Don't stack the plates if you have more than one to prevent edge effects During the overnight incubation the cells will secrete cytokine, which will bind to the primary antibody.
Note: Exceeding 1 h incubation with enzyme conjugate could result in increased background color. The ELISpot technique is not limited to measurement of cytokines; it is also suitable for almost any secreted protein where single-cell analysis is of interest.
Plates pre-coated with the capture antibodies and one-step detection reagents offer additional advantages. Wear disposable gloves and personal protection throughout the entire procedure and thoroughly wash hands after handling the test reagents.
This helps to prevent high background as some reagents can leak through the membrane into the bottom tray of the plate. Use immediately.
Day 3 Remove culture medium with cells. Coat well plate with capture antibody diluted in phosphate buffered saline PBS. Approximately 0. Do not use a plate washer at this stage if available. If spots are not clearly visible, incubate again and check regularly.
Even brief exposure to a mitogenic serum can cause high background while other sera can have suppressive effects. After an appropriate incubation time, cells are removed and the secreted molecule is detected using a detection antibody in a similar procedure to that employed by the ELISA.
Sensitive The ELISpot assay captures the presence of cytokines immediately after secretion, in contrast to measurements that are skewed by receptor binding or protease degradation.