Development and application of an immuoperoxidase monolayer assay for the detection of PRRSV

Development and application of an immuoperoxidase monolayer assay for the detection of PRRSV | 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 and application of an immuoperoxidase monolayer assay for the detection of PRRSV peng LI, Chunxiao GUAN, Liping WANG, Huajian WANG, Guopeng SUN, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4331925/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a highly contagious disease with high morbidity and mortality that affects the global swine industry. So far, there is still a widespread dissemination of PRRSV with obvious genetic variations in swine population, resulting in huge economic losses annually. Therefore, accurate laboratory diagnosis is needed to quickly confirm PRRSV infection. Results An immunoperoxidase monolayer assay (IPMA) was developed for the specific and sensitive detection of PRRSV based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6. The mAb 28F6-based IPMA could specifically detect PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10 − 2.25 TCID 50 /100 µL. There was no significant difference in the detection of PRRSV of different passages with different batches of mAb 28F6, indicating that the IPMA had good repeatability. In addition, the IPMA could recognize a number of PRRSV variants including field strains such as BJ-4, HN07-1, and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. Validation of the IPMA showed that it was in 100% consistency with qRT-PCR on the detection of 108 clinical samples. Conclusions The IPMA could meet the demand for the specific and sensitive detection of PRRSV, which is helpful for accurate monitoring and early warning of PRRSV infections. PRRSV Broad-spectrum monoclonal antibody Immunoperoxidase monolayer assay Specific detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease characterized by high morbidity and mortality in pregnant sows and respiratory system damage in piglets [ 1 ]. It is listed as the second-class infectious disease of pigs in China [ 2 ]. Common clinical manifestations of PRRS included high fever, mental malaise, abortion, premature stillbirth, mummified fetus, and weak fetus [ 3 , 4 ]. The causative agent of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV), which is an enveloped RNA virus belonging to order Nidovirales, family Arteriviridae, genus Arterivirus [ 5 ]. In 1991, the PRRSV strain Lelystad was first isolated in the Netherlands, and later the PRRSV strain ATCC VR-2332 was isolated in the United States in 1992 [ 6 ]. The two strains could cause similar clinical symptoms, but were different in genome homology and pathogenicity [ 7 ]. The Chinese PRRSV strain was first isolated in 1995 and was demonstrated to be the variant of PRRSV strain ATCC VR-2332 [ 8 ]. In 2006, a highly pathogenic PRRSV (HP-PRRSV), characterized by high fever and high mortality, was isolated in China and widely spread later [ 9 ]. Since the end of 2012, PRRSV NADC30-like strains such as JL580 [ 10 ], HnyC15 [ 11 ], HENAN-XINX [ 12 ], and CHSX1401 [ 13 ], have been isolated in China and become the dominant circulating strains of PRRSV in many areas across China [ 14 ]. Therefore, the circulation of classical PRRSV strains, HP-PRRSV strains, and NADC30-like PRRSV strains has brought new challenges to the swine industry in China. Accurate laboratory diagnosis of PRRSV is considered as an effective control measure. At present, a variety of methods have been developed for the detection of PRRSV, such as reverse transcription polymerase chain reaction (RT-PCR) [ 15 ], quantitative reverse transcription polymerase chain reaction (qRT-PCR) [ 16 ], and immunofluorescence assay (IFA) [ 17 ]. RT-PCR and qRT-PCR require complex instruments and skilled operators, which are suitable for laboratory use and are not suitable for clinical large-scale screening. IFA offers high sensitivity and specificity, making it suitable for early diagnosis. However, the presence of intrinsic fluorescent substances in field samples can lead to background signal, which may affect the interpretation of results. Additionally, fluorescent dyes are prone to quenching during storage, making long-term preservation challenging [ 18 ]. In contrast, immuoperoxidase monolayer assay (IPMA) offers relatively simple experimental procedures that do not require expensive equipment or complex laboratory operations [ 19 ]. It is a widely applied immunological technique that can be used for the detection and localization of specific antigens in cells or tissues [ 20 ]. In IPMA, the enzymatic reaction produces a visible pigment deposition on cells, which can be observed under a regular microscope, enabling visual analysis of immune reactions on the cell surface or within the cells. In addition, the cell monolayers used in IPMA can be preserved after fixation, which enabled long-term sample storage for subsequent analysis and follow-up examinations [ 21 ]. Many IPMA methods have been developed for the detection of bacterial and viral infections, as it enabled the visualization of pathogens within infected cells and facilitated the identification and characterization of infections. Zhang et al. established an IPMA for the detection of classical swine fever virus (CSFV) and found that the positive coincidence rate of IPMA with RT-PCR and ELISA reached more than 92% [ 22 ]. Li et al. developed an IPMA to detect intracellular Lawsonella and found a positive coincidence rate of 94.6% with PCR [ 23 ]. A study compared the effectiveness of IPMA in detecting African Swine Fever Virus (ASFV) in macrophage cultures and found that IPMA had a detection rate of 80% as compared with PCR [ 24 ]. The first IPMA method for the detection of PRRSV was established in 1991 by the Central Veterinary Institute of the Netherlands and showed good sensitivity and specificity in the detection of the PRRSV strain Lelystad virus [ 25 ]. Due to the high variability of the virus itself and the recombination of different PRRSV variants in the field, the diagnosis and prevention and control of PRRSV face continued challenges [ 26 ]. To provide an effective IPMA for the detection of different PRRSV field strains and vaccine strains, a specific and sensitive IPMA was developed based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6 in this study. The working conditions of the mAb 28F6-based IPMA were optimized, and the specificity and sensitivity of the IPMA were evaluated. In addition, the IPMA was validated by detecting 108 clinical samples in parallel with qRT-PCR. The results showed that the mAb 28F6-based IPMA has the potential to be used in veterinary clinics for the detection of different PRRSV variants. 2. Materials and Methods 2.1 Antibodies The anti-PRRSV monoclonal antibody (mAb) 28F6 was previously produced by immunizing BALB/c mice with HP-PRRSV HN07-1 (GenBank: KX766378.1) and preserved in our laboratory. 2.2 Cells and viruses Meat Animal Research Center 145 (MARC-145) cells, Vero cells, and porcine kidney epithelial cells (PK15) were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) in a humidified incubator at 37°C and 5% CO 2 . PRRSV vaccine strains HuN4-F112 (Harbin Harvac Biotechnology Co., Ltd,; Harbin, China), JXA1-R (Wuhan Keqian Biology Co., Ltd.; Wuhan, China), TJM-F92 (Sinovet (Jiangsu) Biotechnology Co., Ltd.; Taizhou, China), GDr180 (Guangdong Winsun Bio-pharmaceutical Co., Ltd.; Guangzhou, China), VR2332 (Boehringer Ingelheim Animal Health Operations (China) Co., Ltd.; Taizhou, China), CH-1R (Harbin Weike Biotechnology Co., Ltd.; Harbin, China), and R98 (Qilu Animal Health Products Co., Ltd.; Jinan, China) were bought and propagated on MARC-145 cells. PRRSV field strains BJ-4 (GenBank: KP771761.1), HN07-1, and NADC30-like PRRSV (GenBank: JN654459.1) were kindly provided by the Henan Provincial Key Laboratory of Animal Immunology. Classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV) were previously stored in our laboratory. 2.3 Pretreatment of clinical samples A total of 108 clinical samples of porcine lymph nodes, tonsils, lungs, and spleens were collected in different regions of Henan province, China and kept at -80°C. Before inoculation onto cells, 0.5 g of each sample was fully ground in liquid nitrogen with the addition of 0.5 mL of phosphate buffered saline (PBS) containing penicillin and streptomycin. The supernatant was collected by centrifugation at 5,000 r/min for 10 min at 4°C. 2.4 Establishment of an IPMA Monolayers of MARC-145 cells were grown in 96-well cell culture plates at 37°C in 5% CO 2 atmosphere. The cells were infected with PRRSV for 48 h before fixation in cold methanol for 15 min at room temperature. After the plates were blocked with 5% skimmed milk at 37°C for 1 h, anti-PRRSV mAb 28F6 was added to incubate with the plates at 37°C for 1 h. Then, horseradish peroxidase (HRP)-labelled goat anti-mouse IgG was used as secondary antibodies to incubate with the plates at 37°C for 1 h. Finally, AEC substrate buffer (3-amino-9-ethylcarbazole) was added for color development for 10 min before the addition of PBS to wash the plates and stop the color reaction. The wells were then observed under a light microscope and the appearance of red-brown precipitates in the wells indicated a positive result. 2.5 Viral titration Viral titration was performed on monolayer cells of MARC-145 using IPMA. When the cells were grown to 80% confluence, ten-fold serial dilutions of the viral stock were added respectively and incubated with the cells for 2 h. After the removal of the inoculum, 100 µL of DMEM containing 2% FBS was added to the wells. Then, the cells were kept at 37°C for 48 h. Later, IPMA was performed to ascertain the presence of the virus. The viral titer was calculated using the Reed–Muench method and expressed as 50% tissue culture infectious dose (TCID 50 ). 2.6 Optimization of the working conditions of IPMA The working conditions of the mAb 28F6-based IPMA were optimized in terms of incubation time of the virus, the dilution and incubation time of mAb 28F6, the dilution and incubation time of HRP-labelled goat anti-mouse IgG, and the time for color development. The condition giving clear morphology of stained cells and moderate amount of unstained cells was selected to be optimal. To determine the optimal incubation time of the virus, MARC-145 cells were infected with 10 − 1.69 TCID 50 /100 µL of PRRSV at 37°C in a 5% CO 2 incubator for 24 h, 48 h, 72 h, 96 h, and 120 h. To determine the optimal dilution of the anti-PRRSV mAb 28F6, the infected MARC-145 cells were incubated with mAb 28F6 at dilutions of 1:4×10 2 , 1:8×10 2 , 1:1.6×10 3 , 1:3.2×10 3 , 1:6.4×10 3 , 1:1.28×10 4 , 1:2.56×10 4 , and 1:5.12×10 4 , respectively. The incubation time of mAb 28F6 was set to be 30 min, 60 min, 90 min, and 120 min to determine its optimal reaction time. To determine the optimal dilution of secondary antibodies, goat anti-mouse IgG-HRP was serially diluted from 1:1×10 2 to 1:2×10 2 , 1:4×10 2 , 1:8×10 2 , and 1:1.6×10 3 . The incubation time of secondary antibodies was set to be 30 min, 60 min, 90 min, and 120 min to obtain the optimal reaction. The time for color development was set to be 5 min, 10 min, and 15 min. Each sample was tested in triplicate, and each assay was repeated three times. 2.7 Specificity of the mAb 28F6-based IPMA PRRSV was inoculated onto MARC-145 cells. CSFV or PCV2 were inoculated onto PK-15 cell. PEDV was inoculated onto Vero cells. The infected cells were cultured in 5% CO 2 cell culture incubator at 37°C for 48 h. Then, the mAb 28F6-based IPMA was conducted to determine the specificity of the method. 2.8 Sensitivity of the mAb 28F6-based IPMA The PRRSV virus stock was subjected to a ten-fold serial dilution from 10 − 6.25 to 10 − 2.25 TCID 50 /100 µL, and then inoculated onto MARC-145 cells, respectively. The cells were cultured in 5% CO 2 incubator at 37°C for 48 h. The mAb 28F6-based IPMA was carried out under the optimal conditions to determine the limit of detection of the method. 2.9 Repeatability assay The repeatability of the mAb 28F6-based IPMA was determined by testing PRRSV of the same passages or different passages within a plate or between different plates. Meanwhile, different batches of anti-PRRSV mAb 28F6 were prepared to evaluate the repeatability of the assay. 2.10 Detection of different PRRSV variants by the mAb 28F6-based IPMA The mAb 28F6-based IPMA was used to detect different PRRSV variants including field strains such as BJ-4, HN07-1 and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. The mock-infected cells were used as negative controls. 2.11 Comparison of the IPMA with qRT-PCR The mAb 28F6-based IPMA was used to test 108 clinical samples together with the qRT-PCR developed in our laboratory. The diagnostic sensitivity (DSN), diagnostic specificity (DSP), and accuracy of the IPMA were calculated according to the formula: DSN = TP/(TP + FN) × 100; DSP = TN/(TN + FP) × 100, and accuracy = (TP + TN)/total number of serum samples tested × 100, where TP, FP, TN, and FN represented true-positive, false-positive, true-negative, and false-negative, respectively. 3. Results 3.1 Development and optimization of the mAb 28F6-based IPMA An IPMA was developed for the specific detection of PRRSV based on mAb 28F6 which possessed high-affinity and high-specificity against PRRSV. The working conditions of the mAb 28F6-based IPMA were optimized and the presence of red-brown precipitates in the wells indicated a positive result. The optimal incubation time of the virus was determined to be 48 h (Fig. 1 ). The optimal dilution of mAb 28F6 was determined to be 1:1.28×10 4 (Fig. 2 ), and the optimal incubation time of mAb 28F6 was found to be 90 min (Fig. 3 ). The optimal dilution of the HRP-labeled goat anti-mouse IgG was 1:400 (Fig. 4 ), and its optimal incubation time was determined to be 60 min (Fig. 5 ). The optimal time for color development was determined to be 10 min (Fig. 6 ). 3.2 Specificity of the mAb 28F6-based IPMA The specificity of the mAb 28F6-based IPMA was determined by testing its cross-reactions with CSFV, PCV2, and PEDV. As shown in Fig. 7 , MARC-145 cells infected with PRRSV showed obvious red-brown precipitates, while PK15 cells infected with CSFV or PCV2 were not stained. Moreover, PEDV-infected Vero cells also showed no reaction with mAb 28F6. These results indicated that the mAb 28F6-based IPMA was specific for the detection of PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. PRRSV-infected MARC-145 cells, CSFV-infected PK-15 cells, PCV2-infected PK-15 cells, and PEDV-infected Vero cells were incubated with mAb 28F6 at a dilution of 1:1.28×10 4 for 90 min. Mock-infected cells were used as controls. 3.3 Sensitivity of the mAb 28F6-based IPMA The sensitivity of the mAb 28F6-based IPMA was determined by testing PPRSV inoculum at different viral titers. The PRRSV virus stock with a viral titer of 10 − 6.25 TCID 50 /100 µL was subjected to a 10-fold serial dilution before inoculation onto MARC-145 cell. After the infected cells were cultured in a 5% CO 2 cell incubator at 37°C for 48 h, the mAb 28F6-based IPMA was performed under the optimal conditions. The results showed that the limit of detection of the IPMA reached 10 − 2.25 TCID 50 /100 µL (Fig. 8 ). 3.4 Repeatability of the mAb 28F6-based IPMA The mAb 28F6-based IPMA was used to detect PRRSV of different passages with different batches of mAb 28F6 (Fig. 9 A and 9 B). There was no significant difference in the detection of PRRSV of different passages with different batches of anti-PRRSV mAb 28F6, indicating that the mAb 28F6-based IPMA had good repeatability. 3.5 Detection of different PRRSV variants by IPMA The actual performance of the mAb 28F6-based IPMA was examined by testing its ability to detect different PRRSV variants including BJ-4, HN07-1, NADC30-like strain, HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. The results showed that MARC-145 cells infected with BJ-4, HN07-1, NADC30-like strain, HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, or R98 were all stained as red-brown (Fig. 10 ), indicating that the mAb 28F6-based IPMA can be used for the detection of different PRRSV variants. 3.6 Comparison of IPMA with qRT-PCR To evaluate the performance of mAb 28F6-based IPMA, 108 clinical samples were detected by IPMA and qRT-PCR (Table 1 ). Considering qRT-PCR as a gold standard, the DSN, DSP, and accuracy of IPMA were determined to be 100%, 100%, and 100%, respectively. Table 1 Comparison of IPMA with qRT-PCR IPMA Total Positive Negative qRT-PCR Positive 75 0 75 Negative 0 33 33 Total 75 33 108 Discussion PRRSV has caused significant economic losses to the global swine industry and continues to be a threat worldwide. The current clinical prevalence of PRRSV in China is characterized by complexity and diversity, with multiple types of strains coexisting and the occurrence of recombination between different types of viruses common [27]. It has been demonstrated that the genetic sequence of PRRSV was prone to mutations, resulting in high variability and strain diversity [28]. Meanwhile, the widespread use of various modified live vaccines (MLVs), including the 5-type PRRSV2 MLVs (Ingelvac PRRS MLV and R98) and the 8-type PRRSV2 MLVs (CH-1R, JXA1-R, HuN4-F112, TJM-F92, and GDr180), has led to the continuous emergence of MLV-derived isolates in the Chinese pig population [29]. For the specific and sensitive detection of various PRRSV field strains and vaccine strains, an IPMA was developed in this study based on an anti-PRRSV monoclonal antibody 28F6. To enhance the performance of the mAb 28F6-based IPMA, the working conditions were optimized in terms of the incubation time of the virus, the dilution and incubation time of mAb 28F6, the dilution and incubation time of secondary antibodies, and the time for color development. The key step in the development of an IPMA is the selection of a specific monoclonal antibody that possesses high-affinity against various viral strains. In this study, mAb 28F6 was previously produced by immunizing BALB/c mice with the native PRRSV particles and screened using IPMA. The recognition capability of mAb 28F6 against various PRRSV strains was validated and it was found that it could react with multiple PRRSV variants, indicating that it was a broad-spectrum monoclonal antibody. To improve the sensitivity of the IPMA, the virus was first allowed to proliferate on MARC-145 cells. After optimization, the incubation time of the virus was determined to be 48 h, which was in consistency with the replication dynamics of PRRSV [30]. In addition, the IPMA could specifically detect PRRSV and did not cross-react with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10 -2.25 TCID 50 /100 μL. Previously, VREMAN et al. infected 7.5-week-old piglets with PRRSV at a dose of 10 -6.0 TCID 50 /100 μL and found that viral titers in the blood on the 3 d and 21 d post-infection reached a level of 10 -2.4 TCID 50 /100 μL or higher [31]. Therefore, the mAb 28F6-based IPMA is capable of detecting PRRSV infection in both the early and late stages. Repeatability experiments showed that the mAb 28F6-based IPMA were highly reproducible as demonstrated in the detection of different passages of PRRSV using mAb 28F6 prepared from different batches of ascitic fluid. More important, the mAb 28F6-based IPMA could detect various PRRSV variants including field strains such as BJ-4, HN07-1, and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. Previously, a systematic phylogenetic analysis on 127 strains revealed four prevalent PRRSV lineages currently circulating in China [27]. The mAb 28F6-based IPMA could allow the detection of all these lineages including classical PRRSV, HP-PRRSV, NADC-like PRRSV, and vaccine strains. Meanwhile, the comparison of IPMA with qRT-PCR on the detection of 108 clinical samples showed that the mAb 28F6-based shared the same diagnostic sensitivity and diagnostic specificity with those of qRT-PCR. No discrepant results were obtained with the two methods. In conclusion, an IPMA was developed in this study to help control the spread of various PRRSV variants in the field. The IPMA was based on a broad-spectrum monoclonal antibody (28F6) against PRRSV and it allowed visual examination of the results with simple experimental procedures. The mAb 28F6-based IPMA had good sensitivity and high specificity for the detection of different PRRSV strains. These data indicated that the IPMA would greatly facilitate the large-scale detection of PRRSV vaccine strains and field strains in veterinary clinic. Abbreviations PRRS Porcine reproductive and respiratory syndrome PRRSV Porcine reproductive and respiratory syndrome virus IPMA Immunoperoxidase monolayer assay RT-PCR Reverse transcription polymerase chain reaction IFA Immunofluorescence assay CSFV Classical swine fever virus ASFV African Swine Fever Virus mAb Monoclonal antibody MARC-145 Meat Animal Research Center 145 PCV2 Porcine circovirus type 2 PEDV Porcine epidemic diarrhea virus TCID 50 50% tissue culture infectious dose. Declarations Acknowledgements This work was conducted in The Animal Disease Molecular Diagnosis Engineering Laboratory of Henan Province. Funding This work was supported by grants from The Modern Agricultural Industry Technology System in Henan Province（HARS2212G3）and The Scientific and Technological Breakthrough Foundation of Henan Province (242102110061). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors’ contributions Peng LI and Chunxiao GUAN contributed equally to this work and should be considered co-first authors. Corresponding authors Correspondence to Xingyou LIU or Xuannian WANG. Ethics approval and consent to participate All samples were obtained from dead animals by identification personnel. Therefore, no pigs were artificially euthanized in this study. Consent for publication Not applicable. 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Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccine in neonatal pigs using different adjuvants. Vet Immunol Immunopathol. 2021;232:110170. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4331925","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297772894,"identity":"e5134302-60df-487f-a696-29de6eb023b8","order_by":0,"name":"peng LI","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"peng","middleName":"","lastName":"LI","suffix":""},{"id":297772898,"identity":"4056fc08-917d-4f93-aeca-29dc435be0c7","order_by":1,"name":"Chunxiao GUAN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBADHn7+5gMHPvwgQYuM5IxjiQdn9pCgxcbgQI7xYQ42IpTKRyQ/e/CzjYGH4cCZD4eBlDy/2AH8WgxvpJkb9pxh4GFs7t1wuMCCwXDm7AQCWmYkmEkzVDDwMDOc3XB4Bg9DgsFtglrSv0kzGDDwsDHkPDgMJAlrkZfIgdjCw5DDQJwWA543ZZIgv0hIHDMABrIEYb/It6dvkwCGmL39+ebHHz78sJHnlyZkywEw9R/Gl8CvHGxLA2E1o2AUjIJRMNIBALVHQVXQmnyhAAAAAElFTkSuQmCC","orcid":"","institution":"Xinxiang University","correspondingAuthor":true,"prefix":"","firstName":"Chunxiao","middleName":"","lastName":"GUAN","suffix":""},{"id":297772903,"identity":"b57293e1-27bc-4c31-9b67-080e5ef673c4","order_by":2,"name":"Liping WANG","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"WANG","suffix":""},{"id":297772908,"identity":"92642591-2e49-47b5-a28f-abe0de677632","order_by":3,"name":"Huajian WANG","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"Huajian","middleName":"","lastName":"WANG","suffix":""},{"id":297772912,"identity":"01550ce8-9703-46d0-978f-8ae246991d54","order_by":4,"name":"Guopeng SUN","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"Guopeng","middleName":"","lastName":"SUN","suffix":""},{"id":297772915,"identity":"35b198fc-c673-45df-9b4a-c780ca24985d","order_by":5,"name":"Jinjiao HE","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"Jinjiao","middleName":"","lastName":"HE","suffix":""},{"id":297772919,"identity":"4bcf2838-bc62-4313-b512-b35f00988021","order_by":6,"name":"Xingyou LIU","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"Xingyou","middleName":"","lastName":"LIU","suffix":""},{"id":297772922,"identity":"5d6caee5-f03f-4d44-b1ac-9a76343473a1","order_by":7,"name":"Xuannian WANG","email":"","orcid":"","institution":"Xinxiang University","correspondingAuthor":false,"prefix":"","firstName":"Xuannian","middleName":"","lastName":"WANG","suffix":""}],"badges":[],"createdAt":"2024-04-27 01:26:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4331925/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4331925/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55896935,"identity":"a7d620e1-8ba4-4494-82e7-121e2eb5695f","added_by":"auto","created_at":"2024-05-06 04:22:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10821493,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the optimal incubation time of PRRSV\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV for 24 h (a), 48 h (b),72 h (c),96 h (d), and 120 h (e), respectively. After fixation, the cells were incubated with mAb 28F6 and HRP-labeled goat anti-mouse IgG in turns. Color development was performed for 10 min after the addition of AEC substrate. Mock-infected MARC-145 cells were used as controls (f).\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/45a56dccbf5a10e5a36ed196.png"},{"id":55896934,"identity":"57eb880e-9a50-4a8d-89ad-12087a427393","added_by":"auto","created_at":"2024-05-06 04:22:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17440238,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the optimal dilution of mAb 28F6\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV for 48 h and fixed. mAb 28F6 diluted 1:4×10\u003csup\u003e2\u003c/sup\u003e (a), 1:8×10\u003csup\u003e2\u003c/sup\u003e (b), 1:1.6×10\u003csup\u003e3\u003c/sup\u003e (c), 1:3.2×10\u003csup\u003e3\u003c/sup\u003e (d), 1:6.4×10\u003csup\u003e3\u003c/sup\u003e (e), 1:1.28×10\u003csup\u003e4\u003c/sup\u003e (f), 1:2.56×10\u003csup\u003e4\u003c/sup\u003e (g), and 1:5.12×10\u003csup\u003e4\u003c/sup\u003e (h) were incubated with the cells, respectively. Mock-infected MARC-145 cells were used as controls (i).\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/d994735d2af80c566450b847.png"},{"id":55896938,"identity":"cb792d5a-e984-4ccd-b4e3-4612e6c42dc5","added_by":"auto","created_at":"2024-05-06 04:22:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7597853,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the optimal incubation time of mAb 28F6\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV for 48 h and fixed. Then, IPMA was performed in which the cells were incubated with mAb 28F6 at a dilution of 1:1.28×10\u003csup\u003e4\u003c/sup\u003e for 30 min, 60 min, 90 min, and 120 min, respectively. Mock-infected MARC-145 cells were used as controls.\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/e5294fc323b44b6bfef70c76.png"},{"id":55896936,"identity":"8be56d66-8793-4052-8e94-f9c1d9734c54","added_by":"auto","created_at":"2024-05-06 04:22:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10719217,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the optimal dilution of secondary antibody\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV for 48 h and fixed. Then, IPMA was performed in which HRP-labeled goat anti-mouse IgG diluted 1:1×10\u003csup\u003e2\u003c/sup\u003e (a), 1:2×10\u003csup\u003e2\u003c/sup\u003e (b), 1:4×10\u003csup\u003e2\u003c/sup\u003e (c), 1:8×10\u003csup\u003e2\u003c/sup\u003e (d), and 1:1.6×10\u003csup\u003e3\u003c/sup\u003e (e) were added to incubated with the pretreated cells, respectively. Mock-infected MARC-145 cells were used as controls (f).\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/b73773b132b740a5252cac8b.png"},{"id":55896937,"identity":"2bd6b912-3361-40f7-918e-550e25da553f","added_by":"auto","created_at":"2024-05-06 04:22:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10719724,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the optimal incubation time of secondary antibody\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV for 48 h and fixed. Then, IPMA was performed with the pre-determined conditions. HRP-labeled goat anti-mouse IgG at a dilution of 1:4×10\u003csup\u003e2\u003c/sup\u003e was allowed to incubated with the cells for 30 min, 60 min, and 90 min, respectively. Mock-infected MARC-145 cells were used as controls.\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/aa61114abf4830345af7cafd.png"},{"id":55896940,"identity":"b364ba56-78b6-4438-b692-1aef5edd1bc9","added_by":"auto","created_at":"2024-05-06 04:22:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10765262,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the optimal time for color development\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV for 48 h and fixed. Then, IPMA was performed under the optimal conditions. The cells were stained with AEC substrate for 5 min, 10 min, and 15 min, respectively. Mock-infected MARC-145 cells were used as controls.\u003c/p\u003e","description":"","filename":"Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/91df11d8e811e58280d43659.png"},{"id":55896939,"identity":"0758380b-aebc-46ad-96f7-8ad45410412c","added_by":"auto","created_at":"2024-05-06 04:22:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7993216,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity of the mAb 28F6-based IPMA\u003c/p\u003e\n\u003cp\u003ePRRSV-infected MARC-145 cells, CSFV-infected PK-15 cells, PCV2-infected PK-15 cells, and PEDV-infected Vero cells were incubated with mAb 28F6 at a dilution of 1:1.28×10\u003csup\u003e4\u003c/sup\u003e for 90 min. Mock-infected cells were used as controls.\u003c/p\u003e","description":"","filename":"Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/8b72526ff2a2a81434ed382a.png"},{"id":55897394,"identity":"58e516a0-943f-4a80-9dfa-c87b585b3551","added_by":"auto","created_at":"2024-05-06 04:30:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":10676232,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity of the mAb 28F6-based IPMA\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with PRRSV at viral titers of 10\u003csup\u003e-6.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 μL (a), 10\u003csup\u003e-5.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 μL (b), 10\u003csup\u003e-4.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 μL (c), 10\u003csup\u003e-3.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 μL (d), and 10\u003csup\u003e-2.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 μL (e), respectively. Mock-infected MARC-145 cells were used as controls.\u003c/p\u003e","description":"","filename":"Figures8.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/e90ae58d5fca8f98deff7f1c.png"},{"id":55896943,"identity":"471051b2-b233-44f3-aeed-b5e67d2091f9","added_by":"auto","created_at":"2024-05-06 04:22:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":15214624,"visible":true,"origin":"","legend":"\u003cp\u003eRepeatability of the mAb 28F6-based IPMA\u003c/p\u003e\n\u003cp\u003eA: mAb 28F6 of the same batch was allowed to incubated with MARC-145 cells infected with three batches of PRRSV (PRRSV # 1, PRRSV # 2, PRRSV # 3). The peritoneal fluid of healthy mice was used as a control to incubated with the infected cells. B: Three batches of mAb 28F6 (Antibody 1, Antibody 2, Antibody 3) were allowed to incubate with PRRSV-infected MARC-145 cells. Mock-infected MARC-145 cells were used as controls. Each assay was repeated three times.\u003c/p\u003e","description":"","filename":"Figures9.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/3482667731e9c82dc53bc0ac.png"},{"id":55896942,"identity":"8b5a2c24-df75-4637-9615-cba7ac4947d6","added_by":"auto","created_at":"2024-05-06 04:22:05","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":12089922,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of different PRRSV variants by the mAb 28F6-based IPMA\u003c/p\u003e\n\u003cp\u003eMARC-145 cells were infected with BJ-4, NADC30-like strain, HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98 for 48 h, respectively before IPMA was performed. MARC-145 cells infected with PRRSV HN07-1 were used as positive controls, and mock-infected MARC-145 cells were used as negative controls.\u003c/p\u003e","description":"","filename":"Figures10.png","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/106091725da379aafa10abde.png"},{"id":80792849,"identity":"8fdfde1e-8e5e-4bfc-83b6-2bbcd2f700aa","added_by":"auto","created_at":"2025-04-17 07:03:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":175924733,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4331925/v1/f09acdda-617f-4b95-918e-b9e5e9e0efa9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and application of an immuoperoxidase monolayer assay for the detection of PRRSV","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePorcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease characterized by high morbidity and mortality in pregnant sows and respiratory system damage in piglets [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is listed as the second-class infectious disease of pigs in China [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Common clinical manifestations of PRRS included high fever, mental malaise, abortion, premature stillbirth, mummified fetus, and weak fetus [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe causative agent of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV), which is an enveloped RNA virus belonging to order Nidovirales, family Arteriviridae, genus Arterivirus [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In 1991, the PRRSV strain Lelystad was first isolated in the Netherlands, and later the PRRSV strain ATCC VR-2332 was isolated in the United States in 1992 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The two strains could cause similar clinical symptoms, but were different in genome homology and pathogenicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The Chinese PRRSV strain was first isolated in 1995 and was demonstrated to be the variant of PRRSV strain ATCC VR-2332 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In 2006, a highly pathogenic PRRSV (HP-PRRSV), characterized by high fever and high mortality, was isolated in China and widely spread later [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Since the end of 2012, PRRSV NADC30-like strains such as JL580 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], HnyC15 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], HENAN-XINX [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and CHSX1401 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], have been isolated in China and become the dominant circulating strains of PRRSV in many areas across China [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the circulation of classical PRRSV strains, HP-PRRSV strains, and NADC30-like PRRSV strains has brought new challenges to the swine industry in China.\u003c/p\u003e \u003cp\u003eAccurate laboratory diagnosis of PRRSV is considered as an effective control measure. At present, a variety of methods have been developed for the detection of PRRSV, such as reverse transcription polymerase chain reaction (RT-PCR) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], quantitative reverse transcription polymerase chain reaction (qRT-PCR) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and immunofluorescence assay (IFA) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. RT-PCR and qRT-PCR require complex instruments and skilled operators, which are suitable for laboratory use and are not suitable for clinical large-scale screening. IFA offers high sensitivity and specificity, making it suitable for early diagnosis. However, the presence of intrinsic fluorescent substances in field samples can lead to background signal, which may affect the interpretation of results. Additionally, fluorescent dyes are prone to quenching during storage, making long-term preservation challenging [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, immuoperoxidase monolayer assay (IPMA) offers relatively simple experimental procedures that do not require expensive equipment or complex laboratory operations [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It is a widely applied immunological technique that can be used for the detection and localization of specific antigens in cells or tissues [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In IPMA, the enzymatic reaction produces a visible pigment deposition on cells, which can be observed under a regular microscope, enabling visual analysis of immune reactions on the cell surface or within the cells. In addition, the cell monolayers used in IPMA can be preserved after fixation, which enabled long-term sample storage for subsequent analysis and follow-up examinations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany IPMA methods have been developed for the detection of bacterial and viral infections, as it enabled the visualization of pathogens within infected cells and facilitated the identification and characterization of infections. Zhang et al. established an IPMA for the detection of classical swine fever virus (CSFV) and found that the positive coincidence rate of IPMA with RT-PCR and ELISA reached more than 92% [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Li et al. developed an IPMA to detect intracellular Lawsonella and found a positive coincidence rate of 94.6% with PCR [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A study compared the effectiveness of IPMA in detecting African Swine Fever Virus (ASFV) in macrophage cultures and found that IPMA had a detection rate of 80% as compared with PCR [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe first IPMA method for the detection of PRRSV was established in 1991 by the Central Veterinary Institute of the Netherlands and showed good sensitivity and specificity in the detection of the PRRSV strain Lelystad virus [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Due to the high variability of the virus itself and the recombination of different PRRSV variants in the field, the diagnosis and prevention and control of PRRSV face continued challenges [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo provide an effective IPMA for the detection of different PRRSV field strains and vaccine strains, a specific and sensitive IPMA was developed based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6 in this study. The working conditions of the mAb 28F6-based IPMA were optimized, and the specificity and sensitivity of the IPMA were evaluated. In addition, the IPMA was validated by detecting 108 clinical samples in parallel with qRT-PCR. The results showed that the mAb 28F6-based IPMA has the potential to be used in veterinary clinics for the detection of different PRRSV variants.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Antibodies\u003c/h2\u003e \u003cp\u003eThe anti-PRRSV monoclonal antibody (mAb) 28F6 was previously produced by immunizing BALB/c mice with HP-PRRSV HN07-1 (GenBank: KX766378.1) and preserved in our laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cells and viruses\u003c/h2\u003e \u003cp\u003eMeat Animal Research Center 145 (MARC-145) cells, Vero cells, and porcine kidney epithelial cells (PK15) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin) in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. PRRSV vaccine strains HuN4-F112 (Harbin Harvac Biotechnology Co., Ltd,; Harbin, China), JXA1-R (Wuhan Keqian Biology Co., Ltd.; Wuhan, China), TJM-F92 (Sinovet (Jiangsu) Biotechnology Co., Ltd.; Taizhou, China), GDr180 (Guangdong Winsun Bio-pharmaceutical Co., Ltd.; Guangzhou, China), VR2332 (Boehringer Ingelheim Animal Health Operations (China) Co., Ltd.; Taizhou, China), CH-1R (Harbin Weike Biotechnology Co., Ltd.; Harbin, China), and R98 (Qilu Animal Health Products Co., Ltd.; Jinan, China) were bought and propagated on MARC-145 cells. PRRSV field strains BJ-4 (GenBank: KP771761.1), HN07-1, and NADC30-like PRRSV (GenBank: JN654459.1) were kindly provided by the Henan Provincial Key Laboratory of Animal Immunology. Classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV) were previously stored in our laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Pretreatment of clinical samples\u003c/h2\u003e \u003cp\u003eA total of 108 clinical samples of porcine lymph nodes, tonsils, lungs, and spleens were collected in different regions of Henan province, China and kept at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eBefore inoculation onto cells, 0.5 g of each sample was fully ground in liquid nitrogen with the addition of 0.5 mL of phosphate buffered saline (PBS) containing penicillin and streptomycin. The supernatant was collected by centrifugation at 5,000 r/min for 10 min at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Establishment of an IPMA\u003c/h2\u003e \u003cp\u003eMonolayers of MARC-145 cells were grown in 96-well cell culture plates at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The cells were infected with PRRSV for 48 h before fixation in cold methanol for 15 min at room temperature. After the plates were blocked with 5% skimmed milk at 37\u0026deg;C for 1 h, anti-PRRSV mAb 28F6 was added to incubate with the plates at 37\u0026deg;C for 1 h. Then, horseradish peroxidase (HRP)-labelled goat anti-mouse IgG was used as secondary antibodies to incubate with the plates at 37\u0026deg;C for 1 h. Finally, AEC substrate buffer (3-amino-9-ethylcarbazole) was added for color development for 10 min before the addition of PBS to wash the plates and stop the color reaction. The wells were then observed under a light microscope and the appearance of red-brown precipitates in the wells indicated a positive result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Viral titration\u003c/h2\u003e \u003cp\u003eViral titration was performed on monolayer cells of MARC-145 using IPMA. When the cells were grown to 80% confluence, ten-fold serial dilutions of the viral stock were added respectively and incubated with the cells for 2 h. After the removal of the inoculum, 100 \u0026micro;L of DMEM containing 2% FBS was added to the wells. Then, the cells were kept at 37\u0026deg;C for 48 h. Later, IPMA was performed to ascertain the presence of the virus. The viral titer was calculated using the Reed\u0026ndash;Muench method and expressed as 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Optimization of the working conditions of IPMA\u003c/h2\u003e \u003cp\u003eThe working conditions of the mAb 28F6-based IPMA were optimized in terms of incubation time of the virus, the dilution and incubation time of mAb 28F6, the dilution and incubation time of HRP-labelled goat anti-mouse IgG, and the time for color development. The condition giving clear morphology of stained cells and moderate amount of unstained cells was selected to be optimal.\u003c/p\u003e \u003cp\u003eTo determine the optimal incubation time of the virus, MARC-145 cells were infected with 10\u003csup\u003e\u0026minus;\u0026thinsp;1.69\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026micro;L of PRRSV at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 h, 48 h, 72 h, 96 h, and 120 h. To determine the optimal dilution of the anti-PRRSV mAb 28F6, the infected MARC-145 cells were incubated with mAb 28F6 at dilutions of 1:4\u0026times;10\u003csup\u003e2\u003c/sup\u003e, 1:8\u0026times;10\u003csup\u003e2\u003c/sup\u003e, 1:1.6\u0026times;10\u003csup\u003e3\u003c/sup\u003e, 1:3.2\u0026times;10\u003csup\u003e3\u003c/sup\u003e, 1:6.4\u0026times;10\u003csup\u003e3\u003c/sup\u003e, 1:1.28\u0026times;10\u003csup\u003e4\u003c/sup\u003e, 1:2.56\u0026times;10\u003csup\u003e4\u003c/sup\u003e, and 1:5.12\u0026times;10\u003csup\u003e4\u003c/sup\u003e, respectively. The incubation time of mAb 28F6 was set to be 30 min, 60 min, 90 min, and 120 min to determine its optimal reaction time. To determine the optimal dilution of secondary antibodies, goat anti-mouse IgG-HRP was serially diluted from 1:1\u0026times;10\u003csup\u003e2\u003c/sup\u003e to 1:2\u0026times;10\u003csup\u003e2\u003c/sup\u003e, 1:4\u0026times;10\u003csup\u003e2\u003c/sup\u003e, 1:8\u0026times;10\u003csup\u003e2\u003c/sup\u003e, and 1:1.6\u0026times;10\u003csup\u003e3\u003c/sup\u003e. The incubation time of secondary antibodies was set to be 30 min, 60 min, 90 min, and 120 min to obtain the optimal reaction. The time for color development was set to be 5 min, 10 min, and 15 min. Each sample was tested in triplicate, and each assay was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Specificity of the mAb 28F6-based IPMA\u003c/h2\u003e \u003cp\u003ePRRSV was inoculated onto MARC-145 cells. CSFV or PCV2 were inoculated onto PK-15 cell. PEDV was inoculated onto Vero cells. The infected cells were cultured in 5% CO\u003csub\u003e2\u003c/sub\u003e cell culture incubator at 37\u0026deg;C for 48 h. Then, the mAb 28F6-based IPMA was conducted to determine the specificity of the method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Sensitivity of the mAb 28F6-based IPMA\u003c/h2\u003e \u003cp\u003eThe PRRSV virus stock was subjected to a ten-fold serial dilution from 10\u003csup\u003e\u0026minus;\u0026thinsp;6.25\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;2.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026micro;L, and then inoculated onto MARC-145 cells, respectively. The cells were cultured in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C for 48 h. The mAb 28F6-based IPMA was carried out under the optimal conditions to determine the limit of detection of the method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Repeatability assay\u003c/h2\u003e \u003cp\u003eThe repeatability of the mAb 28F6-based IPMA was determined by testing PRRSV of the same passages or different passages within a plate or between different plates. Meanwhile, different batches of anti-PRRSV mAb 28F6 were prepared to evaluate the repeatability of the assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Detection of different PRRSV variants by the mAb 28F6-based IPMA\u003c/h2\u003e \u003cp\u003eThe mAb 28F6-based IPMA was used to detect different PRRSV variants including field strains such as BJ-4, HN07-1 and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. The mock-infected cells were used as negative controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Comparison of the IPMA with qRT-PCR\u003c/h2\u003e \u003cp\u003eThe mAb 28F6-based IPMA was used to test 108 clinical samples together with the qRT-PCR developed in our laboratory. The diagnostic sensitivity (DSN), diagnostic specificity (DSP), and accuracy of the IPMA were calculated according to the formula: DSN\u0026thinsp;=\u0026thinsp;TP/(TP\u0026thinsp;+\u0026thinsp;FN) \u0026times; 100; DSP\u0026thinsp;=\u0026thinsp;TN/(TN\u0026thinsp;+\u0026thinsp;FP) \u0026times; 100, and accuracy = (TP\u0026thinsp;+\u0026thinsp;TN)/total number of serum samples tested \u0026times; 100, where TP, FP, TN, and FN represented true-positive, false-positive, true-negative, and false-negative, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Development and optimization of the mAb 28F6-based IPMA\u003c/h2\u003e\n\u003cp\u003eAn IPMA was developed for the specific detection of PRRSV based on mAb 28F6 which possessed high-affinity and high-specificity against PRRSV. The working conditions of the mAb 28F6-based IPMA were optimized and the presence of red-brown precipitates in the wells indicated a positive result. The optimal incubation time of the virus was determined to be 48 h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The optimal dilution of mAb 28F6 was determined to be 1:1.28\u0026times;10\u003csup\u003e4\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), and the optimal incubation time of mAb 28F6 was found to be 90 min (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The optimal dilution of the HRP-labeled goat anti-mouse IgG was 1:400 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), and its optimal incubation time was determined to be 60 min (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The optimal time for color development was determined to be 10 min (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Specificity of the mAb 28F6-based IPMA\u003c/h2\u003e\n\u003cp\u003eThe specificity of the mAb 28F6-based IPMA was determined by testing its cross-reactions with CSFV, PCV2, and PEDV. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, MARC-145 cells infected with PRRSV showed obvious red-brown precipitates, while PK15 cells infected with CSFV or PCV2 were not stained. Moreover, PEDV-infected Vero cells also showed no reaction with mAb 28F6. These results indicated that the mAb 28F6-based IPMA was specific for the detection of PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV.\u003c/p\u003e\n\u003cp\u003ePRRSV-infected MARC-145 cells, CSFV-infected PK-15 cells, PCV2-infected PK-15 cells, and PEDV-infected Vero cells were incubated with mAb 28F6 at a dilution of 1:1.28\u0026times;10\u003csup\u003e4\u003c/sup\u003e for 90 min. Mock-infected cells were used as controls.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Sensitivity of the mAb 28F6-based IPMA\u003c/h2\u003e\n\u003cp\u003eThe sensitivity of the mAb 28F6-based IPMA was determined by testing PPRSV inoculum at different viral titers. The PRRSV virus stock with a viral titer of 10\u003csup\u003e\u0026minus;\u0026thinsp;6.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026micro;L was subjected to a 10-fold serial dilution before inoculation onto MARC-145 cell. After the infected cells were cultured in a 5% CO\u003csub\u003e2\u003c/sub\u003e cell incubator at 37\u0026deg;C for 48 h, the mAb 28F6-based IPMA was performed under the optimal conditions. The results showed that the limit of detection of the IPMA reached 10\u003csup\u003e\u0026minus;\u0026thinsp;2.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026micro;L (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Repeatability of the mAb 28F6-based IPMA\u003c/h2\u003e\n\u003cp\u003eThe mAb 28F6-based IPMA was used to detect PRRSV of different passages with different batches of mAb 28F6 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB). There was no significant difference in the detection of PRRSV of different passages with different batches of anti-PRRSV mAb 28F6, indicating that the mAb 28F6-based IPMA had good repeatability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Detection of different PRRSV variants by IPMA\u003c/h2\u003e\n\u003cp\u003eThe actual performance of the mAb 28F6-based IPMA was examined by testing its ability to detect different PRRSV variants including BJ-4, HN07-1, NADC30-like strain, HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. The results showed that MARC-145 cells infected with BJ-4, HN07-1, NADC30-like strain, HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, or R98 were all stained as red-brown (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e), indicating that the mAb 28F6-based IPMA can be used for the detection of different PRRSV variants.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 Comparison of IPMA with qRT-PCR\u003c/h2\u003e\n\u003cp\u003eTo evaluate the performance of mAb 28F6-based IPMA, 108 clinical samples were detected by IPMA and qRT-PCR (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Considering qRT-PCR as a gold standard, the DSN, DSP, and accuracy of IPMA were determined to be 100%, 100%, and 100%, respectively.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of IPMA with qRT-PCR\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIPMA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eqRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\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\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\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\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e108\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePRRSV has caused significant economic losses to the global swine industry and continues to be a threat worldwide. The current clinical prevalence of PRRSV in China is characterized by complexity and diversity, with multiple types of strains coexisting and the occurrence of recombination between different types of viruses common [27]. It has been demonstrated that the genetic sequence of PRRSV was prone to mutations, resulting in high variability and strain diversity [28]. Meanwhile, the widespread use of various modified live vaccines (MLVs), including the 5-type PRRSV2 MLVs (Ingelvac PRRS MLV and R98) and the 8-type PRRSV2 MLVs (CH-1R, JXA1-R, HuN4-F112, TJM-F92, and GDr180), has led to the continuous emergence of MLV-derived isolates in the Chinese pig population [29]. For the\u0026nbsp;specific and sensitive\u0026nbsp;detection of various PRRSV field strains and vaccine strains, an IPMA was developed in this study based on an anti-PRRSV monoclonal antibody 28F6.\u003c/p\u003e\n\u003cp\u003eTo enhance the performance of the mAb 28F6-based IPMA, the working conditions were optimized in terms of the incubation time of the virus, the dilution and incubation time of mAb 28F6, the dilution and incubation time of secondary antibodies, and the time for color development.\u0026nbsp;The key step in the development of an IPMA is the selection of a specific monoclonal antibody that possesses high-affinity against various viral strains. In this study, mAb 28F6 was previously produced by immunizing BALB/c mice with the native PRRSV particles and screened using IPMA. The recognition capability of mAb 28F6 against various PRRSV strains was validated and it was found that it could react with multiple PRRSV variants, indicating that it was a broad-spectrum monoclonal antibody.\u003c/p\u003e\n\u003cp\u003eTo improve the sensitivity of the IPMA, the virus was first allowed to proliferate on MARC-145 cells. After optimization, the incubation time of the virus was determined to be 48 h, which was in consistency with the replication dynamics of PRRSV [30].\u0026nbsp;In addition, the IPMA could specifically detect PRRSV and did not cross-react with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10\u003csup\u003e-2.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026mu;L. Previously, VREMAN et al. infected 7.5-week-old piglets with PRRSV at a dose of 10\u003csup\u003e-6.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026mu;L and found that viral titers in the blood on the 3 d and 21 d post-infection reached a level of 10\u003csup\u003e-2.4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026mu;L or higher [31]. Therefore, the mAb 28F6-based IPMA is capable of detecting PRRSV infection in both the early and late stages. Repeatability experiments showed that the mAb 28F6-based IPMA were highly reproducible as demonstrated in the detection of different passages of PRRSV using mAb 28F6 prepared from different batches of ascitic fluid. More important, the mAb 28F6-based IPMA could detect various PRRSV variants including field strains such as BJ-4, HN07-1, and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and\u0026nbsp;R98. Previously, a systematic phylogenetic analysis on 127 strains revealed four prevalent PRRSV lineages currently circulating in China [27]. The mAb 28F6-based IPMA could allow the detection of all these lineages including classical PRRSV, HP-PRRSV, NADC-like PRRSV, and vaccine strains. Meanwhile, the comparison of IPMA with qRT-PCR on the detection of 108 clinical samples showed that the mAb 28F6-based shared the same diagnostic sensitivity and diagnostic specificity with those of\u0026nbsp;qRT-PCR. No discrepant results were obtained with the two methods.\u003c/p\u003e\n\u003cp\u003eIn conclusion, an IPMA was developed in this study to help control the spread of various PRRSV variants in the field. The IPMA was based on a broad-spectrum monoclonal antibody (28F6) against PRRSV and it allowed visual examination of the results with simple experimental procedures. The mAb 28F6-based IPMA had good sensitivity and high specificity for the detection of different PRRSV strains. These data indicated that the IPMA would greatly facilitate the large-scale detection of PRRSV vaccine strains and field strains in veterinary clinic.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRRS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorcine reproductive and respiratory syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRRSV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorcine reproductive and respiratory syndrome virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunoperoxidase monolayer assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReverse transcription polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunofluorescence assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCSFV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClassical swine fever virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASFV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAfrican Swine Fever Virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emAb\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMonoclonal antibody\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMARC-145\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMeat Animal Research Center 145\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCV2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorcine circovirus type 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEDV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorcine epidemic diarrhea virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCID\u003csub\u003e50\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e50% tissue culture infectious dose.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was conducted in The Animal Disease Molecular Diagnosis Engineering Laboratory of Henan Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from The Modern Agricultural Industry Technology System in Henan Province（HARS2212G3）and The Scientific and Technological Breakthrough Foundation of Henan Province (242102110061).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeng LI and Chunxiao GUAN contributed equally to this work and should be considered co-first authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xingyou LIU or Xuannian WANG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll samples were obtained from dead animals by identification personnel. Therefore, no pigs were artificially euthanized in this study.\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 interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eSchool of Biological Engineering, Xinxiang University, Xinxiang, 453003,China; \u003csup\u003e2\u003c/sup\u003eCollege of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, 453003,China; \u003csup\u003e3\u003c/sup\u003eCollege of Life Science, Henan Normal University, Xinxiang, 453007,China; \u003csup\u003e4\u003c/sup\u003eCollege of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002，China)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmadori M, Razzuoli E. Immune control of prrs: Lessons to be learned and possible ways forward. Front Veterinary Sci. 2014;1:2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Q, Xu H, An T, Cai X, Tian Z, Zhang H. Recent progress in studies of porcine reproductive and respiratory syndrome virus 1 in china. Viruses. 2023;15(7):1528.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho JG, Dee SA. Porcine reproductive and respiratory syndrome virus. Theriogenology. 2006;66(3):655\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlanratmanee E-o, Wongyanin P, Thanawongnuwech R, Tummaruk P. Prevalence of porcine reproductive and respiratory syndrome virus detection in aborted fetuses, mummified fetuses and stillborn piglets using quantitative polymerase chain reaction. J Vet Med Sci. 2015;77(9):1071\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuhn JH, Lauck M, Bailey AL, Shchetinin AM, Vishnevskaya TV, B\u0026agrave;o Y, et al. Reorganization and expansion of the nidoviral family arteriviridae. Arch Virol. 2016;161(3):755\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Zhang W, Xiang L, Leng C, Tian Z, Tang Y, et al. Emergence of novel porcine reproductive and respiratory syndrome viruses (orf5 rflp 1-7-4 viruses) in china. Vet Microbiol. 2018;222:105\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKappes MA, Faaberg KS. Prrsv structure, replication and recombination: Origin of phenotype and genotype diversity. Virology. 2015;479\u0026ndash;480:475\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo B, Chen Z, Liu W, Cui Y, Kong L. 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Open Virol J. 2017;11:59\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang K, Li Y, Duan Z, Guo R, Liu Z, Zhou D, et al. A one-step rt-pcr assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens. Gene. 2013;531(2):199\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian H, Wu J, Shang Y, Cheng Y, Liu X. The development of a rapid sybr one step real-time rt-pcr for detection of porcine reproductive and respiratory syndrome virus. Virol J. 2010;7:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeifke JP, Dauber M, Fichtner D, Lenk M, Polster U, Weiland E, et al. Detection of european porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling. J Comp Pathol. 2001;124(4):238\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakane PK, Pierce GB. Enzyme-labeled antibodies: Preparation and application for the localization of antigens. J Histochem Cytochemistry. 1966;14(12):929\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaegeman A, De Leeuw I, Mostin L, Van Campe W, Aerts L, Vastag M, et al. An immunoperoxidase monolayer assay (ipma) for the detection of lumpy skin disease antibodies. J Virol Methods. 2020;277:113800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuedes RMC, Gebhart CJ, Winkelman NL, Mackie-Nuss RA. A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy. J Vet Diagn Invest. 2002;14(5):420\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Liu W, Chen W, Li C, Xie M, Bu Z. Development of an immunoperoxidase monolayer assay for the detection of antibodies against peste des petits ruminants virus based on bhk-21 cell line stably expressing the goat signaling lymphocyte activation molecule. PLoS ONE. 2016;11(10):e0165088.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHANG Z, WEl L, Guo K, Luo Y. Establishment of immunoperoxidase monolayer assay detecting classical swine fever virus (in chinese). Chin Veterinary Sci. 2018;48(03):275\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLI M, Ll J, ZHOU H, XlAO N, HUI X. Establishment and preliminary application of lawsonia intracellularis ipma antigen detectionmethod based on sod c monoclonal antibody (in chinese). Scientia Agricultura Sinica. 2021;54(20):4478\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfayoa M, Atuhaire DK, Ochwo S, Okuni JB, Majid K, Mwiine FN, et al. Comparison of immunoperoxidase monolayer assay, polymerase chain reaction and haemadsorption tests in the detection of african swine fever virus in cell cultures using ugandan isolates. J Gen Mol Virol. 2014;6(4):36\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWensvoort G, de Kluyver EP, Pol JM, Wagenaar F, Moormann RJ, Hulst MM, et al. Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: A review of mystery swine disease research at lelystad. Vet Microbiol. 1992;33(1\u0026ndash;4):185\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrevisan G, Zeller M, Li G, Zhang J, Gauger P, Linhares DCL. Implementing a user-friendly format to analyze prrsv next-generation sequencing results and associating breeding herd production performance with number of prrsv strains and recombination events. Transbound Emerg Dis. 2022;69(5):e2214\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Z, Chen X, Li R, Qiao S, Zhang G. The prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in china: A molecular epidemiological perspective. Virol J. 2018;15(1):2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRisser J, Ackerman M, Evelsizer R, Wu S, Kwon B, Hammer JM. Porcine reproductive and respiratory syndrome virus genetic variability a management and diagnostic dilemma. Virol J. 2021;18(1):206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen N, Xiao Y, Ye M, Li X, Li S, Xie N, et al. High genetic diversity of chinese porcine reproductive and respiratory syndrome viruses from 2016 to 2019. Res Vet Sci. 2020;131:38\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKombiah S, Senthilkumar D, Kumar M, Sivasankar P, Singh VP, Rajukumar K. Growth kinetics of an indian isolate of highly pathogenic porcine reproductive and respiratory syndrome virus in marc-145 cells. Virusdisease. 2022;33(2):208\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVreman S, Stockhofe-Zurwieden N, Popma-de Graaf DJ, Savelkoul HFJ, Barnier-Quer C, Collin N, et al. Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccine in neonatal pigs using different adjuvants. Vet Immunol Immunopathol. 2021;232:110170.\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":false,"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":"PRRSV, Broad-spectrum monoclonal antibody, Immunoperoxidase monolayer assay, Specific detection","lastPublishedDoi":"10.21203/rs.3.rs-4331925/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4331925/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePorcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a highly contagious disease with high morbidity and mortality that affects the global swine industry. So far, there is still a widespread dissemination of PRRSV with obvious genetic variations in swine population, resulting in huge economic losses annually. Therefore, accurate laboratory diagnosis is needed to quickly confirm PRRSV infection.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAn immunoperoxidase monolayer assay (IPMA) was developed for the specific and sensitive detection of PRRSV based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6. The mAb 28F6-based IPMA could specifically detect PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10\u003csup\u003e\u0026minus;\u0026thinsp;2.25\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/100 \u0026micro;L. There was no significant difference in the detection of PRRSV of different passages with different batches of mAb 28F6, indicating that the IPMA had good repeatability. In addition, the IPMA could recognize a number of PRRSV variants including field strains such as BJ-4, HN07-1, and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. Validation of the IPMA showed that it was in 100% consistency with qRT-PCR on the detection of 108 clinical samples.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe IPMA could meet the demand for the specific and sensitive detection of PRRSV, which is helpful for accurate monitoring and early warning of PRRSV infections.\u003c/p\u003e","manuscriptTitle":"Development and application of an immuoperoxidase monolayer assay for the detection of PRRSV","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-06 04:21:56","doi":"10.21203/rs.3.rs-4331925/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":"aceff7f4-ccec-4c3c-8f54-d9a77e4cfbce","owner":[],"postedDate":"May 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-17T06:54:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-06 04:21:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4331925","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4331925","identity":"rs-4331925","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}