Panida Khunkaewla

Electrochemical detection of the disease marker human chitinase-3-like protein 1 by matching antibody-modified gold electrodes as label-free immunosensors

Tissue inflammation, certain cardiovascular syndromes and the occurrence of some solid tumors are correlated with raised serum concentrations of human chitinase-3-like protein 1 (YKL-40), a mammalian chitinase-like glycoprotein, which has become the subject of current research. Here we report the construction and characterization of an electrochemical platform for label-free immunosensing of YKL-40. Details of the synthesis of YKL-40 and production of anti-YKL-40 immunoglobulin G (IgG) are provided and cross-reactivity tests presented. Polyclonal anti-YKL-40 IgG was immobilized on gold electrodes and the resulting immunosensors were operated in an electrochemical flow system with capacitive signal generation. The strategy offered a wide linear detection range (0.1μg/L to 1mg/L) with correlation coefficients (R(2)) above 0.99 and good sensitivity (12.28±0.27nF/cm(2) per decade of concentration change). Additionally, the detection limit of 0.07±0.01μg/L was well below that of optical enzyme-linked immunosorbent assays (ELISAs), which makes the proposed methodology a promising alternative for YKL-40 related disease studies.

Tuned Amperometric Detection of Reduced β-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer

We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxy-phenylacetic acid ( p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted Osmium(III)-complex modified redox polymer film for analyte quantification. Chains of complexed Os(III)-centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a crosslinked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxyphenylacetic acid (p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in-situ addition or pre-incubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme activation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.

Human Cartilage Chitinase 3-like Protein 2: Cloning, Expression, and Production of Polyclonal and Monoclonal Antibodies for Osteoarthritis Detection and Identification of Potential Binding Partners

Human cartilage chitinase 3-like protein 2 (CHI3L2 or YKL-39) is a member of family-18 glycosyl hydrolases that lacks chitinase activity. YKL-39 is known as a potential marker for the activation of chondrocytes and the progression of osteoarthritis. In this study, we cloned and expressed a functional form of human YKL-39 in the bacterial system. The Escherichia coli expressed YKL-30 was used as immugen for production of anti YKL-39 polyclonal and monoclonal antibodies. Both antibody types were highly selective, reacting only with YKL-39. Isotype mapping identified two hybridoma clones (so called clones 6H11 and 8H3) to be IgM isotype. Dot blot assay showed that the monoclonal antibody was strongly active with the synovial fluid of an osteoarthritis patient, human monocyte, and T lymphocyte cell lines. Database search for protein binding partners gave high hits with several glycoproteins that play particular roles in cartilage tissue scaffolding, connective tissue formation, and cell-cell interactions. In conclusion, anti YKL-39 polyclonal and monoclonal antibodies were raised and tested to be suitable for immunological applications, such as the investigation of the YKL-39 regulating pathway and the development of an immunosensing tool for sensitive detection of cartilage tissue destruction.

Simple and Economical Analytical Voltammetry in 15 μL Volumes: Paracetamol Voltammetry in Blood Serum as a Working Example

Reported is a three-electrode mini-cell for voltammetry in 15 μL solutions. The key device component is a rolled platinum foil of an inverted omega-shaped cross section, which functions as both the electrolyte container and the counter-electrode. The analytical assembly was completed with properly sized working and reference electrodes in the two terminals of the quasi-tubular Pt trough. Its applicability in electrochemical assays of 15 μL solutions was verified by redox mediator voltammetry at graphite and noble metal sensors and by trace lead stripping voltammetry. Real sample analysis was adequate for drug detection in a volunteers blood, drawn before and 1 or 4 h after ingestion of paracetamol. In line with its known pharmacokinetics, lack of drug as well as drug presence and clearance were proven correctly in the three samples. The mini-cell here is easy to assemble and operate, indefinitely reusable, and offers valuable economy in chemical usage and minimal waste. This is primarily a versatile device for electrochemical laboratory analysis of samples that are available only in small quantities, and cost-effective quantitative screens for expensive high-molecular-weight compounds, products of microsynthesis, physiological microdialysis collections, and finger-prick blood sampling are seen as feasible targets.

Biochemical and functional analysis of COS3A, a novel CD63-specific monoclonal antibody.

BACKGROUND Monoclonal antibodies (mAbs) have become essential tools in life science research and in medicine, because of their extreme specificity. Several mAbs against leukocyte surface molecules have been generated in our laboratory. From these, mAb COS3A was selected for biochemical and functional analysis. OBJECTIVE To analyze the properties and function of the mAb COS3A. METHOD Cellular distribution was analyzed by immunofluorescence staining and flow cytometry. Biochemical characterization of the molecular target of COS3A was approached by immunoprecipitation, Western blotting and amino acid sequencing using LC-MS. N-glycosidase F treatment of COS3A-precipitated protein and culture of U937 cells in the presence of tunicamycin before cell lysate preparation were used to study the glycosylation state of proteins. Phagocytosis was examined by flow cytometry. RESULTS MAb COS3A bound specifically to a molecule expressed on the surface of various human hematopoietic cells and cell lines but not on erythrocytes. The antigen had a molecular weight of 30-70 kDa, which was reduced to 25 kDa by elimination of N-linked glycan. LC-MS data and immunoprecipitation indicated that mAb COS3A bound specifically to the CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis. CONCLUSIONS The mAb COS3A recognized the CD63 molecule and strongly diminished granulocyte phagocytosis of E. coli, suggesting that CD63 may play a crucial role in the initial step of phagocytosis. MAb COS3A is, therefore, suitable for both biochemical and functional studies of CD63, and may be used for further study of the mechanism of phagocytosis and also in therapeutic approaches.