Development and characterization of new rat monoclonal antibodies for procalcitonin

Abstract

The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs) for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3, was used as capture antibody. Four mAbs, PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6, were used as detection mAbs, either as Protein G-purified or as biotinylated mAbs. A surface plasmon resonance (SPR) biosensor was used to characterize the antigen-antibody biomolecular interactions. The capture mAb (PROC1 3G3) has an equilibrium dissociation constant (K (D)) of 3.42 x 10(-8) M. All four detection mAbs (PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6) are of high affinity (K (A) = 2.81-6.11 x 10(8) M(-1); K (D) = 1.64-3.56 x 10(-9) M) and have moderate dissociation rate constants (k (d) = 1.70-2.40 x 10(-3) s(-1)). Four different sandwich enzyme-linked immunosorbent assays (ELISAs) with standards of human recombinant (hr) PCT, using PROC1 3G3 as capture mAb and PROC4 mAbs as detection mAbs, respectively, led to highly specific determinations of PCT without cross-reactivities to calcitonin and katacalcin. The lower limits of quantification (LLOQ) for hrPCT (in 40 mM phosphate-buffered saline (PBS), pH 7.6) with these assays ranged from 2.3 to 12.8 microg L(-1). In addition, sandwich ELISAs were set up with biotinylated PROC4 mAbs, and with hrPCT in 4% human serum albumin (diluted 1:10 in 40 mM PBS, including 1:5 (v/v) LowCross Buffer(R)). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 microg L(-1) and were thus much closer together for the different assays. With the latter assay setup (PROC1 3G3 as capture mAb, PROC4 6C6-biotin as detection mAb) a first collection of five serum samples was determined (healthy volunteers, unspiked, and spiked). Recovery rates for the spiked samples ranged from 98.3 to 115.7%. The newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.