Boris B. Dzantiev

Electrochemical immunosensors for determination of the pesticides 2,4-dichlorophenoxyacetic and 2,4,5-tricholorophenoxyacetic acids

Two immunochemical sensors are described for detection of the pesticides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The assay monitors the competitive binding of free pesticide and pesticide-peroxidase conjugate with antibodies immobilized on a graphite electrode, by measurement of peroxidase activity in the immune complexes on the electrode surface. 5-aminosalicyclic acid and H2O2 are used as substrates. An automated potentiometric device measures changes in redox potential during the peroxidase reaction. The assay parameters were optimized to achieve detection limits of 40 ng/ml 2,4-D and 50 ng/ml 2,4,5-T (in both water and serum). The total time required to perform the assay (including electrode regeneration) was 12 min, compared to about 2 h for solid-phase enzyme immunoassays. The electrode life extends to 60 sequential measurements. The sensors therefore appear suitable for medical and ecological applications.

Rapid pretreatment-free immunochromatographic assay of chloramphenicol in milk.

A pretreatment-free immunochromatographic assay for detection of chloramphenicol (CAP) in milk was developed. The assay is based on competition between CAP molecules in the sample and immobilized CAP-protein conjugate for binding to monoclonal anti-CAP antibodies conjugated with colloidal gold particles (average diameter 30nm). The assay is carried out in the course of sample flowing along test strip with immobilized reactants, and its results can be detected by the naked eye or by a photometric device. Effect of the concentration of immunoreactants on assay characteristics was studied. The assay protocol with maximal sensitivity and reliability was optimized using measured values of brightness of lines. Detection limit for CAP is 10ngmL(-1). Assay duration is 10min, and it can be carried out at room temperature without any additional devices and reactants. The developed test strip has been applied to CAP detection in dairy products.

Correlation between the composition of multivalent antibody conjugates with colloidal gold nanoparticles and their affinity.

Interactions between multivalent preparations of antibodies (conjugated with colloidal gold nanoparticles (GNP) as a carrier system) and a multivalent ligand were investigated. The aim of the present study was to reveal the relationship between the affinity of the conjugate and its composition (i.e., the valency). Surface plasmon resonance was applied to study the affinity and the kinetics of the interaction of multivalent conjugates and multivalent virus (on the example of the plum pox virus (PPV)). Three monoclonal antibodies against PPV were prepared. Five GNP preparations with an average particle size in the range from 5 to 60nm (according to electron microscopy measurements) were obtained. The series of preparations allowed us to synthesize GNP-antibody conjugates with different surface areas for immobilization of antibodies, and, consequently, conjugates with different valencies. It was shown that the affinity of the conjugates changes with size of colloidal carriers (i.e. with the valency of the conjugates). The affinity of the virus-antibody interaction (antibodies with affinities of 1.46.10(-8)M and 1.73.10(-8)M) is one to three orders of magnitude lower (depending on the valency of the conjugate) compared to that of the interactions of the virus with GNP conjugates (conjugates with the affinity varying from 1.69.10(-9) to 7.02.10(-12)M and from 2.39.10(-9) to 2.62.10(-11)M, respectively). An increase in the conjugate size leads to an increase in its affinity. The similar trends were observed for the potato virus X.

Immunodetection of herbicide 2,4-dichlorophenoxyacetic acid by field-effect transistor-based biosensors

Abstract An immunosensor method for determination of herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was developed. It is based on the use of the competition between 2,4-D in analyzed probes and 2,4-D-peroxidase conjugate for binding with antibodies being immobilized on porous photo-activated cellulose membranes. These membranes were attached to the gate region of pH-sensitive field transistor (FET) and electrochemical detection of enzyme activity was carried out. Mixture of ascorbic acid (0.1 mM), o-phenylenediamine (1.0 mM), hydrogen peroxide (1.0 mM) was used as substrate solution. The initial rate of pH-shift of the FETs gate region was recorded as measured parameter. The developed system permits to determine 2,4-D in water solutions for concentrations down to 1 ng/ml. This portable and inexpensive immunosensor meets modern requirements as analytical system for ecological monitoring.

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR-protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL(-1)) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16-250 ng mL(-1) its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR-protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).

A new assay format for electrochemical immunosensors: polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by pH-sensitive field-effect transistor.

A new rapid immunotechnique combining separation of reactants by filtration through a porous membrane and potentiometric detection of the bound enzyme label by a pH-sensitive field-effect transistor is proposed. The complexes to be detected are formed by the method described earlier in (Anal. Chem. 71 (1999) 3538), including a homogeneous binding of immunoreactants and a polyanion carrier (polymethacrylate) followed by heterogeneous separation on a membrane incorporating an immobilized polycation (poly-N-vinyl-4-ethylpyridinium). The proposed technique for a sensitive detection of peroxidase label is based on the measurement of pH changes in the optimised substrate solution containing o-phenylenediamine, hydrogen peroxide and ascorbic acid. The antigens studied were herbicide atrazine and hormone testosterone. Their specific detection is realised via competitive binding of free and peroxidase-labelled antigens by antibodies integrating with a (staphylococcal protein A-polyanion) conjugate. The total analysis time is 20-25 min. The range of quantitative detection is 0.2-100 ng ml(-1) for atrazine and 5-300 ng ml(-1) for testosterone. Data scatter of replicate tests varies from 3 to 10%. Application of protein A-polyanion conjugate allows to use the proposed protocol for different antigens without additional treatment of specific antisera.

Development and Comparative Study of Different Immunoenzyme Techniques for Pesticides Detection

Abstract Different ELISA techniques have been developed for the detemination of four widely used pesticides: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), simazine and atrazine. Dependences between the assay scheme and the limiting detectable concentration of the pesticide were studied. The cases of preferential applying of the scheme with immobilized antibodies or one with immobilized pesticide-protein conjugate have been revealed. The following approaches resulting in lowering of ELISA sensitivity were proposed: preliminary incubation of the tested sample with antibodies, immobilization of antibodies via staphylococcal protein A, usage of monovalent fragments of antibodies instead of native ones and chemical modification of the pesticide molecules in the sample. Optimal combinations of these approaches permitted to lower the detection limit of the assays in about 5–30 times. The achieved sensitivities were 3 ng/mL for 2,4-D, 5 ng/mL for 2,4,5-T, 0.05 ng/mL for sima...

Immunochromatographic assay for the detection of ochratoxin A

The detection of mycotoxins—toxic contaminants of fungal origin—is an important problem in the food and feed quality control. An immunochromatographic system was developed for the detection of ochratoxin A (OTA), which is one of the priority contaminants in grain. Monoclonal antibodies against OTA and their conjugates with colloidal gold nanoparticles were prepared. The detection is based on the competition of OTA in a sample and an OTA-protein conjugate immobilized on a test strip for the binding to anti-bodies on the colloidal particle surface. The method was tested in the analysis of plant extracts (maize and barley extracts). It was shown that OTA can be detected in a medium with a high content of an organic solvent (up to 35% of methanol). The disappearance of the line in the test zone is visually detected at OTA concentrations starting from 50 ng/mL. In the case of the video-digital detection of changes in the color intensity of the test zone, the limit of detection of OTA is 5 ng/mL. The duration of the assay is 10 min.

Immunochemical methods of mycotoxin analysis (review)

The review is devoted to comparative characterization of immunochemical methods of detection of mycotoxin, which belongs to one of the priority groups of the food contaminants. It has been shown that the high specificity and the possibility of mycotoxin detection in low concentrations combined with existent diverse equipment allow for considering the immunochemical methods of analysis to be the most promising for wide practical application. The analytical characteristics of the existent developments are presented; the merits and demerits of the different kinds of immunoanalytical systems are compared.

Immunoenzyme assay of nonylphenol: study of selectivity and detection of alkylphenolic non-ionic surfactants in water samples

Immunoenzyme assay (ELISA) is proposed and characterized for determination of alkylphenol ethoxylates, a primary class of manufactured non-ionic surfactants. The assay is based on the obtained polyclonal antibodies against nonylphenol (NP), the main stable intermediate of the decomposition of nonylphenol ethoxylates. A mixture of non-modified branched isomers of NP was applied as hapten coupled to protein carriers by Mannich reaction with the use of formaldehyde. The proposed ELISA format is based on immobilized NP-(soybean trypsin inhibitor) conjugate as a competitor of antigen molecules contained in the tested sample for binding with specific antibodies indirectly labeled via an anti-species immunoperoxidase conjugate. The developed ELISA allows to reveal NP with the limit of detection about 10ngml(-1) and NP-related compounds such as octylphenol, alkylphenoletoxylates, alkylphenolcarboxylates and their halogenated derivatives. The ELISA was applied for assaying polluted water samples, namely influents and effluents from different wastewater treatment plants (WWTP) and tap water. ELISA and chromatographic data demonstrate good correlation (r = 0.94), while ELISA gives higher values. Due to endocrine disrupting and other toxic activities of some metabolites of alkylphenolic non-ionic surfactants, the developed assay may be effectively used in ecological monitoring and sanitary control.