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This study describes the applicability of the fluorescence polarization assay (FPA) based on the use of FITC-labeled oligosaccharide tracers of defined structure for the measurement of active lysozyme in hen egg white. Depending on the oligosaccharide chain length of the tracer, this method detects both the formation of the enzyme-to-tracer complex (because of lectin-like, i.e., carbohydrate-binding action of lysozyme) and tracer splitting (because of chitinase activity of lysozyme). Evaluation of the fluorescence polarization dynamics enables simultaneous measurement of the chitinase and lectin activities of lysozyme, which is crucial for its detection in complex biological systems. Hen egg white lysozyme (HEWL), unlike human lysozyme (HL), formed a stable complex with the chitotriose tracer that underwent no further transformations. This fact allows for easy measurement of the carbohydrate-binding activity of the HEWL. The results of the lysozyme activity measurement for hen egg samples obtained through the FPA correlated with the results obtained using the traditional turbidimetry method. The FPA does not have the drawbacks of turbidimetry, which are associated with the need to use bacterial cells that cannot be precisely standardized. Additionally, FPA offers advantages such as rapid analysis, the use of compact equipment, and standardized reagents. Therefore, the new express technique for measuring the lysozyme activity is applicable for evaluating the complex biomaterial, including for the purposes of food product quality control. [ABSTRACT FROM AUTHOR]

This study focuses on the synthesis of a series of oligo- α -(1→6)-D-galactopyranosides bearing β -D-galactofuranosyl residues at O-2 and/or O-3, which relate structurally to fragments of glucuronoxylomannogalactan (GXMGal) from the fungal pathogen Cryptococcus neoformans that causes severe diseases in immunocompromised patients. The preparation of target compounds is based on the use of a selectively O-protected N-phenyltrifluoroacetimidoyl galactopyranoside donor with an allyl group at O-2, levulinoyl group (Lev) at O-3, pentafluorobenzoyl (PFB) group at O-4, and fluorenylmethoxycarbonyl (Fmoc) group at O-6. The choice of protecting groups for this donor ensures the stereospecific formation of α- (1→6)-glycosidic bonds due to the stereodirecting effect of acyls at O-3, O-4, and O-6. At the same time, this combination of O-substituents permits the selective recovery of free OH groups at O-2, O-3, and O-6 for chain elongation via the introduction of β- D-galactofuranosyl and α- D-galactopyranosyl residues. The reported compounds are obtained as aminopropyl glycosides, which are transformed into biotinylated conjugates for further use as coating antigens in immunological studies. The obtained oligosaccharides were subjected to detailed 13C NMR analysis to show the spatial similarity of the obtained hexasaccharide with the corresponding fragment in the GXMGal chain, making this compound suitable for further immunological studies of C. neoformans. [ABSTRACT FROM AUTHOR]

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Brucellosis is a dangerous zoonotic disease caused by bacteria of the genus Brucella. Diagnosis of brucellosis is based on the detection in animal and human sera of antibodies to the O-polysaccharide of Brucella lipopolysaccharide. The currently employed serodiagnosis of brucellosis relies on the use of the Brucella O-polysaccharide as a diagnostic antigen. However, the existence of bacterial species, which also express O-polysaccharides structurally similar to that of Brucella, may decrease the specificity of the brucellosis detection due to false-positive test results. It has been shown that the efficiency of the test can be significantly improved by using synthetic oligosaccharides that correspond to the so-called M epitope of the Brucella O-antigen. This epitope is characterized by an a-(1^3)-linkage between D-perosamine units and is unique to Brucella. Here we report on an efficient approach to the synthesis of oligosaccharides that model the M epitope of the Brucella O-polysaccharide. The approach is based on the use of the a-(1^3)-linked disaccharide thioglycoside as the key donor block. Its application allowed the straightforward assembly of a set of four protected oligosaccharides, which includes a disaccharide, two trisaccharides, and a tetrasaccharide, in five glycosylation steps. The synthesized oligosaccharides are planned to be used in the development of diagnostic tools for identifying brucellosis in humans and domestic animals, as well as a potential vaccine against it. [ABSTRACT FROM AUTHOR]