High-sensitivity express immunochromatographic method for detection of plant infection by tobacco mosaic virus.

A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object - tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2-4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1-0.2 ml tested solution (extract from 10-20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.

[Development of immunochromatographic test systems for express detection of plant viruses].

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rod-shaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 microg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.

A new method for differential determination of basic natural forms of indolyl-3-acetic acid.

A new ELISA method is proposed for differential quantitative determination of free (indolyl-3-acetic acid; IAA) and bound (indolyl-3-acetyl-L-aspartate) forms of natural auxins. There is similarity in results obtained by this and some traditionally used methods. The standard error of determination of the active form of IAA by our method is 1.5-2.0 times less than that using the traditional method. The method of quantitative differential determination of the main natural auxins does not require preliminary sample preparation, and this shortens assay time. The developed method has been used for practical determination of different forms of endogenous IAA in wheat and dandelion ovaries subjected to minimal treatment. This method can be used to investigate changes in the ratio of various hormonal forms of auxins that differ in their physiological activity in reproductive organs of angiosperms at various stages of reproduction.

Immunochemical approach to the problem of differential determination of natural forms of abscisic acid.

An original modification of the standard ELISA procedure for differential determination of different forms of abscisic acid (ABA) is proposed. It is shown that endogenous forms of ABA may be quantitatively determined in plant tissues subjected to minimal treatment, without purification of the hormones and their chemical modification. The modification has been approved when analyzing changes in the content of different ABA forms in plant tissues differing in physiological activity. Quantitative differential determination of changes in the content of different ABA forms has been performed in ovaries of Triticum aestivum L. and Taraxacum officinale Web. in the period of activity of the ovule (from the moment of its activation to the beginning of division). It is shown that, despite the different types of reproduction in the species studied (amphimixis and apomixis), the time course of changes in the content of different forms of ABA in ovaries is similar, which is suggestive of a correlation between the activity of endogenous hormonal system and chronology of main events (e.g., the beginning of endospermogenesis) of the reproductive cycle.

[A new micromethod for differential quantitative assay of zeatin and zeatin riboside]. / Novyi mikrometod differentsial'nogo kolichestvennogo opredeleniia zeatina i zeatinribozida.

A new method is proposed for differential quantitative assay of two major endogenous cytokinin forms. It is based on determination of two effective parameters-concentrations of zeatin and zeatin riboside--with the use of appropriate antigens as standards. The method can be used for determining cytokinins in small samples of plant tissues without extract fractionation. This study pioneers in quantitation of changes in the hormonal status of ovules and ovaries of Triticum aestivum L. at early stages of embryogeny. A gradual increase in the content of the active and storage forms of the hormones from the ovary to the ovule was revealed.

Changes in cytokinin distribution in the ovule and ovary of Taraxacum officinale Web. in early stages of embryogenesis.

Quantitative changes in the hormonal status of the ovules and ovaries were first studied in Taraxacum officinale Web. at the early stages of embryogenesis. The plant material was analyzed by ELISA using labeled anti-rabbit antibodies. A new procedure for differential and quantitative determination of the main endogenous cytokinins based on the estimation of the effective zeatin and zeatin riboside concentrations from calibration curves constructed using zeatin and zeatin riboside as standard antigens was developed. It was shown that, at the three initial stages of embryogenesis examined, the concentration of zeatin uniformly increased in T. officinale ovules. The concentration of zeatin riboside, conversely, uniformly decreased. However, their total concentration changed insignificantly. A gradual increase in the concentration of the active and storage hormone forms from the ovary to the ovule was shown.

[On differential determination of basic natural forms of cytokinins by and immunoenzyme method]. / O differentsial'nom opredelenii osnovnykh prirodnykh form tsitokininov immunofermentnym metodom.

An immunochemical method for quantitative differential analysis of major types of natural cytokinins in plant tissues subjected to minimal treatment, without purification or chemical modification of hormones, is proposed. This method is recommended for use in biology, medicine, and agriculture for determination of low-molecular-weight compounds having similar chemical structures but various biological activities.

Changes in the hormonal status of the Taraxacum officinale Web. ovary at early stages of embryogenesis.

The hormonal status of the Taraxacum officinale Web. ovary was quantitatively assayed for the first time during early stages of embryogenesis. Apparent concentrations of endogenous cytokinins were measured using two systems of enzyme-linked immunosorbent assay (ELISA). The ELISA systems differed from one another by the specificity for the main endogenous forms of zeatin. The specificity of two heterological ELISA systems based on zeatin- and kinetin-specific antisera was studied. A new immunochemical approach to the problem of differential quantitative determination of natural zeatin forms is suggested. This approach does not require preliminary separation of experimental samples into individual fractions. True concentrations of zeatin and zeatin riboside in the T. officinale ovary were calculated based on the average values of apparent concentrations of endogenous cytokinins. When the embryo sac maturation had been completed, there was a threefold increase in the zeatin riboside concentration within the following 12 h. By the time of the first division of an unfertilized ovicell (i.e., within the next 12 h), there had been a twofold decrease in the zeatin riboside concentration. Therefore, at early stages of division of the unfertilized ovicell the zeatin riboside concentration virtually returned to the initial level. In contrast to zeatin riboside, there was a steady trend toward an increase in the zeatin concentration in the T. officinale ovary. Within the first 12 h and the next 12 h after completion of the embryo sac maturation, the zeatin concentration was increased 1.5-fold and 2-fold, respectively. The results of this work provide a pioneering insight into the dynamics of various natural forms of zeatin during the reproductive process. The immunochemical approach to quantitative monitoring of various natural forms of zeatin and their dynamics during embryogenesis suggested in this work can be extended to similar biological, medical, and agricultural problems of differential determination of low-molecular-weight agents of similar structure but different biological activity.

[Solid-phase immunoenzyme analysis of chloramphenicol in human blood serum]. / Tverdofaznyi immunofermentnyi analiz khloramfenikola v syvorotke krovi cheloveka.

The method of enzyme linked immunosorbent assay (ELISA) for quantitative detection of chloramphenicol (CAP) in human blood serum was developed. Peculiarities of the adsorption on the microtitre plates surface of CAP-ovalbumin conjugate were investigated. Different conditions of competition stage of the analysis were studied. Conditions providing CAP monitoring in human blood serum in the clinical range were optimized. Matrix effect on the assay results was studied. The specificity of the analytical system was investigated and the reagents stability was examined. The method developed permits CAP concentration to be determined in human blood serum, diluted 1/100, in the linear range from 10 to 1000 ng/ml. The assay is characterized by high sensitivity (1 ng/mL) and good reproducibility (CV < 12%), assay time is about 3 hours.

[Development of a solid-phase immunoenzyme analysis of gentamicin in human blood serum]. / Razrabotka tverdofaznogo immunofermentnogo analiza gentamitsina v syvorotke krovi cheloveka.

An enzyme immune test system was designed and optimized for quantitative assay of gentamicin in human sera. Immunospecific reagents i.e. gentamicin conjugates with ovalbumin (for sorption on polysterol plates) and with bovine serum albumin (immunogen) were prepared. Gentamicin specific antisera were isolated and tested. Conditions for the antigen sorption on polysterol plates were determined and optimized. Different regimes of the competition reaction were investigated and conditions for the antibiotic assay in human sera were determined. The assay specificity was studied and the stability of the test system was checked. An experimental lot of the reagent set was manufactured at the ZAO NPP Immunotech and the correlation tests with the use of the fluorescence polarization immunoassay were performed. The set is destined for the assay of 40 samples (in duplicate). The method sensitivity is 1 ng/ml of gentamicin. The range of the detectable concentration is 1 to 32 ng/ml of gentamicin in 1000-fold diluted sera. The assay time is not more than 3 hours. The variation coefficient of the results does not exceed 12 per cent. The shelf-life of the set is 0.5 years when stored at a temperature of 2 to 8 degrees C.