Development of a Rapid and Visual Dual-color Latex Microsphere-based Lateral Flow Immunoassay for the Detection of African swine fever virus antibody

Development of a Rapid and Visual Dual-color Latex Microsphere-based Lateral Flow Immunoassay for the Detection of African swine fever virus antibody | 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 Development of a Rapid and Visual Dual-color Latex Microsphere-based Lateral Flow Immunoassay for the Detection of African swine fever virus antibody Jie Chen, Zhengwang Shi, Yi Ru, Shuaipeng Li, Yuqian Zhu, Juncong Luo, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7261926/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2026 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Abstract African swine fever (ASF), an acute infectious disease with a high mortality rate, poses considerable challenges to the development of the global swine industry and socio-economic stability. At present, given the lack of commercial vaccines or effective therapeutic methods, the primary strategy for its management relies on rapid diagnosis and culling of infected animals. Thus, there is a pressing need to develop sensitive, simple and rapid detection methods. In this study, a latex microsphere (LM)-based dual-color lateral flow immunoassay (LFIA) was designed for the detection of African swine fever virus (ASFV) antibody, which was detected within 15 mins by the naked eye. Protein 72 (p72) was conjugated with red LM (RLM) and Chicken IgY (CIgY) with blue LM (BLM) to achieve dual-color detection. Moreover, no cross-reactivity was observed between the standard sera of ASFV and other common swine pathogens. The method exhibited a sensitivity of 1:1024, comparable with the commercial ELISA kit. Meanwhile, the evaluation of 159 clinical samples yielded a kappa value of 0.955. Regarding stability, the assay could be stored at 4 ℃, room temperature (18–25 ℃), and 37 ℃ for 16, 12, and 10 months, respectively. In addition, the intra- and inter-assay variability showed no significant difference, highlighting the excellent repeatability of the method. Overall, these results demonstrate that the dual-color LM-LFIA offers advantages such as low cost, high sensitivity, strong specificity, outstanding stability, and fast detection speed, making it particularly suitable for point-of-care testing (POCT) of ASF. African swine fever p72 protein latex microsphere dual-color detection lateral flow immunoassay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Key Points · Visually intuitive: Antibody against ASFV can be detected within 15 min, indicated by distinct red (test line) and blue (control line). · High diagnostic accuracy: The assay achieved a Cohen’s kappa value of 0.955 compared to commercial ASFV antibody ELISA kit, indicating excellent agreement and suitability for point-of-care testing (POCT). · Enhanced sensitivity: The assay exhibited a detection limit of 1:1024, matching the performance of a commercial ASFV ELISA kit. Introduction African swine fever (ASF), an acute and highly contagious disease of domestic pigs and wild boar caused by infection with ASFV (Zhao et al. 2023 ), is classified as a notifiable animal disease by the World Organization for Animal Health (WOAH) (Juszkiewicz et al. 2023 ). Its typical clinical manifestations include high fever, dyspnea, red to purple skin lesions, loss of appetite, vomiting, and diarrhea, and the disease is associated with a mortality rate of up to 100% across all age groups, with infected animals harboring the virus for prolonged periods (Sun et al. 2021 ). Notably, ASF was first reported in Kenya, Africa, in 1921 and subsequently spread to Eastern Europe, South America, and Asia over the century (Sánchez-Cordón et al. 2018 ). In August 2018, it was first identified in China (Zhou et al. 2018 ) and has spread to other Southeast Asian countries (Mighell and Ward 2021 ). The rapid spread of ASF has caused devastating impacts on the swine industry worldwide, especially in countries with large-scale pig farming, high pork consumption, and a significant contribution to gross domestic product, such as China (You et al. 2021 ). For example, within two years of the initial ASF outbreak, China experienced 165 outbreaks in 32 provinces across the country, resulting in the death of approximately 1.193 million pigs, which has raised widespread concern (Dixon et al. 2020 ; Gaudreault et al. 2020 ; Zhu et al. 2020 ). At present, no commercial vaccines or effective therapeutic methods are available for the prevention and management of ASF. Consequently, monitoring based on accurate clinical diagnosis and strict biosafety measures, such as delineating quarantine zones of infected areas and emergency culling, remains the primary strategy to control disease spread (Lim et al. 2023 ). Therefore, establishing a rapid, accurate and efficient detection method holds significant implications for the prevention and control of ASF epidemics. ASFV, the sole member of the Asfarviridae family, is an enveloped double-stranded DNA virus with a diameter ranging between 260–300 nm (Liu et al. 2019 ). The ASFV genome is approximately 170–193 kb in size and encodes 68 structural proteins and over 100 non-structural proteins (Ge et al. 2018 ; Wang et al. 2021 ). Among structural proteins, protein 72 (p72) assembles into the icosahedral protein capsid in the form of a homotrimer, accounting for 31 to 33% of the total viral particle mass (Wang 2019 ). It is encoded by the B646L gene and possesses a highly conserved hydrophilic region across different strains. Meanwhile, compared to other structural proteins of ASFV, p72 is more stable and can induce neutralizing antibodies (Munoz and Tabares 2022 ), establishing it as a crucial antigen for subunit vaccines and a promising diagnostic target for serological detection methods (Bergeron et al. 2017 ; Miao et al. 2024 ; Tesfagaber et al. 2024 ; Wang et al. 2022 ; Wang et al. 2024 ; Zhang et al. 2021 ; Zhu et al. 2024 ). Common diagnostic strategies for ASFV infection encompass molecular and serological methods (Hu et al. 2023 ), which predominantly detect viruses, DNA, or anti-ASFV antibodies. Molecular diagnostic approaches are associated with strong specificity, high sensitivity, and rapid diagnosis. ASFV has been present in China for over 6 years. At present, the prevalence of ASFV in China is mainly dominated by genotype I, genotype II, and genotype I/II recombinant strains (Liu et al. 2021 ). However, the limitation of molecular diagnostic methods lies in their inability to accurately identify recovered animals or those infected with attenuated strains, given that viral levels in serum or feces may fall below detectable thresholds. Therefore, serological diagnosis, especially antibody testing, has emerged as a key and necessary complement for pathogen detection. To date, numerous serologic testing strategies, including enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IFA), virus neutralization test (VNT), immunohistochemistry (IHC), chemiluminescent immunoassay (CLIA) etc. have been reported for the detection of antibodies (Schlumberger 2018). However, these methods share several drawbacks, such as being time-consuming, labor-intensive, costly, typically requiring specialized equipment, and some of these methods must be operated in Animal Biosafety Level 3 Laboratories (ABSL-3), depending on the specific pathogen, which limits their applicability and potentially leads to underreporting, especially in resource-limited areas. Therefore, early diagnosis is vital to control the ASF epidemic and further prevent its spread. Similarly, preliminary on-site diagnosis is paramount to promoting the prompt implementation of countermeasures. As a powerful tool for POCT, LFIA meets most of the ASSURED standards (Deng et al. 2018 ; Li et al. 2019 ) and has been extensively applied in various fields, such as disease screening and diagnosis (Ince and Sezgintürk 2022 ; Liu et al. 2023 ; Zhang 2023 ), environmental monitoring (Mao et al. 2024 ; Sicard et al. 2015 ) and food safety (Deng et al. 2022 ; Ma et al. 2024 ; Xu et al. 2023 ; Yin et al. 2022b ). Its critical advantage is that it does not require additional equipment or trained professionals, which can substantially shorten detection time and improve detection efficiency, especially in resource-limited areas. In this study, a dual-color LM-based LFIA (LM-LFIA) was designed and optimized to detect antibodies against ASFV in swine serum. The immunoassay employed a double-antigen sandwich format, where RLM conjugated with p72 and BLM with CIgY as detection probes. This rapid and visual dual-color LM-LFIA method is highly specific, sensitive, reproducible, stable, and demonstrated high consistency with the commercial ELISA kit. Additionally, the LM-LFIA featured simple operation and reduced reaction time, positioning it as an efficient and reliable POCT tool for of ASF. Materials and methods Materials and instruments Latex microspheres (LM) were sourced from Vdo Biotech Co. (Suzhou, China). 2-(N-Morpholino) ethanesulfonicacid (MES), N-hydroxysuccinimide (NHS), 1-Ethyl3-(3-(dimethylamino) propyl) carbodiimide (EDC), Bovine serum albumin (BSA), tween-20, and casein were purchased from Sigma-Aldrich Co. (Shanghai, China). Nitrocellulose (NC) membranes were provided by Merck Millipore (Germany). Sample pads, conjugation pads, absorbent pads, polyvinyl chloride (PVC) backings, and plastic cards were purchased from Jiening Biotechnology Co. (Shanghai, China). Chicken IgY (CIgY) and Goat anti chicken IgY (GCIgY) were acquired from SolarBio Co. (Beijing, China). ASFV antibody detection ELISA kit (Ingenasa, Spain) was obtained from Qingdao RealVet Bio-Technology Co., Ltd. (ASF.K001/5, Qingdao, China). Three ASFV standard positive and -negative sera and 159 clinical serum samples were obtained from the ASF Regional Laboratory of China (Lanzhou). Positive sera for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2) were obtained from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science. Serum positive for foot-and-mouth disease virus serotype O (FMDV-O) was provided by the WOAH/National Foot-and-Mouth Diseases Reference Laboratory. The main equipments used in this study were as follows: pH meter (Mettler Toledo, Switzerland), particle size analyzer (Malvern, England), BioDotXYZ3050 three-dimensional spraying platform and BioDotCM4000 cutting machine (Bio-Dot Scientific Equipment, China). Expression and identification of truncated p72 A truncated (20–303 aa) (Liao et al. 2024 ; Miao et al. 2023 ; Yin et al. 2022a ) B646L gene was artificially synthesized and cloned into the bacterial expression vector pET-28a based on the ASFV CN/GS/2018 genome sequence by Sangon Biotech (Shanghai) Co., Ltd. The recombinant expression plasmid pET-28a-p72 was transformed into BL21 (DE3) competent cells and induced with 0.1 mmol/L isopropyl β- d-thiogalactoside (IPTG) at 37 ℃ for 8 h. The p72 was purified via Ni NTA affinity chromatography using His Bind Resin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its concentration was determined using a BCA protein test kit (Tiangen Biotechnology, China) according to the manufacturer's instructions. Then, the reactivity of the p72 was confirmed by Western blotting and ELISA using ASFV-positive and -negative sera. Preparation of p72@RLM and CIgY@BLM immune probes Briefly, RLM were suspended in cold MES buffer (pH 6.2) containing EDC (10 mg/mL) and NHS (10 mg/mL). After activation, a pre-defined amount of the p72 was added (optimized in section 2.5 of this article). Next, the blocking buffer was added, and the resulting mixture was incubated for another 1 h to block the carboxyl groups that had been activated but not conjugated with proteins. Afterward, the p72@RLM was centrifuged and resuspended in a preservation buffer (pH 7.2) and stored at 4 ℃. The preparation process for CIgY@BLM was similar to that of p72@RLM, except that CIgY was used instead of the p72 and conjugated with BLM. Preparation of dual-color LM-LFIA The p72@RLM and CIgY@BLM were mixed and sprayed onto the conjugating pad. Meanwhile, the p72 and GCIgY were sprayed onto the NC membrane at a speed of 1.0 µL/cm to form the T-line and C-line, respectively. Thereafter, the NC membrane, conjugating pad, sample pad, and absorption pad were sequentially assembled on a PVC backing card, overlapping by 2.5 mm. Lastly, the card was cut into 4-mm-wide strips using a cutting machine and stored in a plastic case for subsequent testing. Optimization of parameters In the present study, ASFV standard positive and negative sera were diluted tenfold with PBS as the positive control (P) and negative control (N). The color change was observed and recorded at 10 ± 3 min following the introduction of the sera to the sample pad. Firstly, RLM with varying particle sizes (200 nm, 300 nm, 400 nm) were conjugated with p72, following which RLM with determined particle sizes were conjugated with different concentrations (12.5, 25, 37.5, 50, 62.5, 75, 87.5, 100 µg/mL) of p72 at room temperature (RT). Sedimentation was observed after 5 min and 24 h of conjugation. The highest concentration that did not induce aggregation after 24 h was considered the optimal protein binding amount. Similarly, BLM were conjugated with different concentrations (12.5, 25, 37.5, 50, 62.5, 75, 87.5, 100 µg/mL) of CIgY and screened. Next, the C-line concentration was kept constant (0.75 mg/mL), the p72 was diluted with PBS to different concentrations (0.25, 0.50, 0.75, 1.00, 1.25, 1.50 mg/mL), and the optimal T-line concentration was determined. Likewise, the T-line concentration was maintained at 0.75 mg/mL, and GCIgY was diluted with PBS to different concentrations (0.25, 0.50, 0.75, 1.00, 1.25, 1.50 mg/mL). Finally, screening was conducted to optimize the proportions (1:9, 2:8, 3:7, 4:6, 5:5, 6:4) of CIgY@BLM and p72@RLM mixtures. Evaluation of LM-LFIA Performance Specificity : The specificity of LM-LFIA was evaluated by detecting ASFV-positive and -negative serum and positive serum samples from common pathogens in swine such as CSFV, PRRSV, FMDV-O, PEDV, PCV-2. Three serum samples were tested for each pathogen, with each test repeated in triplicate. Sensitivity : Sensitivity was assessed using standard positive serum serially diluted in PBS at a 2-fold ratio (from 1:2 to 1:2048), with each dilution being simultaneously detected using the developed LM-LFIA and commercial ELISA kit. Each experiment was repeated three times, and the results were recorded. Ultimately, sensitivity was indirectly evaluated based on the maximum dilution ratio of serum that generated visible T-lines. Repeatability : In this study, intra- and inter-batch repeatability experiments were conducted. Regarding intra-batch repeatability, the same sample was tested three times in parallel using LM-LFIA from the same production batch. Inter-batch repeatability involved measuring the same sample using test strips from three different batches, with each test repeated three times. Stability : Stability is defined as the ability of in vitro diagnostic reagents to maintain consistency in characteristics after storage, transportation, and environmental changes. It plays a crucial role in ensuring the effectiveness of the used reagents. In this study, the developed LM-LFIA was stored at 4 ℃, RT, and 37 ℃, respectively, and three positive and three negative sera were tested monthly. Unused strips were stored under their respective temperature conditions for the subsequent test. Application in clinical samples and validation with commercial ELISA kit A total of 159 clinical serum samples were detected for ASFV-specific antibodies using the LM-LFIA and commercial ELISA kits. Following this, the consistency of the two methods was evaluated by calculating the kappa value. Statistical analysis Data were visualized using GraphPad Prism (version 8.0; GraphPad Software, USA) and OriginPro (version 2024b, OriginLab, USA). Results Expression and identification of truncated p72 The truncated p72 was expressed in an E. coli system and analyzed using SDS-PAGE (Fig. 1 A), revealing high purity with a molecular weight of approximately 34 kDa. Western blot (Fig. 1 B) and indirect ELISA analyses (Fig. 1 C) were carried out with ASFV-positive and -negative sera, demonstrating ideal reactivity. Mechanism of the dual-color LM-LFIA The basic mechanism of LM-LFIA is based on the sandwich structure formed by the antigen-antibody and antibody-secondary antibody reactions. To begin, carboxyl groups on the surface of LMs were activated using NHS and EDC. Then, RLM and BLM were conjugated with the p72 and CIgY, yielding immune probes termed p72@RLM and CIgY@BLM, respectively (Fig. 2 A). Next, p72@RLM and CIgY@BLM were mixed and sprayed on the conjugate pad. At the same time, the p72 and GCIgY were sprayed as the T-line and C-line, respectively, on the NC membrane (Fig. 2 B). Under the action of capillary force, if ASFV antibodies are present in the sample, they specifically bind to p72@RLM to form an immune complex (antibody-p72@RLM) and subsequently migrate along with CIgY@BLM to the NC membrane to be captured by the p72 on the T line. Afterward, the excess p72@RLM, as well as CIgY@BLM, continue to migrate until reaching the absorbent pad, with CIgY@BLM being captured by GCIgY on the C-line to form GCIgY-CIgY@BLM. Finally, the combination of a red T-line and a blue C-line indicates a positive test result. Conversely, in the absence of ASFV antibodies in the sample, only the C-line appears blue, indicating a negative result. If the C line does not appear, regardless of T-line color changes, the result is considered invalid (Fig. 2 C). Parameter Optimization Firstly, the influence of particle size was investigated, as displayed in Fig. 3 , under identical reaction times. The results demonstrated that larger particle sizes resulted in more pronounced color changes at the T-line. However, at a particle size of 400 nm, negative samples also exhibited a weak T-line, indicative of a false positive. Thus, 300 nm was ultimately selected as the optimal particle size. Next, the optimal protein concentration was determined as follows: when different concentrations of p72 were conjugated with RLM, the highest concentration that did not result in significant aggregation after 24 h at RT was 50 µg/mL (Fig. 5 A), which was considered the saturation level for p72 conjugation with LMs. Similarly, the saturation level of CIgY was identified as 25 µg/mL (Fig. 5 B). Afterward, the C-line concentration was kept constant (0.75 mg/mL) while the T-line concentration was altered, resulting in different differentiation between positive and negative sera. The color change was most pronounced at 0.75 mg/mL, beyond which further increases in T-line concentration did not significantly increase color intensity. From a cost-efficiency perspective, a T-line concentration of 0.75 mg/mL was eventually selected (Fig. 6 ). Similarly, using a single-factor control variable method and maintaining the T-line concentration constant (0.75 mg/mL), a significant dose-dependent effect was observed in the range of 0.25 mg/mL to 1.0 mg/mL, signifying that color intensity increased with increasing C-line concentrations. However, at C-line concentrations exceeding 1.0 mg/mL, the color intensity did not increase, consistent with previous observations. Therefore, 1.0 mg/mL is selected as the optimal C-line concentration (Fig. 7 ). In addition, as the proportion of BLM increased and that of RLM decreased (1:9, 2:8, 3:7, 4:6, 5:5, 6:4), the color of the C-line initially deepened, then lightened, and eventually became almost invisible. On the other hand, the T-line became progressively blurred at a ratio below 4:6. Accordingly, the optimal mixing ratio of BLM and RLM was ultimately determined to be 2:8 (Fig. 8 ). To summarize, after extensive screening and validation, the optimal detection conditions for dual color LM-LFIA determined in this study were as follows: RLM and BLM with a particle size of 300 nm conjugated with 50 µg/mL of p72 and 25 µg/mL of CIgY. Moreover, optimal T-line and C-line concentrations were 0.75 mg/mL and 1.0 mg/mL, respectively, with a blue-to-red LM mixing ratio of 2:8. Table 1 Comparison of the dual-color LM-LFIA with commercial ELISA kit ELISA dual-color LM-LFIA Total Negative Positive Negative 110 1 111(69.81%) Positive 2 46 48(30.19%) Total 112(70.44%) 47(29.56%) 159 Kappa value 0.955 Po = (110 ་ 46)/159 = 0.9811; Pe = 0.6981×0.7044 + 0.3019×0.2956 = 0.4917 ་ 0.0892 ༝ 0.5809 Kappa value = (Po − Pe)/(1 − Pe) ༝ (0.9811 − 0.5809)/(1 − 0.5809) ༝ 0.955 Characterization of p72@RLM and CIgY@BLM After conjugated with p72, the properties such as particle size and surface charge of LM were examined by dynamic light scattering. Firstly, the hydrodynamic diameter was increased after conjugation (Fig. 4 A, 4 B), which were 322 ± 6.1 nm (RLM), 509 ± 13.8 nm (p72@RLM), 327 ± 8.3 nm (BLM), and 554 ± 17.4 nm (CIgY@BLM). Meanwhile, the zeta potential varied distinctly (Fig. 4 C) from − 22.10 ± 6.11 mV (RLM) to -10.43 ± 0.51 mV (p72@RLM) and from − 33.57 ± 7.78 mV (BLM) to -7.81 ± 0.51 mV (CIgY@BLM). All the results above demonstrated that the conjugation between protein and LM was successful. Evaluation of the performance of the dual-color LM-LFIA Sensitivity, specificity, repeatability, and stability evaluations of the LM-LFIA were separately conducted, as depicted in Fig. 9 – 11 and Table 2 . All C-lines were blue, confirming the validity of the results. Specifically, the T-line was observable at a dilution ratio of positive serum of 1:1024, indicating that the sensitivity of this LM-LFIA was 1:1024 (Fig. 9 A). The same positive serum was detected using a commercial ELISA kit, and the results were highly consistent (Fig. 9 B). Besides, no cross-reactivity was noted with the serum of several common pathogens in pigs, highlighting the strong specificity of this method (Fig. 10 ). Furthermore, serum samples were detected using LM-LFIA prepared from the same batch and three different batches. As anticipated, the results were consistent in both inter- and intra-batch evaluations, with evident differentiation between positive and negative samples, indicating the excellent repeatability of this method (Fig. 11 ). Noteworthily, the LM-LFIA maintained its detection performance at storage temperatures of 37 ℃ for 10 months, RT for 12 months, and 4 ℃ for 16 months (Table 2 ), demonstrating outstanding stability. Table 2 Stability of the dual-color LM-LFIA. Storage Time(month) Storage Temperature 37℃ RT(18-25℃) 4℃ 0 ＋ ＋ ＋ 1 ＋ ＋ ＋ 2 ＋ ＋ ＋ 3 ＋ ＋ ＋ 4 ＋ ＋ ＋ 5 ＋ ＋ ＋ 6 ＋ ＋ ＋ 7 ＋ ＋ ＋ 8 ＋ ＋ ＋ 9 ＋ ＋ ＋ 10 ＋ ＋ ＋ 11 － ＋ ＋ 12 － ＋ ＋ 13 － － ＋ 14 － － ＋ 15 － － ＋ 16 － － ＋ 17 － － － 18 － － － Notes: ＋:The positive and negative samples can be distinguished clearly; －: The positive and negative samples can NOT be distinguished clearly. Application in clinical samples compared with commercial ELISA kit A total of 159 clinic serum samples were simultaneously analyzed using the dual-color LM-LFIA and a commercial ELISA kit, and the kappa value between them was 0.955 (Table 1 ), suggesting that the developed dual-color LM-LFIA demonstrated high accuracy. Discussion As is well documented, ASF is a highly contagious and devastating disease that has caused considerable economic losses to the global pig farming industry and concurrently restricted international trade (Schmidhuber et al. 2020 ). Indeed, it is considered one of the most critical diseases affecting pigs (VanderWaal and Deen 2018 ). In 2018, after the disastrous outbreak of ASF in China, the world's largest pork producer, pig mortality rates sharply rose, leading to widespread panic among farmers, a severe supply shortage in the pig market, a sharp rise in pig prices, and a significant imbalance between supply and demand in the meat market. Concomitantly, due to the rise in international agricultural product prices further inflates the cost of pig feed and contributes to the continued volatility in pig prices. At present, ascribed to the lack of commercial vaccines and effective drugs, the mortality rate of ASF remains as high as 100%, with disease control contingent upon the timely detection of infected animals. Considering the complex clinical symptoms, there is an urgent need for rapid and accurate on-site detection and the rapid culling of infected pigs for its prevention and control. More importantly, early and accurate detection of antibodies is crucial for the diagnosis and monitoring of ASF. As a widely used POCT method, LFIA offers multiple advantages and can achieve rapid on-site detection. Currently, several LFIAs for the detection of ASFV have been reported, for example, Wan (Wan et al., 2022) has coupled p30 and p72 proteins with traditional colloidal gold, and the advantage of which is that it can determine whether the infection stage is early (p30) or late (p72) through a single test. However, since colloidal gold itself only has one color, the readability needs to be improved. The dual color LM-LFIA we developed exhibits a clear visual distinction between C-line (blue) and T-line (red), making it more readable. Meanwhile, due to the improvement of the method, the stability at 4 ℃ has been increased from 7 months to 16 months, greatly extending the shelf life of the test strips. Besides, there is also antigen detection LFIA based on p30 and p72 antibodies, with sensitivities of up to 10 ng (p30) and 20 ng (p72), respectively (Wang et al., 2024 ). Unlike antibody detection, this is more suitable in the early stages of infection at which the viral load is higher. However, the production of detectable antibodies usually takes 7–10 days, which indicates from another perspective that antigen detection and antibody detection can be combined to extend the detection window and to improve the accuracy of diagnostic. Compared with traditional colloidal gold, LMs are filled with oil-soluble color dye, with minimal differences between batches. Besides, their surface is frequently modified with carboxyl groups, which can be activated and covalently bound to the amino groups of proteins, forming stable immune probes characterized by strong tolerance to pH. Meanwhile, LMs have abundant colors that can be visually distinguished by the naked eye without the need for additional equipment, making the use of related products more convenient. There are also studies utilizing LM for ASFV detection, such as LFIA constructed with monoclonal antibodies against p72 protein to detect ASFV (Sastre et al., 2016), with a detectable viral load of 10 4 HAU, comparable to that of commercial ELISA kit. The LFIA method we established involves conjugating p72 protein with LM and targeting ASFV antibodies. Although they both use LM as a mediator and p72 as a target, their detection purposes are different, which can precisely assist in the diagnosis of ASF from different prospectives.The advantages of detecting antibodies are mainly reflected in two aspects. Firstly, extending detection window and traceability, which means it has the ability to detect evidence of infection once the virus is no longer present at detectable levels. In ASF, pigs typically develop detectable antibodies about 7–10 days post-infection, which can last for months or even years (Oh et al., 2021). By contrast, antigen or DNA-based detection (such as PCR) only works when the virus is present in sufficient quantities. Once an infected pig has cleared the virus, antigen testing will turn negative, but antibody testing can still identify that the animal had been infected (a retrospective “trace-back” of exposure) (Li et al., 2023). The antibody’s relative stability in the animal (persistent titers) provides a longer diagnostic window for finding these “invisible” infections after the virus itself has disappeared. Secondly, detecting subclinical or recovered cases. The severity of ASF infection varies, although highly virulent strains typically kill pigs before an antibody response occurs, low- or moderate- virulence ASFV strains can result in pigs surviving the infection and becoming carriers with antibodies. In endemic areas or scenarios where attenuated strains circulate, some pigs may be asymptomatic or only mildly ill, yet they seroconvert (develop antibodies). Antigen tests (e.g. PCR or antigen ELISA) might fail to detect these cases if the virus is present at low levels or has been cleared from blood and tissues. Antibody detections can identify pigs that were infected and recovered or had a subclinical infection. For example, Vietnamese researchers have confirmed that after an acute ASF outbreak, the serum antibody levels of all recovered animals remain at high levels for a long time (497 d) without sustained viral infection (Oh et al., 2021). Objectively, detection performance and cost are in a state of trade-off, and it is challenging to achieve both. Generally, superior detection performance is frequently linked to higher costs, attributed to the use of more active biomaterials (antigens or antibodies) (Geng et al. 2022 ), nanomaterials independently designed and synthesized using in-situ growth methods (Hu and Ding 2023 ), commercialized signal amplification materials (Fang et al. 2022 ; Liu et al. 2022 ; Zhang and Guo 2022 ), and the extensive research and development cycles required for precision instruments and platforms incorporating intelligent and digital devices (Cao et al. 2022 ; Chen et al. 2022 ; Li et al. 2022 ; Lu et al. 2020 ; Magiati et al. 2018 ; Rong et al. 2019 ). Hence, a compromise must be made from the perspective of clinical practical applications. In addition, Immunoglobulin of Yolk (IgY) refers to a specific polyclonal antibody derived from the yolk of laying hens via immune injection. Under normal circumstances, the molecular weight of CIgY is roughly 180 kDa, and it exhibits a structure and function similar to mammalian IgG. Compared with rabbits and sheep commonly used for generating polyclonal antibodies, its advantages primarily lie in lower cost, stronger stability, and weaker non-specific reactions (Dou et al. 2022 ). Specifically, CIgY has a higher yield and lower cost compared to mammalian IgG, given that the cost of raising chickens and building coops is lower than that of mammals. Moreover, compared to mammals, chickens have a faster antibody production cycle and yield high titers of antibodies from eggs as early as the 25th day. Since blood collection is not required, only eggs need to be harvested, which is also more in line with animal welfare considerations. Secondly, IgY demonstrates superior stability and can maintain high biological activity within the pH range of 4–11. Of note, it can also retain biological activity when stored at 4 ℃ for 6 months. Moreover, given that chickens are not mammals, they are more likely to synthesize high-affinity antibodies against mammalian antigens. Their IgY does not bind to mammalian Fc receptors and does not activate the mammalian complement system, resulting in lower immunogenicity and cross-reactivity. Conclusions Herein, a novel dual-color (blue in the C-line and red in the T-line) LM-LFIA was established through a series of systematic investigations for the rapid and sensitive detection of ASFV antibodies in serum in this study. It demonstrated high specificity, satisfactory sensitivity (1:1024), robust stability (can be stored at room temperature for 12 months), comparable detection sensitivity and accuracy (kappa value = 0.955) to commercial ELISA kits, and shorter detection time by over 10 times compared to ELISA. Nonetheless, some limitations of this study cannot be overlooked. To begin, compared with fluorescence detection methods (FITC), LM-LFIA can exclusively provide qualitative results that can be visualized with the naked eye and cannot quantify them. Nevertheless, fluorescence materials generally have relatively higher costs, and fluorescence detection requires additional equipment for data acquisition and interpretation, which partly increases costs and complexity. Overall, LM-LFIA offers a promising approach for enhancing the sensitivity of LFIA. In summary, the developed dual-color LM-LFIA provides a fast, economical, and portable POCT tool suitable for on-site application, which is conducive to the early and specific detection of ASFV antibodies, thereby attenuating ASFV transmission to uninfected animals and facilitating the collection of epidemiological research data from remote areas. Taken together, LM-LFIA is a valuable tool for ASFV epidemic monitoring and control. Declarations Funding This work was supported by the National Key R&D Program of China (2021YFD1800100), The open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2024KJ14), the STI 2030-Major Projects (2023ZD0404301), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202302 and CAAS-ASTIP-2024-LVRI), the science and Technology Major Project of Gansu Province (22ZD6NA012), the Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2023SDZG02), China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs (CARS-35), Science and Technology Plan Project of Gansu Province (21IR7RA024), the Strategic Priority Research Program of Project of the National Center of Technology Innovation for Pigs (NCTIP-XD/C03), and the Major Science and Technology Project of Gansu Province (23ZDNA007). Acknowledgements We thank Home for Researchers editorial team (www.home-for-researchers.com) for the language editing service. Availability of data and materials All data generated or analysed during this study are included in this published article. Ethics approval Experiments involving animals were approved by Lanzhou Veterinary Research Institute. Consent for publication Not applicable. Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Bergeron HC, Glas PS, Schumann KR (2017) Diagnostic specificity of the African swine fever virus antibody detection enzyme-linked immunosorbent assay in feral and domestic pigs in the United States. 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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-7261926","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498657933,"identity":"158dc162-858c-4dbf-ae7a-a9ec9734db16","order_by":0,"name":"Jie 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6","display":"","copyAsset":false,"role":"figure","size":448487,"visible":true,"origin":"","legend":"\u003cp\u003eScreening for optimal T-line concentration\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/543436f5fc8d2c0d943a636c.png"},{"id":89080394,"identity":"b98b3189-ba3d-4c71-af47-219b14b5f1cd","added_by":"auto","created_at":"2025-08-14 12:57:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":404780,"visible":true,"origin":"","legend":"\u003cp\u003eScreening for optimal C-line concentration\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/6a9155cc495ef15c4c97a8af.png"},{"id":89080388,"identity":"63cd75a2-27c8-4513-8531-a54f0d2e3f17","added_by":"auto","created_at":"2025-08-14 12:57:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":392068,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of optimal mixing ratios of CIgY@BLM and p72@RLM\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/f009669b14ad8c268572e839.png"},{"id":89081676,"identity":"5a974614-5ad1-4fb3-97bf-c11c20e1bf6f","added_by":"auto","created_at":"2025-08-14 13:13:19","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":243236,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity of the dual-color LM-LFIA\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/a88ac51f18a9f91b91edaf40.png"},{"id":89079413,"identity":"84e2fc9f-6f1e-4123-9a95-7aefb664b9cc","added_by":"auto","created_at":"2025-08-14 12:49:19","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":497435,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity of the dual-color LM-LFIA\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/823ac79220f6c7b6fd1bc567.png"},{"id":89080725,"identity":"dd1e5a52-4e1f-475c-9b9f-96c38fba6cb8","added_by":"auto","created_at":"2025-08-14 13:05:19","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":474630,"visible":true,"origin":"","legend":"\u003cp\u003eRepeatability of the dual-color LM-LFIA\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/9639e15b20b7f651c7cfbd81.png"},{"id":102785449,"identity":"b03480f2-1836-4a70-bf05-adc4a8560a5c","added_by":"auto","created_at":"2026-02-16 16:06:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5880752,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/84de156d-b78c-43d7-b42c-7694c7ce9084.pdf"},{"id":89079409,"identity":"b8a50b58-a0ab-4797-80c8-fa66ed9ef7e1","added_by":"auto","created_at":"2025-08-14 12:49:19","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1285100,"visible":true,"origin":"","legend":"","description":"","filename":"SDSPAGEp72line12forfinaluseline34forrepeat.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/5660e71b4a639d048340421b.jpg"},{"id":89079439,"identity":"b297e7c6-7eae-47c2-8a20-19bc4abd6fdc","added_by":"auto","created_at":"2025-08-14 12:49:20","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24618966,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlotsp72merge.tif","url":"https://assets-eu.researchsquare.com/files/rs-7261926/v1/465a6096398ce006fc7c6e1b.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a Rapid and Visual Dual-color Latex Microsphere-based Lateral Flow Immunoassay for the Detection of African swine fever virus antibody","fulltext":[{"header":"Key Points","content":"\u003cp\u003e\u003cstrong\u003e·\u003c/strong\u003e\u003cstrong\u003eVisually intuitive:\u0026nbsp;\u003c/strong\u003eAntibody against ASFV can be detected within 15 min, indicated by distinct red (test line) and blue (control line).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e·\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;High diagnostic accuracy:\u003c/strong\u003e The assay achieved a Cohen’s kappa value of 0.955 compared to commercial ASFV antibody ELISA kit, indicating excellent agreement and suitability for point-of-care testing (POCT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e·\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Enhanced sensitivity:\u0026nbsp;\u003c/strong\u003eThe assay exhibited a detection limit of 1:1024, matching the performance of a commercial ASFV ELISA kit.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAfrican swine fever (ASF), an acute and highly contagious disease of domestic pigs and wild boar caused by infection with ASFV (Zhao et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), is classified as a notifiable animal disease by the World Organization for Animal Health (WOAH) (Juszkiewicz et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Its typical clinical manifestations include high fever, dyspnea, red to purple skin lesions, loss of appetite, vomiting, and diarrhea, and the disease is associated with a mortality rate of up to 100% across all age groups, with infected animals harboring the virus for prolonged periods (Sun et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, ASF was first reported in Kenya, Africa, in 1921 and subsequently spread to Eastern Europe, South America, and Asia over the century (S\u0026aacute;nchez-Cord\u0026oacute;n et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In August 2018, it was first identified in China (Zhou et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and has spread to other Southeast Asian countries (Mighell and Ward \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The rapid spread of ASF has caused devastating impacts on the swine industry worldwide, especially in countries with large-scale pig farming, high pork consumption, and a significant contribution to gross domestic product, such as China (You et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, within two years of the initial ASF outbreak, China experienced 165 outbreaks in 32 provinces across the country, resulting in the death of approximately 1.193\u0026nbsp;million pigs, which has raised widespread concern (Dixon et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gaudreault et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At present, no commercial vaccines or effective therapeutic methods are available for the prevention and management of ASF. Consequently, monitoring based on accurate clinical diagnosis and strict biosafety measures, such as delineating quarantine zones of infected areas and emergency culling, remains the primary strategy to control disease spread (Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, establishing a rapid, accurate and efficient detection method holds significant implications for the prevention and control of ASF epidemics.\u003c/p\u003e\u003cp\u003eASFV, the sole member of the \u003cem\u003eAsfarviridae\u003c/em\u003e family, is an enveloped double-stranded DNA virus with a diameter ranging between 260\u0026ndash;300 nm (Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The ASFV genome is approximately 170\u0026ndash;193 kb in size and encodes 68 structural proteins and over 100 non-structural proteins (Ge et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among structural proteins, protein 72 (p72) assembles into the icosahedral protein capsid in the form of a homotrimer, accounting for 31 to 33% of the total viral particle mass (Wang \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is encoded by the \u003cem\u003eB646L\u003c/em\u003e gene and possesses a highly conserved hydrophilic region across different strains. Meanwhile, compared to other structural proteins of ASFV, p72 is more stable and can induce neutralizing antibodies (Munoz and Tabares \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), establishing it as a crucial antigen for subunit vaccines and a promising diagnostic target for serological detection methods (Bergeron et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Miao et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tesfagaber et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCommon diagnostic strategies for ASFV infection encompass molecular and serological methods (Hu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which predominantly detect viruses, DNA, or anti-ASFV antibodies. Molecular diagnostic approaches are associated with strong specificity, high sensitivity, and rapid diagnosis. ASFV has been present in China for over 6 years. At present, the prevalence of ASFV in China is mainly dominated by genotype I, genotype II, and genotype I/II recombinant strains (Liu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the limitation of molecular diagnostic methods lies in their inability to accurately identify recovered animals or those infected with attenuated strains, given that viral levels in serum or feces may fall below detectable thresholds. Therefore, serological diagnosis, especially antibody testing, has emerged as a key and necessary complement for pathogen detection.\u003c/p\u003e\u003cp\u003eTo date, numerous serologic testing strategies, including enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IFA), virus neutralization test (VNT), immunohistochemistry (IHC), chemiluminescent immunoassay (CLIA) etc. have been reported for the detection of antibodies (Schlumberger 2018). However, these methods share several drawbacks, such as being time-consuming, labor-intensive, costly, typically requiring specialized equipment, and some of these methods must be operated in Animal Biosafety Level 3 Laboratories (ABSL-3), depending on the specific pathogen, which limits their applicability and potentially leads to underreporting, especially in resource-limited areas. Therefore, early diagnosis is vital to control the ASF epidemic and further prevent its spread. Similarly, preliminary on-site diagnosis is paramount to promoting the prompt implementation of countermeasures.\u003c/p\u003e\u003cp\u003eAs a powerful tool for POCT, LFIA meets most of the ASSURED standards (Deng et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and has been extensively applied in various fields, such as disease screening and diagnosis (Ince and Sezgint\u0026uuml;rk \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), environmental monitoring (Mao et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sicard et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and food safety (Deng et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yin et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Its critical advantage is that it does not require additional equipment or trained professionals, which can substantially shorten detection time and improve detection efficiency, especially in resource-limited areas.\u003c/p\u003e\u003cp\u003eIn this study, a dual-color LM-based LFIA (LM-LFIA) was designed and optimized to detect antibodies against ASFV in swine serum. The immunoassay employed a double-antigen sandwich format, where RLM conjugated with p72 and BLM with CIgY as detection probes. This rapid and visual dual-color LM-LFIA method is highly specific, sensitive, reproducible, stable, and demonstrated high consistency with the commercial ELISA kit. Additionally, the LM-LFIA featured simple operation and reduced reaction time, positioning it as an efficient and reliable POCT tool for of ASF.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eMaterials and instruments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLatex microspheres (LM) were sourced from Vdo Biotech Co. (Suzhou, China). 2-(N-Morpholino) ethanesulfonicacid (MES), N-hydroxysuccinimide (NHS), 1-Ethyl3-(3-(dimethylamino) propyl) carbodiimide (EDC), Bovine serum albumin (BSA), tween-20, and casein were purchased from Sigma-Aldrich Co. (Shanghai, China). Nitrocellulose (NC) membranes were provided by Merck Millipore (Germany). Sample pads, conjugation pads, absorbent pads, polyvinyl chloride (PVC) backings, and plastic cards were purchased from Jiening Biotechnology Co. (Shanghai, China). Chicken IgY (CIgY) and Goat anti chicken IgY (GCIgY) were acquired from SolarBio Co. (Beijing, China). ASFV antibody detection ELISA kit (Ingenasa, Spain) was obtained from Qingdao RealVet Bio-Technology Co., Ltd. (ASF.K001/5, Qingdao, China). Three ASFV standard positive and -negative sera and 159 clinical serum samples were obtained from the ASF Regional Laboratory of China (Lanzhou). Positive sera for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2) were obtained from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science. Serum positive for foot-and-mouth disease virus serotype O (FMDV-O) was provided by the WOAH/National Foot-and-Mouth Diseases Reference Laboratory. The main equipments used in this study were as follows: pH meter (Mettler Toledo, Switzerland), particle size analyzer (Malvern, England), BioDotXYZ3050 three-dimensional spraying platform and BioDotCM4000 cutting machine (Bio-Dot Scientific Equipment, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression and identification of truncated p72\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA truncated (20\u0026ndash;303 aa) (Liao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Miao et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yin et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e) \u003cem\u003eB646L\u003c/em\u003e gene was artificially synthesized and cloned into the bacterial expression vector pET-28a based on the ASFV CN/GS/2018 genome sequence by Sangon Biotech (Shanghai) Co., Ltd. The recombinant expression plasmid pET-28a-p72 was transformed into BL21 (DE3) competent cells and induced with 0.1 mmol/L isopropyl β- d-thiogalactoside (IPTG) at 37 ℃ for 8 h. The p72 was purified via Ni NTA affinity chromatography using His Bind Resin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its concentration was determined using a BCA protein test kit (Tiangen Biotechnology, China) according to the manufacturer's instructions. Then, the reactivity of the p72 was confirmed by Western blotting and ELISA using ASFV-positive and -negative sera.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of p72@RLM and CIgY@BLM immune probes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBriefly, RLM were suspended in cold MES buffer (pH 6.2) containing EDC (10 mg/mL) and NHS (10 mg/mL). After activation, a pre-defined amount of the p72 was added (optimized in section 2.5 of this article). Next, the blocking buffer was added, and the resulting mixture was incubated for another 1 h to block the carboxyl groups that had been activated but not conjugated with proteins. Afterward, the p72@RLM was centrifuged and resuspended in a preservation buffer (pH 7.2) and stored at 4 ℃. The preparation process for CIgY@BLM was similar to that of p72@RLM, except that CIgY was used instead of the p72 and conjugated with BLM.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of dual-color LM-LFIA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe p72@RLM and CIgY@BLM were mixed and sprayed onto the conjugating pad. Meanwhile, the p72 and GCIgY were sprayed onto the NC membrane at a speed of 1.0 \u0026micro;L/cm to form the T-line and C-line, respectively. Thereafter, the NC membrane, conjugating pad, sample pad, and absorption pad were sequentially assembled on a PVC backing card, overlapping by 2.5 mm. Lastly, the card was cut into 4-mm-wide strips using a cutting machine and stored in a plastic case for subsequent testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptimization of parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the present study, ASFV standard positive and negative sera were diluted tenfold with PBS as the positive control (P) and negative control (N). The color change was observed and recorded at 10\u0026thinsp;\u0026plusmn;\u0026thinsp;3 min following the introduction of the sera to the sample pad.\u003c/p\u003e\u003cp\u003eFirstly, RLM with varying particle sizes (200 nm, 300 nm, 400 nm) were conjugated with p72, following which RLM with determined particle sizes were conjugated with different concentrations (12.5, 25, 37.5, 50, 62.5, 75, 87.5, 100 \u0026micro;g/mL) of p72 at room temperature (RT). Sedimentation was observed after 5 min and 24 h of conjugation. The highest concentration that did not induce aggregation after 24 h was considered the optimal protein binding amount. Similarly, BLM were conjugated with different concentrations (12.5, 25, 37.5, 50, 62.5, 75, 87.5, 100 \u0026micro;g/mL) of CIgY and screened. Next, the C-line concentration was kept constant (0.75 mg/mL), the p72 was diluted with PBS to different concentrations (0.25, 0.50, 0.75, 1.00, 1.25, 1.50 mg/mL), and the optimal T-line concentration was determined. Likewise, the T-line concentration was maintained at 0.75 mg/mL, and GCIgY was diluted with PBS to different concentrations (0.25, 0.50, 0.75, 1.00, 1.25, 1.50 mg/mL). Finally, screening was conducted to optimize the proportions (1:9, 2:8, 3:7, 4:6, 5:5, 6:4) of CIgY@BLM and p72@RLM mixtures.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of LM-LFIA Performance\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpecificity\u003c/b\u003e: The specificity of LM-LFIA was evaluated by detecting ASFV-positive and -negative serum and positive serum samples from common pathogens in swine such as CSFV, PRRSV, FMDV-O, PEDV, PCV-2. Three serum samples were tested for each pathogen, with each test repeated in triplicate. \u003cb\u003eSensitivity\u003c/b\u003e: Sensitivity was assessed using standard positive serum serially diluted in PBS at a 2-fold ratio (from 1:2 to 1:2048), with each dilution being simultaneously detected using the developed LM-LFIA and commercial ELISA kit. Each experiment was repeated three times, and the results were recorded. Ultimately, sensitivity was indirectly evaluated based on the maximum dilution ratio of serum that generated visible T-lines. \u003cb\u003eRepeatability\u003c/b\u003e: In this study, intra- and inter-batch repeatability experiments were conducted. Regarding intra-batch repeatability, the same sample was tested three times in parallel using LM-LFIA from the same production batch. Inter-batch repeatability involved measuring the same sample using test strips from three different batches, with each test repeated three times. \u003cb\u003eStability\u003c/b\u003e: Stability is defined as the ability of in vitro diagnostic reagents to maintain consistency in characteristics after storage, transportation, and environmental changes. It plays a crucial role in ensuring the effectiveness of the used reagents. In this study, the developed LM-LFIA was stored at 4 ℃, RT, and 37 ℃, respectively, and three positive and three negative sera were tested monthly. Unused strips were stored under their respective temperature conditions for the subsequent test.\u003c/p\u003e\u003cp\u003e\u003cb\u003eApplication in clinical samples and validation with commercial ELISA kit\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 159 clinical serum samples were detected for ASFV-specific antibodies using the LM-LFIA and commercial ELISA kits. Following this, the consistency of the two methods was evaluated by calculating the kappa value.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were visualized using GraphPad Prism (version 8.0; GraphPad Software, USA) and OriginPro (version 2024b, OriginLab, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression and identification of truncated p72\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe truncated p72 was expressed in an \u003cem\u003eE. coli\u003c/em\u003e system and analyzed using SDS-PAGE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), revealing high purity with a molecular weight of approximately 34 kDa. Western blot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) and indirect ELISA analyses (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) were carried out with ASFV-positive and -negative sera, demonstrating ideal reactivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanism of the dual-color LM-LFIA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe basic mechanism of LM-LFIA is based on the sandwich structure formed by the antigen-antibody and antibody-secondary antibody reactions. To begin, carboxyl groups on the surface of LMs were activated using NHS and EDC. Then, RLM and BLM were conjugated with the p72 and CIgY, yielding immune probes termed p72@RLM and CIgY@BLM, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Next, p72@RLM and CIgY@BLM were mixed and sprayed on the conjugate pad. At the same time, the p72 and GCIgY were sprayed as the T-line and C-line, respectively, on the NC membrane (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Under the action of capillary force, if ASFV antibodies are present in the sample, they specifically bind to p72@RLM to form an immune complex (antibody-p72@RLM) and subsequently migrate along with CIgY@BLM to the NC membrane to be captured by the p72 on the T line. Afterward, the excess p72@RLM, as well as CIgY@BLM, continue to migrate until reaching the absorbent pad, with CIgY@BLM being captured by GCIgY on the C-line to form GCIgY-CIgY@BLM. Finally, the combination of a red T-line and a blue C-line indicates a positive test result. Conversely, in the absence of ASFV antibodies in the sample, only the C-line appears blue, indicating a negative result. If the C line does not appear, regardless of T-line color changes, the result is considered invalid (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParameter Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, the influence of particle size was investigated, as displayed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, under identical reaction times. The results demonstrated that larger particle sizes resulted in more pronounced color changes at the T-line. However, at a particle size of 400 nm, negative samples also exhibited a weak T-line, indicative of a false positive. Thus, 300 nm was ultimately selected as the optimal particle size. Next, the optimal protein concentration was determined as follows: when different concentrations of p72 were conjugated with RLM, the highest concentration that did not result in significant aggregation after 24 h at RT was 50 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), which was considered the saturation level for p72 conjugation with LMs. Similarly, the saturation level of CIgY was identified as 25 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Afterward, the C-line concentration was kept constant (0.75 mg/mL) while the T-line concentration was altered, resulting in different differentiation between positive and negative sera. The color change was most pronounced at 0.75 mg/mL, beyond which further increases in T-line concentration did not significantly increase color intensity. From a cost-efficiency perspective, a T-line concentration of 0.75 mg/mL was eventually selected (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Similarly, using a single-factor control variable method and maintaining the T-line concentration constant (0.75 mg/mL), a significant dose-dependent effect was observed in the range of 0.25 mg/mL to 1.0 mg/mL, signifying that color intensity increased with increasing C-line concentrations. However, at C-line concentrations exceeding 1.0 mg/mL, the color intensity did not increase, consistent with previous observations. Therefore, 1.0 mg/mL is selected as the optimal C-line concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). In addition, as the proportion of BLM increased and that of RLM decreased (1:9, 2:8, 3:7, 4:6, 5:5, 6:4), the color of the C-line initially deepened, then lightened, and eventually became almost invisible. On the other hand, the T-line became progressively blurred at a ratio below 4:6. Accordingly, the optimal mixing ratio of BLM and RLM was ultimately determined to be 2:8 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). To summarize, after extensive screening and validation, the optimal detection conditions for dual color LM-LFIA determined in this study were as follows: RLM and BLM with a particle size of 300 nm conjugated with 50 \u0026micro;g/mL of p72 and 25 \u0026micro;g/mL of CIgY. Moreover, optimal T-line and C-line concentrations were 0.75 mg/mL and 1.0 mg/mL, respectively, with a blue-to-red LM mixing ratio of 2:8.\u003c/p\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of the dual-color LM-LFIA with commercial ELISA kit\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eELISA\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003edual-color LM-LFIA\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e110\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e111(69.81%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48(30.19%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e112(70.44%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e47(29.56%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e159\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eKappa value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.955\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003ePo = (110 ་ 46)/159\u0026thinsp;=\u0026thinsp;0.9811;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003ePe = 0.6981\u0026times;0.7044\u0026thinsp;+\u0026thinsp;0.3019\u0026times;0.2956\u0026thinsp;=\u0026thinsp;0.4917 ་ 0.0892 ༝ 0.5809\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eKappa value = (Po\u0026thinsp;\u0026minus;\u0026thinsp;Pe)/(1\u0026thinsp;\u0026minus;\u0026thinsp;Pe) ༝ (0.9811\u0026thinsp;\u0026minus;\u0026thinsp;0.5809)/(1\u0026thinsp;\u0026minus;\u0026thinsp;0.5809) ༝ 0.955\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of p72@RLM and CIgY@BLM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter conjugated with p72, the properties such as particle size and surface charge of LM were examined by dynamic light scattering. Firstly, the hydrodynamic diameter was increased after conjugation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), which were 322\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 nm (RLM), 509\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8 nm (p72@RLM), 327\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3 nm (BLM), and 554\u0026thinsp;\u0026plusmn;\u0026thinsp;17.4 nm (CIgY@BLM). Meanwhile, the zeta potential varied distinctly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) from \u0026minus;\u0026thinsp;22.10\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11 mV (RLM) to -10.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 mV (p72@RLM) and from \u0026minus;\u0026thinsp;33.57\u0026thinsp;\u0026plusmn;\u0026thinsp;7.78 mV (BLM) to -7.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 mV (CIgY@BLM). All the results above demonstrated that the conjugation between protein and LM was successful.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of the performance of the dual-color LM-LFIA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSensitivity, specificity, repeatability, and stability evaluations of the LM-LFIA were separately conducted, as depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. All C-lines were blue, confirming the validity of the results. Specifically, the T-line was observable at a dilution ratio of positive serum of 1:1024, indicating that the sensitivity of this LM-LFIA was 1:1024 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). The same positive serum was detected using a commercial ELISA kit, and the results were highly consistent (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB). Besides, no cross-reactivity was noted with the serum of several common pathogens in pigs, highlighting the strong specificity of this method (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). Furthermore, serum samples were detected using LM-LFIA prepared from the same batch and three different batches. As anticipated, the results were consistent in both inter- and intra-batch evaluations, with evident differentiation between positive and negative samples, indicating the excellent repeatability of this method (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). Noteworthily, the LM-LFIA maintained its detection performance at storage temperatures of 37 ℃ for 10 months, RT for 12 months, and 4 ℃ for 16 months (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), demonstrating outstanding stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp; \u003c/strong\u003eStability of the dual-color LM-LFIA.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"444\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"104\"\u003e\n\u003cp\u003eStorage Time(month)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" width=\"340\"\u003e\n\u003cp\u003eStorage Temperature\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e37℃\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eRT(18-25℃)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4℃\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003e＋\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003e＋\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e＋\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003e＋\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e－\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes: ＋:The positive and negative samples can be distinguished clearly;\u003c/p\u003e\n\u003cp\u003e－: The positive and negative samples can NOT be distinguished clearly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApplication in clinical samples compared with commercial ELISA kit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 159 clinic serum samples were simultaneously analyzed using the dual-color LM-LFIA and a commercial ELISA kit, and the kappa value between them was 0.955 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that the developed dual-color LM-LFIA demonstrated high accuracy.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs is well documented, ASF is a highly contagious and devastating disease that has caused considerable economic losses to the global pig farming industry and concurrently restricted international trade (Schmidhuber et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Indeed, it is considered one of the most critical diseases affecting pigs (VanderWaal and Deen \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In 2018, after the disastrous outbreak of ASF in China, the world's largest pork producer, pig mortality rates sharply rose, leading to widespread panic among farmers, a severe supply shortage in the pig market, a sharp rise in pig prices, and a significant imbalance between supply and demand in the meat market. Concomitantly, due to the rise in international agricultural product prices further inflates the cost of pig feed and contributes to the continued volatility in pig prices. At present, ascribed to the lack of commercial vaccines and effective drugs, the mortality rate of ASF remains as high as 100%, with disease control contingent upon the timely detection of infected animals. Considering the complex clinical symptoms, there is an urgent need for rapid and accurate on-site detection and the rapid culling of infected pigs for its prevention and control. More importantly, early and accurate detection of antibodies is crucial for the diagnosis and monitoring of ASF. As a widely used POCT method, LFIA offers multiple advantages and can achieve rapid on-site detection. Currently, several LFIAs for the detection of ASFV have been reported, for example, Wan (Wan et al., 2022) has coupled p30 and p72 proteins with traditional colloidal gold, and the advantage of which is that it can determine whether the infection stage is early (p30) or late (p72) through a single test. However, since colloidal gold itself only has one color, the readability needs to be improved. The dual color LM-LFIA we developed exhibits a clear visual distinction between C-line (blue) and T-line (red), making it more readable. Meanwhile, due to the improvement of the method, the stability at 4 ℃ has been increased from 7 months to 16 months, greatly extending the shelf life of the test strips. Besides, there is also antigen detection LFIA based on p30 and p72 antibodies, with sensitivities of up to 10 ng (p30) and 20 ng (p72), respectively (Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Unlike antibody detection, this is more suitable in the early stages of infection at which the viral load is higher. However, the production of detectable antibodies usually takes 7\u0026ndash;10 days, which indicates from another perspective that antigen detection and antibody detection can be combined to extend the detection window and to improve the accuracy of diagnostic. Compared with traditional colloidal gold, LMs are filled with oil-soluble color dye, with minimal differences between batches. Besides, their surface is frequently modified with carboxyl groups, which can be activated and covalently bound to the amino groups of proteins, forming stable immune probes characterized by strong tolerance to pH. Meanwhile, LMs have abundant colors that can be visually distinguished by the naked eye without the need for additional equipment, making the use of related products more convenient. There are also studies utilizing LM for ASFV detection, such as LFIA constructed with monoclonal antibodies against p72 protein to detect ASFV (Sastre et al., 2016), with a detectable viral load of 10\u003csup\u003e4\u003c/sup\u003e HAU, comparable to that of commercial ELISA kit. The LFIA method we established involves conjugating p72 protein with LM and targeting ASFV antibodies. Although they both use LM as a mediator and p72 as a target, their detection purposes are different, which can precisely assist in the diagnosis of ASF from different prospectives.The advantages of detecting antibodies are mainly reflected in two aspects. Firstly, extending detection window and traceability, which means it has the ability to detect evidence of infection once the virus is no longer present at detectable levels. In ASF, pigs typically develop detectable antibodies about 7\u0026ndash;10 days post-infection, which can last for months or even years (Oh et al., 2021). By contrast, antigen or DNA-based detection (such as PCR) only works when the virus is present in sufficient quantities. Once an infected pig has cleared the virus, antigen testing will turn negative, but antibody testing can still identify that the animal had been infected (a retrospective \u0026ldquo;trace-back\u0026rdquo; of exposure) (Li et al., 2023). The antibody\u0026rsquo;s relative stability in the animal (persistent titers) provides a longer diagnostic window for finding these \u0026ldquo;invisible\u0026rdquo; infections after the virus itself has disappeared. Secondly, detecting subclinical or recovered cases. The severity of ASF infection varies, although highly virulent strains typically kill pigs before an antibody response occurs, low- or moderate- virulence ASFV strains can result in pigs surviving the infection and becoming carriers with antibodies. In endemic areas or scenarios where attenuated strains circulate, some pigs may be asymptomatic or only mildly ill, yet they seroconvert (develop antibodies). Antigen tests (e.g. PCR or antigen ELISA) might fail to detect these cases if the virus is present at low levels or has been cleared from blood and tissues. Antibody detections can identify pigs that were infected and recovered or had a subclinical infection. For example, Vietnamese researchers have confirmed that after an acute ASF outbreak, the serum antibody levels of all recovered animals remain at high levels for a long time (497 d) without sustained viral infection (Oh et al., 2021). Objectively, detection performance and cost are in a state of trade-off, and it is challenging to achieve both. Generally, superior detection performance is frequently linked to higher costs, attributed to the use of more active biomaterials (antigens or antibodies) (Geng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), nanomaterials independently designed and synthesized using \u003cem\u003ein-situ\u003c/em\u003e growth methods (Hu and Ding \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), commercialized signal amplification materials (Fang et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang and Guo \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and the extensive research and development cycles required for precision instruments and platforms incorporating intelligent and digital devices (Cao et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Magiati et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rong et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hence, a compromise must be made from the perspective of clinical practical applications.\u003c/p\u003e\u003cp\u003eIn addition, Immunoglobulin of Yolk (IgY) refers to a specific polyclonal antibody derived from the yolk of laying hens via immune injection. Under normal circumstances, the molecular weight of CIgY is roughly 180 kDa, and it exhibits a structure and function similar to mammalian IgG. Compared with rabbits and sheep commonly used for generating polyclonal antibodies, its advantages primarily lie in lower cost, stronger stability, and weaker non-specific reactions (Dou et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Specifically, CIgY has a higher yield and lower cost compared to mammalian IgG, given that the cost of raising chickens and building coops is lower than that of mammals. Moreover, compared to mammals, chickens have a faster antibody production cycle and yield high titers of antibodies from eggs as early as the 25th day. Since blood collection is not required, only eggs need to be harvested, which is also more in line with animal welfare considerations. Secondly, IgY demonstrates superior stability and can maintain high biological activity within the pH range of 4\u0026ndash;11. Of note, it can also retain biological activity when stored at 4 ℃ for 6 months. Moreover, given that chickens are not mammals, they are more likely to synthesize high-affinity antibodies against mammalian antigens. Their IgY does not bind to mammalian Fc receptors and does not activate the mammalian complement system, resulting in lower immunogenicity and cross-reactivity.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHerein, a novel dual-color (blue in the C-line and red in the T-line) LM-LFIA was established through a series of systematic investigations for the rapid and sensitive detection of ASFV antibodies in serum in this study. It demonstrated high specificity, satisfactory sensitivity (1:1024), robust stability (can be stored at room temperature for 12 months), comparable detection sensitivity and accuracy (kappa value = 0.955) to commercial ELISA kits, and shorter detection time by over 10 times compared to ELISA. Nonetheless, some limitations of this study cannot be overlooked. To begin, compared with fluorescence detection methods (FITC), LM-LFIA can exclusively provide qualitative results that can be visualized with the naked eye and cannot quantify them. Nevertheless, fluorescence materials generally have relatively higher costs, and fluorescence detection requires additional equipment for data acquisition and interpretation, which partly increases costs and complexity. Overall, LM-LFIA offers a promising approach for enhancing the sensitivity of LFIA.\u003c/p\u003e\u003cp\u003eIn summary, the developed dual-color LM-LFIA provides a fast, economical, and portable POCT tool suitable for on-site application, which is conducive to the early and specific detection of ASFV antibodies, thereby attenuating ASFV transmission to uninfected animals and facilitating the collection of epidemiological research data from remote areas. Taken together, LM-LFIA is a valuable tool for ASFV epidemic monitoring and control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2021YFD1800100), The open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2024KJ14), the STI 2030-Major Projects (2023ZD0404301), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202302 and CAAS-ASTIP-2024-LVRI), the science and Technology Major Project of Gansu Province (22ZD6NA012), the Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2023SDZG02), China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs (CARS-35), Science and Technology Plan Project of Gansu Province (21IR7RA024), the Strategic Priority Research Program of Project of the National Center of Technology Innovation for Pigs (NCTIP-XD/C03), and the Major Science and Technology Project of Gansu Province (23ZDNA007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Home for Researchers editorial team (www.home-for-researchers.com) for the language editing service.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAll data generated or analysed during this study are included in this published article.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments involving animals were approved by Lanzhou Veterinary Research Institute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBergeron HC, Glas PS, Schumann KR (2017) Diagnostic specificity of the African swine fever virus antibody detection enzyme-linked immunosorbent assay in feral and domestic pigs in the United States. 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Anal Chim Acta 1140:30\u0026ndash;40\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"African swine fever, p72 protein, latex microsphere, dual-color detection, lateral flow immunoassay","lastPublishedDoi":"10.21203/rs.3.rs-7261926/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7261926/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAfrican swine fever (ASF), an acute infectious disease with a high mortality rate, poses considerable challenges to the development of the global swine industry and socio-economic stability. At present, given the lack of commercial vaccines or effective therapeutic methods, the primary strategy for its management relies on rapid diagnosis and culling of infected animals. Thus, there is a pressing need to develop sensitive, simple and rapid detection methods. In this study, a latex microsphere (LM)-based dual-color lateral flow immunoassay (LFIA) was designed for the detection of African swine fever virus (ASFV) antibody, which was detected within 15 mins by the naked eye. Protein 72 (p72) was conjugated with red LM (RLM) and Chicken IgY (CIgY) with blue LM (BLM) to achieve dual-color detection. Moreover, no cross-reactivity was observed between the standard sera of ASFV and other common swine pathogens. The method exhibited a sensitivity of 1:1024, comparable with the commercial ELISA kit. Meanwhile, the evaluation of 159 clinical samples yielded a kappa value of 0.955. Regarding stability, the assay could be stored at 4 ℃, room temperature (18\u0026ndash;25 ℃), and 37 ℃ for 16, 12, and 10 months, respectively. In addition, the intra- and inter-assay variability showed no significant difference, highlighting the excellent repeatability of the method. Overall, these results demonstrate that the dual-color LM-LFIA offers advantages such as low cost, high sensitivity, strong specificity, outstanding stability, and fast detection speed, making it particularly suitable for point-of-care testing (POCT) of ASF.\u003c/p\u003e","manuscriptTitle":"Development of a Rapid and Visual Dual-color Latex Microsphere-based Lateral Flow Immunoassay for the Detection of African swine fever virus antibody","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 12:49:14","doi":"10.21203/rs.3.rs-7261926/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0918fb6f-08af-4908-b3a1-a3e28f406b95","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:03:06+00:00","versionOfRecord":{"articleIdentity":"rs-7261926","link":"https://doi.org/10.1007/s00253-025-13694-w","journal":{"identity":"applied-microbiology-and-biotechnology","isVorOnly":false,"title":"Applied Microbiology and Biotechnology"},"publishedOn":"2026-02-11 15:58:51","publishedOnDateReadable":"February 11th, 2026"},"versionCreatedAt":"2025-08-14 12:49:14","video":"","vorDoi":"10.1007/s00253-025-13694-w","vorDoiUrl":"https://doi.org/10.1007/s00253-025-13694-w","workflowStages":[]},"version":"v1","identity":"rs-7261926","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7261926","identity":"rs-7261926","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}