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.

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.

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.

Protein-based APIs represent a big group of modern therapeutics. Their characterization involves complex analytical protocols which require special methods, especially in the case when the protein drug is included into tablet dosage forms. Although the fluorescence polarization assay (FPA) is not currently regulated by many national Pharmacopeias, it represents a promising approach for protein drug standardization, considering their rapid, sensitive, and automatable detection suitable for high-throughput analysis and real-time quality control. To evaluate the applicability of FPA for the analysis of protein drugs in tablets, the quantifying of lysozyme in tablet dosage forms was studied by this method with the use of a fluorescently labeled synthetic chitooligosaccharide tracer. It was shown that this approach overcomes the limitations of the conventional turbidimetric assay of lysozyme determination, which is labor-intensive and relies on unstable reagents. Measurements were performed with both portable and stationary fluorescence polarization readers. Commercial tablets from five manufacturers containing lysozyme (20 mg) and pyridoxine hydrochloride (10 mg) together with other excipients were analyzed. The FPIA method showed a linear range of 5.0–70 µg/mL, with specificity confirmed by the absence of interference from excipients. Accuracy, evaluated by standard addition (10–20 mg), yielded recoveries of 100.2–106.0%. Placebo spiked with lysozyme at 80–120% of nominal content demonstrated recoveries of 98.0–100.1%, with RSD (n = 6) not exceeding 13.7%, indicating good precision. The developed method enables reliable lysozyme quantification in tablets, offering speed, simplicity, and robustness, and shows its suitability for the routine quality control of protein-containing dosage forms including the enzyme ones.

Hen egg white lysozyme (HEWL) is a well-characterized antibacterial enzyme widely used in research and industry. However, little is known about the functional properties of lysozyme from other bird species, such as pheasants. This study aimed to compare the muramidase and chitinase activities of HEWL with the lysozyme from pheasant egg white (PEWL). Egg white samples were obtained from ten hens and ten pheasants. Lysozyme activity was assessed by two methods: a turbidimetric assay using Micrococcus luteus cell suspension to measure bacteriolytic (muramidase) activity and a fluorescence polarization assay (FPA) using FITC-labeled tri- and chitopentaoside substrates to measure the chitinase activity. The observed dissociation rate constant (k_obs) was derived from the time-dependent FP signal. Turbidimetric measurements showed that PEWL possessed bacteriolytic activity approximately 30% lower than that of HEWL under the same conditions. However, FPA revealed a striking functional difference: whereas HEWL only bound the chitotrioside substrate without hydrolyzing it, PEWL was able to hydrolyze chitotriose. Both enzymes readily hydrolyzed the chitopentaoside substrate. The hydrolysis rate of the chitopentaoside was 50–70 times higher than that of the chitotrioside for PEWL. Sequence comparison identified six amino acid substitutions in PEWL (Phe3 → Tyr, His15 → Leu, Gln41 → His, Asn77 → His, Gln121 → Asn, Ile124 → Thr) relative to HEWL, which likely alter the conformation of subsites A–C and enable productive binding of the shorter oligosaccharide. Thus, despite high sequence similarity, pheasant lysozyme exhibits a unique functional property—the ability to hydrolyze chitotriose into N-acetylglucosamine. These findings expand functions of avian lysozymes and highlight the potential of alternative bird species as sources of enzymes with novel properties.