# Combining magnetically isolated CD45 cells with serum maintains intact drug responsiveness for ELISpot analysis in clinical trials

## TL;DR

A new method combines isolated immune cells with serum to maintain drug responsiveness in ELISpot analysis for clinical trials.

## Contribution

A novel protocol preserves drug effects in ELISpot analysis by combining magnetically isolated CD45 cells with subject serum.

## Key findings

- TNF-α inhibition by infliximab is preserved in serum but lost after traditional cell isolation.
- Combining serum with CD45 cells maintains drug responsiveness in ELISpot analysis.
- The protocol is suitable for limited blood volumes in clinical trials.

## Abstract

Enzyme-linked immunosorbent spot analysis is frequently used to investigate immune responsiveness during clinical trials. However, ELISpot classically utilizes peripheral blood mononuclear cell isolates from whole blood, requiring relatively high blood draw volumes and removing both granulocytes and bound drug. Here, we describe a novel protocol whereby CD45 cells are magnetically isolated from human whole blood and co-incubated with serum isolated from the same subject. Infliximab is a well characterized anti-tumor necrosis factor α (TNF-α) antibody in clinical use since the late 1990s. We demonstrated that TNF-α inhibition by infliximab in spiked whole blood is lost on peripheral blood mononuclear cell isolation but remains in serum, and that combining serum from infliximab spiked whole blood with magnetically isolated CD45 immune cells inhibited PMA/ionomycin-stimulated TNF-α secretion. This novel protocol has important implications for enzyme-linked immunosorbent spot analysis in clinical trials in which blood volume is limited, and keeping drug responses intact provides critical information.