Development and characterization of new rat monoclonal antibodies for procalcitonin

Krämer, Petra M.; Gouzy, Marie-Françoise; Keß, Melanie; Kleinschmidt, Ulrike; Kremmer, Elisabeth
October 2008
Analytical & Bioanalytical Chemistry;Oct2008, Vol. 392 Issue 4, p727
Academic Journal
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 × 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 × 108 M−1; K D = 1.64–3.56 × 10–9 M) and have moderate dissociation rate constants ( k d = 1.70–2.40 × 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 µg 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®). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 µg 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.


Related Articles

  • Towards a platform PBPK model to characterize the plasma and tissue disposition of monoclonal antibodies in preclinical species and human. Shah, Dhaval; Betts, Alison // Journal of Pharmacokinetics & Pharmacodynamics;Feb2012, Vol. 39 Issue 1, p67 

    The objectives of the following investigation were (1) development of a physiologically based pharmacokinetic (PBPK) model capable of characterizing the plasma and tissue pharmacokinetics (PK) of nonspecific or antigen specific monoclonal antibodies (mAbs) in wild type, FcRn knockout, tumor...

  • SPR imaging biosensor for podoplanin: sensor development and application to biological materials. Gorodkiewicz, Ewa; Charkiewicz, Radoslaw; Rakowska, Alicja; Bajko, Paulina; Chyczewski, Lech; Niklinski, Jacek // Microchimica Acta;Feb2012, Vol. 176 Issue 3/4, p337 

    Podoplanin (PDP) is a small transmembrane protein and widely present in various specialized cells throughout the human body. It is a specific marker for identification of lymphatic vessel and a candidate marker for cancer stem cells in squamous cell carcinoma of the lung. We report on method for...

  • Characterization of Therapeutic Monoclonal Antibodies Reveals Differences Between In Vitro and In Vivo Time-Course Studies. Yin, Sheng; Pastuskovas, Cinthia; Khawli, Leslie; Stults, John // Pharmaceutical Research;Jan2013, Vol. 30 Issue 1, p167 

    Purpose: To examine and determine the sites and the kinetics of IgG1 mAb modifications from both in vitro (rat plasma and PBS) and in vivo (rat model) time-course studies. Methods: A comprehensive set of protein characterization methods, including RPLC/MS, LC-MS/MS, iCIEF, capSEC, and CE-SDS...

  • Reference intervals of serum tartrate-resistant acid phosphatase type 5b activity measured with a novel assay in Japanese subjects. Yoshiki Nishizawa; Masaaki Inaba; Mitsukazu Ishii; Hiroyuki Yamashita; Takami Miki; Hitoshi Goto; Sinsuke Yamada; Osamu Chaki; Kentaro Kurasawa; Yoshiko Mochizuki // Journal of Bone & Mineral Metabolism;May2008, Vol. 26 Issue 3, p265 

    Abstract  Among the isotypes of serum tartrate-resistant acid phosphatase (TRACP), only type 5b (TRACP-5b) is derived from osteoclasts, and it is necessary to develop an assay specific for this TRACP-5b for evaluation of osteoclastic activity. Recently, a novel assay system for TRACP-5b...

  • Epitope mapping of human Fas using peptide phage display. S. Abbasova; Zh. Shcheprova; A. Laman; A. Shepelyakovskaya; L. Baidakova; I. Rodionov; N. Kushlinskii // Bulletin of Experimental Biology & Medicine;Oct2007, Vol. 144 Issue 4, p515 

    Abstract  Sandwich EIA for measurement of soluble Fas was developed on the basis of SA-7 and SA-8 monoclonal antibodies to full-length human Fas. The threshold sensitivity of the test system is 0.3 ng/ml. Several isoforms of soluble Fas were identified. The structure of SA-7 and SA-8...

  • Application of PBPK modeling to predict monoclonal antibody disposition in plasma and tissues in mouse models of human colorectal cancer. Abuqayyas, Lubna; Balthasar, Joseph // Journal of Pharmacokinetics & Pharmacodynamics;Dec2012, Vol. 39 Issue 6, p683 

    This investigation evaluated the utility of a physiologically based pharmacokinetic (PBPK) model, which incorporates model parameters representing key determinants of monoclonal antibody (mAb) target-mediated disposition, to predict, a priori, mAb disposition in plasma and in tissues, including...

  • Mice and monoclonal antibody production. Edwards, Keith // Animals Today;Aug-Oct97, Vol. 5 Issue 3, p15 

    Discusses issues related to the use of rats in the production of monoclonal antibodies (mAbs) in Australia. Uses of mAbs; Suffering associated with ascites and monoclonal antibody production in animals; Development of a gas-permeable tissue culture bag by the Alternatives Research and...

  • Radiotoxicity of h-R3 monoclonal antibody labeled with[sup 188] Re administered intracerebrally in rats. Gonzalez, B; Casaco, A; Alvarez, P; Leon, M; Arteaga, M; Leon, A; Santana, E; Bada, A; Figueredo, R; Hernández, R; Iznaga-Escobar, N; González, F; Perez, R // Human & Experimental Toxicology;Dec2000, Vol. 19 Issue 12, p684 

    Brain tumors are often incurable despite current aggressive treatmentmodalities. Regionalintracerebral administration of labeled monoclonal antibodies Mabs) can maximize the radioisotope and Mab concentration to tumor sites while reducing systemic toxicity, h-R3 is a humanized antiepidermal...

  • Prevalence of Monoclonal Gammopathy of Undetermined Significance. Kyle, Robert A.; Therneau, Terry M.; Rajkumar, S. Vincent; Larson, Dirk R.; Plevak, Matthew F.; Offord, Janice R.; Dispenzieri, Angela; Katzmann, Jerry A.; Melton, L. Joseph // New England Journal of Medicine;3/30/2006, Vol. 354 Issue 13, p1362 

    Background: The prevalence of monoclonal gammopathy of undetermined significance (MGUS), a premalignant plasma-cell disorder, among persons 50 years of age or older has not been accurately determined. We used sensitive laboratory techniques to ascertain the prevalence of MGUS in a large...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics