Enzyme Linked Immunosorbent Assay for the Quantification of Ipilimumab in Human Serum, Plasma, Cerebrospinal Fluid, and Maternal Milk

Immune checkpoint inhibitors (ICI) such as the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor ipilimumab have become part of the standard of care for different types of cancer. Pharmacokinetic-pharmacodynamic dose rationalization studies are needed to limit the toxicity and economic costs, and to increase the efficacy of ipilimumab treatment. For this purpose, we developed and validated an enzyme-linked immunosorbent assay (ELISA) for the accurate and sensitive determination of ipilimumab concentrations in serum, plasma, cerebrospinal fluid (CSF), and maternal milk. The assay is based on the specific capture of ipilimumab by immobilized CTLA-4 and chemiluminescent detection by anti-IgG1 coupled with horseradish peroxidase. The lower limit of quantification of ipilimumab in the investigated biological matrices is 5ng/mL. The ELISA method showed long-term sample stability for at least one year at -80°C and was successfully cross-validated with Ultra-Performance Liquid Chromatography coupled with tandem Mass Spectrometry. The ELISA method is reliable, relatively inexpensive and can be used in serum, plasma, CSF, and maternal milk from patients treated with ipilimumab as evidenced by the analysis of real clinical samples.