Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear-flow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this "active" assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1-1 pg/mL for water and to 1 pg/mL for food samples.

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.

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide‐binding sites were shown to be located inside T‐lymphocytes, macrophages and neuroblastoma cells, but not inside B‐cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1‐3 cells with K fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D‐configuration of isoglutamine residue are important.

Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the virulence of a strain for the purpose of diphtheria agent monitoring. To determine the LTP of diphtheria agents, an immunoassay based on monoclonal antibodies (mAbs) has been developed. A pair of mAbs specific to the fragment B of DT was selected, which makes it possible to detect DT in a sandwich ELISA with a detection limit of DT less than 1 ng/mL. Sandwich ELISA was used to analyze 218 liquid culture supernatants of high-, low- and non-toxigenic strains of various corynebacteria. It was shown that the results of ELISA are in good agreement with the results of PCR and the Elek test for the tox gene and DT detection, respectively. The diagnostic sensitivity of the assay was approximately 99%, and specificity was 100%. It has been found that strains of C. ulcerans, on average, produce 10 times less DT than C. diphtheriae. The mAbs used in the ELISA proved to be quite discriminatory and could be further used for the design of the LFIA, a method that can reduce the labor and cost of laboratory diagnosis of diphtheria.

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