Immunodetection of Streptococcus uberis pathogen in raw milk.

Streptococcus uberis is a major mastitis-causing environmental pathogen, which rapid immunodetection has not been possible due to the absence of specific anti-Str. uberis antibodies. Recently, a specific antibody against the Str. uberis adhesion molecule (SUAM) has been designed. In the present study, the specificity and affinity of this antibody towards SUAM antigenic region SAPVYLGVSTE and Str. uberis cells are characterized, using experimental and in silico bioinformatic methods. The selectivity studies and bioinformatic analyses revealed high specificity of the antibody towards Str. uberis. The Kd value of SAPVYLGVSTE/anti-Str. uberis antibody complex was 27 ±â€¯6 nM, indicating the applicability of this antibody for the detection of Str. uberis. The anti-Str. uberis antibody was used as a specific biorecognition element of a biosensor for the detection of Str. uberis bacteria in phosphate buffer and in milk and these analyses took less than 20 min. The Str. uberis biosensor was also tested in the milk of cows suffering from mastitis and the obtained results were in good agreement with the conventional identification of this pathogen by microbiological plating.

Design and production of antibodies for the detection of Streptococcus uberis.

Streptococcus uberis (S. uberis) is an important environmental pathogen causing mastitis in dairy cattle. Mastitis or mammary gland infection is the most common disease in milking cows and cause significant economic burden to farmers due to the reduction of the amount of milk and its quality. The development of rapid analytical methods for the determination of mastitis-causing pathogens in milk is of utmost importance for the early identification of the causes of mastitis and the beginning of timely treatment of cows. Combining in silico bioinformatic analysis and solid phase peptide synthesis using Fmoc chemistry, we have made two different peptides to mimic the adhesion protein of S. uberis, which is promoting the attachment of bacteria to epithelial cells. After purification with RP-HPLC, the peptides were conjugated with a larger carrier protein (KLH) and used for immunization of rabbits to produce specific antibodies. The separation of anti-S. uberis antibodies from rabbit blood antisera was carried out with affinity chromatography, using the synthetic peptides as affinity ligands. The purified antibodies showed high affinity and specificity towards S. uberis and were used for rapid bio-recognition and identification of S. uberis with an immunobiosensing system.

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme.

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40±15) mM in freshly prepared solution and (0.28±0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43±0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4 °C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.

Analyzing the biosensor signal in flows: studies with glucose optrodes.

Responses of enzymatic bio-optrodes in flow regime were studied and an original model was proposed with the aim of establishing a reliable method for a quick determination of biosensor signal parameters, applicable for biosensor calibration. A dual-optrode glucose biosensor, comprising of a glucose bio-optrode and a reference oxygen optrode, both placed into identical flow channels, was developed and used as a model system. The signal parameters of this biosensor at different substrate concentrations were not dependent on the speed of the probe flow and could be determined from the initial part of the biosensor transient phase signal, providing a valuable tool for rapid analysis. In addition, the model helped to design the biosensor system with reduced impact of enzyme inactivation to the system stability (20% decrease of the enzyme activity lead to only a 1% decrease of the slope of the calibration curve) and hence significantly prolong the effective lifetime of bio-optrodes.