Figure 4.

Direct and indirect LBA-LC-MS/MS assay formats for ADA detection. (A) shows the direct LBA-LC-MS/MS assay format for detection of ADAs, where samples are pre-incubated with the biotinylated drug to form a biotinylated drug-ADA complex and then captured by the streptavidin magnetic beads. Subsequently, nondrug-specific proteins are removed from the beads by washes. The bound ADAs are eluted by an acidic buffer and followed by neutralization. Thereafter, the eluted antibodies undergo reduction, alkylation, and digestion with trypsin to obtain ADA tryptic peptides. The signature peptides derived from ADAs are chromatographically separated and then detected by measuring each signature peptide under a specific SRM mode; (B) shows the indirect LBA-LC-MS/MS assay format for detection of ADAs, where excess drugs are spiked into the serum sample to saturate the ADA binding sites, followed by using a mouse anti-drug monoclonal antibody immobilized on magnetic beads to capture the ADA-drug complex. Thereafter, the ADAs are directly measured by the ADA’s signature peptides.