The heavy chain of spent laying hens was 6.60 0.20 pixels/unit, and the light chain was 4.60 0.10 pixels/unit, with the ratio of heavy Canertinib (CI-1033) chain to light chain being 1.430.10 pixels/unit. IgY from spent and new laying hens demonstrated effective hemagglutination against cat erythrocytes in the A blood group. == Conclusions: == This study demonstrated the potential of using spent laying hens Rabbit Polyclonal to hCG beta to produce IgY, with significant implications for future research, immunotherapy, and diagnostic applications, despite the observed reduction in egg production compared with new laying hens. Keywords:antibody production, diagnosis, immunoglobulin Y antibody, immunological assays, spent laying hens == Introduction == Antibodies serve as important tools with numerous applications in life science and translational research in medicine. Polyclonal and monoclonal antibodies, each with their own advantages and limitations, are important to research [1]. Among them, immunoglobulin (Ig) Y, a polyclonal antibody found predominantly in egg yolk, has significance in diagnostic applications. Although primarily identified in avian species, IgY shares structural similarities with mammalian Canertinib (CI-1033) IgG antibodies [2]. In avian biology, IgY plays an important role in passive immunity during the early life stages of birds, protecting embryos through efficient transfer to egg yolks [3]. IgY technology has gained considerable attention over recent decades, particularly for its sensitive detection of biomolecules and its ability to be applied to human and animal diagnoses [4]. Remarkably, hens have the potential to produce antibodies, making them a practical source of antibodies. High antibody production yields in hen eggs fulfill not only ethical considerations, such as the principle of the 3Rs concept (reduction, replacement, and refinement) but also offer economic advantages over conventional animal antibody production [2, 46]. Notably, the noninvasive isolation of IgY antibodies from egg yolks distinguished hen-based production methods from other animal systems, mitigating ethical concerns associated with conventional antibody retrieval methods through blood collection. Moreover, hens have lower maintenance costs than other commonly used animal models, including mice and rabbits [7]. Despite these advantages, IgY yields from hens are comparable to those from larger animals, such as goats and sheep, with an estimated production of 1825 g of IgY per year per hen [3,8]. Furthermore, specific IgY antibodies constitute a significant portion, ranging from 1% to 10% of the total antibodies produced by hens [3]. However, various factors, including antigen dosage, immunogenicity, and molecular weight, influence antibody production in each hen [2,3]. IgY has been utilized in various applications to improve both human and veterinary health, including immunodiagnostics [3, 4, 911], immunotherapy [12,13], passive immunization [14,15], neutralization of toxins from venomous animals [16,17] and bacteria [8, 1820], and production of functional foods [21,22]. Despite its potential, the expansion of the IgY product range has been relatively slow because of infrastructure costs, the pricing of raw antibodies or ready-to-use products, and limited market demand [23]. Canertinib (CI-1033) Nevertheless, IgY production remains cost effective, particularly when extracted from egg yolks [24]. To optimize IgY production costs and increase efficiency in the use of hens for antibody production, a study should be conducted comparing the use of spent and new laying hens. A previous study by Marzecet al. [25] indicated that both the functional and physicochemical properties of fresh eggs remain unaffected by hen age, making spent laying hens an interesting option for IgY production. This study aimed to determine the feasibility of using spent laying hens to produce polyclonal IgY. Furthermore, by comparing outcomes between spent and new laying hens, the study assessed the impact of antigen injection on egg and IgY production and the application of IgY for hemagglutination potential using cat red blood cells (RBCs). == Materials and Methods == == Ethical approval == This study was approved by the Institutional Animal Care and Use Committee of Kasetsart University (protocol number ACKU64-VET-018) and was in accordance with the principles of the 3Rs concept. == Study period and location == The study was conducted from July 2021 to December 2021. Eight hens were enrolled in the present study at the Animal Facility of the Faculty of Veterinary Medicine, Kasetsart University, Bang Khen, Bangkok, Thailand. == Ghost cell preparation == Blood samples from two cats (A and B blood groups) and two dogs (dog erythrocyte antigen.