Direct Enzyme-Linked Immunosorbent Assay (ELISA) Protocol

Enzyme-linked immunosorbent assay (ELISA) has become a cutting-edge topic in the field of analytical chemistry. It is a special reagent analytical method, which is a new type of immunoassay technique based on immuno-enzymatic techniques.

Antigen encapsulation

• Dilute the antigen with PBS or carbonate buffer to a final concentration of 20 μg/ml. Pipette 50 μl of the diluted antigen onto a PVC plate and make serial dilutions as required.
• Cover the plate with the membrane and incubate for 2 hours at room temperature or overnight at 4°C. The incubation time should be optimized. The incubation time should be optimized.
• Pour off the incubation solution and wash the plate twice with PBS, 200 μl per well. Pour off the wash solution by flicking it on the sink and tap the plate on a paper towel to remove the residual liquid.

Blocking

• Block the remaining protein binding sites on the plate with PBS buffer containing 5% skimmed milk powder or 5% serum, 200 μl per well, or substitute with other blocking agents such as Block ACE, and BSA (bovine serum albumin).
• Cover the plate with the membrane and incubate for at least 2 hours at room temperature or overnight at 4°C.
• Wash the plate twice with PBS.

Incubation with antibodies

• Add antibody, 100 μl per well, and dilute to optimal concentration. Prepare with the sealing solution before use.
• Cover the plate with the membrane and incubate at room temperature for 2 hours, the incubation time should be optimized. Usually, 2 hours of incubation is sufficient to obtain a strong signal, but if the signal obtained is weak, incubate at 4°C overnight to obtain a stronger signal.
• Wash the plate 4 times with PBS.

Detection

• Add substrate with a multichannel pipette, 100 μl (or 50 μl) per well.
• After a sufficiently dark color is displayed, add termination solution (if necessary), 100 μl per well.

• For formal tests, the test conditions should be controlled by positive control and negative control respectively, and the samples to be examined should be made in duplicate to ensure the accuracy of the experimental results.
• Sometimes the background is high, indicating that there is a non-specific reaction, which can be closed by sheep serum, rabbit serum, or BSA.

Biofluids