Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry

Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry Nikolay Saushkin, Jeanne Samsonova, Galina Presnova, Maya Rubtsova, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3982114/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 µm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High, medium or low antibody titre level for each of three infections could be quickly assessed visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation. post-vaccinal antibodies serological control multiplex assay Newcastle disease infectious bronchitis bursal disease Figures Figure 1 Figure 2 Figure 3 Introduction Infectious poultry diseases such as avian influenza, Newcastle disease and others can affect a large number of birds in a short time due to the high stocking density (Tsiouris et al. 2015 ). The vaccination of industrial poultry against hazardous pathogens is the most efficient approach for the prevention of the spread of infectious diseases (Kaiser 2010 ). Regular control of the flock's immunosafety is carried out by assessing the level of post-vaccinal antibodies in chicken serum. This helps to minimize the potential risk of economic damage associated with disease and death of the bird (Kaspers et al. 2022 ). Microplate ELISA is a well-established technique that is extensively employed for a diverse range of analytical purposes. However, in general, it is intended to detect one antigen. The same holds true for ELISA test systems routinely used for post-vaccinal antibodies control. The necessity of assessing humoral immunity to multiple diseases makes it relevant and financially viable to employ analytical systems that permit the simultaneous detection of antibodies against several pathogens in one blood (serum) sample. Moreover, there is a demand for the development of rapid diagnostic test systems that would allow monitoring vaccination not only in the laboratory, but in field conditions where poultry is kept since getting data quickly and adjusting the livestock vaccination schedule is crucial for a poultry farm. Today, microarrays are used to detect multiple antigens simultaneously, including DNA, proteins, peptides, and antibodies (Aparna and Tetala 2023 ). This format of analysis can significantly shorten operation time for large-scale monitoring also reducing the amount of specific reagents used. Some new methods based on microarrays for anti-pathogen chicken antibodies detection are proposed, however, the number of papers regarding this topic is minimal. To detect antibodies to the chicken infectious bursal disease virus, a membrane microchip with chemiluminescent detection was developed (Yan et al. 2018 ). Subsequently, the principle of the described system was implemented for qualitative simultaneous determination of antibodies against pathogens of avian influenza, Newcastle disease and infectious bronchitis with visual detection (Li et al. 2021 ). A similar principle was described in (Xiao et al. 2019 ), where the antibodies to certain subtypes of avian influenza virus was studied by immobilizing different types of virus protein (haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein) onto the chip surface. A glass-based protein chip for the simultaneous determination of antibodies against four chickens pathogens has been developed (Wang et al. 2010 ). A multiplex immunoassay using the Luminex 200 system for the determination of antibodies against poultry pathogens was described in a few works (Pinette et al. 2014 ; Wang et al. 2018 , 2019 ). Although Luminex system has many advantages for simultaneous detection of several antigens, it should be noted that it requires special high-cost detecting equipment. All microarray methods described to date for the simultaneous determination of several anti-pathogen chicken antibodies, as well as other animals, for example, pigs (Wu et al. 2020 ), are mainly qualitative tests. Whereas, ELISA provides a semi-quantitative or quantitative evaluation of antibody titres in a flock, which is crucial for the livestock immunization programme. The qualitative control of antibodies is not sufficient for evaluation of flock immunization status, and the data about antibody titre increasing is essential to reduce the dangers of disease outbreaks. In this regard, the concept of a gradient immunoassay, which is comprised of a few analytical zones with varying concentrations of binding reagent that correspond to distinct detection limits of the target analyte, is very promising for quick semi-quantitative results evaluation. The number of lines/zones to be formed (N) is determined by the number of concentration ranges of the target analyte (N + 1). In this case, the target analyte level (concentration range) is assessed by the number of coloured test lines/zones. This principle was recently realized in lateral flow immunoassay for various antigens (Serebrennikova et al. 2017 , 2019 ; Zhang et al. 2020 ; Li et al. 2022 ; Yang et al. 2023 ). In this work, we developed a gradient multiplex analytical system as an immunochip-in-a well format that allows the simultaneous semi-quantitative detection of post-vaccinal antibodies against three infectious chicken diseases (Newcastle disease, infectious bronchitis and bursal disease) in one probe. The principle of multiplex immunoassay (microarray) was combined with gradient approach for the determination of high, medium and low levels of post-vaccinal antibodies. Issues of the optimal assay conditions were considered. Materials and methods Tween 20 was from Amresco, USA, and dextransulphate (MW 6500–10000) was from Sigma, USA. Inorganic salts were obtained from Chimmed, Russia. Ready-to-use substrate solution containing 3,3’,5,5’-teramethylbenzidine (TMB) was supplied by UniversiTest, Russia. The following buffers were used: 0.01M K-phosphate (K 2 HPO 4 -KH 2 PO 4 ) 0.15M NaCl pH 6.0-7.3 (PBS) and PBS supplied with 0.05–0.1% Tween 20, pH 6.0-7.4 (PBST). Goat anti-chicken IgY polyclonal antibodies – horseradish peroxidase conjugate (anti-IgY Ab-HRP) was obtained from Imtek, Russia. Inactivated virus suspensions (Newcastle disease, infectious bronchitis, bursal disease), chicken sera (SPF and post-vaccinal) were provided by SPE AVIVAC, Russia. IgY was purified from SPF-chicken sera by sodium sulfate precipitation. ELISA diagnostic test kits (IDEXX, USA) were used to detect antibodies to Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV) in chicken serum. Microlon 600 Strip Plates, High Binding (Greiner Bio-One, USA) were used for ELISA experiments and immunochip printing. Purification of a virus suspension Virus suspensions were purified by dialysis against distilled water at room temperature for 2–3 hours. After centrifugation of 1 ml solution (10 min 3000 g) the pellet was resuspended in 100 µL PBS pH 6.0 containing 0.04% NaN 3 and stored at + 4°C. ELISA procedure Microtiter plate wells were covered by viral antigens (150 µl per well, PBS pH 6.0) overnight and then washed with PBST. Post-vaccinal chicken sera were diluted 1:500 with PBST (pH 6.0, 0.1% Tween 20) and incubated in microtiter plate wells (100 µl) for 30 min at room temperature. After washing anti-IgY Ab-HRP was added to wells (100 µl) and incubated for another 30 min at room temperature. After incubation and plate washing the substrate solution (100 µl) was added to each well. The colour reaction was stopped after 10–15 min with 100 µl 0.1M H 2 SO 4 and the result was evaluated on Anthos 2010 (Austria) spectrophotometer at 450–620 nm. Gradient immunochip for the detection of post-vaccinal antibodies in chickens For immunochip printing all reagents were diluted in PBS pH 6.0 supplied with 0.02% Tween 20. Purified virus solutions in certain dilution were spotted on the bottom of strip plate wells with the help of the LabNext Xact II microarray system (USA) and then dried for 30 min at 37 o C. An array of 36 spots (6x6, d = 400µm each) was formed by five concentrations of each viral antigen (in duplicates), negative control spots (printing buffer, n = 2) and two positive control spots (IgY 5 µg/ml n = 2 and IgY 10 µg/ml n = 2). The concentration of each viral antigen was decreased gradually from left to right in a row (Fig. 1 ). Post-vaccinal chicken sera were diluted 1:500 with PBST and incubated in microtiter plate wells for 30 min at room temperature. After washing with PBST anti-IgY Ab-HRP (1/1500 dilution in PBST) was added to wells and incubated for 30 min at room temperature. The plate was washed with PBST and then TMB substrate solution containing 0.5% dextran sulfate was used for colour development of microspots. Immunochip results were assessed visually or with the help of smartphone. The plate was also scanned (Epson Perfection V700 Photo, Seiko Epson, Japan) and the colour intensity of microspots was evaluated with ImageJ software (USA). Results and discussion The design of the gradient immunochip (the 6*6 spots array on a well bottom) was based on three rows of viral antigens, namely NDV (upper row), IBV (middle row), IBDV (bottom row), and the concentration of each viral antigen used was decreased gradually from spot to spot left to right in a row (Fig. 1 ). The number of coloured spots after completion of analyses of chicken sera should indicate the level of post-vaccinal antibodies to the particular infection (low, medium or high). First, all components of the multiplex test system were characterized by ELISA, including the optimization of reagents concentration and assay conditions, pH value, and composition of absorption and reaction buffers. The NDV, IBV and IBDV antigens as major components for immunochip development and fabrication were purified by dialysis against distilled water, which was the most effective among other approaches used (Online Resource Fig. S1 ). The resulting NDV, IBV and IBDV antigen preparations were sufficiently pure to be utilized as coating antigens, however, an unspecific reaction towards anti-IgY Ab-HRP was observed. To optimize antigen absorption conditions, a range of buffers was investigated across pH value from 4.0 to 9.6. The best result in terms of low unspecific binding and signal development was obtained for slightly acidic buffer (pH 6.0) (Online Resource Fig. S2). As a result, PBS buffer with pH 6.0 was chosen as an optimal for all three viral antigens absorption taking into account the difference of resulting optical density between hyperimmune serum, negative control serum (SPF-chicken serum) and conjugate control (Online Resource Fig. S3). Chicken IgY has a strong ability to absorb on polystyrene wells due to its hydrophobicity, so, particular attention should be paid to the choice of reaction buffer (Miers et al. 1983 ). The presence of detergent (Tween 20) was necessary to reduce IgY unspecific binding during the first 30-min incubation of absorbed antigens with chicken sera (Online Resource Fig. S4). In terms of lower unspecific binding the most favorable results were obtained for PBS pH 6.0 with optimal concentration of Tween 20 to be 0.1%. We found that the combination of particular buffer composition for viral antigens absorption and assay steps, together with the necessary level of detergent in chicken serum dilution buffer provided low unspecific binding of IgY and HRP conjugate and improve the differentiation of post-vaccinal chicken sera by different titre groups. The chosen optimal conditions were used to create a gradient immunochip for multiplex test system (Fig. 1 ). High binding polystyrene ELISA plate was used as a standardized solid support for a well-based immunochip. To ensure adequate colour intensity of microspots, the NDV, IBV, and IBDV antigen concentration was increased ten times and HRP conjugate concentration was increased two times against ELISA conditions. An array of 6*6 spots was placed on flat bottom of a well, where NDV, IBV and IBVD antigens were absorbed in gradually decreased concentrations (in duplicates) from left to right (Fig. 1 ). The gradient assay required the choice of NDV, IBV, and IBVD antigen concentrations that would meet certain threshold intensities of the immunochip microspots during a subsequent immunochemical reaction with virus-specific antibodies. Therefore, each viral antigen concentrations were chosen to provide a different number of coloured circular zones depending on the titre of specific antibodies present in the test sample (post-vaccinal chicken serum). An illustration of this selection for the NDV antigen is presented in Fig. 2 . As a result of NDV, IBV, and IBVD antigen dilution optimisation, it was possible to correlate the number of developed coloured spots with the titre value of the anti-NDV, IBV or IBDV antibodies in the analysed chicken serum, i.e. evaluate the result semi-quantitatively. To create positive control, a purified IgY of chicken SPF-serum was used. An assessment of the intensity of coloured microspots when varying the concentration of absorbed IgY demonstrated the possibility of quantitative determination at least 1–2 µg/ml chicken antibodies on the surface (Online Resource Fig. S5). A variety of chicken sera was selected to cover the full spectrum of anti-NDV, IBV and IBVD post-vaccinal antibody titres. Comparable data were obtained using commercial ELISA kit and the developed gradient multiplex assay in immunochip-in-a well format for 63 post-vaccinal chicken sera simultaneously for three infections in one probe (Fig. 3 ). Quick assessment of immunochip results can be done with the help of smartphone to enlarge or make photo of a coloured array after analysis (Fig. 3 A and B). It should be noted that SPF-sera showed no visible unspecific reaction and no coloured microspots were observed (n = 20). The design of the multiplex assay makes it possible to assess the immune status of chickens semi-quantitatively by the number of observed coloured microspots (Fig. 1 ). As a result, the post-vaccinal antibody titres (ELISA results) and corresponding number of coloured microspots (gradient immunochip results) can be subdivided into a few ranges, namely zero, low, medium and high: no specific anti-pathogen antibodies (NDV, IBV and IBDV) – no coloured microspots; 0–1 coloured microspot correspond to antibody titre up to 1000, 2–3 coloured microspots – antibody titres from 1000 to 3000; 3–4 coloured microspots – antibody titres 3000–8000, 5 coloured microspots - high and very high level of immune response, antibody titres higher than 8000 (Fig. 3 , Online Resource Table S1 ). The developed gradient multiplex analysis can be utilized to quickly semi-qiantitatively distinguish tested sera based on the level of post-vaccinal antibodies (titre group) and evaluate the immunity status of flocks on poultry farms following preventive measures against Newcastle disease, infectious bronchitis and bursal disease. The method that has been developed possesses advantages in comparison to arrays that provide a yes/no answer (Wang et al. 2010 ; Yan et al. 2018 ; Li et al. 2021 ) and analytical systems that necessitate costly equipment, such as the Luminex method (Pinette et al. 2014 ; Wang et al. 2018 , 2019 ). Conclusion The principle of immunochip-in-a well multianalysis permits simultaneous detection of post-vaccinal antibodies to the infections of choice. Semi-quantitative assessment of the elevation of antibody titres is especially important for the rapid monitoring of poultry immunity. Moreover, the proposed gradient multiplex approach makes it possible to create arrays of target pathogen antigens, taking into account the epizootic situation in the region (poultry farms) and the vaccination schemes used. The development of scientific and methodological approaches for the multiplex determination of several types of post-vaccinal antibodies is of great importance for poultry farming, due to the high economic importance of both the industry itself and the importance of measures to maintain the health of the livestock. The use of digital scanning systems and software processing of the results can provide rapid simultaneous assessment of antibody titres to several infections, reducing the cost of samples testing by several times. At the same time, the developed approaches and analytical systems can be used for livestock monitoring in other areas of veterinary medicine and agriculture. Declarations Competing Interests None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Ethical statement All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Consent to participate Not applicable. Consent to publish Not applicable. Funding This work was supported by the Russian Science Foundation (project no 22-74-00018). Author Contribution All authors contributed to the study conception and design. Experiment, data collection and analysis were performed by J.V.S., N.Yu.S., G.V.P and M.Yu.R. M.Yu.R. and A.P.O. critically reviewed the manuscript. The first draft of the manuscript was written by N.Yu.S and J.V.S. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability Data is provided within the manuscript. References Aparna GM, Tetala KKR (2023) Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays. 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Supplementary Files Supplimentaryinformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 Apr, 2024 Reviews received at journal 20 Mar, 2024 Reviewers agreed at journal 09 Mar, 2024 Reviewers invited by journal 03 Mar, 2024 Editor assigned by journal 27 Feb, 2024 Submission checks completed at journal 27 Feb, 2024 First submitted to journal 23 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3982114","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":275193916,"identity":"a266cd22-acc2-4ecd-a6e5-3bf688724679","order_by":0,"name":"Nikolay Saushkin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYHACNsYGBhsefiDrwAMStKTJSTYAtSSQoOWwscEBIJMoLfIz0p89nMFwOHHztcMPgbbcS2wgpMXgRo654QaG9MRtt9MMgFqKidAikcMm+YDBGqglAaQlgbAWkMOAWpgTN89O/0CcFoYbCWaSGxicjQ2kc4i0xeDMGzPJGcBAlridU3AgwSDBmLDD2oEO6wFF5ez0zR8+VCTIEnYYCDD+g1tKlPpRMApGwSgYBYQAALgDQIaFaTB+AAAAAElFTkSuQmCC","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":true,"prefix":"","firstName":"Nikolay","middleName":"","lastName":"Saushkin","suffix":""},{"id":275193917,"identity":"e4d740a9-1e64-4bf7-b5b2-964efba4a31e","order_by":1,"name":"Jeanne Samsonova","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Jeanne","middleName":"","lastName":"Samsonova","suffix":""},{"id":275193918,"identity":"a369ab26-c54e-4f4e-b955-7405da03e941","order_by":2,"name":"Galina Presnova","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Galina","middleName":"","lastName":"Presnova","suffix":""},{"id":275193919,"identity":"496580df-2b42-4203-98cb-3fed20ba49a4","order_by":3,"name":"Maya Rubtsova","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Rubtsova","suffix":""},{"id":275193920,"identity":"4c45dba9-4438-4422-bd50-1288331eb089","order_by":4,"name":"Alexander Osipov","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Osipov","suffix":""}],"badges":[],"createdAt":"2024-02-23 14:21:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3982114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3982114/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51809628,"identity":"29c8751d-a2fb-4a05-bbfe-83084843932a","added_by":"auto","created_at":"2024-02-29 12:03:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":108458,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral sheme of NDV-IBV-IBDV immunochip design, performance and assessment. Configuration of gradient immunochip based on three viral antigens (five gradually decreasing concentration from left to right each). AG1 – NDV, AG2 – IBV, AG3 – IBDV. Negative control (K-) – printing buffer, positive control 1 (K1+) – chicken IgY 5 µg/ml, positive control 2 (K2+) – chicken IgY 10 µg/ml\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3982114/v1/67bf3900b8bf482256993ecf.png"},{"id":51809629,"identity":"12b7db23-a4f7-4a2d-acbd-d8b9e56773a9","added_by":"auto","created_at":"2024-02-29 12:03:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69873,"visible":true,"origin":"","legend":"\u003cp\u003eTitration curves of post-vaccinal chicken sera in immunochip format (NDV antigen). ELISA anti-NDV antibody titres: serum 1 – 18259, serum 2 – 13016, serum 3 – 4524, serum 4 - 2137, serum 5 –1377. Arrows indicate NDV antigen dilutions to compose the gradient immunochip\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3982114/v1/22be6680ccb6f4abd98bc365.png"},{"id":51809630,"identity":"75703bec-f9df-4329-9c01-2e8cf972fe50","added_by":"auto","created_at":"2024-02-29 12:03:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307131,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of quantitative ELISA results (titres) and semi-quantitative results of the gradient immunochip analysis (number of coloured microspots) for 63 post-vaccinal chicken sera (NDV, IBV and IBDV). An example of gradient immunochip visual result after reaction with TMB: A – SPF serum, B – hyperimmnune serum (NDV – 5 coloured microspots, IBV – 2-3 coloured microspots, IBD – 5 coloured microspots)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3982114/v1/60f89b7c12293f7225e55cc9.png"},{"id":51809783,"identity":"ce6c7d5d-8354-48a4-82cb-272f4d625ea2","added_by":"auto","created_at":"2024-02-29 12:11:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":555327,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3982114/v1/2b432209-84be-43c8-ae35-00d046295fc0.pdf"},{"id":51809632,"identity":"4580f33e-8809-4bac-a2ae-efc34e77fabd","added_by":"auto","created_at":"2024-02-29 12:03:29","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":573610,"visible":true,"origin":"","legend":"","description":"","filename":"Supplimentaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3982114/v1/a9a8774b7e5e227ba220e447.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfectious poultry diseases such as avian influenza, Newcastle disease and others can affect a large number of birds in a short time due to the high stocking density (Tsiouris et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The vaccination of industrial poultry against hazardous pathogens is the most efficient approach for the prevention of the spread of infectious diseases (Kaiser \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Regular control of the flock's immunosafety is carried out by assessing the level of post-vaccinal antibodies in chicken serum. This helps to minimize the potential risk of economic damage associated with disease and death of the bird (Kaspers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroplate ELISA is a well-established technique that is extensively employed for a diverse range of analytical purposes. However, in general, it is intended to detect one antigen. The same holds true for ELISA test systems routinely used for post-vaccinal antibodies control. The necessity of assessing humoral immunity to multiple diseases makes it relevant and financially viable to employ analytical systems that permit the simultaneous detection of antibodies against several pathogens in one blood (serum) sample. Moreover, there is a demand for the development of rapid diagnostic test systems that would allow monitoring vaccination not only in the laboratory, but in field conditions where poultry is kept since getting data quickly and adjusting the livestock vaccination schedule is crucial for a poultry farm. Today, microarrays are used to detect multiple antigens simultaneously, including DNA, proteins, peptides, and antibodies (Aparna and Tetala \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This format of analysis can significantly shorten operation time for large-scale monitoring also reducing the amount of specific reagents used. Some new methods based on microarrays for anti-pathogen chicken antibodies detection are proposed, however, the number of papers regarding this topic is minimal. To detect antibodies to the chicken infectious bursal disease virus, a membrane microchip with chemiluminescent detection was developed (Yan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Subsequently, the principle of the described system was implemented for qualitative simultaneous determination of antibodies against pathogens of avian influenza, Newcastle disease and infectious bronchitis with visual detection (Li et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A similar principle was described in (Xiao et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where the antibodies to certain subtypes of avian influenza virus was studied by immobilizing different types of virus protein (haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein) onto the chip surface. A glass-based protein chip for the simultaneous determination of antibodies against four chickens pathogens has been developed (Wang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A multiplex immunoassay using the Luminex 200 system for the determination of antibodies against poultry pathogens was described in a few works (Pinette et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although Luminex system has many advantages for simultaneous detection of several antigens, it should be noted that it requires special high-cost detecting equipment. All microarray methods described to date for the simultaneous determination of several anti-pathogen chicken antibodies, as well as other animals, for example, pigs (Wu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), are mainly qualitative tests. Whereas, ELISA provides a semi-quantitative or quantitative evaluation of antibody titres in a flock, which is crucial for the livestock immunization programme. The qualitative control of antibodies is not sufficient for evaluation of flock immunization status, and the data about antibody titre increasing is essential to reduce the dangers of disease outbreaks. In this regard, the concept of a gradient immunoassay, which is comprised of a few analytical zones with varying concentrations of binding reagent that correspond to distinct detection limits of the target analyte, is very promising for quick semi-quantitative results evaluation. The number of lines/zones to be formed (N) is determined by the number of concentration ranges of the target analyte (N\u0026thinsp;+\u0026thinsp;1). In this case, the target analyte level (concentration range) is assessed by the number of coloured test lines/zones. This principle was recently realized in lateral flow immunoassay for various antigens (Serebrennikova et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this work, we developed a gradient multiplex analytical system as an immunochip-in-a well format that allows the simultaneous semi-quantitative detection of post-vaccinal antibodies against three infectious chicken diseases (Newcastle disease, infectious bronchitis and bursal disease) in one probe. The principle of multiplex immunoassay (microarray) was combined with gradient approach for the determination of high, medium and low levels of post-vaccinal antibodies. Issues of the optimal assay conditions were considered.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eTween 20 was from Amresco, USA, and dextransulphate (MW 6500\u0026ndash;10000) was from Sigma, USA. Inorganic salts were obtained from Chimmed, Russia. Ready-to-use substrate solution containing 3,3\u0026rsquo;,5,5\u0026rsquo;-teramethylbenzidine (TMB) was supplied by UniversiTest, Russia.\u003c/p\u003e \u003cp\u003eThe following buffers were used: 0.01M K-phosphate (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e-KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) 0.15M NaCl pH 6.0-7.3 (PBS) and PBS supplied with 0.05\u0026ndash;0.1% Tween 20, pH 6.0-7.4 (PBST).\u003c/p\u003e \u003cp\u003eGoat anti-chicken IgY polyclonal antibodies \u0026ndash; horseradish peroxidase conjugate (anti-IgY Ab-HRP) was obtained from Imtek, Russia. Inactivated virus suspensions (Newcastle disease, infectious bronchitis, bursal disease), chicken sera (SPF and post-vaccinal) were provided by SPE AVIVAC, Russia. IgY was purified from SPF-chicken sera by sodium sulfate precipitation.\u003c/p\u003e \u003cp\u003eELISA diagnostic test kits (IDEXX, USA) were used to detect antibodies to Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV) in chicken serum.\u003c/p\u003e \u003cp\u003eMicrolon 600 Strip Plates, High Binding (Greiner Bio-One, USA) were used for ELISA experiments and immunochip printing.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePurification of a virus suspension\u003c/h2\u003e \u003cp\u003eVirus suspensions were purified by dialysis against distilled water at room temperature for 2\u0026ndash;3 hours. After centrifugation of 1 ml solution (10 min 3000 g) the pellet was resuspended in 100 \u0026micro;L PBS pH 6.0 containing 0.04% NaN\u003csub\u003e3\u003c/sub\u003e and stored at +\u0026thinsp;4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eELISA procedure\u003c/h2\u003e \u003cp\u003eMicrotiter plate wells were covered by viral antigens (150 \u0026micro;l per well, PBS pH 6.0) overnight and then washed with PBST. Post-vaccinal chicken sera were diluted 1:500 with PBST (pH 6.0, 0.1% Tween 20) and incubated in microtiter plate wells (100 \u0026micro;l) for 30 min at room temperature. After washing anti-IgY Ab-HRP was added to wells (100 \u0026micro;l) and incubated for another 30 min at room temperature. After incubation and plate washing the substrate solution (100 \u0026micro;l) was added to each well. The colour reaction was stopped after 10\u0026ndash;15 min with 100 \u0026micro;l 0.1M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and the result was evaluated on Anthos 2010 (Austria) spectrophotometer at 450\u0026ndash;620 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGradient immunochip for the detection of post-vaccinal antibodies in chickens\u003c/h2\u003e \u003cp\u003eFor immunochip printing all reagents were diluted in PBS pH 6.0 supplied with 0.02% Tween 20. Purified virus solutions in certain dilution were spotted on the bottom of strip plate wells with the help of the LabNext Xact II microarray system (USA) and then dried for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC. An array of 36 spots (6x6, d\u0026thinsp;=\u0026thinsp;400\u0026micro;m each) was formed by five concentrations of each viral antigen (in duplicates), negative control spots (printing buffer, n\u0026thinsp;=\u0026thinsp;2) and two positive control spots (IgY 5 \u0026micro;g/ml n\u0026thinsp;=\u0026thinsp;2 and IgY 10 \u0026micro;g/ml n\u0026thinsp;=\u0026thinsp;2). The concentration of each viral antigen was decreased gradually from left to right in a row (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Post-vaccinal chicken sera were diluted 1:500 with PBST and incubated in microtiter plate wells for 30 min at room temperature. After washing with PBST anti-IgY Ab-HRP (1/1500 dilution in PBST) was added to wells and incubated for 30 min at room temperature. The plate was washed with PBST and then TMB substrate solution containing 0.5% dextran sulfate was used for colour development of microspots. Immunochip results were assessed visually or with the help of smartphone. The plate was also scanned (Epson Perfection V700 Photo, Seiko Epson, Japan) and the colour intensity of microspots was evaluated with ImageJ software (USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThe design of the gradient immunochip (the 6*6 spots array on a well bottom) was based on three rows of viral antigens, namely NDV (upper row), IBV (middle row), IBDV (bottom row), and the concentration of each viral antigen used was decreased gradually from spot to spot left to right in a row (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of coloured spots after completion of analyses of chicken sera should indicate the level of post-vaccinal antibodies to the particular infection (low, medium or high). First, all components of the multiplex test system were characterized by ELISA, including the optimization of reagents concentration and assay conditions, pH value, and composition of absorption and reaction buffers. The NDV, IBV and IBDV antigens as major components for immunochip development and fabrication were purified by dialysis against distilled water, which was the most effective among other approaches used (Online Resource Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The resulting NDV, IBV and IBDV antigen preparations were sufficiently pure to be utilized as coating antigens, however, an unspecific reaction towards anti-IgY Ab-HRP was observed. To optimize antigen absorption conditions, a range of buffers was investigated across pH value from 4.0 to 9.6. The best result in terms of low unspecific binding and signal development was obtained for slightly acidic buffer (pH 6.0) (Online Resource Fig. S2). As a result, PBS buffer with pH 6.0 was chosen as an optimal for all three viral antigens absorption taking into account the difference of resulting optical density between hyperimmune serum, negative control serum (SPF-chicken serum) and conjugate control (Online Resource Fig. S3).\u003c/p\u003e \u003cp\u003eChicken IgY has a strong ability to absorb on polystyrene wells due to its hydrophobicity, so, particular attention should be paid to the choice of reaction buffer (Miers et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). The presence of detergent (Tween 20) was necessary to reduce IgY unspecific binding during the first 30-min incubation of absorbed antigens with chicken sera (Online Resource Fig. S4). In terms of lower unspecific binding the most favorable results were obtained for PBS pH 6.0 with optimal concentration of Tween 20 to be 0.1%. We found that the combination of particular buffer composition for viral antigens absorption and assay steps, together with the necessary level of detergent in chicken serum dilution buffer provided low unspecific binding of IgY and HRP conjugate and improve the differentiation of post-vaccinal chicken sera by different titre groups.\u003c/p\u003e \u003cp\u003eThe chosen optimal conditions were used to create a gradient immunochip for multiplex test system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). High binding polystyrene ELISA plate was used as a standardized solid support for a well-based immunochip. To ensure adequate colour intensity of microspots, the NDV, IBV, and IBDV antigen concentration was increased ten times and HRP conjugate concentration was increased two times against ELISA conditions. An array of 6*6 spots was placed on flat bottom of a well, where NDV, IBV and IBVD antigens were absorbed in gradually decreased concentrations (in duplicates) from left to right (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The gradient assay required the choice of NDV, IBV, and IBVD antigen concentrations that would meet certain threshold intensities of the immunochip microspots during a subsequent immunochemical reaction with virus-specific antibodies. Therefore, each viral antigen concentrations were chosen to provide a different number of coloured circular zones depending on the titre of specific antibodies present in the test sample (post-vaccinal chicken serum). An illustration of this selection for the NDV antigen is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As a result of NDV, IBV, and IBVD antigen dilution optimisation, it was possible to correlate the number of developed coloured spots with the titre value of the anti-NDV, IBV or IBDV antibodies in the analysed chicken serum, i.e. evaluate the result semi-quantitatively. To create positive control, a purified IgY of chicken SPF-serum was used. An assessment of the intensity of coloured microspots when varying the concentration of absorbed IgY demonstrated the possibility of quantitative determination at least 1\u0026ndash;2 \u0026micro;g/ml chicken antibodies on the surface (Online Resource Fig. S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA variety of chicken sera was selected to cover the full spectrum of anti-NDV, IBV and IBVD post-vaccinal antibody titres. Comparable data were obtained using commercial ELISA kit and the developed gradient multiplex assay in immunochip-in-a well format for 63 post-vaccinal chicken sera simultaneously for three infections in one probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Quick assessment of immunochip results can be done with the help of smartphone to enlarge or make photo of a coloured array after analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). It should be noted that SPF-sera showed no visible unspecific reaction and no coloured microspots were observed (n\u0026thinsp;=\u0026thinsp;20). The design of the multiplex assay makes it possible to assess the immune status of chickens semi-quantitatively by the number of observed coloured microspots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As a result, the post-vaccinal antibody titres (ELISA results) and corresponding number of coloured microspots (gradient immunochip results) can be subdivided into a few ranges, namely zero, low, medium and high: no specific anti-pathogen antibodies (NDV, IBV and IBDV) \u0026ndash; no coloured microspots; 0\u0026ndash;1 coloured microspot correspond to antibody titre up to 1000, 2\u0026ndash;3 coloured microspots \u0026ndash; antibody titres from 1000 to 3000; 3\u0026ndash;4 coloured microspots \u0026ndash; antibody titres 3000\u0026ndash;8000, 5 coloured microspots - high and very high level of immune response, antibody titres higher than 8000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Online Resource Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The developed gradient multiplex analysis can be utilized to quickly semi-qiantitatively distinguish tested sera based on the level of post-vaccinal antibodies (titre group) and evaluate the immunity status of flocks on poultry farms following preventive measures against Newcastle disease, infectious bronchitis and bursal disease. The method that has been developed possesses advantages in comparison to arrays that provide a yes/no answer (Wang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and analytical systems that necessitate costly equipment, such as the Luminex method (Pinette et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe principle of immunochip-in-a well multianalysis permits simultaneous detection of post-vaccinal antibodies to the infections of choice. Semi-quantitative assessment of the elevation of antibody titres is especially important for the rapid monitoring of poultry immunity. Moreover, the proposed gradient multiplex approach makes it possible to create arrays of target pathogen antigens, taking into account the epizootic situation in the region (poultry farms) and the vaccination schemes used. The development of scientific and methodological approaches for the multiplex determination of several types of post-vaccinal antibodies is of great importance for poultry farming, due to the high economic importance of both the industry itself and the importance of measures to maintain the health of the livestock. The use of digital scanning systems and software processing of the results can provide rapid simultaneous assessment of antibody titres to several infections, reducing the cost of samples testing by several times. At the same time, the developed approaches and analytical systems can be used for livestock monitoring in other areas of veterinary medicine and agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eNone of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical statement\u003c/h2\u003e \u003cp\u003eAll applicable international, national and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to publish\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Russian Science Foundation (project no 22-74-00018).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Experiment, data collection and analysis were performed by J.V.S., N.Yu.S., G.V.P and M.Yu.R. M.Yu.R. and A.P.O. critically reviewed the manuscript. The first draft of the manuscript was written by N.Yu.S and J.V.S. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eData is provided within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAparna GM, Tetala KKR (2023) Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays. 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Anal Bioanal Chem 412:8135\u0026ndash;8144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-020-02944-7\u003c/span\u003e\u003cspan address=\"10.1007/s00216-020-02944-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"post-vaccinal antibodies, serological control, multiplex assay, Newcastle disease, infectious bronchitis, bursal disease","lastPublishedDoi":"10.21203/rs.3.rs-3982114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3982114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d\u0026thinsp;=\u0026thinsp;400 \u0026micro;m each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High, medium or low antibody titre level for each of three infections could be quickly assessed visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation.\u003c/p\u003e","manuscriptTitle":"Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 12:03:24","doi":"10.21203/rs.3.rs-3982114/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-14T07:21:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-20T10:19:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265f78df-d2a8-4139-bd5a-08bb4c69a391","date":"2024-03-10T01:49:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-03T14:35:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-27T14:30:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-27T14:30:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2024-02-23T14:18:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"91caa4f1-3e81-432f-875c-05fcfb4c0b10","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-22T18:29:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 12:03:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3982114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3982114","identity":"rs-3982114","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}