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. 2025 Aug 18;11(1):2546245. doi: 10.1080/20565623.2025.2546245

Using IgY for diagnosis of respiratory infections: practical application in COVID-19

Cícero Matheus Lima Amaral a, Valdester Cavalcante Pinto Júnior a, Daniel Freire Lima a, Hellen paula Silva da Costa b, Danúbio Andrade Bezerra Farias a, João Xavier da Silva Neto b, Luiz Francisco Wemmenson Gonçalves Moura a, Bruno Bezerra da Silva a, Ednardo Rodrigues Freitas c, Maurício Fraga Van Tilburg d, Maria Izabel Florindo Guedes d,✉
PMCID: PMC12363533  PMID: 40823946

Abstract

Aim

The COVID-19 pandemic underscores the need for expanded diagnostic tools to combat respiratory pathogens with pandemic potential, particularly in developing countries. This study aimed to create a Dot Blotting test utilizing IgY antibodies for acute respiratory infection diagnosis, with COVID-19 as the disease model.

Methods

Leghorn chickens were immunized with precipitated SARS-CoV-2 virus, and IgY antibodies were purified via ammonium sulfate precipitation and titrated by ELISA. Dot Blotting detected viral antigens in saliva samples, demonstrating efficacy comparable to ELISA tests.

Results

The IgY antibody was successfully produced and purified, obtaining a titration of 1:16,000. The ability of IgY to detect SARS-CoV-2 in clinical saliva samples showed promising results in terms of accuracy (91.3%), sensitivity (92.5%), specificity (90.0%), positive predictive value (PPV) (90.2%), negative predictive value (NPV) (92.3%), and Cohen’s Kappa (0.825).

Conclusion

Chicken antibodies proved effective for early and accurate diagnosis of respiratory infections, including COVID-19. This study validates the efficacy of chicken antibodies in diagnosing respiratory infections, supporting pandemic response in developing nations. Expanding diagnostic capabilities is crucial for combating respiratory pathogens.

Keywords: Immunodiagnosis, immunoglobulins, IgY technology, public health, respiratory tract diseases

PLAIN LANGUAGE SUMMARY

The COVID-19 pandemic showed that we need better and faster ways to find out if someone has a virus that affects the lungs. This is very important, especially in countries that don’t have many medical resources. In this study, scientists made a simple lab test called Dot Blotting. It helps to check if someone has a lung infection like COVID-19. To make this test, they used special antibodies from chickens. The chickens were safely given a small amount of the virus, and then their eggs were used to get the antibodies. These antibodies, called IgY, can help find the virus in a person’s saliva (spit). The test worked well and gave similar results to other standard lab tests. It was right about 91% of the time, which means it is a reliable test. The chicken antibodies were good at finding the virus early, which helps doctors give the proper care sooner. This new test can help many people, especially in places where tests are hard to find. Making more straightforward and low-cost tests like this is crucial to fight future virus outbreaks.

HIGHLIGHTS

  1. The chickens were immunized with the SARS-CoV-2 virus to produce anti-SARS-CoV-2 IgY.

  2. The anti-SARS-CoV-2 IgY antibody detected the presence of the SARS-CoV-2 virus in saliva samples.

  3. The anti-SARS-CoV-2 IgY performed well in Dot Blotting to diagnose COVID-19 in saliva, similar to its application in ELISA.

  4. Chicken antibodies are effective for diagnosing respiratory infections, potentially aiding developing countries.

GRAPHICAL ABSTRACT

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1. Introduction

The onset of the COVID-19 pandemic, caused by SARS-CoV-2, impacted millions of people worldwide and caused significant disruption to society and the economy in the early 2020s [1]. This pandemic also exposed the limitations of the global health supply chain, directly impacting low-income countries [2].

Accurate diagnosis is a cornerstone in effectively managing respiratory pandemics, enabling prompt implementation of control measures like isolation and contact tracing [3]. Although molecular tests, such as RT-qPCR, are considered the gold standard for diagnosis, their high costs and the demand for infrastructure still limit their widespread application in public health emergencies. Antigen detection tests are a pragmatic alternative [4].

Antigen detection tests depend on precisely identifying an antigen by a labeled molecule. The simplicity of performing these tests allows them to be conducted in field settings or environments with limited infrastructure, having simple transport logistics and enabling mass testing with faster results [4]. Antibodies, the main molecules used in antigen detection tests, are proteins of the adaptive immune response, typically produced after animal immunization. The chosen immunogen, animal species, and purification process directly impact the cost-effectiveness of antibody-based tests [5]. Chicken-derived IgY antibodies from egg yolk have gained prominence as diagnostic molecules due to their unique characteristics.

The use of immunoglobulin Y (IgY) in antigen detection tests has gained attention due to its efficacy, ease of purification from chicken eggs, and advantageous characteristics such as high yield (50 to 100 mg per egg), chemical stability (stable at a pH of 3.5 to 11.0), and thermal stability (stable up to 70 °C). These properties make IgY an excellent diagnostic resource, particularly for countries with limited infrastructure [5]. Additionally, IgY has demonstrated efficacy in diagnosing various viral infections, including influenza (H1N1), MERS-CoV, and SARS-CoV [6].

In This context, this study aimed to develop a Dot Blot-based diagnostic test for COVID-19 using saliva samples. The aim was to create a diagnostic tool for acute respiratory infections using COVID-19 as the model.

2. Materials and methods

2.1. Immunogen production

The Central Public Health Laboratory of Ceará (LACEN) collected a positive clinical sample for SARS-CoV-2 in March 2020 at a hospital unit, as described by Holshue et al. [7]. The nasopharyngeal swab was taken on the third day after the onset of symptoms, and the molecular diagnosis was confirmed by the RT-qPCR test (SARS-CoV-2 Molecular Kit (E)—Bio-Manguinhos).

For viral isolation, Vero E6 cells were cultured and infected following the protocol described by Harcourt et al. [8]. Wells showing cytopathic effects were subjected to confirmatory tests using RT-qPCR and sequencing of part of the genome. The supernatant from infected Vero cells was titrated using the plate-forming unit (PFU) technique, following the protocol previously described by Mendoza et al. [9].

To prepare the virus for animal immunizations, we inactivated the SARS-CoV-2 cell culture supernatant using a thermal process (65 °C for 30 minutes). The viral particles were concentrated by precipitation with PEG 6000 [10] and used as the immunogen for IgY production. Finally, immunization with SARS-CoV-2 precipitated at 10^5PFU/mL was standardized.

2.2. Production and purification of polyclonal IgY antibodies from chickens

2.2.1. Ethical aspects

IgY was produced by immunizing chickens, and all animal handling experiments were conducted at the Federal University of Ceará (UFC). The Animal Research Ethics Committee of UFC, Brazil, reviewed and approved all experiments under protocol 174402111.

2.2.2. Immunization protocol

Two twenty-week-old White Leghorn hens were individually housed in cages, with ad libitum access to feed and water. After the acclimation period, five eggs were collected before immunization to produce pre-immune IgY as a control. Then, the chickens were intramuscularly immunized in the breast region with 3 mL of a mixture containing SARS-CoV-2 virus precipitated from cell culture with PEG 6000 at a concentration of 10^5 PFU/mL (≈ 40 μg). An aluminum hydroxide solution at 0.2 M was used as an adjuvant in a 1:1 (v/v) ratio. The exact amounts of precipitated virus and adjuvant solution were used as booster doses on the 14th and 28th day after the initial immunization.

Immune egg collection started five days after the last booster dose. The eggs were collected daily for two weeks, stored at 4 °C, and then purified in a single batch.

2.2.3. Polyclonal IgY purification

The polyclonal IgY was extracted according to Tilburg et al. [11] and Araújo [12]. Briefly, the eggs were cracked open to collect the yolks, followed by dilution in nine volumes of ultrapure water. To delipidate the samples, the pH of the mixtures was adjusted to 5.0, followed by a freeze-thaw step. Afterward, the delipidated samples were centrifuged at 800 g for 40 minutes at 4 °C for supernatant collection.

Next, the pH was adjusted to 7.4, and ammonium sulfate was slowly added to a final concentration of 20% (w/v). The solution was centrifuged at 2000 g for 20 minutes, and the precipitate was collected and resuspended in 20 mM PBS (pH 7.4) at 1/10 of the initial volume. The ammonium sulfate precipitation step was repeated once more. To remove the ammonium sulfate, the samples were thoroughly washed with PBS and centrifuged using an Amicon® 30 MW tube (3000 g for 30 minutes). This final step was repeated twice, and the final sample was stored at -20 °C.

2.4. Antibodies titration

An indirect ELISA (Enzyme-Linked Immunosorbent Assay) was performed to titrate the IgY antibodies. First, the SARS-CoV-2 virus precipitated from the cell culture was diluted in buffer (0.1 M sodium carbonate, pH 9.5) for coating the microplates (Sigma, M9410). The plates were incubated overnight at 4 °C. After coating, the plates were washed three times with PBS-T (0.05% Tween® 20, Sigma, P9416) and blocked with 5% (w/v) nonfat milk powder in PBS for one hour at 37 °C. The polyclonal anti-SARS-CoV-2 IgY antibody and pre-immune IgY were serially diluted (1:2 to 1:524288) to a final volume of 100 μL/well and incubated for one hour at 37 °C. After another wash step, a secondary antibody (goat anti-IgY conjugated to peroxidase, Sigma A9792, 1:5000) was used for detection after incubation for another hour at 37 °C. Following a final washing step, 100 μL of 3,3’,5,5’-Tetramethylbenzidine (TMB) solution (Life TechnologyThermo, 34028) was added to each well, followed by incubation 20 minutes in the dark. The absorbance of each well at 650 nm was quantified using a microplate reader (Synergy™ 2, Biotek). Absorbance values were considered positive when their absorbance met at least double the absorbance of the respective pre-immune dilution [13].

2.5. Development and diagnostic performance evaluation of the anti-SARS-CoV-2 IgY Dot Blot Assay, with comparison to its application in ELISA

2.5.1. Ethical aspects

This study followed the Declaration of Helsinki and was reviewed and approved by the ethical committee of the Hospital Universitário Walter Cantídio (approval number: CAAE: 43505120.0.0000.5045). The participants in this study signed an informed consent form before providing saliva, which was confirmed by RT-qPCR testing (SARS-CoV-2 Molecular Kit (E)—Bio-Manguinhos). Saliva samples collected in 2018 and stored in the Central Public Health Laboratory of Ceará (LACEN) biobank were used as pre-pandemic anonymized controls.

2.5.2. Anti-SARS-CoV-2 IgY Dot Blot Assay (DBA)

To assess the diagnostic performance of the DBA, saliva samples from 160 human subjects were collected. These samples were subdivided into two groups based on their RT-qPCR diagnostic status:

  • Positive samples (n = 80): Patients with SARS-CoV-2 detected by RT-qPCR (SARS-CoV-2 Molecular Kit (E) – Bio-Manguinhos) at the moment of collection.

  • Negative samples (n = 80): Patients with SARS-CoV-2 undetected by RT-qPCR at collection.

To conduct the DBA, strips of nitrocellulose membrane (Amersham™ ProtranTM, GE Healthcare) were individually coated with a 2 µL droplet of saliva sample and incubated at 37 °C for 30 minutes. Next, the membranes were blocked (20 mM PBS, pH 7.4, containing 5% skimmed milk) for 30 minutes at 37 °C. Then, the membranes were incubated with the produced polyclonal IgY anti-SARS-CoV-2 antibody (diluted at 1:10000) for 30 minutes. Following a wash step, the membranes were incubated with the secondary antibody (goat anti-IgY conjugated to peroxidase, Sigma A9792, 1:5000) for 30 minutes at 37 °C. Viral antigens were detected by immersing the test membranes in a solution of DAB (3,3’-diaminobenzidine tetrahydrochloride) at a concentration of 333 µg/mL for approximately 15 minutes.

The SARS-CoV-2 virus purified from cell culture was used as the positive control, and sterile distilled water was used as the negative control. As previously mentioned, the positive and negative patients were confirmed by RT-qPCR testing using the commercial kit (SARS-CoV-2 Molecular Kit (E)—Bio-Manguinhos).

2.5.3. Anti-SARS-CoV-2 IgY Enzyme Linked Immunosorbent Assay (ELISA)

The IgY was evaluated for its recognition capacity through an indirect ELISA (Enzyme-Linked Immunosorbent Assay). For this purpose, 50 samples from RT-qPCR-positive patients and 35 samples from RT-qPCR-negative patients (previously used in the DBA test) were utilized. As a positive control, precipitated SARS-CoV-2 virus was used, and a pool of pre-pandemic negative saliva samples was employed as a negative control. The samples were diluted in coating buffer (0.1 M sodium carbonate, pH 9.5) to sensitize each well of the microplates (Sigma, M9410) with a final protein concentration of 3 µg in a total volume of 100 µL per well. The plates were incubated overnight at 4 °C. After sensitization, the plates were washed three times with PBS-T (0.05% Tween® 20, Sigma, P9416) and then blocked with 5% (w/v) nonfat dry milk in PBS for one hour at 37 °C, followed by three additional washes with PBS-T. The anti-SARS-CoV-2 IgY polyclonal antibody was used as the primary antibody at a concentration of 1:500, with a final volume of 100 μL/well, and incubated for one hour at 37 °C. After another washing step, 100 μL of secondary antibody (anti-IgY goat conjugated with peroxidase, Sigma A9792, 1:5000) was added and incubated for one hour at 37 °C. The reaction was developed with o-phenylenediamine dihydrochloride (OPD; Thermo Scientific™, 34006), and the absorbance was read at 450 nm using a Synergy™ 2 plate reader (Biotek) without halting the reaction with acid.

2.5.4. Evaluation of the limit of detection (LOD) of the anti-SARS-CoV-2 IgY antibody in saliva samples

The supernatant of the SARS-CoV-2 cell culture was spiked into a saliva sample and subjected to serial dilutions with healthy saliva, obtaining final concentrations ranging from 105 PFU/mL to 0.19 × 100 PFU. These samples were analyzed by indirect ELISA according to the procedures described in section 2.4.

2.6. Data analysis

All data was analyzed using Prism software version 8.0 (GraphPad Software, Inc, La Jolla, CA, USA), IBM SPSS Statistics 29.0.2.0 (IBM, Armonk, NY), and Excel 2019 (Microsoft, Redmond, WA). The Mann-Whitney test was applied to compare the positive and negative ELISA groups, which assessed the ability to recognize antigens in saliva. The cutoff value was calculated based on the absorbance results from the ELISA assays, establishing a threshold to distinguish positive from negative samples, as described by Lardeux et al. [14]. The accuracy, sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Cohen’s Kappa coefficient for both the Anti-SARS-CoV-2 IgY ELISA and DBA methods were calculated according to Buderer [15].

3. Results

This study describes the production and purification of polyclonal IgY against the SARS-CoV-2 virus for diagnostic purposes. In the electrophoretic profile under reducing conditions, we observed the distinction between the light (approximately 25 kDa) and heavy chains (approximately 67–70 kDa) of IgY (Figure 1A). The IgY concentration obtained was 1.9 mg/mL in the evaluated purification batch, corresponding to the average anti-SARS-CoV-2 IgY content of the eggs collected after the final immunization, representing approximately 14 eggs (2 weeks).

Figure 1.

Figure 1.

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Purification and titration of anti-SARS-CoV-2 IgY antibodies. (A) Purification of IgY anti-SARS-CoV-2 antibodies. Electrophoretic profile of IgY purified with ammonium sulfate on a 12% SDS-PAGE gel. MW: molecular weight. IgY: 7.5 µg of purified IgY. (B) Analysis of specific IgY anti-SARS-CoV-2 antibody titers. The dilution is expressed as a negative base-2 logarithm. The secondary antibody used for both anti-SARS-CoV-2 IgY and pre-immune IgY was chicken anti-IgG (Sigma, A9792) (1:5000 in PBS). IgY antibody extracted from pre-immune eggs was used as a negative control (NC).

Indirect ELISA titred the pre-immune and anti-SARS-CoV-2 polyclonal IgY against viral particles from cell culture. The IgY antibody titer reached approximately 1:16,000, indicating robust IgY immunoreactivity against SARS-CoV-2, with the ability to bind to the antigen even at very low concentrations (Figure 1B).

The ability to detect SARS-CoV-2 in clinical saliva samples was assessed and compared using the DBA methodology. The anti-SARS-CoV-2 IgY exhibited the ability to identify antigens in saliva samples that tested positive for SARS-CoV-2, as confirmed by the RT-qPCR test (Figure 2A).

Figure 2.

Figure 2.

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Utilization of IgY Anti-SARS-CoV-2 Antibodies in Dot-Blotting Methodology. (A) Dot-blotting test for the detection of viral antigens in saliva. Four saliva samples from individuals positive for RT-qPCR (Row A: A1, A2, A3, and A4), four saliva samples sourced from the pre-pandemic period (Row B: B1, B2, B3, and B4), four aliquots of distilled water as a negative control (Row C: C1, C2, C3, and C4), and four aliquots of SARS-CoV-2 virus precipitated from cell culture as a positive control (Row D: D1, D2, D3, and D4). (B) Diagnostic performance table of IgY anti-SARS-CoV-2 antibodies application in dot-blotting methodology for saliva sample antigen detection. Infected: Saliva samples with SARS-CoV-2 detected by RT-qPCR. Uninfected: Saliva samples with SARS-CoV-2 not detected by RT-qPCR.

Following the promising performance observed in applying the anti-SARS-CoV-2 IgY produced through the DBA methodology, a larger-scale test was conducted to ascertain the sensitivity and specificity of this diagnostic application. In this test, 160 saliva samples were evaluated, comprising 80 positive and 80 negative for the presence of SARS-CoV-2 (Figure 2B). The performance parameters of this test are shown below.

To confirm the performance of the anti-SARS-CoV-2 IgY antibody in diagnosing the virus in saliva samples and its application in the DBA methodology, we evaluated the IgY antibody using the ELISA methodology. The results presented in Figure 3A reveal a significant difference between the 50 positive samples and the 35 negative samples, with p < 0.0001 according to the Mann-Whitney test, demonstrating the IgY’s ability to distinguish infected individuals from healthy ones using ELISA methods, similar to the DBA (Figure 3A). The anti-SARS-CoV-2 IgY showed the ability to accurately identify antigens in saliva samples, which were confirmed positive for SARS-CoV-2 by the RT-qPCR test, as highlighted in the table (Figure 3B).

Figure 3.

Figure 3.

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ELISA Using IgY Anti-SARS-CoV-2 for the Detection of SARS-CoV-2 in Saliva Samples. (A) Results of ELISA Using IgY Anti-SARS-CoV-2 for the Detection of SARS-CoV-2 in 50 Positive and 35 Negative Saliva Samples. The distributions were significantly different as calculated by the Mann-Whitney U test, p < 0.0001. (B) Diagnostic performance table of IgY anti-SARS-CoV-2 antibodies application in ELISA methodology. Infected: Saliva samples with SARS-CoV-2 detected by RT-qPCR. Uninfected: Saliva samples with SARS-CoV-2 not detected by RT-qPCR.

Furthermore, we compared the performance of the Dot Blot and ELISA methods. In Table 1, we observed the precision, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Kappa coefficients of Cohen for both tests, using RT-PCR as the gold standard. The results show high concordance between the methods and RT-PCR, with the DBA demonstrating slight superiority in some parameters.

Table 1.

Performance comparison between Dot Blot and ELISA methods using IgY anti-SARS-CoV-2 antibodies.

  Accuracy Sensivity Specificity PPV NPV Cohen’s kappa
Dot Blot 91.3% 92.5% 90.0% 90,2% 92.3% 0.82
Elisa 88.2% 88.0% 88.7% 91,7% 83.8% 0.76
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Cohen’s Kappa coefficient was calculated for both tests using RT-PCR as the gold standard. The evaluated parameters include accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).

To assess the sensitivity of the produced antibodies, we established the detection limit of the IgY anti-SARS-CoV-2 antibody through an indirect ELISA assay using serial dilutions of the virus in saliva samples. The detection curve showed a progressive reduction in absorbance at 650 nm in response to decreasing viral concentrations (Figure 4). We determined the detection limit based on the lowest viral concentration that exceeded the established cutoff value. The detection limit was 1.953125 × 102 PFU, demonstrating the ability of IgY anti-SARS-CoV-2 to identify low viral loads.

Figure 4.

Figure 4.

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Determination of the Limit of Detection of the IgY Anti-SARS-CoV-2 Antibody. Graph representing the detection curve of the anti-SARS-CoV-2 IgY antibody as a function of the serial dilution of the SARS-CoV-2 virus using an indirect ELISA assay. The dotted line represents the cutoff value established based on negative control samples, as described in section 2.8. The lowest virus concentration that resulted in a signal above the cutoff was considered the assay’s detection limit. Saliva samples negative for COVID-19, confirmed by RT-qPCR and without added virus, were used as a negative control (NC).

4. Discussion

In this preliminary study, we demonstrated the diagnostic potential of an IgY-based DBA under resource-limited conditions. Using the SARS-CoV-2 virus as a model, IgY production was achieved with minimal precipitated virus for hen immunization. The antibodies used for DBA production were obtained through a cost-effective and high-yield process.

We observed a higher titration of anti-SARS-CoV-2 IgY in our study compared to reports from other authors. Artman et al. [13] immunized chickens with 100 μg of recombinant S1 spike glycoprotein (S1) using the poultry adjuvant Montanide ISA 70 VG, which resulted in a 1:10,000 titration of IgY polyclonal antibodies. Similarly, Ge et al. [16] used 250 μg of S1 protein in a mixture with Freund’s adjuvant and achieved a 1:10,000 titration. These titration results were lower than those in our study, which used specific rather than complete antigens to immunize the chickens.

Immunizing animals with the complete SARS-CoV-2 virus exposes them to all viral antigens, offering advantages over models targeting a single protein [17]. Although focusing on specific antigens using proteins or viral fragments is feasible, the S protein and its variants increase the risk of false negatives [18]. Producing polyclonal antibodies targeting multiple viral epitopes, like anti-SARS-CoV-2 IgY, is a promising strategy to mitigate this issue. This approach reduces the risk of immune escape by new variants and enhances binding capacity to a broader range of viral variants, improving neutralization efficacy. Moreover, producing specific antibodies, such as monoclonal antibodies, is expensive, technically demanding, and highly specific. This represents a disadvantage considering the frequent viral mutations of COVID-19, increasing the risk of variant escape from immune recognition [13,19].

IgY antibodies exhibit structural and functional characteristics that provide significant advantages over mammalian immunoglobulin G (IgG), particularly in diagnostic contexts. Unlike IgG, IgY antibodies do not interact with protein A and G produced by Staphylococcus aureus and group G streptococci, thus preventing false positives in clinically contaminated samples, as these bacterial proteins bind to the Fc region of mammalian IgG. Furthermore, IgY does not bind to rheumatoid factor or Fc receptors on immune cells, significantly reducing the risk of cross-reactivity and nonspecific binding in immunoassays [20,21].

When applied in the DBA methodology, the anti-SARS-CoV-2 IgY antibodies exhibited a sensitivity, specificity, NPV, and PPV of approximately 90%, demonstrating better performance than in the ELISA assay. These results highlight the antibody’s effectiveness in detecting SARS-CoV-2, suggesting its utility as a reliable and accurate tool for COVID-19 diagnosis, even in resource-limited settings. In their investigation, Kivrane et al. [22] immunized mice with the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 virus spike protein to develop a lateral flow assay for detecting SARS-CoV-2 in saliva samples. The Lateral Flow Assay used a combination of antibodies, including commercial rabbit anti-spike PAbs and mouse anti-rRBD PAbs, yielding a sensitivity of 26.5%, specificity of 58.1%, PPV of 50.0%, NPV of 33.3%, and diagnostic accuracy of 38.7%. This diminished performance could result from using specific antigens, which limit the range of identifiable viral epitopes, and the animal model used for antibody production.

In another study, the DBA methodology was used for diagnosing SARS-CoV-2 through a serological test utilizing the flow-through dot-blot assay, which yielded a sensitivity and specificity of 98.8% and 98%, respectively, with overall PPV and NPV of 99.6% and 99% [23]. While these figures surpass those of the anti-SARS-CoV-2 IgY, the results of our study were deemed satisfactory, particularly considering the more straightforward, low-tech production process. Through this test, we demonstrated the feasibility of detecting SARS-CoV-2 without the need to produce specific purified antigens, bypassing complex and costly processes that require infrastructure, which is beyond the reach of most of the world’s population.

Hagbom et al. [24] compared two rapid antigen immunochromatographic tests for detecting SARS-CoV-2 in saliva: the Rapid Response™ COVID-19 Antigen Rapid Test Cassette for oral fluids and the DIAGNOS™ COVID-19 Antigen Saliva Test. The DIAGNOS method exhibited a sensitivity of 50.0%, while the Rapid Response showed 38.9%. Both tests demonstrated high specificity (100%) and PPV of 100%. For NPV, DIAGNOS achieved 82.0%, and Rapid Response reached 78.9%. When comparing these results with those obtained from IgY antibodies, we observed superior outcomes in the parameters of sensitivity (92,5%) and NPV (92,3%), with slightly lower but still near-ideal results in specificity (90%), further emphasizing the diagnostic potential of the proposed model when compared to commercial tests.

IgY has also demonstrated successful application in detecting another coronavirus, showing high sensitivity to the N protein of SARS-CoV in serum and nasopharyngeal aspirate [25]. Their study aimed to devise a rapid IgY test utilizing an immunoswab, incorporating mouse monoclonal antibodies alongside a commercial antibody linked to peroxidase. While our study did not undertake mass testing, we presented significant findings with a substantial sample size, unlike Kammila et al. [25], who not only omitted performance assessments of the diagnostic model but also utilized monoclonal antibodies, thereby complicating production and escalating costs. The DBA employing anti-SARS-CoV-2 IgY holds promise for developing countries as they can locally produce their diagnostic components, getting past the high costs associated with multinational suppliers and shortages during periods of heightened demand [26].

The viral loads of SARS-CoV-2 in saliva samples from COVID-19 patients typically range from 1 × 101 PFU/mL to 4.6 × 104 PFU/mL, influenced by factors such as disease severity, time since symptom onset, and individual immune response [27]. The detection limit established for the anti-SARS-CoV-2 IgY (1.95 × 102 PFU) produced in this study covers a significant portion of this range. However, this result did not consistently impact the performance parameters, as we obtained robust results compared to the gold standard RT-qPCR, highlighting the practical potential of the produced antibody for clinical application.

Our findings highlight the potential of IgY as a robust alternative for epidemic and pandemic diagnosis due to its rapid implementation and scalability. We can generate and purify antibodies within weeks post-immunization, enabling a swift response to emerging pathogens. While initial results are promising, further assessing the antibodies’ application across more extensive and diverse populations is essential to validate COVID-19 diagnosis. Furthermore, a thorough evaluation of cross-reactivity and optimization of quality control standards are crucial steps forward.

5. Conclusion

In this study, we demonstrate that the application of IgY antibodies can recognize SARS-CoV-2 viral antigens in saliva samples. The IgY technology showed strong performance results for COVID-19 diagnosis using the Dot Blotting methodology, employing a low-cost, low-tech, high-yield production protocol, which was confirmed with similar results in the ELISA methodology. This study shows that chicken antibodies are effective for the early and accurate diagnosis of respiratory infections, including COVID-19, and can aid the response of developing countries to future pandemics.

Acknowledgments

The authors would like to thank the Oswaldo Cruz Foundation (FIOCRUZ) Ceará, the Ceará Foundation for Support of Scientific and Technological Development (FUNCAP), the National Council for Scientific and Technological Development (CNPq), the Funding Authority for Studies and Projects (FINEP) of the Ministry of Science, Technology, and Innovation (MCT), and the Coordination for the Improvement of Higher Education Personnel (CAPES), institutions and funding agencies that made this work possible.

Author contributions

C.M.L.A. contributed to the conception and design of the study, acquisition of data, or analysis and interpretation of data, and drafted the article or critically revised it for important intellectual content. V.C.P.J., D.F.L., H.P.S.C., D.A.B.F., J.X.S.N., and L.F.W.G. contributed to the conception and design of the study, or acquisition of data, or analysis and interpretation of data. B.B.S., E.R.F., M.F.V.T., and M.I.F.G. drafted the article or critically revised it for important intellectual content and approved the final version to be submit-ted. All authors read and approved the final version of the manuscript.

Disclosure statement

The authors declare no potential conflicts of interest, financial or non-financial, regarding this study. No funding or support was received from organizations or individuals that could influence the research outcome or interpretation. All authors reviewed and approved the manuscript, ensuring its integrity and objectivity.

Data availability statement

The data supporting the findings of this study are available upon request from the corresponding author, AMARAL CML. However, the data are not publicly available as a patent has been requested at the Brazilian National Institute of Industrial Property (INPI) under application number BR 10 2021 006097 2.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data supporting the findings of this study are available upon request from the corresponding author, AMARAL CML. However, the data are not publicly available as a patent has been requested at the Brazilian National Institute of Industrial Property (INPI) under application number BR 10 2021 006097 2.

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