Development of a Creatinine ELISA and an Amperometric Antibody-Based Creatinine Sensor with a Detection Limit in the Nanomolar Range

• Published in: Analytical chemistry (Washington) Vol. 72; no. 5; pp. 916 - 921
• Main Authors: Benkert, Alexander, Scheller, Frieder, Schössler, Werner, Hentschel, Christian, Micheel, Burkhard, Behrsing, Olaf, Scharte, Gudrun, Stöcklein, Walter, Warsinke, Axel
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.03.2000
• Subjects: Biochemistry; Biological and medical sciences; Biosensors; Biotechnology; Fundamental and applied biological sciences. Psychology; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Methods. Procedures. Technologies; Miscellaneous. Technology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Proteins; Sensors; Various methods and equipments; Creatinine; Biological fluid; Biochemical analysis; Monoclonal antibody; Immunosensor; Immunological method; Analysis method; Biosensor; Clinical biology; Amperometry; Serum; ELISA assay; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9909047

Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology. Antibodies from various clones have been characterized with BIAcore 2000 with respect to the dissociation constant and specificity. Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74 μM) and the highest specificity for creatinine and were chosen for the development of a competitive ELISA and an amperometric creatinine sensor. The creatinine sensor was constructed by fixing a creatinine-modified membrane on the top of a platinum working electrode which was then incorporated into a stirred electrochemical measuring cell. For creatinine determination the creatinine-containing sample was incubated with B90-AH5 and anti-IgG(mouse)−glucose oxidase conjugate and applied to the measuring cell. After a washing step glucose was added and the produced hydrogen peroxide was registrated at E appl = +600 mV vs Ag/AgCl. The measuring range was 0.01−10 μg/mL. The highest sensitivity for creatinine was achieved at 330 ng/mL (3 μM) and the lower detection limit at 4.5 ng/mL (40 nM). This is far below the relevant clinical range, which is 5−17 μg/mL (44−150 μM) and allows a reliable determination of very low creatinine concentrations in serum, where standard methods cannot be applied. After each measurement the sensor was regenerated with 10 mM HCl without any loss in binding activity.