Ewa Gorodkiewicz

Cystatin C: a kidney function biomarker.

Renal function is essential for homeostasis. The kidneys play important pleiotropic roles including removal of metabolic waste products and maintenance of water-electrolyte balance and blood pressure. Early diagnosis of renal dysfunction and institution of appropriate therapy are vital to survival. Unfortunately, common indicators of renal function lack necessary sensitivity and specificity. Recent evidence has, however, suggested that cystatin C (cysC) may be useful as a marker for glomerular filtration. CysC is a protein belonging to a group of cysteine proteases inhibitors produced primarily by nucleated cells. Due to low molecular weight and positive pI, it is easily filtered. Moreover, its serum concentration is independent of gender, age, or muscle mass, i.e., typical confounders in assessing glomerular filtration rate (GFR). This chapter discusses the structure and biologic function of cysC, its role as an indicator of GFR, and the most frequently used methods for its determination.

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

Surface plasmon resonance imaging biosensor for cathepsin G based on a potent inhibitor: development and applications.

A specific surface plasmon resonance imaging (SPRI) array biosensor for the determination of the enzymatically active cathepsin G (CatG) has been developed. For this purpose, a specific interaction between an inhibitor immobilized onto a chip surface and CatG in an analyzed solution was used. The MARS-115 CatG peptidyl inhibitor containing the 1-aminoalkylphosphonate diaryl ester moiety at the C terminus and N-succinamide with a free carboxylic function was synthesized and covalently immobilized onto the gold chip surface via the thiol group (cysteamine). Atomic force microscopy was used for the observation of surface changes during the subsequent steps of chip manufacture. Optimal detection conditions were chosen. High specificity of synthesized inhibitor to CatG was proved. The precision, as well as the accuracy, was found to be well suited to enzyme determination. The sensor application for the determination of CatG in white blood cells and saliva was shown for potential diagnosis of leukemia and oral cavity diseases during the early stages of those pathological states.

Cathepsin D serum and urine concentration in superficial and invasive transitional bladder cancer as determined by surface plasmon resonance imaging

Determination of cathepsin D (Cat D) concentration in serum and urine may be useful in the diagnosis of bladder cancer. The present study included 54 healthy patients and 68 patients with bladder cancer, confirmed by transurethral resection or cystectomy. Cat D concentration was determined using a surface plasmon resonance imaging biosensor. Cat D concentration in the serum of bladder cancer patients was within the range of 1.3–5.59 ng/ml, while for healthy donors it was within the range of 0.28–0.52 ng/ml. In urine, the Cat D concentration of bladder cancer patients was within the range of 1.35–7.14 ng/ml, while for healthy donors it was within the range of 0.32–0.68 ng/ml. Cat D concentration may represent an efficient tumor marker, as its concentration in the serum and urine of transitional cell carcinoma patients is extremely high when compared with healthy subjects.

Surface plasmon resonance imaging biosensors for aromatase based on a potent inhibitor and a specific antibody: Sensor development and application for biological material

AbstractAromatase (ARO) is an enzyme with potential diagnostic significance. Aberrant expression of aromatase in tissues is associated with a number of pathological conditions, including tumor of the breast, ovary, testes, liver, adrenal cortex and uterus, as well as endometriosis.Two methods for the highly selective determination of ARO concentration in human tissues by using of two different biosensors co-operating with the surface plasmon resonance imaging technique (SPRI) have been developed. One of the developed biosensors contains immobilised rabbit polyclonal antibody specific for aromatase (Y-ARO), while the other contains immobilized ARO inhibitor-exemestane (E-ARO). Both biosensors specifically bound ARO from analyzed samples. The analytically useful dynamic response range of both biosensors is between 0.3 and 5.0 ng mL−1. The detection limit (3S.D.) of both biosensors is 90 pg mL−1. Standard deviation of both biosensors is 1%. Recoveries of ARO spikes are between 97 and 108% for both biosensors under model conditions and for real samples. Albumin and alkaline phosphatase are tolerated for both biosensors up to 10,000 fold excess.

Podoplanin serum and urine concentration in transitional bladder cancer

BACKGROUND Podoplanin (PDP) is a mucin - a type of transmembrane protein expressed in numerous tissues during ontogeny and in adult animals, including the brain, heart, kidney, osteoblasts and lymphoid organs. OBJECTIVE The aim of this study was to determine podoplanin concentration in the blood serum and urine of patients with bladder cancer. Quantifying podoplanin concentration and its correlation with various clinicopathological parameters may be useful for more accurate predictions and identifying high-risk patients. METHODS The present study included 82 patients with bladder cancer confirmed by transurethral resection or cystectomy and 27 healthy volunteers. The Surface Plasmon Resonance Imaging biosensor was applied for the detection of podoplanin in the serum and urine samples. RESULTS Significant differences in serum and urine podoplanin concentration levels were observed between bladder cancer patients. The statistically significant higher values of PDP were detected in serum of patients with invasive, more aggressive, larger, multifocal tumors. CONCLUSIONS The association between podoplanin concentration and clinicopathological features indicates that it might be useful while making therapeutic decisions.

Concentration of UHCL1 in the Serum of Children with Acute Appendicitis, Before and After Surgery, and Its Correlation with CRP and Prealbumin

ABSTRACT Ubiquitin-mediated protein degradation plays a crucial role in various cellular processes, including signal transduction, cell differentiation, and stress response. Ubiquitin C-terminal hydrolase 1 (UCHL1) is a unique deubiquitinating enzyme that has both hydrolase and ligase activities. The aim of this study was the determination of UCHL1 concentration in serum of children with appendicitis, before and after the surgery. Material and methods: 42 children with acute appendicitis, who were managed at the Pediatric Surgery Department, between 2013 and 2014, were randomly included into the study (age 9 months up to 14 years, mean age 2.5 + 1 years). There were 15 girls and 27 boys. 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Exclusion criteria were: severe preexisting infections, immunological or cardiovascular diseases that required long-term medication, and complicated cases of appendicitis with perforation of appendix and/or peritonitis. Results: The UCHL1 concentrations in the blood plasma of patients with acute appendicitis, were highest before the surgery, and were above the range of concentrations measured in controls, the difference was statistically significant. The UCHL1 concentration measured 24 and 72 h after the operation, slowly decreased over time, and still did not reach the normal range, when compared with the concentration measured in controls (p < 0.05). Conclusions: UCHL1 concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation. The method of operation—classic open appendectomy, or laparoscopic appendectomy, does not influence the general trend in UCHL1 concentration in children with appendicitis. There is strong negative correlation between prealbumin and UCHL1 concentrations.

SPRI biosensors for quantitative determination of matrix metalloproteinase-2

The aim of this study was to develop a new, label-free, highly selective Surface Plasmon Resonance Imaging biosensor for the quantitative determination of matrix metalloproteinase-2. Matrix metalloproteinase-2 specific inhibitor, ARP 101, was used as the receptor, the main part of the biosensor, which bound the enzyme from the sample. The analytical response signal (linear part of the calibration curve) of the developed biosensor was in the range of 1.0–100.0 ng ml−1. Its detection limit was 3.9 ng ml−1, and the limit of quantification was 7.7 ng ml−1. The selectivity, precision and accuracy of the new biosensor were acceptable. The biosensor was used for the quantitative determination of matrix metalloproteinase-2 in the blood plasma of healthy persons and in the plasma of burned children, with good results (good tolerance for potential interferents). Also, to validate the new biosensor, the measurements of plasma matrix metalloproteinase-2 concentration in the biological samples by Enzyme-Linked Immunosorbent Assay were conducted. It was found that the correlation between these two methods was good.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in serum of children after thermal injury

PURPOSE The study aims to determinate concentrations of ubiquitin C-terminal hydrolase 1 (UCHL1), which hydrolyzes amino acids from ubiquitin and cleave di-ubiquitins, in serum of children after thermal injury. PATIENTS/METHODS 42 children scalded by hot water, managed at the Department of Pediatric Surgery, with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2.5±1 years). Blood plasma UCHL1 concentration was assessed in 2-6h, 12-16h, 3d, 5d, and 7d after injury using surface plasmon resonance imaging biosensor. 18 healthy subjects admitted for planned surgeries served as controls. RESULTS The UCHL1 concentration in the blood plasma of patients with thermal injuries reached its peak 12-16h after thermal injury and slowly decreased over time, and still did not reach the normal range on the 7th day after thermal injury. Mean concentrations of UCHL1 after thermal injury were above the range measured in controls (0.12ng/ml): 2-6h after injury - 5.59ng/dl, 12-16h after injury - 9.16ng/dl, 3 days after injury - 6.94ng/dl, 5 days after 5.41ng/dl, 7 days after injury - 4.09ng/dl. CONCLUSIONS We observed sudden increase in the concentration of UCHL1 2-16h after thermal injury with the slow decrease in the UCHL1 concentration over the time. UCHL1 concentration was proportional to the severity of the burn. Further studies are needed to determine the mechanisms by which UCHL1 contributes to metabolic response following thermal injury.

Bladder cancer detection using a peptide substrate of the 20S proteasome.

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2‐aminobenzoic acid (ABZ)‐Val‐Val‐Ser‐Tyr‐Ala‐Met‐Gly‐Tyr(3‐NO2)‐NH2, is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 105 m−1·s−1) and a high kcat (8 s−1). Using this peptide, we identified chymotrypsin‐like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.