xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2-to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3−200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.

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By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high-affinity cell surface GMDPbiding sites (& 350 PM). PhotoaBinity labeling followed by SDS-PAGE enabled us to identify 32-34 and 38 kDa proteins inside these cells that bound GMDP specifically.

We performed a three-locus phylogenetic analysis of Fusarium strains presumably capable of trichothecene production, which were deposited in the Russian national collections. The intra- and interspecific polymorphism of partial sequences of the translation elongation factor 1 alpha (TEF1α) gene and two genes from the trichothecene cluster TRI5 and TRI14 was studied. A study of 60 strains of different origins using DNA markers confirmed, and in the case for several strains, clarified their taxonomic characteristics. As a result, a strain of F. commune (F-900) was identified in Russia for the first time. Furthermore, the strain F-846 proved to be phylogenetically distinct from any of the known Fusarium species. F. equiseti strains from Northwest Russia were found to belong to the North European group (I), whereas a strain from the North Caucasus – to the South European one (II). Partial TRI14 sequences from 9 out of 12 species were determined for the first time. Their comparative analysis demonstrated a relatively high level of intraspecific variability in F. graminearum and F. sporotrichioides, but no correlation between the sequence polymorphism and the geographic origin of the strains or their chemotype was found. Specific chemotypes of trichothecene B producers were characterized using two primer sets. The chemotyping results were verified by HPLC.

A b s t r a c tAscomycetous fungi of the genus Fusarium, which was separated as a single taxonomic group in 1809 by Heinrich Friedrich Link, are distributed worldwide and have different ecological niches. They have been found in soil and plants as endophytes, saprotrophs and parasites. Infections of agricultural plants, caused by Fusarium fungi, lead to annual damage, estimated in hundred million dollars. In addition to decrease harvest quality and quantity, Fusarium fungi are able to produce toxic metabolites (fusariotoxins). The consumption of fusariotoxin-contaminated winter grain led to deaths of tens of thousands of people in Volga and Ural regions in 1930s-1940s. These factors make necessary investigation of morphology, biochemistry and genetics of those organisms. Nowadays there is no universal taxonomic system of the genus Fusarium, so it is not possible to identify an isolate to species level in many cases. The high variability of morphological structures, and, on the other side, their similarity for closely related species, are major problems for researchers which use classical methods for identification and systematization of this group of fungi. Today, the molecular methods, based on the use of specific DNA sequence, are playing an increasingly important role in Fusarium systematics. The application of this approach has led to establishment of a number of new species in the genus. T. Yli-Mattila et al. (2009, 2011 used multilocus phylogenetic analysis to describe the novel species F. sibiricum and F. ussurianum which were found in Siberia and Far East and proved to be morphologically similar to F. graminearum and F. langsethiae, respectively. The authors of this paper identified the species F. torulosum (morphologically similar to F. avenaceum) in Russia for the first time based on the use of DNA markers. It is no less important that the analysis of interand intraspecific polymorphism makes possible the development of highly-specific assays for molecular detection of Fusarium fungi, including ones which infect plants and produce mycotoxins. The use of these assays (for example, ones described by M. Nicolaisen et al., 2009 and A.A. Stakheev et al., 2011) allows not only to detect the contamination of plants by mycotoxin producers, but also to estimate the quantity of their content in a probe. At the same time, a lot of questions are still unanswered: particularly, simultaneous application of different species concepts (morphological, biological and phylogenetic) makes it difficult to develop universal taxonomic system of the genus; also the most informative and reliable marker, which would reflect the evolutionary history of the genus, has not been found. The main achievements and problems of Fusarium phylogenetics, and perspectives of the application of molecular approach for studies of this group of organisms are discussed in this review.

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

Detection of staphylococcal toxins presents a great interest for medical diagnostics. Screening of clinical samples for the presence of several types of staphylococcal toxins using traditional methods-biological tests on animals or cell cultures as well as ELISA-is laborious. Multiplex detection methods would simplify testing. We have designed an xMAP-based assay to detect three staphylococcal toxins-enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin (TSST)-in cultural supernatants obtained from different strains of Staphylococcus aureus. The limits of detection of SEA, SEB, and TSST multiplex detection in S. aureus growth medium were 10, 1,000, and 5 pg/mL, respectively. Fifty-nine samples of S. aureus cultural supernatants were tested with the xMAP assay. The developed assay has proved highly effective detection of the natural toxins in the samples obtained due to bacterial cells cultivation. In prospect, the developed test system can be used in clinical diagnostics and in monitoring of foodstuffs and environmental objects.

Hybrid hybridomas (tetradomas), producing bispecific monoclonal antibodies (bmabs) binding to both interleukin 2 and horseradish peroxidase were obtained by fusing IL 2-specific and HRP-specific hybridomas. Parental hybridomas were labelled prior to the fusion with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate. Bifluorescent (tetradoma) cells were sorted out using fluorescence activated cell sorter. Two clones, designated D8C1/G and H7C11/H were shown to secrete bmabs over at least 6 months growth in culture. Bmabs have been purified from mouse ascites by ammonium sulfate precipitation and affinity chromatography on immobilized peptide, modelling corresponding IL 2 epitope. The purity of antibodies obtained was characterized by capillary electrophoresis. The possibility of using these antibody preparations in rIL 2 analysis was evaluated in two types of EIA: direct and competitive solid-phase EIA. Both assays had similar sensitivity of about 1.5-4 ng IL 2 per ml.