A biosensor for determination of the circulating biomarker CA125/MUC16 by Surface Plasmon Resonance Imaging.

CA125/MUC16 is an ovarian tumor cell marker widely used as a biomarker in epithelial ovarian carcinoma. CA125/MUC16 is also used for evaluation of the ROMA (Risk of Ovarian Malignancy Algorithm) value. In this work, a Surface Plasmon Resonance Imaging (SPRI) biosensor for circulating CA125/MUC16 has been developed. The anti-MUC16 antibody was attached to a gold chip via a cysteamine linker. The EDS/NHS protocol was used for the covalent attachment of the antibody. The developed biosensor is specific for CA125/MUC16, and exhibits good recovery and acceptable precision. Its linear response range (2.2-150 U/ml) is well suited to determination of the marker in the blood serum of a healthy control group and, after appropriate dilution, of patients with ovarian cancer. CA125/MUC16 was determined in two series of real samples: blood serum from patients with ovarian cancer and endometrial cysts. The method was validated by parallel determination of the samples using the chemiluminescent Architect i2000 method.

20S proteasome in the blood plasma of boys with cryptorchidism.

PURPOSE: To evaluate the concentration of 20S proteasome in the blood plasma of boys with cryptorchidism. METHODS: Patients-50 boys aged 1-4 years (median = 2.4 years) with unilateral cryptorchidism. The control group-50 healthy, age-matched boys (aged 1-4 years, median = 2.1 years), admitted for planned herniotomy. In our study, we used a novel technique Surface PLASMON RESONANCE Imaging. RESULTS: The median concentration of 20S proteasome in the blood plasma of boys with cryptorchidism was 2.5-fold higher than in boys with inguinal hernia. We noticed statistically significant difference between 20S proteasome levels in boys with cryptorchidism up to 2 years old and above 2 years old. CONCLUSIONS: We believe that the 20S proteasome concentrations in the blood plasma of boys with cryptorchidism reflect the heat-induced apoptosis of germ cells.

Immunoproteasome in the blood plasma of children with acute appendicitis, and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors.

The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). Twenty-seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P < 0·05). There was no statistical difference between immunoproteasome, 20S proteasome and UCHL1 concentrations in children operated on laparoscopically and children after classic appendectomy. The immunoproteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation may thus be helpful in the assessment of the efficacy of treatment. The method of operation - classic open appendectomy or laparoscopic appendectomy - does not influence the general trend in immunoproteasome concentration in children with appendicitis.

Determination of collagen type IV by Surface Plasmon Resonance Imaging using a specific biosensor.

Serum collagen type IV (COLIV) is a promising tumor marker. High COLIV concentrations have been found in the serum of patients with colorectal, gastric, lung, liver and breast cancers. The aim of this work was to develop a biosensor for use with the Surface Plasmon Resonance Imaging (SPRI) technique for COLIV determination. The biosensor consists of glass covered with gold and immobilized monoclonal mouse anti-human collagen type IV antibody via cysteamine linker. The biosensor works selectively within a dynamic response range between 10 and 300 ng mL-1, with LOD 2.4 ng mL-1 and LOQ 8 ng mL-1. The precision of determination is 4.7% at a 150 ng mL-1 COLIV spike and 8.0% at a 20 ng mL-1 spike, with recoveries of 101% and 106% respectively. A 100-fold excess of collagen I, albumin, laminin and fibronectin is tolerated. The average COLIV blood plasma concentration of healthy donors determined by the developed method was 69 ± 10 ng mL-1, while the median of six results available in the literature was approximately 80 ng mL-1. The average COLIV blood plasma concentration of breast cancer patients was 360 ± 68 ng mL-1, showing the high potential of COLIV as a marker of this type of cancer.

SPR imaging biosensor for determination of laminin-5 as a potential cancer marker in biological material.

A new method for the selective determination of laminin-5 concentration using a biosensor and surface plasmon resonance imaging (SPRI) technique is presented. A biosensor based on the specific interaction of laminin-5 with rabbit polyclonal antibody was constructed. The analytically useful dynamic response range of the biosensor is between 0.014 and 0.1 ng mL(-1). The detection limit is 4 pg mL(-1). The potential influence of interferences on the SPRI signal was investigated, and the high selectivity of the biosensor was confirmed. In order to demonstrate the potential application of the biosensor, laminin-5 concentration in blood plasma was determined. The results were compared with the laminin-5 concentration obtained by the commercial enzyme-linked immunosorbent assay (ELISA) kit. A comparison of results from healthy donors obtained by SPRI measurement and ELISA indicates that they are close and shows good agreement with the data reported in the literature. The plasma samples of bladder cancer patients gave higher concentration measured with specific biosensor than by ELISA assay. The study shows the clear difference in concentration of laminin-5 in healthy humans and patients with bladder cancer. Extensive clinical studies using the newly developed method can result in an increase in the use of laminin-5 as a potential cancer marker.

Surface Plasmon Resonance Imaging (SPRI) sensor for cystatin determination based on immobilized papain.

A Surface Plasmon Resonance Imaging (SPRI) sensor has been developed for specific determination of cystatin. The sensor contains immobilised papain, which binds cystatin from solution. Papain activated with N-Hydroxysuccinimide (NHS) and N-Ethyl-N'-(3-dimethyl aminopropyl)carbodiimide (EDC) was immobilized on an amine-modified gold surface. Cysteamine was used for modification of the gold surface. Papain concentration and the pH of interaction were optimised. A concentration of papain of 1.5 µg mL(-1) and a pH of 6.5 were selected as optimal. The specificity of interaction was verified by the lack of interaction with human albumin. The sensor's dynamic response range is between 0 and 0.6 mg µL(-1), and the detection limit is 0.09 µg mL(-1). The results were validated by comparison with the PETIA (particle enhanced immunoturbidimetric assay) method showing good agreement. A calibration curve of chicken egg white cystatin or Cystatin C was used. In order to demonstrate the sensor's potential, cystatin C was determined in blood plasma, urine and saliva, showing good agreement with data reported in the literature. The results for cystatin concentration in the blood plasma of people suffering from leukaemia were found to be below the normal level of cystatin.

Surface Plasmon Resonance Imaging sensor for cathepsin determination based on immobilized cystatin.

A specific SPRI sensor for cathepsin determination based on the interaction between immobilized cystatin and cathepsins has been developed. All cathepsins form the same calibration curve. The sensor dynamic response range is between 0.5 and 2.0 ng ml(-1) and the detection limit is equal to 0.1 ng ml(-1).

In Vitro Interaction of Lithium on Phospholipids in Human Erythrocytes.

Lithium salts are used in the treatment of mania and as prophylaxis against manic depressive disorder. The aim of these studies was the in vitro investigation of the effect of lithium on phospholipids of human erythrocyte membranes. Erythrocytes were treated with lithium for 1 h. Phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylocholine (PC) were separated from erythrocyte ghosts and determined by HPLC. Blood samples from healthy adults were investigated. A very strong decrease in PC content in erythrocyte membranes due to lithium in vitro treatment was found, as well as a statistically significant increase in PI content.

The Surface Plasmon Resonance Imaging sensor for papain based on immobilized cystatin.

The Surface Plasmon Resonance Imaging (SPRI) sensor for papain determination based on the interaction between the immobilized cystatin and aqueous papain solution has been developed. The development of the sensor is a stage in the development of the sensor for the determination of cystein proteinases of the papain group. Cystatin was immobilized onto a gold chip by means of a thiol underlayer (cysteamine) and EDS/NHS reaction. Conditions of cystatin- papain interaction were optimized (pH, time of interaction and cystatin concentration). The developed sensor works within the range of 1 - 10 ng ml(-1). However, the precision of measurements (RSD equal to 45%) should be improved. The response of the sensor to papain is specific. This was checked using BSA as a reference.

The mechanism of daunorubicin-induced inhibition of prolidase activity in human skin fibroblasts and its implication to impaired collagen biosynthesis.

One of the recognized side effects accompanying antineoplastic anthracyclines administration is poor wound healing, resulting from impairement of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [E.C. 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. We have found that daunorubicin (DNR) induced coordinately inhibition of prolidase activity (IC50 = 0.3 microM) and collagen biosynthesis (IC50 = 1 microM) in cultured human skin fibroblasts. The decrease in prolidase activity due to the treatment of confluent cells with DNR was not accompanied by any differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. Since prolidase is metaloprotease, requiring manganese for catalytic activity and anthracyclines are known as a chelators of divalent cations we considered that the chelating ability of anthracyclines may be an underlying mechanism for daunorubicin-induced inhibition of prolidase activity. In order to determine the ability of DNR to form complex with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that DNR forms stable complex with manganese (II) and the composition of the complex of DNR with Mn (II) was calculated as 3:1. The constant stability value for the investigated complex was calculated as [beta(av) = (1.74 +/- 0.01) 10(23). The strong ability of DNR to chelate manganese may explain the potential mechanism for inhibition of prolidase activity, subsequently collagen biosynthesis and poor wound healing in patients administered DNR.