Enzyme-Linked ImmunoSpot Assay (ELISpot) Protocol

Enzyme-linked immunoSpot assay (ELISpot) is an immunoassay established by replacing the polystyrene micro-reaction plate commonly used in the immune-enzymatic solid-phase carrier method with a cellulose membrane. It not only retains the advantages of conventional ELISA but also makes up for the shortcomings of antigens or antibodies not firmly encapsulated in the carrier. Therefore, it is characterized by high sensitivity, high specificity, small number of tested samples, material saving, no need for special instruments, simple and easy to determine the results, and easy to store for a long time.

• Cut a piece of nitrocellulose membrane or mixed cellulose membrane according to the number of samples. Use a circular hole punch (agar plate punch) with a diameter of 3-5 mm to press traces on the smooth surface of the membrane in sequence as the spotting site. Soak the membrane in distilled water, and then remove it to dry naturally at room temperature after it is completely wetted.
• Pipette 1-2 μl (or dip a drop into a glass rod or capillary tube) of the examined antigenic solution separately with a micropipette, drop it into the circle, and dry it naturally. To increase the amount of antigen adsorption, it can also be in the spot sample drying, and then a second spot sample. At the same time for negative and positive antigen control.
• Place the membrane in a flat dish or a small pool, add sealer, and soak at 37°C for 30-60 min. Remove and set for a moment, rinse twice with the ishing solution, and air dry.
• Make an appropriate dilution of AFMcAb or OFPcAb with a holding solution. Add to the reaction cell and cover the membrane at 37°C for 2 h.
• Rinse the membrane 3 times with ishing solution for 3 min each time and air dry.
• Lyophilized horseradish peroxidase-labeled rabbit anti-BALB/c mouse antibody is used as a 1:80 dilution with a holding solution, and the membrane is covered at 37°C for 1 h.
• Rinse the membrane 3 times with ishing solution for 3 min each time and air dry.
• The membrane is immersed in the substrate solution and stained for 5-15 min away from light.
• Rinse the filter membrane with distilled water and air dry.
• Under normal conditions for negative and positive controls, the presence of a brown spot within the circle of the membrane is considered positive, and colorless is considered negative. The membrane is dry and can be stored for a long time.

• The sample volume should not be too large so as not to overflow the indentation ring and cause mixing of the samples. If you want to increase the amount of sample adsorption, you can dry the sample at one time, and then carry out the second sample.
• After the membrane is encapsulated, it must be closed indeed, to prevent the non-specific adsorption of antigens or antibodies, and to avoid the membrane background staining being too deep.
• When the result is determined, it should be repeatedly compared with the negative and positive control spots, and try to overcome the error caused by naked eye observation.

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