Ekaterina Igorevna Dementieva

Gel-based microarrays in clinical diagnostics in Russia

Immobilization of molecular probes in 3D hydrogel elements provides some essential advantages compared with conventional flat surfaces. In this article, an integrated technology based on the use of low-density microarrays comprised of hemispherical gel elements, developed at the Engelhardt Institute of Molecular Biology (Moscow, Russia) for various applications will be reviewed. The structure of the gel can be adapted for immobilization of virtually any biological molecules in a natural hydrophilic environment. The discrimination between matching and mismatching duplexes of nucleic acids in these conditions is more reliable than on conventional flat surfaces, minimizing the number of elements needed to detect specific sequences. Protein molecules immobilized in hydrogel-based biochips better preserve their biological properties. As described in this article, such biochips were successfully applied for laboratory diagnostics in a wide variety of clinical conditions involving the identification of bacterial and viral pathogens, cancer-related mutations and protein tumor markers.

Development of hydrogel biochip for in vitro allergy diagnostics

A hydrogel biochip was developed for the simultaneous quantitative determination of sIgE for 21 allergens and total IgE in human serum. The biochips are manufactured by photoinduced copolymerization of different molecules (allergens and antibodies) with gel-forming monomers resulting in the formation of three-dimensional hydrogel elements (1nl gel drops). After incubation of the biochip with the serum, the results are visualized using fluorescently labeled anti-IgE antibodies. Using biochips, serum samples from allergic patients and healthy donors were analyzed and good correlation with the results obtained using commercial EIA test systems of generally recognized quality (Dr. Fooke Laboratorien GmbH, Germany) was observed.

Development of a biochip with an internal calibration curve for quantitating two forms of the prostate-specific antigen

Three-dimensional gel-based biological microchips were developed for simultaneous quantitation of total (PSAtot) and free (PSAfree) forms of the prostate-specific antigen in human serum in the “one patient, one biochip” format. A method not demanding construction of calibration curves prior to the assay was applied to quantitation of PSAtot and PSAfree. In addition to gel elements with immobilized antibodies against PSAtot and PSAfree, the biochip contains elements with immobilized PSA at different concentrations, forming an internal calibration curve. Data are processed and interpreted with the special-purpose ImaGelAssay program. The sensitivity of the assay is 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. The variation coefficient for measurements with one biochip series does not exceed 10%. The correlation coefficients between the estimates obtained for human sera by the biochip assay and by conventional ELISA were 0.988 for PSAtot and 0.987 for PSAfree.

Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

Mass spectrometry-based analysis techniques are widely applied in proteomics. This study presents a novel method for quantitative multiplex candidate protein profiling. It applies immunocapture of differentially labeled protein complements on hydrogel antibody arrays and subsequent quantification by MS. To make this approach quantitative a labeling approach was devised. The impact of labeling on the antibody/antigen interaction was assessed in detail by surface plasmon resonance. Owing to there solution by mass more than two protein samples can be compared simultaneously. Direct labeling of crude samples such as sera was developed and so enables the absolute quantification of target proteins straight from crude samples without a protein purification step. It was used to measure the concentration of apolipoprotein A-1 in serum. This method has been termed A2M2S for Affinity Array sand MALDI Mass Spectrometry.

Protein Microchips in Quantitative Assays for Tumor Markers

Diagnosing malignant tumors is a major problem in oncology. The use of microchips in clinical testing makes it possible to detect several tumor markers in parallel without consuming large volumes of samples and expensive reagents. The goal of this study was to develop a quantitative immunoassay for some markers of commonly occurring tumors using three-dimensional hydrogel-based protein microchips designed at the Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences. Microchips with immobilized antibodies were effective in detecting a number of tumor markers at clinically relevant concentrations in patient and donor sera.

Drug Resistance Mechanisms in Bacteria Causing Sexually Transmitted Diseases and Associated with Vaginosis

Here, we review sexually transmitted diseases (STDs) caused by pathogenic bacteria and vaginal infections which result from an overgrowth of opportunistic bacterial microflora. First, we describe the STDs, the corresponding pathogens and the antimicrobials used for their treatment. In addition to the well-known diseases caused by single pathogens (i.e., syphilis, gonococcal infections, and chlamydiosis), we consider polymicrobial reproductive tract infections (especially those that are difficult to effectively clinically manage). Then, we summarize the biochemical mechanisms that lead to antimicrobial resistance and the most recent data on the emergence of drug resistance in STD pathogens and bacteria associated with vaginosis. A large amount of research performed in the last 10–15 years has shed light on the enormous diversity of mechanisms of resistance developed by bacteria. A detailed understanding of the mechanisms of antimicrobials action and the emergence of resistance is necessary to modify existing drugs and to develop new ones directed against new targets.

Protein microchips: analysis of the expression of the recombinant barstar.

Protein microchips are becoming an effective tool for basic research and practical applications. However, widespread use of protein microchips is limited by sophisticated technology of their production, which is due to the necessity of maintaining the biological function of proteins during microchip manufacturing. The manufacturing of three-dimensional hydrogel protein microchips on the basis of polymerization immobilization technology developed in the Institute of Molecular Biology, Russian Academy of Sciences, is described and the proteins are shown to maintain their biochemical properties. The hydrogel protein microchips were used for the first time for quantitative assay of the expression of recombinant proteins in cells that opens wide perspectives for the application of protein chips in proteomics.

Tetracycline resistance of Neisseria gonorrhoeae in Russia, 2015–2017

The objective of this study was to estimate the tetracycline resistance level in the modern population of Neisseria gonorrhoeae in the Russian Federation, where this drug was removed from the treatment regimen for gonococcal infections in 2003. A total of 401 isolates collected between 2015 and 2017 were analyzed for genetic markers (chromosomal porB, rpsJ and mtrR gene mutations and the plasmid-located tetM gene) involved in tetracycline resistance. Antibiotic susceptibility testing revealed that 19% of the strains were tetracycline resistant (MIC > 1 mg/L) and that 10% of the strains had intermediate susceptibility (0.5 < MIC ≤ 1 mg/L). Various combinations of mutations identified in the rpsJ (Val57Met/Leu), porB (Gly120Lys/Asp/Asn/Thr and Ala121/Asp/Asn/Gly), and mtrR (-35 del A) genes resulted in MIC increases of up to 1.47 mg/L (geometric mean value). The presence of the tetM gene was detected in 29 strains, including 18 tetM genes of the American type and 11 of the Dutch type. The tetM gene was associated with a strong increase in resistance (MIC > 8 mg/L). One N. gonorrhoeae isolate was found to carry a defective tetM gene with an AG deletion at position 1239-1240, а new stop codon was introduced that caused a defect in TetM protein synthesis and decrease in the tetracycline resistance. Phylogenetic trees constructed using N. gonorrhoeae NG-MAST and tetM loci were compared. Complex relationship was observed between the N. gonorrhoeae sequence type and the tetM plasmid type. Partial recovery of N. gonorrhoeae tetracycline susceptibility was observed relative to the proportion of isolates with resistance detected ten years ago (75%). However, the current levels of tetracycline resistance still preclude the renewed use of these drugs for gonococcal infection therapy.