The use of antimicrobial growth promoters (AGPs) is banned because of problems associated with drug residues in animal products and increased bacterial resistance. The immunization of chickens with specific antigens is a promising strategy for generating specific antibodies that can target a wide range of antibiotic-resistant bacteria and can be used as an alternative to antibiotics. Immunoglobulin Y (IgY) antibodies in a polyclonal antibody (pAb) format, when administered orally, modulate the ruminal microbiome and maintain animal health and performance; however, there are concerns pertaining to protein impurities and biotin concentrations in the samples. Signal amplification strategies involving the noncovalent interaction of biotin with streptavidin is extensively used in diagnosis and scientific research, particularly in enzyme-linked immunosorbent assays (ELISAs). However, the high concentrations of biotin in samples, especially in those derived from rich sources such as egg yolk, can pose challenges and potentially harm the accuracy of diagnostic tests and protein concentration measurements. This study aimed to evaluate the influence of biotin on the measurement of IgY in freeze-dried egg yolk samples obtained from immunized laying hens using immunoassays with biotin–avidin/streptavidin. The detection of IgY in yolk samples using ELISA with streptavidin–biotin binding could lead to misdiagnosis due to biotin interference; the level of interference varies with the specific assay conditions and the concentration of biotin in the yolk samples. An ELISA without streptavidin–biotin binding is advisable to avoid interactions between biotin and target proteins, prevent biotin interference with the results, and achieve more reliable and accurate results.
Antimicrobial resistance (AMR) is a global health crisis with significant consequences. Approximately 700,000 fatalities are attributed to AMR annually. By 2050, AMR could lead to approximately 10 million deaths per year and global social costs of USD 100 trillion, underscoring the wide-ranging impact of AMR [1]. The use and misuse of in-feed antibiotics in animal agriculture as growth promoters in poultry and livestock have raised significant concerns related to drug residues in animal products and the development of AMR [2].
There is a growing interest in immunoglobulin Y (IgY), which is abundant in the egg yolks of immunized laying hens, as an alternative to antibiotics for various diagnostic, therapeutic, and research applications [3]. Antibodies against Streptococcus equinus, Lactobacillus spp., Fusobacterium necrophorum, Escherichia coli, Clostridium aminophilum, sticklandii, Peptostreptococcus, Salmonella enteritidis, Salmonella enterica, Salmonella typhimurium, Staphylococcus aureus, Helicobacter pylori, Pseudomonas aeruginosa, rotavirus, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and yeasts have been studied [4,5,6,7,8,9].
IgY antibodies can be obtained in larger quantities from the egg yolks of immunized laying hens compared to those obtained from other traditional sources, such as serum from rats, rabbits, goats, and sheep [10]. Unlike traditional mammalian antibody production, which could involve blood collection that can be stressful, other invasive procedures, and even sacrificing the animal [11], this non-invasive egg collection and IgY extraction from egg yolk is cost-effective and ethical and is a sustainable source of antibodies [12]. Furthermore, egg yolk antibodies do not interact with rheumatoid factors in the serum of mammals and do not bind to proteins A and G, mammalian Fc receptors, or mammalian complements [13]. IgY antibodies are stable and resistant to degradation, allowing for long-term storage and retained activity through different manufacturing steps. Dried IgY batches can maintain their biological activity for several years [14,15].
The oral administration of IgY as an alternative to antibiotics or for microbiome modulation, especially in the polyclonal antibody (pAb) format, often does not require IgY purification for large-scale production. The use of polyethylene glycol (PEG), such as PEG6000, for protein collection from supernatants is a common method for removing fat from samples; however, it can sometimes result in protein impurities and high levels of biotin in the samples, which can be problematic in certain applications [16]. When using IgY without extensive purification, it is important to consider the high level of biotin naturally present in egg yolks, 988–1050 ng/g [17,18], because this could lead to potential biotin interference in assays; it is necessary to consider appropriate strategies to mitigate its impact. Biotin interference is a recognized challenge in immunoassays using streptavidin–biotin-binding interactions [19]; it can affect the accuracy of the results in both research applications and human diagnostics. Approximately 85% of chemiluminescence immunoassays are based on biotin–avidin/streptavidin and are used by more than two-thirds of laboratories in China [20,21]. All laboratories using signal amplification through biotin–streptavidin interactions face the threat of misdiagnosis due to biotin interference caused by excessive biotin consumption [22,23].
This study aimed to evaluate the influence of biotin on IgY quantification in unpurified freeze-dried egg yolk samples obtained from antigen-inoculated hens using biotin–avidin/streptavidin.
Streptococcus equinus was used as an antigen to produce yolk antibodies. The stock culture was thawed and grown on blood plates for 24–48 h at 38 °C under semianaerobic conditions. Colonies were collected by scratching, and they were aseptically transferred to a broth tube containing 2.5 mL of growth medium. The bacteria were grown in a basal medium (39 °C) that contained (per liter) 22 mmol glucose, 1.7 mmol K 2 HPO 4, 2.1 mmol KH 2 PO 4, 3.6 mmol (NH 4)2 SO 4, 8.3 mmol NaCl, 0.75 mmol MgSO 4·7H 2 O, 0.43 mmol CaCl 2·2H 2 O, 2.8 mmol cysteine hydrochloride, 38 mmol Na 2 CO 3, 5 mg/mL casamino acids (Difco), 10 mg/mL Trypticase, and 5 g yeast extract. A 1 L borosilicate bottle was used as the container for preparing the growth medium, which was autoclaved for 15 min at 121 °C and at a pressure of 1 atmosphere. A 40% glucose solution was added at room temperature to prevent caramelization. The pH of the medium was adjusted to 6–7 using either 1 M NaOH or 1 M HCl as required.
The turbidity of the culture was checked and compared to the McFarland standards to obtain a final density of 5 × 10 9 cells/mL, and the culture was transferred to an Erlenmeyer flask containing 50 mL of culture media. When the culture achieved turbidity at 0.5 McFarland, it was transferred to an Erlenmeyer flask with 150 mL of culture media with CO 2, which was sealed and incubated for 48 h at 38 °C. The homogenized content of the Erlenmeyer flask was filtered through four layers of cheesecloth. The solid in the filter was washed with 0.9% saline, and the total filtrate was transferred to centrifuge containers. The containers with the filtrate were balanced in pairs and centrifuged at 1000× g for 10 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in 150 mL of McDougall’s solution. The content was redistributed in centrifuge containers and centrifuged at 11,250× g for 20 min at 4 °C. The supernatant from the second centrifugation was discarded, and the precipitate was transferred to a single container using a spatula and as little deionized water as possible to obtain the final bacterial pellet. The bacterial pellet was resuspended in phosphate-buffered saline (PBS) (pH 7.4) to obtain 5.3 × 10 8 colony-forming units/mL of S. equinus and a turbidity of 0.5 McFarland. To this solution, 4% formaldehyde (18.5%) was added, which was followed by 30% Imject Alum Adjuvant (TermoFisher Scientific, Life Tech Brasil, Itapevi, Sao Paulo, Brazil). The controls were prepared using the same amounts of PBS, formaldehyde, and adjuvant without bacterial pellets. For antigen adsorption, the adjuvant was added slowly, which was followed by constant agitation for 4 h. These immunologic response inductors were transferred to sterile serum bottles, capped, and stored at 4 °C until further use.
White Leghorn hens (25-week-old) were divided into two groups. Solution (500 µL), with or without antigen, was injected deeply into the pectoral muscles of each group every 14 days for 56 days. Eggs were collected weekly, broken, and the shells, yolks, and egg whites were separated. The yolk was subjected to freeze drying and delipidation, according to Akita and Nakai [20], to concentrate the IgY and biotin in the aqueous protein fraction. The lyophilized yolk sample (1 g) was weighed in a 15 mL Falcon tube; 6 mL of phosphate-buffered saline (PBS) and 0.210 g of polyethylene glycol (PEG) 6000 were added. The mixture was vortexed for 1 min and incubated at 4 °C with shaking for 10 min. The samples were centrifuged twice at 4 °C, at 10,000 rpm, for 20 min. The precipitate consisted of solids and fat; one fraction of yellow fat floated above the supernatant, and the transparent supernatant in the middle contained biotin and proteins. The supernatant containing protein and biotin was separated from the solids and fat and centrifuged again; the supernatant was collected using a needle and syringe. The protein fraction was filtered using a funnel and paper filter (40 µm) to remove the suspended insoluble solid residue particles and yellow fat and to clarify the sample for the analysis of IgY and biotin using ‘IDK Biotin ELISA,’ manufactured by Immunodiagnostik AG (Bensheim, Germany). Crude IgY from non-immunized hens was used as the control.
To evaluate the interference of biotin in the detection of Immunoglobulin Y (IgY, an antibody class found in chicken yolk), two commercial kits for sandwich enzyme-linked immunosorbent assay (ELISA) and two non-commercial plates specific to this study were used. These commercial kits, used for quantitatively detecting IgY in various biological samples for various purposes, come with all the necessary reagents, including antibodies, substrates, and standards, along with detailed instructions for conducting the assay. The capture antibody was added at a concentration of 0.5–4 µg/mL (pre-coated plate), while they usually use detection antibodies at 0.5–1 µg/mL. In these commercial ELISA kits, the detection antibodies are nonspecific; freeze drying makes the proteins and biotin in the sample very concentrated; therefore, a high dilution factor (1:1,000,000) is required to ensure that the sample reading signal is within the values of the standard curves.
The commercial test used was ECH0032 from FineTest, in which two specific antibodies were used to “sandwich” IgY between them. One antibody, known as the capture antibody (anti-IgY, usually 0.5–4 µg/mL), was immobilized on a solid surface (e.g., the surface of a microplate well); it binds specifically to IgY. Diluted test samples and standards (0.1 each) were added to the pre-coated plates. The plates were sealed with a cover and incubated at 37 °C for 90 min. The cover was removed, the solution was discarded, and the plate was washed twice with Wash Buffer. Subsequently, 0.1 mL of biotin-conjugated detection antibody was added to the wells, and the plate was sealed with a cover and incubated at 37 °C for 90 min. The cover was removed, and the plate was washed thrice with Wash Buffer; the wash buffer was allowed to stand in the wells for 1 min each time. Horseradish peroxidase (HRP–streptavidin) was added to each well, and the plate was sealed with a cover and incubated at 37 °C for 30 min. Subsequently, the cover was removed, and the plate was washed five times with wash buffer, and the wash buffer was allowed to stand in the wells for 2 min. Then, 90 µL of TMB (3,3′,5,5′ tetramethylbenzidine), the substrate for HRP, was added into each well, and the plate was incubated at 37 °C in the dark for 15–30 min to visualize the HRP enzymatic reaction. Finally, 50 μL of the stop solution was added to each well and mixed thoroughly. The OD 450 was recorded immediately after adding the stop solution using a spectrophotometer, Biochrom EZ Read 400 Microplate Reader from Holliston, MA, USA. The calibration curve obtained from the serial dilution of the standard is shown in [Figure 1](https://www.frankenthalerfoundation.org
The IRKTAH1109 test from Innovative Research Incorporation was used to compare the results for IgY concentration obtained through the interaction of biotin with streptavidin without signal amplification. In the IRKTAH1109 assay, the IgY in the samples reacted with the anti-IgY antibodies adsorbed on the surface of polystyrene microtiter wells. After removing the unbound proteins by washing, an enzyme–antibody conjugate (0.1 mL) with horseradish peroxidase (HRP) was added to each well, and these anti-IgY antibodies conjugated with the previously bound IgY to form complexes. The plates were washed, and 100 µL of TMB (3,3′,5,5′ tetra-methylbenzidine), the substrate for HRP, was added into each well; the plates were incubated in the dark at room temperature for 10 min to visualize the HRP enzymatic reaction. Finally, 100 μL of the stop solution was added into each well and mixed thoroughly. The OD 450 was recorded immediately after the addition of the stop solution using a spectrophotometer. The calibration curve obtained from the serial dilution of the standard is shown in [Figure 2](https://www.frankenthalerfoundation.org
The interference of biotin in specific antibody detection was evaluated with two-plate trapped antigen-enzyme linked immunoso
