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Kremmer, E. ; Meyer, K.* ; Grässer, F.A.* ; Flatley, A. ; Kösters, M.* ; Luppa, P.B.* ; Krämer, P.M.

A new strategy for the development of monoclonal antibodies for the determination of human procalcitonin in serum samples.

Anal. Bioanal. Chem. 402, 989-995 (2012)

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Procalcitonin (PCT)-a diagnostic serum parameter for bacterial infection and sepsis-is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC(50)) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7 μg L(-1) (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R (2): assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.

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