Hydroxyapatite-Gold Modified Screen-Printed Carbon Electrode for Selective SARS-CoV-2 Antibody Immunosensor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or coronavirus disease 2019 (COVID-19), is still spreading worldwide; therefore, the need for rapid and accurate detection methods remains relevant to maintain the spread of this infectious disease. Electrochemical immunosensors are an alternative method for the rapid detection of the SARS-CoV-2 virus. Herein, we report the development of a screen-printed carbon electrode immunosensor using a hydroxyapatite-gold nanocomposite (SPCE/HA-Au) directly spray-coated with the immobilization receptor binding domain (RBD) Spike to increase the conductivity and surface electrode area. The HA-Au composite synthesis was optimized using the Box-Behnken method, and the resulting composite was characterized by UV-vis spectrophotometry, TEM-EDX, and XRD analysis. The specific interaction of RBD Spike with immunoglobulin G (IgG) antibodies was evaluated by differential pulse voltammetry and electrochemical impedance spectroscopy methods in a [Fe(CN)6]4-/3- solution redox system. The IgG was detected with a detection limit of 0.0561 pg mL-1, and the immunosensor had selectivity and stability of 103-122% and was stable until week 7 with the influence of storage conditions. Also, the immunosensor was tested using real samples from human serum, where the results were confirmed using the chemiluminescent microparticle immunoassay (CMIA) method and showed satisfactory results. Therefore, the developed electrochemical immunosensor can rapidly and accurately detect SARS-CoV-2 antibodies.

Chicken Enterococcus faecalis-induced immunoglobulin Y as a prophylactic and therapeutic agent against streptococcosis in red tilapia (Oreochromis hybrid).

Background and Aim: Streptococcosis is a common bacterial disease in red tilapia, in which Enterococcus faecalis infection has not been widely reported. This study aimed to evaluate the efficacy of pellets that contain chicken E. faecalis-induced immunoglobulin Y (IgY) to treat and prevent streptococcosis in red tilapia. Materials and Methods: We conducted a 28-day study for immunoprophylaxis and immunotherapy, each using four groups with two replications: Healthy control fish (KS), non-IgY pellets (PA and TA), pellets with 25% egg yolk containing E. faecalis-induced IgY (PB and TB), and pellets with 50% egg yolk containing E. faecalis-induced IgY(PC and TC). Indirect enzyme-linked immunosorbent assay was performed on prototype pellets produced with an IgY suspension at 1.63 mg/mL as the standard optical density curve. For the immunoprophylaxis study, pellets of 3% of the average body weight of the experimental fish (0.50 g per fish per day) were given daily until day 14 before the challenge test with E. faecalis (2.1 × 109 Colony-forming unit/mL peroral) on day 15. The data from the observation period on days 15-28 were analyzed. For the immunotherapy study, pellets of 3% of the average body weight (0.50 g per fish per day) were given daily for 21 days (days 8-28) 7 day spost-infection. The data from the immunotherapy study were collected during the observation period on days 8-28. Statistical analysis was performed on non-specific immune variables: Total leukocytes, monocytes, lymphocytes, neutrophils, phagocytic activity, and macrophage capacity; and the semi-quantitative distribution of melanomacrophage centers (MMCs) in the lymphoid organs, such as spleen and liver. Photomacrographic data were analyzed descriptively and qualitatively by comparing the healing process and clinical signs found between experiments in the immunotherapy study. Results: The pellet with 50% egg yolk with an IgY at 2.43 mg/g pellet, 3% of body weight once daily, was the best formula on experimental fish. The administration of this formulation can also increase non-specific immunity and the distribution of MMCs in the spleen and liver with a survival rate of 55% for 14 days of challenge period in the immunoprophylaxis study and 70% for 21 days of therapy period in the immunotherapy study. Conclusion: Immunoglobulin Y can be a prophylactic and therapeutic agent against streptococcal infections caused E. faecalis in red tilapia with an optimum dosage of 2.43 mg/g pellet.

Development of a Single-Chain Variable Fragment of CR3022 for a Plasmonic-Based Biosensor Targeting the SARS-CoV-2 Spike Protein.

Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the "new normal" period, where people's activity has bounced back, followed by the easing of travel policy restrictions. The lesson learned is that the wide availability of accurate and rapid testing procedures is crucial to overcome possible outbreaks in the future. Therefore, many laboratories worldwide have been racing to develop a new point-of-care diagnostic test. To aid continuous innovation, we developed a plasmonic-based biosensor designed explicitly for portable Surface Plasmon Resonance (SPR). In this study, we designed a single chain variable fragment (scFv) from the CR3022 antibody with a particular linker that inserted a cysteine residue at the second position. It caused the linker to have a strong affinity to the gold surface through thiol-coupling and possibly become a ready-to-use bioreceptor toward a portable SPR gold chip without purification steps. The theoretical affinity of this scFv on spike protein was -64.7 kcal/mol, computed using the Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method from the 100 ns molecular dynamics trajectory. Furthermore, the scFv was produced in Escherichia coli BL21 (DE3) as a soluble protein. The binding activity toward Spike Receptor Binding Domain (RBD) SARS-CoV-2 was confirmed with a spot-test, and the experimental binding free energy of -10.82 kcal/mol was determined using portable SPR spectroscopy. We hope this study will be useful in designing specific and low-cost bioreceptors, particularly early in an outbreak when the information on antibody capture is still limited.

Preclinical Evaluation of Chicken Egg Yolk Antibody (IgY) Anti-RBD Spike SARS-CoV-2-A Candidate for Passive Immunization against COVID-19.

The coronavirus disease 2019 (COVID-19) has become a substantial threat to the international health sector and the global economy. As of 26 December 2021, the number of mortalities resulting from COVID-19 exceeded 5.3 million worldwide. The absence of an effective non-vaccine treatment has prompted the quest for prophylactic agents that can be used to combat COVID-19. This study presents the feasibility of chicken egg yolk antibody (IgY) anti-receptor-binding domain (RBD) spike SARS-CoV-2 as a strong candidate to neutralize the virus for application in passive immunization. For the purpose of preclinical studies, we radiolabeled IgY anti-RBD spike SARS-CoV-2 with radionuclide iodine-131. This allowed us to evaluate several biological characteristics of IgY in vitro, in vivo, and ex vivo. The preclinical data suggest that IgY anti-RBD spike SARS-CoV-2 could specifically bind to the SARS-CoV-2 antigens; however, little uptake was observed in normal cells (MRC-5) (<2%). Furthermore, the ex vivo biodistribution study revealed that IgY predominantly accumulated in the trachea of normal mice compared to other organs. We also found that IgY possessed a good safety profile when used as an intranasal agent. Taken together, we propose that IgY anti-RBD spike SARS-CoV-2 has the potential for application in passive immunization against COVID-19.

Development of lateral flow assay based on anti-IBDV IgY for the rapid detection of Gumboro disease in poultry.

The poultry industry faces serious problems against infectious diseases, including Gumboro, which is caused by contagious bursal disease virus (IBDV). IBDV infects the bursa of Fabricius (BF), a lymphoid organ for controlling the B-cell maturation. Thus, it can trigger the secondary infection's vulnerability, leading to the high mortality and morbidity of the chicken. Moreover, managing the Gumboro post-outbreaks also requires considerable time and costs. Besides vaccination programs, the early detection of IBDV is vital as an outbreak control strategy. The most popular diagnostic tool is a lateral flow immunoassay or a rapid test that meets ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users) criteria. In this study, the lateral flow immunoassay was successfully developed based on anti-IBDV IgY as the bio receptor. Anti-IBDV IgY was successfully isolated from Isa Brown's egg yolk. The detection system showed an acceptable affinity against the inactivated IBDV sample (1.5 × 103 TCID50). In addition, it did not react with avian influenza and Newcastle disease viruses, demonstrating a good specificity of the test.