Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 μM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity.

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden’s method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

Cyclic β-(1→2)-d-glucan (CβG) is a unique carbohydrate derivative produced by pathogenic bacteria of the genus Brucella. Controversial opinions regarding the use of CβG in brucellosis diagnosis have been published. Herein we report the first synthesis of spacered oligo-β-(1→2)-d-glucosides related to the fragments of CβG and containing up to 5 glucose units. Although the desired di- and trisaccharides could be obtained using standard methods, the synthesis of tetra- and pentasaccharides required substantial effort. The obtained oligosaccharides exhibited complex dependencies of their NMR spectral data on the chain length, explained by a tendency to form helical structures. Despite high production of the CβG by Brucella, antibodies to β-(1→2)-d-glucosides detected in human sera are not related to the brucellosis. However, the antibodies to CβG were raised after immunization by BSA-conjugate of penta-β-(1→2)-d-glucoside and allowed detection of CβG, which opens a way towards the development of the new brucellosis diagnostic kit.

The storage duration and hatchability of eggs largely depend on the lysozyme content in egg whites; therefore, determining the lysozyme status is important for characterizing their quality. For the first time, a fast and accurate method for determining the active lysozyme in egg whites has been proposed to establish the lysozyme status of eggs using the fluorescence polarization assay and synthetic chitooligosaccharide conjugates with a fluorescent label without sample preparation. The egg whites of hens, black hens, chukars, quails, ducks, geese, turkeys, peacocks, and ostriches were studied. Samples of egg whites from hens, black hens, chukars, and quails demonstrate the possibility of measuring the lysozyme activity. Samples of hen and black hen eggs from a farm showed approximately the same enzymatic activity of lysozyme. A relatively higher enzymatic activity was demonstrated by the samples from quail egg whites; however, a wide range of data was observed among the eggs. Chitooligosaccharide conjugates demonstrate that they bind only to C-type lysozyme, and no interaction with G-type lysozyme has been shown. Lysozyme activity in the egg whites of duck, goose, turkey, peacock, and ostrich eggs has not been detected by using the obtained chitooligosaccharide tracers, which may be related to the structural features of lysozyme in different bird species. Thus, the method of fluorescence polarization (FP), using fluorescently labeled chitopentaoside to determine the lysozyme status, can be used to characterize hen, black hen, chukar, and quail eggs, which will allow for the selection of a batch of eggs with a high content of active lysozyme, for example, for long-term storage.

β-(1→2)-Glucans constitute a relatively rare and substantially underexplored class of polysaccharides. Of particular interest is the macrocyclic β-(1→2)-glucan from Brucella (CβG), which regarded as a promising diagnostic marker of brucellosis. However, the development of CβG-based diagnostic system requires careful consideration of potential interference from short β-(1→2)-linked gluco-oligosaccharides present in various bacterial commensals and pathogens. To address this challenge, we performed the stereoselective synthesis of a representative series of trisaccharide antigens bearing di-β-(1→2)-glucoside motifs. Several strategies for β-selective insertion of the di-β-(1→2)-glucoside block were evaluated, including approaches employing picolylated and silylated donors as well as donors lacking stereodirecting groups at C-2. Optimal results were achieved using nonparticipating thioglycoside donors activated by a FeCl ₃ /TCCA promoter system in acetonitrile. The method proved to be widely applicable to a range of monosaccharide acceptors, affording the target trisaccharides with excellent β-selectivity. The applicability of prepared oligosaccharides for profiling carbohydrate specificity of antibodies was demonstrated by large-scale screening of human donor sera.