C for 10 minutes.

12. Termination: Add 100 μL/well of 1M sulfuric acid solution to terminate color development.

13. Microplate Reader Reading: Use a microplate reader to measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm with a correction wavelength of 630 nm, and read within 5 minutes after adding the termination solution.

14. Result Judgment:

1) The OD value of each standard and sample must be subtracted from the OD value of the zero well. If duplicate wells are set, the average value should be taken;

2) Using the concentration of the standard as the abscissa and the OD value as the ordinate, use professional curve-making software to perform four-parameter fitting (4-PL), such as Origin, ELISACalc, etc. Calculate the corresponding fitted concentration from the standard curve based on the OD value of the sample, and then multiply by the dilution factor to obtain the measured concentration of the sample.

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