Immunoaffinity LC–MS/MS Quantification of the Sepsis Biomarker Procalcitonin Using Magnetic- and Polystyrene-Bead Immobilized Polyclonal Antibodies

Procalcitonin (PCT) is a biomarker for bacterial sepsis,
and accurate
quantification of PCT is critical for sepsis diagnosis and treatment.
Immunological PCT quantification methods are routinely used in clinical
laboratories, yet there is a need for harmonization of PCT quantification
protocols. An orthogonal method to clinical immunological assays,
such as LC–MS/MS, is required. In this study, a highly sensitive
and robust immunoaffinity LC-MRM quantitative method for detecting
procalcitonin in human serum has been developed. An initial comparison
of immunocapture of PCT with a polyclonal anti-PCT antibody immobilized
on polystyrene nanoparticles (Latex) and magnetic beads demonstrated
superior performance with magnetic beads. Three tryptic PCT peptides
from the N- and C-terminal regions of PCT were selected for LC–MS/MS
quantification. For PCT quantification, an LLOQ of 0.25 ng/mL of PCT
in human serum was achieved using a sample volume of 1 mL. The method’s
trueness and precision consistently lie within the 15% margin. The
parallel measurement of three PCT peptides may allow future differentiation
of intact PCT vs other PCT forms originating from potential degradation,
processing, or polymorphisms. An established and validated LC-MRM-based
quantification of PCT will be relevant as an orthogonal method for
harmonization and standardization of clinical assays for PCT.