Бровко Федор Александрович

Избранные публикации

  1. Shlyapnikov Y.M., Shlyapnikova E.A., Simonova M.A., Shepelyakovskaya A.O., Brovko F.A., Komaleva R.L., Grishin E.V., Morozov V.N. (2012). Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins. Anal. Chem. 84 (13), 5596–603 [+]

    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.

  2. Shepelyakovskaya A.O., Laman A.G., Lomonosova A.V., Fursova K.K., Savinov G.V., Vertiev Y.V., Brovko F.A., Grishin E.V. (2011). Effect of the format of antibodies on their specificity. Mol. Immunol. 49 (3), 433–40 [+]

    The influence of alterations in the format of antibodies on their specificity has been examined. To analyze the role of Ig constant regions in recognizing antigens, a comparison was made of the specificities of full-scale murine monoclonal antibodies and scFv single-chain miniantibodies obtained from the latter with regard to a group of closely related protein antigens - Staphylococcus enterotoxins. It was found that in the scFv format the specificity and affinity of miniantibodies diminished as compared to the full-scale ones. Specificity of antibodies may be enhanced by transforming them into full-scale antibodies. Moreover it was shown that miniantibodies within a phage particle generated from combinatorial phage libraries possess greater specificity to the antigen, however during the subsequent transformation to soluble scFv antibodies their specificity diminishes.

  3. Rubina A.Y., Filippova M.A., Feizkhanova G.U., Shepeliakovskaya A.O., Sidina E.I., Boziev K.M., Laman A.G., Brovko F.A., Vertiev Y.V., Zasedatelev A.S., Grishin E.V. (2010). Simultaneous Detection of Seven Staphylococcal Enterotoxins: Development of Hydrogel Biochips for Analytical and Practical Application. Anal. Chem. , [+]

    A method of simultaneous analysis of staphylococcal enterotoxins using hydrogel-based microarrays (biochips) has been developed. The method allows simultaneous quantitative detection of seven enterotoxins: A, B, C1, D, E, G, and I in a single sample. The development of the method included expression and purification of recombinant toxins, production of panels of monoclonal antibodies (mAbs) against the toxins, and design and manufacturing of an experimental biochip for the screening of mAbs and selection of optimal pairs of primary and secondary antibodies for each toxin. The selected mAbs have high affinity toward their targets and no cross-reactivity with unrelated enterotoxins. Finally, a diagnostic biochip was designed for quantitative analysis of the toxins, and the analytical protocols were optimized. The sensitivity of the detection reached 0.1-0.5 ng/mL, depending on the type of enterotoxin. The evaluation of the resulting biochip using spiked food samples demonstrated that the sensitivity, specificity, and reproducibility of the proposed test system fully satisfy the requirements for traditional immunoanalytical systems. The diagnostic biochips manufactured on reflecting metal-coated surfaces shortened the time of analysis from 17 to 2 h without loss of sensitivity. The method was successfully tested on samples of food and biological media.