J.V. Samsonova

Competitive ELISA of Chloramphenicol: Influence of Immunoreagent Structure and Application of the Method for the Inspection of Food of Animal Origin

An indirect competitive ELISA for the detection of chloramphenicol (CAP) in food of animal origin (milk, meat, eggs) is described. Influence of immunoreagent structure and composition on the assay sensitivity and specificity was investigated. Two CAP derivatives were used for conjugation with proteins: CAP succinate and a diazo derivative of CAP. Molar incorporation of CAP into the coating conjugates was also varied. To eliminate matrix effect on the assay results, a special casein-containing buffer was used for milk samples, whereas for meat and egg samples a 50-fold dilution of the buffer extracts was needed. The method developed permits CAP concentrations to be determined in the range 0.08100 μg 1−1. The detection limit is 0.08 μg kg−1. Recovery in different food samples averages between 70 and 130%. The method can be applied for inspection of food of animal origin for CAP residues.

Chemiluminescent multiassay of pesticides with horseradish peroxidase as a label

Competitive chemiluminescent immunoassay based on a combination of five antibodies was used in a combination with neural network to identify and estimate amounts of three cross-reacting s-triazines (atrazine, terbythylazine and ametryn). Antibodies with different cross-reactivity towards s-triazines were immobilized in separate wells an eight-well microtiter strip. Training of neural networks was carried out with four different learning procedures. The best topology for the data measured was a net with two hidden layers with ten neurons in the first and 15 in the second layer trained with the Schmidhuber method. s-Triazine classification of environmental samples containing various analyte mixtures was correct in 70-100% of all cases depending on the type of analyte. The test developed can be proposed as an alternative field test for multianalyte environmental monitoring.

A Critical Review of Screening Methods for the Detection of Chloramphenicol, Thiamphenicol, and Florfenicol Residues in Foodstuffs

This article gives an extensive overview of the wide range of analytical procedures developed for the detection of amphenicol antibiotic residues (chloramphenicol, thiamphenicol, and florfenicol) in many different types of foodstuffs (milk, meat, eggs, honey, seafood). Screening methods such as microbial inhibition methods, antibody-based immunoassays using conventional and biosensor-based detection systems, and some methods based on alternative recognition systems are described. The relative advantages and disadvantages of these methods are discussed and compared. The current status and future trends and developments in the need for accurate and rapid detection of this group of antimicrobials are also discussed.

Enzyme-linked immunosorbent assay of chlorampenicol in foodstuff

A test-system based on enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chloramphenicol (CAP) in foodstuff has been developed. The detection limit of the method was 0.05 μg/l. The procedures for milk samples preparation of various fat content and chicken muscles were optimized. Before the analysis milk was diluted 5-fold with a buffer. The detection limit for milk was 0.3 μg/l; recoveries varied from 74 to 118%. Two protocols for chicken muscles preparation were elaborated; extraction with buffer (the express method) and extraction with acetonitrile. The detection limits of CAP in chicken muscles were 0.5 and 0.3 μg/kg, respectively; recovery values were 71–107% and 95–115%, respectively. The results of residual amounts of CAP detection in foodstuff by ELISA and HPLC-MS were in good correlation.

A new dried milk sampling technique and its application for progesterone detection in cows.

A new method for milk sample collection and storage, based on a dried milk sampling technique, is proposed. The method includes application of a whole milk sample to a porous membrane followed by drying. One hundred whole milk samples (dried and liquid) taken on day 21 post insemination were analysed for progesterone by ELISA and results for both dried and liquid samples were well correlated (r=0.911). Milk progesterone ELISA accuracy for pregnancy diagnosis in cows was 87%.

Lateral Flow Immunoassay for Progesterone Detection

A new express method based on lateral flow immunoassay (LFIA) for progesterone detection was developed. To increase the assay sensitivity an enzyme label (horse-radish peroxidase) was used instead of colloidal gold. An optimal assay format was chosen and the influence of a range of buffer supplements (detergents, proteins and sucrose) was investigated by enzyme-linked immunosorbent assay (ELISA). Linear range of LFIA was between 2 and 40 ng/mL in buffer. Limit of detection was 2 ng/mL, assay time was within 15 min.

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone.

The sensitive BRET system for the homogeneous immunoassay of a low‐molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)—the “red” mutant with λmax.em = 590 nm (RedLuc) and the “green” mutant with λmax.em = 550 nm (GreenLuc)—were tested as the donors. The water‐soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF–Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL−1.

Detection of bovine leukemia virus by the polymerase chain reaction in dried blood spots using a membrane system of a new format

Detection of bovine leukemia virus in dried blood spots by the polymerase chain reaction (PCR) was performed using a new format of membrane system for sample collection. The analysis results of liquid and dried samples of bovine whole blood were entirely consistent with each other and with the data of a veterinary laboratory. It was found that any part of a membrane strip with size of 0.5 × 0.5 cm containing an applied sample can be used for detection of a viral DNA copy. Provirus DNA in dried blood spots was stable for 8 days at 37°C and for 24 hours at 60°C. The new system for the sampling of whole blood in the form of dried spots can be used for sampling in field conditions for subsequent laboratory analysis.

Comparison of PCR and ELISA methods for the detection of bovine leucosis in dried blood spots

PCR and ELISA methods for the detection of bovine leucosis in dried blood spots on porous membranes were compared. Dry samples were analyzed through real-time PCR, using several diagnostic test systems. Nineteen and 20 samples were identified as positive by PCR and ELISA, respectively. Fourteen of these samples were identified as positive by both methods. When using PCR and ELISA, 26 samples were identified as positive for leucosis, which amounted to 47% of the total number of tested samples. The results of the analysis of dried and native samples were in good agreement. The obtained results showed that whole blood sampling in the form of dried spots applied on membrane can be used as a convenient and reliable way to obtain dry samples of biological fluids with the purpose of screening herds for infectious diseases, in particular for bovine leucosis.

Enzyme-linked immunosorbent assay for bisphenol A: Assay optimization and its application for surface water analysis

A range of ELISAs in indirect and direct formats for the determination of bisphenol A (BPA) was developed. Bisphenol A carboxymethyl ether (BPA-CME), (BPA-CPE) and 4,4-bis(4-hydroxyphenyl)valeric acid were coupled with bovine serum albumin (BSA) (immunogens for the production of polyclonal antibodies), ovalbumin (OVA) and horseradish peroxidase. In general, the indirect assay was more sensitive and specific than the direct one. Using heterologous combinations of immunoreagents in the indirect assay allowed to increase assay sensitivity and specificity. The highest sensitivity was obtained for the antibodies produced against a conjugate of BPA-CPE with BSA and a conjugate of 4,4-bis(4-hydroxyphenyl)valeric acid with OVA. The detection limit of BPA in phosphate buffer was 0.03 ng mL−1. The assay developed was also the most specific towards BPA. Maximum cross-reactivity values did not exceed 11% for 4-cumylphenol, 5% for bisphenol E and 2% for bisphenol S. Finally, the developed assay was used to analyze surface water samples spiked with known amount of BPA. The assay showed good recovery values (85–109%) for surface water with mineralization level lower than 500 mg L−1.