With the wide application of enzyme-linked immunoassay (ELISA) technology in the field of medicine and biology, new breakthroughs have been made in the study of in vitro detection of various cytokines and antibodies. In the study of immune response mechanism, enzyme-linked immunosorbent assay (ELISA) was commonly used to detect free cytokines (CK) or antibodies in body fluids, but due to the different half-life of free circulating antibodies or CK, they were constantly metabolized in body fluids or bound to the target organs, which could not reflect the level of antibodies and CK in the body accurately. In 1980s, according to the basic principle of ELISA technology, foreign researchers established solid-phase enzyme-linked immunospot technology (ELISPOT) for in vitro detection of specific antibody-secreting cells and CK-secreting cells.

Operation method

ELISPOT assay

Materials and Instruments

Antigen
PBS PRMI 1640 medium Ammonium chloride
PVDF membrane 96-well plate

Move

1. Dissolve the antigen in PBS at 30 μg/ml and add 100 μl/well to a 96-well sterilized plate with a PVDF membrane base overnight;2. On the second day, after aspirating the coating solution, add 100 μl of PRMI 1640 medium with 5% FCS and block for 1 hour at 37℃;3. Preparation of splenocyte suspensions (single splenic lymphocyte suspensions prepared by removing red blood cells with ammonium chloride);4. Starting from 1 × 106/well, make different concentration gradients by dilution of 1:3 well by well, and make 3 duplicate wells, and incubate at 37 ℃ for 5 hours;5. Wash with PBS 3-5 times and incubate with biotinylated anti-mouse IgG secondary antibody for 30 min;6. Wash 3-5 times with PBS and incubate with streptavidin-labeled alkaline phosphatase for 30 min;7. Wash with PBS for 3-5 times, use the substrate BCIP/NBT to develop the color, observe the color reaction under the microscope, and terminate the reaction promptly after the color is developed;8. Count the number of spots in each well and calculate the number of antibody-secreting cells per ¹⁰⁶ spleen cells.

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