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Introduction: Toxoplasmosis is caused by the opportunistic, cosmopolitan protozoan Toxoplasma gondii is one of the most common parasitoses in the world. This parasite can pose a threat to people with immunodeficiency but also to the fetus, since the invasion can lead to miscarriages. Moreover, this parasite can contribute to economic losses in livestock farming. These problems lead to the implementation of new, safe solutions for the development of effective toxoplasmosis immunoprophylaxis., Methods: In this work, newly produced recombinant trivalent chimeric proteins of T. gondii , based on SAG1-SAG2 recombinant chimeric antigen that differ in one terminal antigenic component, were tested in terms of their ability to induce an effective post-vaccination response. Antigens were tested in vitro to assess their ability to elicit APC cells response and further mice of the C3H/HeOuJ strain were immunized using those antigens, to evaluate their immunogenicity and immunoprotective effect in vivo . Two weeks after the last dose mice were either sacrificed to assess selected parameters of the immune response or infected with T. gondii DX strain to determine the degree of protection one month later., Results: The results of serological tests revealed a high level of serum IgG antibodies specific for the native T. gondii TLA antigens. TLA-stimulated splenocytes produced cytokines that are important in inhibiting protozoal invasion. Additionally, CD3 + CD4 + and CD3 + CD8 + T cell subpopulations of splenocytes were analysed by flow cytometry. One month after experimental infection mice were sacrificed, and their brains were isolated to count T. gondii tissue cyst. Immunization of mice with recombinant trivalent chimeric proteins of T. gondii resulted in reduction of tissue cyst burden rates reaching even 74%., Discussion: The obtained results demonstrate strong immunogenicity of the studied proteins and will allow to select candidates for further research aimed at increasing the immunoprotective properties of experimental vaccines against toxoplasmosis based on T. gondii chimeric antigens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chyb, Ferra, Kawka, Skwarecka, Dziadek and Gatkowska.)

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti- Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

This study reports a novel impedimetric immunosensor for protein D detection in purified and bacterial (Haemophilus influenzae, Hi) samples. The detection was based on antigen recognition by anti-protein D antibodies (apD) immobilised at the maze-like boron-doped carbon nanowall electrodes (B:CNW). The B:CNW electrodes were synthesised, and their surface was characterised by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) methods. The sensor was prepared in a two-step procedure: apD were covalently linked on the previously modified B:CNW electrodes using diazonium salt. Modification steps were controlled by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. The immunosensor exhibited excellent electrochemical performance, stability, satisfactory sensitivities, and linear ranges for antigen detection. Protein D was detected down to 2.39 × 10 2 fg/mL with a linear range extending from 3.37 × 10 -11 to 3.37 × 10 -3 μg/mL (in purified sample). Next, Hi's LOD was 5.20 × 10 2 CFU/mL with a linear range of 8.39 × 10 1 -8.39 × 10 3 CFU/mL. Selectivity studies showed no reaction with negative samples as Streptococcus pyogenes, Streptococcus pneumoniae or Bordetella parapertussis bacteria. Therefore, the new approach is suitable for rapid and quantitative detection of Hi, and is a good candidate for further tests on clinical samples., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This paper presents the development and comparison of label-free electrochemical immunosensors based on screen-printed gold and glassy carbon (GC) disc electrodes for efficient and rapid detection of respiratory syncytial virus (RSV). Briefly, the antibody specific to the F protein of RSV was successfully immobilized on modified electrodes. Antibody coupling on the Au surface was conducted via 4-aminothiophenol (4-ATP) and glutaraldehyde (GA). The GC surface was modified with poly-L-lysine (PLL) for direct anti-RSV conjugation after EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide) activation. Electrochemical characterizations of the immunosensors were carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). GC-based immunosensors show a dynamic range of antigen detection from 1.0 × 10 5 PFU/mL to 1.5×10 7 PFU/mL, more than 1.0 × 10 5 PFU/mL to 1.0 × 10 7 PFU/mL for the Au-based sensor. However, the GC platform is less sensitive and shows a higher detection limit (LOD) for RSV. The limit of detection of the Au immunosensor is 1.1 × 10 3 PFU/mL, three orders of magnitude lower than 2.85 × 10 6 PFU/mL for GC. Thus, the Au-based immunosensor has better analytical performance for virus detection than a carbon-based platform due to high sensitivity and very low RSV detection, obtained with good reproducibility.

The present work describes an impedimetric immunosensor for Pseudomonas syringae pv. lachrymans ( Psl ) detection. This pathogen infects many crop species causing considerable yield losses, thus fast and cheap detection method is in high demand. In the assay, the gold disc electrode was modified with 4-aminothiophenol (4-ATP), glutaraldehyde (GA), and anti-Psl antibodies, and free-sites were blocked with bovine serum albumin (BSA). Sensor development was characterized by cyclic voltammetry (CV) and antigen detection by electrochemical impedance spectroscopy (EIS) measurements. Seven analyzed strains of Psl were verified as positive by the reference method (PCR) and this immunoassay, proving sensor specificity. Label-free electrochemical detection was in the linear range 1 × 10 3 -1.2 × 10 5 CFU/mL (colony-forming unit) with an R 2 coefficient of 0.992 and a detection limit (LOD) of 337 CFU/mL. The sensor did not interfere with negative probes like buffers and other bacteria. The assay was proven to be fast (10 min detection) and easy in preparation. The advantage was the simplicity and availability of the verified analyte (whole bacteria) as the method does not require sample pretreatment (e.g., DNA isolation). EIS biosensing technique was chosen as one of the simplest and most sensitive with the least destructive influence on the probes compared to other electrochemical methods.