Indirect ELISA Protocol

The steps for an indirect enzyme-linked immunosorbent assay (ELISA) are the same as for a direct ELISA, except for the additional washing step and the type of antibody added after removing the buffer. Indirect ELISA requires two antibodies, a detection primary antibody that adheres to the target protein and an enzyme-linked secondary antibody that is complementary to the primary antibody.

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 primary and secondary antibodies

• Add 100 μl of diluted primary antibody to each well.
• Incubate the membrane-covered plates at room temperature for 2 hours, the incubation time needs to 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.
• Add labeled secondary antibody, 100 μl per well. dilute to the optimal concentration and mix with the sealing solution before use.
• Cover the plate with membrane and incubate at room temperature for 1-2 hours.
• 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.
• The absorbance value (optical density) is read by the enzyme marker.

• Good purity is required for the adsorption of antibodies (or antigens) onto the surface of solid phase carriers. When adsorbed, the PH is generally required to be between 9.0 and 9.6.
• Adsorption temperature, time, and protein amount also have a certain impact, generally used at 4°C, 18 to 24 hours.

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