A Rapid and Sensitive Lanthanide-Based Fluorescent Microsphere Immunochromatographic Test for Quantitative Detection of infectious bursal disease virus (IBDV) in Poultry

A Rapid and Sensitive Lanthanide-Based Fluorescent Microsphere Immunochromatographic Test for Quantitative Detection of infectious bursal disease virus (IBDV) 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 Research Article A Rapid and Sensitive Lanthanide-Based Fluorescent Microsphere Immunochromatographic Test for Quantitative Detection of infectious bursal disease virus (IBDV) in Poultry Yuxin Wu, Zekai Zhang, Haiqi Zhao, Lizhong Miao, Weiqin Meng, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8346765/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Infectious bursal disease virus (IBDV) is a major immunosuppressive pathogen threatening global poultry production. The emergence of very virulent (vvIBDV) and novel variant strains (nVarIBDV) has increased immune escape and reduced vaccine efficacy, underscoring the need for rapid and reliable diagnostic tools. This study developed a simple, rapid, and sensitive fluorescent microsphere immunochromatographic test strip (FM-ICTS) for quantitative detection of IBDV antigen. The FM-ICTS was constructed using lanthanide fluorescent microspheres conjugated to an anti-VP2 monoclonal antibody within a double-antibody sandwich lateral flow format. Reaction conditions were optimized, and a quantitative standard curve was established using serial dilutions of inactivated IBDV (B87 strain). The assay generated stable fluorescence signals within 15 minutes and showed high analytical sensitivity, detecting IBDV antigen at dilutions up to 1:5120. No cross-reactivity with CAV, ALV, AIV, or MDV was observed, confirming excellent specificity. Repeatability tests demonstrated low coefficients of variation (< 10%). When evaluated using 50 clinical samples, FM-ICTS exhibited 97.5% concordance with the national standard RT-PCR method and accurately identified vvIBDV, clIBDV, and nVarIBDV strains. Overall, the developed FM-ICTS provides a rapid, sensitive, specific, and quantitative method suitable for field diagnostics and vaccine quality monitoring, supporting timely prevention and control of IBD. Infectious bursal disease virus rapid antigen detection fluorescent microspheres lateral flow immunochromatography quantitative assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Infectious bursal disease (IBD), one of the most impactful immunosuppressive diseases in the poultry sector, has caused substantial economic losses to the poultry industry in China and worldwide [Mundt E et al 1995]. The causative agent, infectious bursal disease virus (IBDV), is a unique double-stranded RNA virus composed of two distinct genomic segments [Müller H et al 1979]. Among these, segment A encodes the VP2 protein, which not only serves as a major structural component of the virus but also plays a critical role in virulence determination, antigenic variation, and cytophilia [Brandt M et al 2001, Coulibaly F et al 2005, Letzel T et al 2007]. Baylis et al. reported that the vp2 gene contains a highly variable region, particularly between amino acid positions 206 and 350 [Bayliss CD et al 1990]. Infection with IBDV leads to pronounced atrophy of the bursa of Fabricius, a key immune organ in young chickens, resulting in severe immunosuppression. This immunocompromised state increases susceptibility to secondary infections and markedly reduces vaccine efficacy, posing a significant economic burden to the poultry industry [Saif YM 1991]. The classical infectious bursal disease virus (clIBDV) was first identified in the United States in 1957. In 1987, a mutant strain emerged that was capable of evading immunity induced by vaccines based on the classical strain [Jackwood DH 1987]. Shortly thereafter, very virulent IBDV (vvIBDV) appeared and rapidly spread worldwide, causing high mortality and raising significant concern [Pikuła A et al, 2020]. More recently, the poultry industry has faced additional challenges with the emergence of novel variant IBDV (nVarIBDV). Studies have demonstrated that nVarIBDV can escape immune protection provided by vaccines developed against clIBDV and vvIBDV [Wang Y et al, 2021; Hou B, et al, 2022]. However, the development of new vaccines requires a relatively long time. Therefore, there is an urgent need to establish rapid and effective diagnostic tools to accurately distinguish infected flocks from uninfected ones, thereby helping to reduce disease transmission. Currently, several approaches are available for the clinical detection of IBDV, including the agar gel precipitation test (AGPT) and reverse transcription-polymerase chain reaction (RT-PCR). Although each method offers specific diagnostic value, they also have notable limitations. Many existing techniques are time-consuming, labor-intensive, and require skilled personnel as well as costly instruments and equipment. The detection procedures can be particularly complex [Wang C et al, 2023; Howie R and Thorsen J, 1981]. These challenges restrict the widespread application of such methods in grassroots farms and resource-limited settings, making timely clinical diagnosis of IBDV difficult. Due to its speed and simplicity, lateral flow immunoassay (LFIA) particularly the colloidal gold immunochromatographic assay (GICA) is widely used in clinical settings for the diagnosis of numerous diseases [Barbosa Júnior WL et al, 2015; Dinga DK et al, 2023]. However, as GICA has become more broadly applied and extensively studied, several limitations have become evident. These include subjective interpretation due to naked-eye observation and relatively low detection sensitivity [Dong S et al 2023]. Moreover, GICA provides only qualitative results and cannot deliver quantitative measurements. To overcome these constraints, innovative nanomaterials such as fluorescent dye embedded microspheres are increasingly being employed to enhance sensitivity and enable more reliable detection. Time-resolved fluorescent microspheres (TRFM) are composed of trace lanthanide elements (e.g., Eu³⁺, Tb³⁺, Sm³⁺, Dy³⁺) embedded within a microsphere matrix. Upon excitation by a UV light source, these microspheres emit light with distinct fluorescent characteristics. Their long fluorescence lifetime, large Stokes shift, and high fluorescence intensity significantly improve detection sensitivity and accuracy, making TRFM particularly effective for identifying substances present at low concentrations [Li Z et al, 2021; Wang H et al, 2021; Dong S et al 2023]. Conventional immunochromatographic techniques are valued for their high-throughput capabilities. When fluorescent microspheres (FM) are incorporated, they form fluorescent microsphere immunochromatography (FMIA), which not only preserves high-throughput detection but also markedly improves sensitivity for more refined diagnostic applications [; Cho U et al, 2020]. A key advantage of FMIA is its ability to quantitatively assess viral load by measuring fluorescence signal intensity using a fluorescence analyzer, thereby enhancing both the accuracy and reliability of the results. In this study, we employed fluorescent microspheres as highly efficient fluorescent probes and covalently coupled an anti-IBDV monoclonal antibody (IBD-mAb) specific to IBDV onto the surface of the microspheres using a precise chemical coupling technique. This approach enabled the construction of a fluorescent microsphere-based immunochromatographic test strip (FM-ICTS) following a double-antibody sandwich principle. Accordingly, our study aimed to develop a sensitive and efficient detection method for IBDV by leveraging the specific antibody antigen antibody recognition mechanism. 2. Materials and Methods 2.1 Clinical samples and strains A total of 50 clinical samples and 40 IBDV-negative control samples were collected from the Shandong Provincial Poultry Industry and Technology System at Shandong Binzhou Animal Science and Veterinary Medicine Academy. The IBDV vaccine strain B87 was obtained from the China National Institutes for Food and Drug Control (Beijing, China). 2.2 Preparation of fluorescent microsphere probes To activate the fluorescent microspheres, 0.4 mL of borate buffer (0.05 M, pH 8.2) and 15 µL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 10 mg/mL; Sigma Aldrich) were added sequentially to 100 µL of fluorescent microspheres (365/610 nm, 1% solids; Nanjing Microdetection Bio-Technology Co., Ltd., Nanjing, China). The mixture was gently shaken at room temperature for 15 min. The microspheres were then centrifuged at 12,000 rpm for 10 min at 10°C to remove the supernatant and resuspended in 0.5 mL of the same borate buffer. After sonication for 1 min, 40 µg of monoclonal antibody-1 (mAb-1; Luoyang Gushuo Bio-Technology Co., Ltd., Henan, China) was added, and the mixture was incubated on a shaker for 2 h. Bovine serum albumin (BSA) was then added to a final concentration of 1% and incubated for an additional 2 h. Finally, the microspheres were centrifuged, resuspended in 0.1% BSA in borate buffer (0.05 M, pH 8.2), sonicated again, and stored at 4°C until further use. 2.3 Composition and implementation of FM-ICTS The FM-ICTS was assembled on a plastic base plate comprising a sample pad, conjugate pad, absorbent pad, and nitrocellulose (NC) membrane (Shanghai Jieyi Bio-Technology Co., Ltd., Shanghai, China). The mAb-1–fluorescent microsphere complex was applied to the conjugate pad using a BioDot-XYZ3210 film sprayer (Irvine, CA, USA). Goat anti-mouse IgG and anti-IBDV mAb-2 were immobilized on the NC membrane as the control line (C-line) and test line (T-line), respectively. The strips were cut to 4.0 × 80 mm, packaged, and stored dry until use. Bursa and spleen tissues from IBDV-infected chickens were homogenized, lysed in PBS (0.01 M, pH 7.2–7.4), and the supernatant mixed with sample diluent (PBS with 0.3% BSA and 0.2% Triton X-100). A 65 µL aliquot was applied to each strip, allowing the sample to migrate. IBDV antigens bound to mAb-1 were captured at the T-line, producing a fluorescence signal measured by a fluorescence immunoassay analyzer (Nanjing Microdetection Bio-Technology Co., Ltd., Nanjing, China), while excess mAb-1 reached the C-line as a control. Weak or absent C-line signals indicate mishandling, strip failure, or sample interference. The reaction process of FM-ICTS during detection is described in (Fig-1) below. 2.4 Determination of FM-ICTS reaction conditions To ensure FM-ICTS accuracy, positive and negative samples were diluted and added to the sample wells. Fluorescence of the T- and C-lines was measured every 2 minutes at room temperature, and the T/C ratio was calculated. The interpretation time was defined when the T/C ratio stabilized. For T-line optimization, mAb-2 was coated at 0.2, 0.4, 0.6, and 0.8 mg/mL, while the C-line antibody was fixed at 1 mg/mL. Samples were diluted 1:50, and the concentration yielding the highest T/C ratio for positive samples was selected as optimal. 2.5 Modelling of FM-ICTS quantitative testing For quantitative IBDV detection using FM-ICTS, the B87 vaccine strain (10⁴.⁵ TCID₅₀/0.1 mL), inactivated with β-propiolactone, was used as the standard. Two-fold serial dilutions (2¹–2¹²) were applied to the sample wells, and T/C values were measured. A standard curve was generated, and the coefficient of determination (R²) was calculated to assess the fit. 2.6 Performance evaluation of FM-ICTS To determine the FM-ICTS threshold, 40 IBDV-negative samples validated by the national RT-PCR standard were tested. T/C values were measured, and the threshold was set as the mean (X) plus three standard deviations (X + 3SD). Sensitivity, specificity, and repeatability were assessed in three experiments. Positive samples were serially diluted (1:10–1:10,240) to determine the detection limit against the threshold. Samples containing other avian viruses (CAV, ALV, AIV, MDV) evaluated specificity. Repeatability was tested using IBDV-positive samples diluted 1:10–1:320. 3. Results 3.1 Determination of FM-ICTS reaction conditions Fluorescence intensities of the T- and C-lines and the corresponding T/C ratios were measured every 2 minutes. All values stabilized at 15 minutes, which was selected as the optimal interpretation time (Fig. 2 A). Evaluation of different mAb-2 coating concentrations showed that 0.4 mg/mL produced the strongest and most stable T-line signals for positive samples while maintaining minimal background for negatives (Fig. 2 B). 3.2 Establishment of quantitative detection A standard curve was constructed using two-fold serial dilutions (2²–2¹²) of inactivated IBDV (B87 strain). A nonlinear regression model was obtained with a high goodness of fit (R² = 0.9861), defining the quantitative detection range (Fig. 3 A). At extremely high viral concentrations (2¹ dilution), T/C values decreased due to the hook effect. 3.3 Performance evaluation of FM-ICTS 3.3.1. Threshold Forty RT-PCR–negative samples were tested, yielding a cutoff value of 0.0152, calculated as mean T/C + 3SD. Samples above this value were considered positive (Fig. 3 B). (A) Fitted curve for virus dilutions in the range 2²–2¹², showing the relationship between T/C values and (log_2 \(\:\left(c\right)\) (virus dilution). Shaded areas represent 95% confidence and prediction intervals. (B) T/C values of 40 negative samples used to define the detection threshold. The box shows the interquartile range (25th–75th percentiles), whiskers represent 1.5× the interquartile range, black squares indicate the mean, and purple dots represent individual samples. 3.3.2. Sensitivity Serial dilutions of inactivated IBDV showed a limit of detection at 1:5120, which remained above the cutoff. Dilutions at 1:10240 produced T/C values below the threshold (Fig. 4 B), consistent with the visual results shown under UV illumination (Fig. 4 A). Compared with ELISA [ De Herdt P et al, 2005] and RT-PCR [Wang C et al, 2023], FM-ICTS demonstrated higher sensitivity, highlighting its capability to detect IBDV even at very low concentrations. 3.3.3. Specificity Testing against CAV, ALV, AIV, and MDV confirmed that only IBDV-positive samples generated T-line fluorescence above the threshold. No cross-reactivity was observed (Fig. 5 A–B). 3.3.4. Repeatability Across three replicate assays for each dilution (1:10–1:320), all coefficient-of-variation (CV) values were below 25%, with a maximum CV of 8.31% and an average of 4.88%. 3.4 Evaluation of FM-ICTS clinical testing capabilities A total of 50 clinical samples were tested using FM-ICTS and the national standard RT-PCR assay. RT-PCR detected 40 positives, while FM-ICTS detected 38. Sequencing confirmed 23 vvIBDV, 9 clIBDV, and 8 nVarIBDV among RT-PCR–positive samples. The overall concordance rate between FM-ICTS and RT-PCR was 97.5% (Table 1). Table.1 Comparison of the national standard RT-PCR method and FM-ICTS results Positive/sample Positive rate/% Compliance rate (%) National standard RT-PCR method 40 80 97.5% FM-ICTS 38 76% 4. Discussion Infectious bursal disease (IBD) continues to pose a substantial threat to poultry health and productivity worldwide. The epidemiological landscape of IBDV has become increasingly complex due to the emergence of very virulent strains (vvIBDV) and novel variant strains (nVarIBDV), which often escape immune protection conferred by conventional vaccines [Jackwood and Saif, 1987; Pikuła et al., 2020; Hou et al., 2022]. The VP2 hypervariable region (HVR), which dictates antigenicity and virulence, evolves rapidly and complicates diagnostic accuracy [Bayliss et al., 1990; Brandt et al., 2001]. Timely field diagnosis is therefore essential for containing outbreaks, guiding vaccination strategies, and supporting surveillance programs. However, existing methods such as reverse transcription-polymerase chain reaction (RT-PCR), ELISA, and agarose gel precipitation (AGPT) require trained personnel, laboratory infrastructure, and longer turnaround times [Howie & Thorsen, 1981; Wang et al., 2023]. Furthermore, while colloidal gold immunochromatographic assays (GICA) provide rapid field testing, they have a shortcoming of low sensitivity and subjective visual interpretation [Barbosa Júnior et al., 2015; Dong et al., 2023]. The present study addresses these challenges by developing a time-resolved fluorescent microsphere–based immunochromatographic test strip (FM-ICTS), leveraging the advantages of lanthanide fluorescent probes. These microspheres offer extended fluorescence lifetimes, large stokes shifts, and high signal stability [Li et al., 2021; Cho & Chen, 2020], enabling quantitative and sensitive detection suited for field and resource-limited settings. The optimization of reaction conditions is crucial for the reliability of rapid diagnostic assays. The determination of a 15-minute optimal interpretation time demonstrates that the antigen–antibody interaction reaches equilibrium rapidly under the established assay format. This is consistent with other lanthanide-based LFIA systems, which typically show stable signal formation within 10–20 minutes due to efficient fluorescent probe–antigen interactions [Wang H. et al., 2019]. Optimizing the T-line antibody concentration to 0.4 mg/mL ensured strong binding affinity while avoiding steric hindrance or nonspecific adsorption. Proper antibody density is essential to maintain signal-to-noise ratios and prevent weak-positive masking—challenges often seen in traditional GICA methods [Dong et al., 2023]. A key advancement of this study is the establishment of a quantitative relationship between viral load and fluorescence intensity. The high goodness of fit (R² = 0.9861) observed for the nonlinear standard curve illustrates that T/C fluorescence values strongly correlate with viral concentration across the dilution range of 2²–2¹². This aligns with prior findings showing that lanthanide-based immunochromatography supports robust quantitative relationships between fluorescence intensity and antigen load [Li et al., 2021; Wang H. et al., 2019]. The observed hook effect at extremely high viral concentrations (2¹ dilution) mirrors previously reported antigen-excess phenomena in immunoassays, where the T-line signal saturates or decreases at high antigen levels [Chen et al., 2022]. This reinforces the need for controlled sample dilution—particularly for tissue homogenates or high-titer vaccine material to maintain quantitative accuracy. Importantly, the system’s effective detection range corresponds well with viral loads encountered in field samples and commercial vaccines, highlighting its practical utility for both diagnostics and vaccine quality monitoring. Using negative samples to establish an objective threshold (T/C = 0.0152) addresses the main limitation of colloidal gold–based assays subjective visual interpretation [Barbosa Júnior et al., 2015]. Quantitative thresholds reduce operator bias and ensure consistent interpretation across laboratories. Such threshold-based systems are recommended for high-throughput surveillance and on-farm rapid decision-making. In terms of analytical performance, the FM-ICTS demonstrated superior sensitivity compared to traditional methods. The assay successfully detected IBDV antigen at dilutions up to 1:5120, substantially higher than that of many ELISA and RT-PCR methods reported for IBDV antigen detection [De Herdt et al., 2005; Wang C. et al., 2023]. This high level of sensitivity is attributed to the high fluorescence intensity of the microspheres, which allows for the identification of substances at lower concentrations than colloidal gold assays [Dinga et al., 2023; Dong et al., 2023]. Early detection is crucial for limiting flock-level immunosuppression and associated secondary infections [Saif, 1991]. The absence of cross-reactivity with CAV, ALV, AIV, and MDV confirms the strong epitope specificity of the anti-VP2 monoclonal antibodies used. This is consistent with VP2’s unique structural and antigenic determinants [Letzel et al., 2007; Coulibaly et al., 2005]. The distinct cutoff threshold established in this study (T/C > 0.0152) further removes the subjectivity associated with naked-eye observations in GICA [22], providing a standardized metric for result interpretation Specificity is a paramount requirement for field diagnostics, particularly given the prevalence of co-infections in poultry flocks. IBDV is frequently found alongside other immunosuppressive or oncogenic viruses such as Chicken Anemia Virus (CAV), Avian Leukosis Virus (ALV), and Marek’s Disease Virus (MDV) [Chen W. et al., 2022; Eladl et al., 2020]. The FM-ICTS showed no cross-reactivity with these common avian pathogens, confirming that the assay relies on a highly specific antibody-antigen recognition mechanism. The assay’s low coefficient of variation (average 4.88%) demonstrates excellent intra-assay precision and aligns with accepted reproducibility standards for lateral flow diagnostics [Villa et al., 2018]. Such consistency is necessary for quantitative field assays that may be used repeatedly for flock monitoring or vaccine batch quality control. The high concordance (97.5%) between FM-ICTS and national standard RT-PCR results highlights the system’s reliability for field-level diagnosis. Importantly, FM-ICTS successfully identified vvIBDV, clIBDV, and nVarIBDV strains, supporting its broad reactivity across diverse genotypes. Given the ongoing emergence of novel reassortant strains with enhanced pathogenicity and immune escape potential [Pikuła et al., 2020; Wang Y. et al., 2021], a rapid assay that reliably detects multiple genotypes is particularly valuable. Furthermore, the ability to quantify viral load provides additional advantages not available from conventional qualitative test strips supporting disease staging, vaccine take assessment, and monitoring of viral shedding patterns. 5. Conclusions In this study, we developed a rapid quantitative method for detecting IBDV by integrating lanthanide fluorescent microspheres with LFIA technology. The FM-ICTS demonstrated strong performance, showing high sensitivity, specificity, and stability. The assay can be completed within 15 minutes, significantly faster than many conventional methods while remaining simple to operate, cost-effective, and suitable for routine use in laboratories and farms. This provides valuable technical support for rapid screening and diagnosis of IBDV in clinical settings. The method also shows promise for applications such as monitoring IBDV vaccine quality. Furthermore, because the system uses monoclonal antibody–based fluorescent microspheres, it can be adapted for detecting other viruses simply by changing the specific monoclonal antibodies used, highlighting its broad utility. Declarations Author Contributions : Yuxin Wu: conceptualization; methodology; data curation; visualization; writing-original draft; Zekai Zhang: data curation; validation; methodology Haiqi Zhao: investigation; conceptualization Lizhong Miao: resources; project administration Weiqin Meng: investigation; methodology Shuhong Sun: supervision; visualization Qingqing Xu: conceptualization; methodology Ashenafi Kiros Wubshet: methodology , data curation, writing-review and editing Jinliang Wang: visualization; resources Zhiqiang Shen: conceptualization; formal analysis; supervision Na Tang: project administration; funding acquisition; writing-review and editing Funding : This work was supported by the National Key Research and Development Program of China (Grant No.2023YFE0106100), Shandong Provincial Poultry Industry and Technology System (Grant No. SDAIT-11-16) and Grant No. WSR2023042 Data Availability Statement : Data will be available on request. 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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-8346765","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589063284,"identity":"ed96fd64-53a8-4361-8aef-66ee10b52728","order_by":0,"name":"Yuxin Wu","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271000, China","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Wu","suffix":""},{"id":589063285,"identity":"39245fa1-b9dd-440a-9c09-e357f24a6d51","order_by":1,"name":"Zekai Zhang","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Zekai","middleName":"","lastName":"Zhang","suffix":""},{"id":589063286,"identity":"b870b8fd-eaa9-4ab0-bb4d-ea4954c47762","order_by":2,"name":"Haiqi Zhao","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Haiqi","middleName":"","lastName":"Zhao","suffix":""},{"id":589063287,"identity":"d8a356ad-f709-49b4-942b-c32e6be036f5","order_by":3,"name":"Lizhong Miao","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Lizhong","middleName":"","lastName":"Miao","suffix":""},{"id":589063288,"identity":"dc0ec74d-df2c-40f1-9d32-3aa31b905aae","order_by":4,"name":"Weiqin Meng","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Weiqin","middleName":"","lastName":"Meng","suffix":""},{"id":589063289,"identity":"e1459fa9-58b1-4cf7-89c6-8001079bf2b8","order_by":5,"name":"Shuhong Sun","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271000, China","correspondingAuthor":false,"prefix":"","firstName":"Shuhong","middleName":"","lastName":"Sun","suffix":""},{"id":589063290,"identity":"3317dc73-c99b-409b-8a4f-ad7a9632ec43","order_by":6,"name":"Qingqing Xu","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Qingqing","middleName":"","lastName":"Xu","suffix":""},{"id":589063291,"identity":"49509ee3-cf1b-4f3f-87aa-eb2808291cfa","order_by":7,"name":"Ashenafi Kiros Wubshet","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Ashenafi","middleName":"Kiros","lastName":"Wubshet","suffix":""},{"id":589063292,"identity":"6d9d2eb6-b670-423a-9698-55d454d7103a","order_by":8,"name":"Jinliang Wang","email":"","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":false,"prefix":"","firstName":"Jinliang","middleName":"","lastName":"Wang","suffix":""},{"id":589063293,"identity":"adac63c2-ae41-4b80-aedd-2c8f7e93ca98","order_by":9,"name":"Zhiqiang Shen","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271000, China","correspondingAuthor":false,"prefix":"","firstName":"Zhiqiang","middleName":"","lastName":"Shen","suffix":""},{"id":589063294,"identity":"428330cc-030c-436d-9d2b-cf6652192c1b","order_by":10,"name":"Na Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYPACiXrG/uYDBz78IEItD4SySWCecSzx4Mwe4rWkJbA35Bgf5mAjQos9+9nDrwtqDufxNpz5cBhogjy/2AECtvDkpVnPOHa4WLK5d8PhAgsGw5mzEwg5LMfMmIftMOPGhrMbDs/gYUgwuE1IC/8boJZ/hxn3H8h5cJiHjRgtEjnGj3nb0hIbG3IYiNRy440ZM2+fjTHjjGMGwECWIOwX9v4c48883yTkgFH5+MOHHzby/NIEtAABmwQSRwKnMmTA/IEoZaNgFIyCUTByAQD+T0fYGKj2qwAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong Binzhou institute of Animal husbandry \u0026 Veterinary Science, Bingzhou 256600, China","correspondingAuthor":true,"prefix":"","firstName":"Na","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2025-12-12 14:38:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8346765/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8346765/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102433376,"identity":"43d0a1e7-ce56-4431-85fc-17c4a1a8b048","added_by":"auto","created_at":"2026-02-11 15:41:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":275104,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of FM-ICTS for IBDV reaction process during detection. The strip includes a sample well, conjugate pad, nitrocellulose membrane with test (T) and control (C) lines, and absorbent pad. Sample mixed with buffer flows laterally, where IBDV binds fluorescent mAb-1. Complexes are captured on the T-line, excess conjugates bind the C-line, and fluorescence is visualized under UV or measured by a fluorescence analyzer. (Created in BioRender. Wu, Y. (2024) BioRender. com/f14s606)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8346765/v1/3fda26efe24a8f8445c919e9.png"},{"id":102433464,"identity":"b16badcf-a4bb-426d-a464-29893dd3de8b","added_by":"auto","created_at":"2026-02-11 15:42:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":281577,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of FM-ICTS reaction conditions. (A) Fluorescence intensity of the C-line (dark blue circles) and T-line (light blue squares) and the T/C ratio (red triangles) were measured over time. Both C- and T-line signals increased, while the T/C ratio stabilized after 15 min, indicating completion of the immunoreaction. (B) Optimization of T-line concentration. Positive (striped) and negative (solid) samples were tested at varying mAb-2 concentrations. The highest T/C ratio with strong stability was observed at 0.4 mg/mL.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8346765/v1/7e118f951a42a9ac367dafea.png"},{"id":102433463,"identity":"e58dd640-a0a2-4236-80a4-7c046651122b","added_by":"auto","created_at":"2026-02-11 15:42:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":324185,"visible":true,"origin":"","legend":"\u003cp\u003eFM-ICTS interpretation and quantification criteria.\u003c/p\u003e\n\u003cp\u003e(A) Fitted curve for virus dilutions in the range 2²–2¹², showing the relationship between T/C values and (log_2(c) (virus dilution). Shaded areas represent 95% confidence and prediction intervals. (B) T/C values of 40 negative samples used to define the detection threshold. The box shows the interquartile range (25th–75th percentiles), whiskers represent 1.5× the interquartile range, black squares indicate the mean, and purple dots represent individual samples.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8346765/v1/1c423b627e4de83a793f5d3c.png"},{"id":102433597,"identity":"399951dc-8622-4f9f-8285-3f1d33db37b1","added_by":"auto","created_at":"2026-02-11 15:42:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":263171,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity assessment of FM-ICTS. (A) Detection results under a portable UV lamp. (B) Corresponding T/C values at each tested concentration.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8346765/v1/232fb697feed957cb101fe0c.png"},{"id":102433469,"identity":"b02ac2a2-464e-4a02-85a9-051240d236da","added_by":"auto","created_at":"2026-02-11 15:42:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":254966,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity assessment of FM-ICTS. (A) Specificity results visualized under a portable UV lamp. (B) Corresponding T/C values for each tested antigen.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8346765/v1/c82c8001cba599033f5e4a1a.png"},{"id":102433663,"identity":"5c13d23f-9d1e-423d-b25c-0319dd6a8db9","added_by":"auto","created_at":"2026-02-11 15:42:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2306329,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8346765/v1/6ff192e3-e797-4ba5-854c-cebf819b7ba3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Rapid and Sensitive Lanthanide-Based Fluorescent Microsphere Immunochromatographic Test for Quantitative Detection of infectious bursal disease virus (IBDV) in Poultry","fulltext":[{"header":"1. Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInfectious bursal disease (IBD), one of the most impactful immunosuppressive diseases in the poultry sector, has caused substantial economic losses to the poultry industry in China and worldwide [Mundt E et al 1995]. The causative agent, infectious bursal disease virus (IBDV), is a unique double-stranded RNA virus composed of two distinct genomic segments [M\u0026uuml;ller H et al 1979]. Among these, segment A encodes the VP2 protein, which not only serves as a major structural component of the virus but also plays a critical role in virulence determination, antigenic variation, and cytophilia [Brandt M et al 2001, Coulibaly F et al 2005, Letzel T et al 2007]. Baylis et al. reported that the vp2 gene contains a highly variable region, particularly between amino acid positions 206 and 350 [Bayliss CD et al 1990]. Infection with IBDV leads to pronounced atrophy of the bursa of Fabricius, a key immune organ in young chickens, resulting in severe immunosuppression. This immunocompromised state increases susceptibility to secondary infections and markedly reduces vaccine efficacy, posing a significant economic burden to the poultry industry [Saif YM 1991].\u003c/p\u003e \u003cp\u003eThe classical infectious bursal disease virus (clIBDV) was first identified in the United States in 1957. In 1987, a mutant strain emerged that was capable of evading immunity induced by vaccines based on the classical strain [Jackwood DH 1987]. Shortly thereafter, very virulent IBDV (vvIBDV) appeared and rapidly spread worldwide, causing high mortality and raising significant concern [Pikuła A et al, 2020]. More recently, the poultry industry has faced additional challenges with the emergence of novel variant IBDV (nVarIBDV). Studies have demonstrated that nVarIBDV can escape immune protection provided by vaccines developed against clIBDV and vvIBDV [Wang Y et al, 2021; Hou B, et al, 2022]. However, the development of new vaccines requires a relatively long time. Therefore, there is an urgent need to establish rapid and effective diagnostic tools to accurately distinguish infected flocks from uninfected ones, thereby helping to reduce disease transmission.\u003c/p\u003e \u003cp\u003eCurrently, several approaches are available for the clinical detection of IBDV, including the agar gel precipitation test (AGPT) and reverse transcription-polymerase chain reaction (RT-PCR). Although each method offers specific diagnostic value, they also have notable limitations. Many existing techniques are time-consuming, labor-intensive, and require skilled personnel as well as costly instruments and equipment. The detection procedures can be particularly complex [Wang C et al, 2023; Howie R and Thorsen J, 1981]. These challenges restrict the widespread application of such methods in grassroots farms and resource-limited settings, making timely clinical diagnosis of IBDV difficult.\u003c/p\u003e \u003cp\u003eDue to its speed and simplicity, lateral flow immunoassay (LFIA) particularly the colloidal gold immunochromatographic assay (GICA) is widely used in clinical settings for the diagnosis of numerous diseases [Barbosa J\u0026uacute;nior WL et al, 2015; Dinga DK et al, 2023]. However, as GICA has become more broadly applied and extensively studied, several limitations have become evident. These include subjective interpretation due to naked-eye observation and relatively low detection sensitivity [Dong S et al 2023]. Moreover, GICA provides only qualitative results and cannot deliver quantitative measurements. To overcome these constraints, innovative nanomaterials such as fluorescent dye embedded microspheres are increasingly being employed to enhance sensitivity and enable more reliable detection.\u003c/p\u003e \u003cp\u003eTime-resolved fluorescent microspheres (TRFM) are composed of trace lanthanide elements (e.g., Eu\u0026sup3;⁺, Tb\u0026sup3;⁺, Sm\u0026sup3;⁺, Dy\u0026sup3;⁺) embedded within a microsphere matrix. Upon excitation by a UV light source, these microspheres emit light with distinct fluorescent characteristics. Their long fluorescence lifetime, large Stokes shift, and high fluorescence intensity significantly improve detection sensitivity and accuracy, making TRFM particularly effective for identifying substances present at low concentrations [Li Z et al, 2021; Wang H et al, 2021; Dong S et al 2023]. Conventional immunochromatographic techniques are valued for their high-throughput capabilities. When fluorescent microspheres (FM) are incorporated, they form fluorescent microsphere immunochromatography (FMIA), which not only preserves high-throughput detection but also markedly improves sensitivity for more refined diagnostic applications [; Cho U et al, 2020]. A key advantage of FMIA is its ability to quantitatively assess viral load by measuring fluorescence signal intensity using a fluorescence analyzer, thereby enhancing both the accuracy and reliability of the results.\u003c/p\u003e \u003cp\u003eIn this study, we employed fluorescent microspheres as highly efficient fluorescent probes and covalently coupled an anti-IBDV monoclonal antibody (IBD-mAb) specific to IBDV onto the surface of the microspheres using a precise chemical coupling technique. This approach enabled the construction of a fluorescent microsphere-based immunochromatographic test strip (FM-ICTS) following a double-antibody sandwich principle. Accordingly, our study aimed to develop a sensitive and efficient detection method for IBDV by leveraging the specific antibody antigen antibody recognition mechanism.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Clinical samples and strains\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA total of 50 clinical samples and 40 IBDV-negative control samples were collected from the Shandong Provincial Poultry Industry and Technology System at Shandong Binzhou Animal Science and Veterinary Medicine Academy. The IBDV vaccine strain B87 was obtained from the China National Institutes for Food and Drug Control (Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of fluorescent microsphere probes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo activate the fluorescent microspheres, 0.4 mL of borate buffer (0.05 M, pH 8.2) and 15 \u0026micro;L of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 10 mg/mL; Sigma Aldrich) were added sequentially to 100 \u0026micro;L of fluorescent microspheres (365/610 nm, 1% solids; Nanjing Microdetection Bio-Technology Co., Ltd., Nanjing, China). The mixture was gently shaken at room temperature for 15 min. The microspheres were then centrifuged at 12,000 rpm for 10 min at 10\u0026deg;C to remove the supernatant and resuspended in 0.5 mL of the same borate buffer. After sonication for 1 min, 40 \u0026micro;g of monoclonal antibody-1 (mAb-1; Luoyang Gushuo Bio-Technology Co., Ltd., Henan, China) was added, and the mixture was incubated on a shaker for 2 h. Bovine serum albumin (BSA) was then added to a final concentration of 1% and incubated for an additional 2 h. Finally, the microspheres were centrifuged, resuspended in 0.1% BSA in borate buffer (0.05 M, pH 8.2), sonicated again, and stored at 4\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Composition and implementation of FM-ICTS\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe FM-ICTS was assembled on a plastic base plate comprising a sample pad, conjugate pad, absorbent pad, and nitrocellulose (NC) membrane (Shanghai Jieyi Bio-Technology Co., Ltd., Shanghai, China). The mAb-1\u0026ndash;fluorescent microsphere complex was applied to the conjugate pad using a BioDot-XYZ3210 film sprayer (Irvine, CA, USA). Goat anti-mouse IgG and anti-IBDV mAb-2 were immobilized on the NC membrane as the control line (C-line) and test line (T-line), respectively. The strips were cut to 4.0 \u0026times; 80 mm, packaged, and stored dry until use.\u003c/p\u003e \u003cp\u003eBursa and spleen tissues from IBDV-infected chickens were homogenized, lysed in PBS (0.01 M, pH 7.2\u0026ndash;7.4), and the supernatant mixed with sample diluent (PBS with 0.3% BSA and 0.2% Triton X-100). A 65 \u0026micro;L aliquot was applied to each strip, allowing the sample to migrate. IBDV antigens bound to mAb-1 were captured at the T-line, producing a fluorescence signal measured by a fluorescence immunoassay analyzer (Nanjing Microdetection Bio-Technology Co., Ltd., Nanjing, China), while excess mAb-1 reached the C-line as a control. Weak or absent C-line signals indicate mishandling, strip failure, or sample interference. The reaction process of FM-ICTS during detection is described in (Fig-1) below.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of FM-ICTS reaction conditions\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo ensure FM-ICTS accuracy, positive and negative samples were diluted and added to the sample wells. Fluorescence of the T- and C-lines was measured every 2 minutes at room temperature, and the T/C ratio was calculated. The interpretation time was defined when the T/C ratio stabilized.\u003c/p\u003e \u003cp\u003eFor T-line optimization, mAb-2 was coated at 0.2, 0.4, 0.6, and 0.8 mg/mL, while the C-line antibody was fixed at 1 mg/mL. Samples were diluted 1:50, and the concentration yielding the highest T/C ratio for positive samples was selected as optimal.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Modelling of FM-ICTS quantitative testing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor quantitative IBDV detection using FM-ICTS, the B87 vaccine strain (10⁴.⁵ TCID₅₀/0.1 mL), inactivated with β-propiolactone, was used as the standard. Two-fold serial dilutions (2\u0026sup1;\u0026ndash;2\u0026sup1;\u0026sup2;) were applied to the sample wells, and T/C values were measured. A standard curve was generated, and the coefficient of determination (R\u0026sup2;) was calculated to assess the fit.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Performance evaluation of FM-ICTS\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo determine the FM-ICTS threshold, 40 IBDV-negative samples validated by the national RT-PCR standard were tested. T/C values were measured, and the threshold was set as the mean (X) plus three standard deviations (X\u0026thinsp;+\u0026thinsp;3SD).\u003c/p\u003e \u003cp\u003eSensitivity, specificity, and repeatability were assessed in three experiments. Positive samples were serially diluted (1:10\u0026ndash;1:10,240) to determine the detection limit against the threshold. Samples containing other avian viruses (CAV, ALV, AIV, MDV) evaluated specificity. Repeatability was tested using IBDV-positive samples diluted 1:10\u0026ndash;1:320.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Determination of FM-ICTS reaction conditions\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFluorescence intensities of the T- and C-lines and the corresponding T/C ratios were measured every 2 minutes. All values stabilized at 15 minutes, which was selected as the optimal interpretation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Evaluation of different mAb-2 coating concentrations showed that 0.4 mg/mL produced the strongest and most stable T-line signals for positive samples while maintaining minimal background for negatives (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Establishment of quantitative detection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA standard curve was constructed using two-fold serial dilutions (2\u0026sup2;\u0026ndash;2\u0026sup1;\u0026sup2;) of inactivated IBDV (B87 strain). A nonlinear regression model was obtained with a high goodness of fit (R\u0026sup2; = 0.9861), defining the quantitative detection range (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). At extremely high viral concentrations (2\u0026sup1; dilution), T/C values decreased due to the hook effect.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Performance evaluation of FM-ICTS\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Threshold\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eForty RT-PCR\u0026ndash;negative samples were tested, yielding a cutoff value of 0.0152, calculated as mean T/C\u0026thinsp;+\u0026thinsp;3SD. Samples above this value were considered positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e(A) Fitted curve for virus dilutions in the range 2\u0026sup2;\u0026ndash;2\u0026sup1;\u0026sup2;, showing the relationship between T/C values and (log_2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(c\\right)\\)\u003c/span\u003e\u003c/span\u003e (virus dilution). Shaded areas represent 95% confidence and prediction intervals. (B) T/C values of 40 negative samples used to define the detection threshold. The box shows the interquartile range (25th\u0026ndash;75th percentiles), whiskers represent 1.5\u0026times; the interquartile range, black squares indicate the mean, and purple dots represent individual samples.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Sensitivity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSerial dilutions of inactivated IBDV showed a limit of detection at 1:5120, which remained above the cutoff. Dilutions at 1:10240 produced T/C values below the threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), consistent with the visual results shown under UV illumination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared with ELISA [ De Herdt P et al, 2005] and RT-PCR [Wang C et al, 2023], FM-ICTS demonstrated higher sensitivity, highlighting its capability to detect IBDV even at very low concentrations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. Specificity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTesting against CAV, ALV, AIV, and MDV confirmed that only IBDV-positive samples generated T-line fluorescence above the threshold. No cross-reactivity was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4. Repeatability\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAcross three replicate assays for each dilution (1:10\u0026ndash;1:320), all coefficient-of-variation (CV) values were below 25%, with a maximum CV of 8.31% and an average of 4.88%.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Evaluation of FM-ICTS clinical testing capabilities\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA total of 50 clinical samples were tested using FM-ICTS and the national standard RT-PCR assay. RT-PCR detected 40 positives, while FM-ICTS detected 38. Sequencing confirmed 23 vvIBDV, 9 clIBDV, and 8 nVarIBDV among RT-PCR\u0026ndash;positive samples. The overall concordance rate between FM-ICTS and RT-PCR was 97.5% (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTable.1 Comparison of the national standard RT-PCR method and FM-ICTS results\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive/sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive rate/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompliance rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNational standard RT-PCR method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e97.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFM-ICTS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInfectious bursal disease (IBD) continues to pose a substantial threat to poultry health and productivity worldwide. The epidemiological landscape of IBDV has become increasingly complex due to the emergence of very virulent strains (vvIBDV) and novel variant strains (nVarIBDV), which often escape immune protection conferred by conventional vaccines [Jackwood and Saif, 1987; Pikuła et al., 2020; Hou et al., 2022]. The VP2 hypervariable region (HVR), which dictates antigenicity and virulence, evolves rapidly and complicates diagnostic accuracy [Bayliss et al., 1990; Brandt et al., 2001].\u003c/p\u003e \u003cp\u003eTimely field diagnosis is therefore essential for containing outbreaks, guiding vaccination strategies, and supporting surveillance programs. However, existing methods such as reverse transcription-polymerase chain reaction (RT-PCR), ELISA, and agarose gel precipitation (AGPT) require trained personnel, laboratory infrastructure, and longer turnaround times [Howie \u0026amp; Thorsen, 1981; Wang et al., 2023]. Furthermore, while colloidal gold immunochromatographic assays (GICA) provide rapid field testing, they have a shortcoming of low sensitivity and subjective visual interpretation [Barbosa J\u0026uacute;nior et al., 2015; Dong et al., 2023].\u003c/p\u003e \u003cp\u003eThe present study addresses these challenges by developing a time-resolved fluorescent microsphere\u0026ndash;based immunochromatographic test strip (FM-ICTS), leveraging the advantages of lanthanide fluorescent probes. These microspheres offer extended fluorescence lifetimes, large stokes shifts, and high signal stability [Li et al., 2021; Cho \u0026amp; Chen, 2020], enabling quantitative and sensitive detection suited for field and resource-limited settings.\u003c/p\u003e \u003cp\u003eThe optimization of reaction conditions is crucial for the reliability of rapid diagnostic assays. The determination of a 15-minute optimal interpretation time demonstrates that the antigen\u0026ndash;antibody interaction reaches equilibrium rapidly under the established assay format. This is consistent with other lanthanide-based LFIA systems, which typically show stable signal formation within 10\u0026ndash;20 minutes due to efficient fluorescent probe\u0026ndash;antigen interactions [Wang H. et al., 2019]. Optimizing the T-line antibody concentration to 0.4 mg/mL ensured strong binding affinity while avoiding steric hindrance or nonspecific adsorption. Proper antibody density is essential to maintain signal-to-noise ratios and prevent weak-positive masking\u0026mdash;challenges often seen in traditional GICA methods [Dong et al., 2023].\u003c/p\u003e \u003cp\u003eA key advancement of this study is the establishment of a quantitative relationship between viral load and fluorescence intensity. The high goodness of fit (R\u0026sup2; = 0.9861) observed for the nonlinear standard curve illustrates that T/C fluorescence values strongly correlate with viral concentration across the dilution range of 2\u0026sup2;\u0026ndash;2\u0026sup1;\u0026sup2;. This aligns with prior findings showing that lanthanide-based immunochromatography supports robust quantitative relationships between fluorescence intensity and antigen load [Li et al., 2021; Wang H. et al., 2019].\u003c/p\u003e \u003cp\u003eThe observed hook effect at extremely high viral concentrations (2\u0026sup1; dilution) mirrors previously reported antigen-excess phenomena in immunoassays, where the T-line signal saturates or decreases at high antigen levels [Chen et al., 2022]. This reinforces the need for controlled sample dilution\u0026mdash;particularly for tissue homogenates or high-titer vaccine material to maintain quantitative accuracy. Importantly, the system\u0026rsquo;s effective detection range corresponds well with viral loads encountered in field samples and commercial vaccines, highlighting its practical utility for both diagnostics and vaccine quality monitoring. Using negative samples to establish an objective threshold (T/C\u0026thinsp;=\u0026thinsp;0.0152) addresses the main limitation of colloidal gold\u0026ndash;based assays subjective visual interpretation [Barbosa J\u0026uacute;nior et al., 2015]. Quantitative thresholds reduce operator bias and ensure consistent interpretation across laboratories. Such threshold-based systems are recommended for high-throughput surveillance and on-farm rapid decision-making.\u003c/p\u003e \u003cp\u003eIn terms of analytical performance, the FM-ICTS demonstrated superior sensitivity compared to traditional methods. The assay successfully detected IBDV antigen at dilutions up to 1:5120, substantially higher than that of many ELISA and RT-PCR methods reported for IBDV antigen detection [De Herdt et al., 2005; Wang C. et al., 2023]. This high level of sensitivity is attributed to the high fluorescence intensity of the microspheres, which allows for the identification of substances at lower concentrations than colloidal gold assays [Dinga et al., 2023; Dong et al., 2023]. Early detection is crucial for limiting flock-level immunosuppression and associated secondary infections [Saif, 1991].\u003c/p\u003e \u003cp\u003eThe absence of cross-reactivity with CAV, ALV, AIV, and MDV confirms the strong epitope specificity of the anti-VP2 monoclonal antibodies used. This is consistent with VP2\u0026rsquo;s unique structural and antigenic determinants [Letzel et al., 2007; Coulibaly et al., 2005]. The distinct cutoff threshold established in this study (T/C\u0026thinsp;\u0026gt;\u0026thinsp;0.0152) further removes the subjectivity associated with naked-eye observations in GICA [22], providing a standardized metric for result interpretation\u003c/p\u003e \u003cp\u003eSpecificity is a paramount requirement for field diagnostics, particularly given the prevalence of co-infections in poultry flocks. IBDV is frequently found alongside other immunosuppressive or oncogenic viruses such as Chicken Anemia Virus (CAV), Avian Leukosis Virus (ALV), and Marek\u0026rsquo;s Disease Virus (MDV) [Chen W. et al., 2022; Eladl et al., 2020]. The FM-ICTS showed no cross-reactivity with these common avian pathogens, confirming that the assay relies on a highly specific antibody-antigen recognition mechanism.\u003c/p\u003e \u003cp\u003eThe assay\u0026rsquo;s low coefficient of variation (average 4.88%) demonstrates excellent intra-assay precision and aligns with accepted reproducibility standards for lateral flow diagnostics [Villa et al., 2018]. Such consistency is necessary for quantitative field assays that may be used repeatedly for flock monitoring or vaccine batch quality control. The high concordance (97.5%) between FM-ICTS and national standard RT-PCR results highlights the system\u0026rsquo;s reliability for field-level diagnosis. Importantly, FM-ICTS successfully identified vvIBDV, clIBDV, and nVarIBDV strains, supporting its broad reactivity across diverse genotypes. Given the ongoing emergence of novel reassortant strains with enhanced pathogenicity and immune escape potential [Pikuła et al., 2020; Wang Y. et al., 2021], a rapid assay that reliably detects multiple genotypes is particularly valuable. Furthermore, the ability to quantify viral load provides additional advantages not available from conventional qualitative test strips supporting disease staging, vaccine take assessment, and monitoring of viral shedding patterns.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, we developed a rapid quantitative method for detecting IBDV by integrating lanthanide fluorescent microspheres with LFIA technology. The FM-ICTS demonstrated strong performance, showing high sensitivity, specificity, and stability. The assay can be completed within 15 minutes, significantly faster than many conventional methods while remaining simple to operate, cost-effective, and suitable for routine use in laboratories and farms. This provides valuable technical support for rapid screening and diagnosis of IBDV in clinical settings.\u003c/p\u003e \u003cp\u003eThe method also shows promise for applications such as monitoring IBDV vaccine quality. Furthermore, because the system uses monoclonal antibody\u0026ndash;based fluorescent microspheres, it can be adapted for detecting other viruses simply by changing the specific monoclonal antibodies used, highlighting its broad utility.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Yuxin Wu: conceptualization; methodology; data curation; visualization; writing-original draft; Zekai Zhang: data curation; validation; methodology Haiqi Zhao: investigation; conceptualization Lizhong Miao: resources; project administration Weiqin Meng: investigation; methodology Shuhong Sun: supervision; visualization Qingqing Xu: conceptualization; methodology Ashenafi Kiros Wubshet: methodology , data curation, \u0026nbsp;writing-review and editing Jinliang Wang: visualization; resources Zhiqiang Shen: conceptualization; formal analysis; supervision Na Tang: project administration; funding acquisition; writing-review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by the National Key Research and Development Program of China (Grant No.2023YFE0106100), Shandong Provincial Poultry Industry and Technology System (Grant No. SDAIT-11-16) and Grant No. WSR2023042\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e: Data will be available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e: This article does not contain any studies with animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMundt E, M\u0026uuml;ller H. Complete Nucleotide Sequences of 5\u0026prime;- and 3\u0026prime;-Noncoding Regions of Both Genome Segments of Different Strains of Infectious Bursal Disease Virus. Virology. 1995;209(1):10\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller H, Scholtissek C, Becht H. The genome of infectious bursal disease virus consists of two segments of double-stranded RNA. J Virol. 1979 ;31(3):584\u0026ndash;589.\u003c/li\u003e\n\u003cli\u003eBrandt M, Yao K, Liu M, et al. Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus. J Virol. 2001;75(24):11974\u0026ndash;11982.\u003c/li\u003e\n\u003cli\u003eCoulibaly F, Chevalier C, Gutsche I, et al. The Birnavirus Crystal Structure Reveals Structural Relationships among Icosahedral Viruses. Cell. 2005;120(6):761\u0026ndash;772.\u003c/li\u003e\n\u003cli\u003eLetzel T, Coulibaly F, Rey FA, et al. Molecular and structural bases for the antigenicity of VP2 of infectious bursal disease virus. J Virol. 2007;81(23):12827\u0026ndash;12835.\u003c/li\u003e\n\u003cli\u003eBayliss CD, Spies U, Shaw K, et al. A comparison of the sequences of segment A of four infectious bursal disease virus strains and identification of a variable region in VP2. J Gen Virol. 1990;71 (Pt 6):1303\u0026ndash;1312.\u003c/li\u003e\n\u003cli\u003eSaif YM. Immunosuppression induced by infectious bursal disease virus. Veterinary Immunology and Immunopathology. 1991;30(1):45\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eJackwood DH, Saif YM. Antigenic diversity of infectious bursal disease viruses. Avian Dis. 1987;31(4):766\u0026ndash;770.\u003c/li\u003e\n\u003cli\u003ePikuła A, Śmietanka K, Perez LJ. Emergence and expansion of novel pathogenic reassortant strains of infectious bursal disease virus causing acute outbreaks of the disease in Europe. Transbound Emerg Dis. 2020;67(4):1739\u0026ndash;1744.\u003c/li\u003e\n\u003cli\u003eWang Y, Jiang N, Fan L, et al. Identification and Pathogenicity Evaluation of a Novel Reassortant Infectious Bursal Disease Virus (Genotype A2dB3). Viruses. 2021;13(9):1682.\u003c/li\u003e\n\u003cli\u003eHou B, Wang C-Y, Luo Z-B, et al. Commercial vaccines used in China do not protect against a novel infectious bursal disease virus variant isolated in Fujian. Vet Rec. 2022;191(10): e1840.\u003c/li\u003e\n\u003cli\u003eWang C, Hou B, Shao G, et al. Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China. Viruses. 2023;15(7):1453.\u003c/li\u003e\n\u003cli\u003eHowie R, Thorsen J. An enzyme-linked immunosorbent assay (ELISA) for infectious bursal disease virus. Can J Comp Med. 1981;45(1):51\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eBarbosa J\u0026uacute;nior WL, Ramos de Ara\u0026uacute;jo PS, Dias de Andrade L, et al. Rapid Tests and the Diagnosis of Visceral Leishmaniasis and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome Coinfection. Am J Trop Med Hyg. 2015;93(5):967\u0026ndash;969.\u003c/li\u003e\n\u003cli\u003eDinga DK, Kasprzycka E, Assun\u0026ccedil;\u0026atilde;o IP, et al. High brightness red emitting polymer beads for immunoassays: Comparison between trifluoroacetylacetonates of Europium. Front Chem. 2023;11.\u003c/li\u003e\n\u003cli\u003eDong S, Meng W, Mo L, et al. [Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein]. Sheng Wu Gong Cheng Xue Bao. 2023;39(12):4915\u0026ndash;4926.\u003c/li\u003e\n\u003cli\u003eLi Z, Liu Q, Li Y, et al. One-step polymerized lanthanide-based polystyrene microsphere for sensitive lateral flow immunoassay. Journal of Rare Earths [Internet]. 2021;39(1):11\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eWang H, Guan J, Liu X, et al. Rapid detection of avian leukosis virus using a fluorescent microsphere immunochromatographic test strip assay. Poult Sci. 2019;98(12):6492\u0026ndash;6496.\u003c/li\u003e\n\u003cli\u003eDong S, Meng W, Yang Z, et al. Development of a sensitive immunochromatographic method using lanthanide fluorescent microsphere for rapid test for PPRV antibody. J Virol Methods. 2023; 321:114809.\u003c/li\u003e\n\u003cli\u003eCho U, Chen JK. Lanthanide-Based Optical Probes of Biological Systems. Cell Chemical Biology. 2020;27(8):921\u0026ndash;936.\u003c/li\u003e\n\u003cli\u003eJiang H, Du M, Ke D. A Rapid Quantitative Determination Method of AFP Concentration with Gold Immunochromatographic Strip. Journal of Computers [Internet]. 2012 ;7(12).\u003c/li\u003e\n\u003cli\u003eLi Y, Zeng N, Du M. A Novel Image Methodology for Interpretation of Gold Immunochromatographic Strip. Journal of Computers [Internet]. 2011;6(3):540\u0026ndash;547.\u003c/li\u003e\n\u003cli\u003eChen M, Tian Y, Zhang D, et al. An improved method for rapid identification of hook effect samples in HBsAg quantitative assay. Journal of Virological Methods [Internet]. 2022; 309:114606.\u003c/li\u003e\n\u003cli\u003eCao Z, Yi L, Liu X, et al. Rapid lateral flow immunoassay for fluorescence detection of canine distemper virus (CDV). Front Vet Sci [Internet].2024 ;11:1413420.\u003c/li\u003e\n\u003cli\u003eDe Herdt P, Jagt E, Paul G, et al. Evaluation of the enzyme-linked immunosorbent assay for the detection of antibodies against infectious bursal disease virus (IBDV) and the estimation of the optimal age for IBDV vaccination in broilers. Avian Pathol. 2005;34(6):501\u0026ndash;504.\u003c/li\u003e\n\u003cli\u003eEladl AH, Mosad SM, El-Shafei RA, et al. Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV. Comp Immunol Microbiol Infect Dis. 2020; 72:101505.\u003c/li\u003e\n\u003cli\u003eEl-Azm KIA, Hamed MF, Matter A, et al. Molecular and pathological characterization of natural co-infection of poultry farms with the recently emerged Leucocytozoon caulleryi and chicken anemia virus in Egypt. Trop Anim Health Prod. 2022;54(2):91.\u003c/li\u003e\n\u003cli\u003eWang H, Li W, Zheng SJ. Advances on Innate Immune Evasion by Avian Immunosuppressive Viruses. Front Immunol. 2022; 13:901913.\u003c/li\u003e\n\u003cli\u003eChen W, Chen S, Nie Y, et al. Synergistic Immunosuppression of Avian Leukosis Virus Subgroup J and Infectious Bursal Disease Virus Is Responsible for Enhanced Pathogenicity. Viruses. 2022;14(10):2312.\u003c/li\u003e\n\u003cli\u003eVilla C, Costa J, Gondar C, et al. Effect of food matrix and thermal processing on the performance of a normalised quantitative real-time PCR approach for lupine (Lupinus albus) detection as a potential allergenic food. Food Chem. 2018; 262:251\u0026ndash;259.\u003c/li\u003e\n\u003c/ol\u003e\n"}],"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":"poultry-science-and-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Poultry Science and Management](https://poultrysciencemanagement.biomedcentral.com/)","snPcode":"44364","submissionUrl":"https://submission.springernature.com/new-submission/44364/3","title":"Poultry Science and Management","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Infectious bursal disease virus, rapid antigen detection, fluorescent microspheres, lateral flow immunochromatography, quantitative assay","lastPublishedDoi":"10.21203/rs.3.rs-8346765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8346765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInfectious bursal disease virus (IBDV) is a major immunosuppressive pathogen threatening global poultry production. The emergence of very virulent (vvIBDV) and novel variant strains (nVarIBDV) has increased immune escape and reduced vaccine efficacy, underscoring the need for rapid and reliable diagnostic tools. This study developed a simple, rapid, and sensitive fluorescent microsphere immunochromatographic test strip (FM-ICTS) for quantitative detection of IBDV antigen. The FM-ICTS was constructed using lanthanide fluorescent microspheres conjugated to an anti-VP2 monoclonal antibody within a double-antibody sandwich lateral flow format. Reaction conditions were optimized, and a quantitative standard curve was established using serial dilutions of inactivated IBDV (B87 strain). The assay generated stable fluorescence signals within 15 minutes and showed high analytical sensitivity, detecting IBDV antigen at dilutions up to 1:5120. No cross-reactivity with CAV, ALV, AIV, or MDV was observed, confirming excellent specificity. Repeatability tests demonstrated low coefficients of variation (\u0026lt;\u0026thinsp;10%). When evaluated using 50 clinical samples, FM-ICTS exhibited 97.5% concordance with the national standard RT-PCR method and accurately identified vvIBDV, clIBDV, and nVarIBDV strains. Overall, the developed FM-ICTS provides a rapid, sensitive, specific, and quantitative method suitable for field diagnostics and vaccine quality monitoring, supporting timely prevention and control of IBD.\u003c/p\u003e","manuscriptTitle":"A Rapid and Sensitive Lanthanide-Based Fluorescent Microsphere Immunochromatographic Test for Quantitative Detection of infectious bursal disease virus (IBDV) in Poultry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 15:39:25","doi":"10.21203/rs.3.rs-8346765/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-15T17:27:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T14:34:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T00:11:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T02:26:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T16:05:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T10:36:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312747126728744996404759714386579338232","date":"2026-02-18T12:46:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167425605766743326325104956079494120367","date":"2026-02-13T00:20:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292854808071103308449655325009578184905","date":"2026-02-12T11:17:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14384202034973703316129887421280781925","date":"2026-02-11T16:47:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253371488402820583927601423063061014497","date":"2026-02-11T06:38:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T16:17:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-15T10:42:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-15T10:41:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Poultry Science and Management","date":"2025-12-12T14:23:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"poultry-science-and-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Poultry Science and Management](https://poultrysciencemanagement.biomedcentral.com/)","snPcode":"44364","submissionUrl":"https://submission.springernature.com/new-submission/44364/3","title":"Poultry Science and Management","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9cfa4be3-626d-4abc-82e3-a6d482dc58cc","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-18T10:24:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 15:39:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8346765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8346765","identity":"rs-8346765","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}