Step-by-step guide to ELISpot

Published: September 7, 2023

Updated: November 27, 2023

The enzyme-linked immunospot (ELISpot) assay is a sensitive method for quantification of the number of analyte secreting cells. This is an in-depth guide for each step of the ELISpot protocol, including tips and tricks, key details to think about, and best practice.

ELISpot is an immunoassay used to quantify analyte-secreting cells. Cytokines, immunoglobulins, or other target proteins secreted by cells are captured by specific antibodies immediately after secretion and throughout the stimulation process. This instant capture makes ELISpot an extremely sensitive sandwich assay, down to the single-cell level.

The assay principle is straightforward: cells are cultured in the bottom of each well on a membrane surface coated with a specific capture antibody and in the presence or absence of stimuli. Thus, target proteins secreted by the cells are captured immediately. After an appropriate incubation time, cells are removed and the secreted analyte is detected using a detection antibody. By using a substrate with a precipitating product, the end result is visible spots on the membrane surface. Each spot corresponds to the foot-print of an individual protein-secreting cell.

The general steps of an ELISpot assay

ELISpot assay principle

A cornerstone of ELISpot is high-quality matched pairs of capture and detection monoclonal antibodies (mAbs) that have been developed and selected for the particular application. One might expect that an ELISA sandwich pair of antibodies would work in ELISpot, but our experience is that you need a mAb pair optimized for the task for reliable results from an ELISpot assay.

Having worked with ELISpot for over three decades, we can offer many tips to fine-tune and maximize the success of your assay. We will discuss these suggestions in this guide, starting where the assay begins – with selecting the ELISpot plate – and continue through the 7 general steps of the assay.

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