Abstract
Background
Pseudorabies (PR) is a highly contagious disease, and it causes significant economic losses to the global swine industry. Vaccination plays an important role in the prevention and control of pseudorabies virus (PRV). To evaluate vaccine efficacy, there is a need for a quick and straight forward method to monitor PRV-induced antibody levels in practice.
Results
A time-resolved fluorescence immunochromatographic (TRFIC) strip was developed for the serological detection of PRV gB antibodies in swine. Following systematic analysis and evaluation, the assay demonstrated a high degree of correlation with established reference method. The positive and negative coincidence rates between the TRFIC strip and ELISA were 96.8% and 94.2%, respectively. Furthermore, comprehensive analytical and comparative assessments revealed that the TRFIC strip exhibited no cross-reactivity with antibodies against other porcine pathogens.
Conclusion
Given its high specificity, sensitivity, and convenience, the TRFIC strip is suitable for on-site detection of PRV gB antibodies and can serve as a valuable tool for monitoring PRV immune status in animal populations.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12917-025-04913-7.
Keywords: PRV, gB, Time-resolved fluorescence immunochromatographic strip, ELISA
Background
Pseudorabies (PR) is caused by pseudorabies virus (PRV), which has been lasting for nearly 200 years since the first case occurred. It displays different symptoms at distinct growth phases after being infected with PRV, including the reproductive failure of sows, fatal encephalitis and 100% mortality of newborn pigs, and respiratory distress and growth block of young pigs [1–3].
With the extensive application of vaccines, PRV was well controlled for a period of time. However, due to the variation of PRV strain, PRV re-emerged and spread quickly in some countries. Although researchers have been deeply committed to the design of vaccines and the development of diagnostic methods, it remains a significant infectious disease and affects the global swine industry extensively. Moreover, PRV infection might cause encephalitis and endophthalmitis in humans. Using metagenomic next-generation sequencing, some studies identified the specific sequences of PRV within patients’ tissues. Additionally, a human-originated PRV strain, hSD-1/2019, was isolated from the cerebrospinal fluid of an individual diagnosed with acute encephalitis [4–6].
Pseudorabies virus (PRV), a member of the Alphaherpesvirinae subfamily within the Herpesviridae family [7], is an enveloped double-stranded DNA virus with a genome size of approximately 150 kb that encodes around 70 proteins [8]. The glycoproteins on the viral envelope are crucial for immune responses and interactions between the virus and host cells during PRV infection [9]. 11 PRV glycoproteins have been identified, and gB, gC, and gD are the primary protective antigens which can induce the production of neutralizing antibodies and virus-specific cellular immune responses in swine [10].
Vaccination plays a critical role in the prevention and control of PRV in developing countries. To evaluate vaccine efficacy, there is a need for a quick and straightforward method to monitor PRV-induced antibody levels in routine field applications. Currently, enzyme-linked immunosorbent assay (ELISA), particularly monoclonal antibody blocking ELISA, is widely used for detecting PRV antibodies. Although ELISA offers accurate and sensitive results, it demands specialized equipment, professional personnel, and testing procedure for at least 2 h. In recent years, immunochromatographic assays (ICAs) have gained prominence as vital tools for rapid veterinary diagnostics due to their safety, precision, high sensitivity, and ease of operation [3, 11]. Colloidal gold nanoparticles (CGs) as one type of the ICAs, are frequently utilized upon their cost-effectiveness [12–14]. Other alternative fluorescent materials including fluorescent dyes, quantum dots (QDs), and nanoparticles are increasingly applied in point-of-care testing [15]. Time-resolved fluorescent microspheres (TRFM), which incorporate lanthanide complexes, enhance sensitivity by addressing issues such as background fluorescence and instability associated with other particles, which offer advantages such as prolonged fluorescent signals, improved sensitivity, and favorable linearity [16–18]. An appropriate amount of the lanthanide complexes Europium (Eu) (III) in the carboxylate-modified polystyrene microspheres can improve the efficiency of fluorescent labeling, and the emission fluorescence can be detected after 400 µs [19]. Additionally, the polystyrene shell protects the internal chelate from external influences, enhancing overall stability [20, 21]. In this study, a simple and rapid time-resolved fluorescence immunochromatography (TRFIC) for PRV gB antibody detection was developed for the immunized antibody evaluation.
Materials and methods
Gene, cell, and serum samples
PRV gB genome sequence of HeNLH/2017 stain is available in National Center for Biotechnology Information (NCBI) GenBank (MT775883). Spodoptera frugiperda 21 insect cells (sf21) were grown in Sf-900II medium at 27 °C. All serum samples used in this study were preserved and provided by the Key Laboratory of Animal Immunology of Henan Academy of Agricultural Sciences.
Antigen for use in the TRFIC strip
The gB encoding sequence derived from PRV HeNLH/2017 strain (Genbank ID: MT775883) was optimized and cloned into the pFastBac1 vector by Sangon Biotech (China). The expression and purification of the gB protein were operated according to previous report [22]. Then, the purified protein were analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot.
Preparation of TRFM-labeled antigen
To prepare TRFM-based immunoprobes, time-resolved fluorescent microspheres (TRFMs) were coupled with gB antigen through covalent linkage (Fig. 1A). The coupling method was moderately modified based upon previous work carried out by J. Chen as follows [23]: 10 µL of TRFMs (MF02, -COOH, 1% w/v, 200 nm, Made New, China) were first incubated with 200 µL of MES buffer (0.05 M) and homogenized using ultrasound. Next, added 90 µL of NHS (0.1 mg/mL) and 75 µL of EDC (0.1 mg/mL) to activate the carboxyl groups on the TRFMs surface and incubated for 30 min, followed by centrifugation at 12,000 rpm for 25 min to isolate the activated TRFMs. Last, the probes were resuspended in 200 µL of boric acid buffer (BB, 0.05 M, pH 8.0), which contained an optimized concentration of gB antigen, then gently oscillated at room temperature (RT) for 30 min to ensure efficient conjugation. 1 mL of blocking solution was added to block any residual active sites. Consequently, the resulting TRFM-gB complex was centrifuged to remove unreacted proteins and resuspended, the sediment was dissolved in 200 µL of phosphate buffer (PB, 0.5 M, pH 7.4) and stored at 4 ℃.
Fig. 1.
Schematic illustration of the structure and principle of the TRFIC strip. A, The EDC-mediated coupling of TRFMs with gB protein. B, The structural components and assemble diagram of the TRFIC strip. C, Working principle and results determination schematic diagram of the TRFIC strip. D, Results analysis methods of the TRFIC strip. (i) Visual detection using a UV lamp. (ii) The fluorescence intensity of the T line and C line scanning with a fluorescence immunity analyzer
Assembly of the TRFIC strip
The TRFIC strip was constructed using a PVC adhesive backplane, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad, which were precisely assembled in a predefined sequence to ensure optimal immunoassay performance. Specifically, Staphylococcal protein A (SPA), which has the ability to bind specifically to the Fc region of mammalian antibodies, as well as anti-gB monoclonal antibody (mAb) IgG, were diluted using PBS buffer (0.01 M, pH 7.4) at concentrations of 0.8 mg/mL and 1.0 mg/mL, and coated on the NC membrane corresponding to the test (T) line and the quality control (C) line which were located in the central region of the adhesive card, together with conjugate pad and absorbent pad overlaid 2 mm space with the NC membrane (Fig. 1B). The assembled test pad was cut into 3.0 mm-wide sections and stored under dry and sealed conditions.
Detection procedure and principle of the TRFIC strip
The TRFIC strip employs an indirect immunoassay reaction mode (Fig. 1C). Simply, serum samples diluted 200-fold with normal saline are added to the sample pad, as the solution migrates toward the absorbent pad, PRV gB-specific antibodies present in the sample bind to the TRFM-gB antigen conjugate, forming a TRFM-antigen-antibody complex. The complex reacts with SPA immobilized at T line on the NC membrane, resulting in fluorescence signal generation, which suggests a positive result. The absence of the T line indicates a negative result. As a control, C line consistently visible regardless of presence of anti-PRV gB antibodies. With in a certain range, the fluorescence intensity (FI) of the T line is positively correlated with the concentration of anti-PRV gB antibodies in the sample. Following incubation at room temperature for 10–15 min, the results can be interpreted via visual inspection under a UV lamp or quantitatively analyzed by measuring FI using a fluorescence immunoassay analyzer.(Fig. 1D).
Determination of cut-off value
To determine the cut-off value, swine PRV gB-antibody negative sera (n = 36) were tested using the TRFIC strip, and the T lines were quantitatively analyzed by a fluorescence immunity analyzer. Statistically, at the 99.9% confidence level, a sample was considered positive if its FI value was greater than or equal to the mean FI value (X) of the negative samples plus two standard deviations (2SD) [24]. Therefore, the value of X + 2SD was established as the threshold for distinguishing between negative and positive results. Specifically, a serum sample was classified as positive when its FI value was ≥ X + 2SD, and as negative when its FI value was < X + 2SD.
Validation of limit of detection (LOD) of the TRFIC strip
To evaluate the LOD of the LFIC strip, an anti-PRV positive sample was serially diluted two-fold ranging from 1:200 to 1:204,800. Each individual TRFIC strip was then individually applied to a corresponding diluted sample and incubated for 10–15 min. Following the development of color on both the C line and T line, the results were detected using UV lamp and fluorescence immunity analyzer, simultaneously.
Cross-reactivity of the TRFIC strip
The cross-reactivity of the TRFIC strip was evaluated using antibody positive sera of classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), Meanwhile, PRV gB antibody positive and negative sera were used as control. Results of T lines were read under a UV lamp within 15 min.
Comparative detection by multiple methods
A total of 147 swine serum samples were utilized for comparative detection. These samples were diluted with normal saline at a ratio of 1:200, then detected by the TRFIC strip. For comparative analysis, all sera samples were simultaneously tested using the PRV gB Ab ELISA kit (IDEXX, USA) in accordance with the manufacturer’s instructions. In addition, a quantum dots-based fluorescent immunochromatographic (QDs-FIC) strip, previously developed by our research team, was employed as an alternative method for evaluation. The coincidence rates of the TRFIC strip were then compared with those obtained from the ELISA and QDs-FIC strip, respectively.
Results
Expression and purification of recombinant proteins gB
The PRV gB gene was cloned into a recombinant baculovirus vector, and the corresponding recombinant protein was expressed in SF21 insect cells. Following expression, the culture medium was harvested via centrifugation, and the supernatant was purified using Ni-NTA agarose beads. As illustrated in Fig. 2, the mature gB protein comprises three subunits: gBa (a small, uncleaved fragment of gB), gBb, and gBc (generated through furin-mediated cleavage and linked by disulfide bonds). This structural organization arises from the presence of a furin cleavage motif at residues 441 RRARR 445 [25]. SDS-PAGE analysis indicated that treatment with DTT disrupted the disulfide bonds, leading to the appearance of three distinct bands corresponding to gBa, gBb, and gBc (Fig. 2A). The identity of the gB protein was further confirmed by Western blot analysis employing His-tag-specific monoclonal antibodies. The results revealed that the His-tag was present on gBa and gBc but not on gBb (Fig. 2B).
Fig. 2.
Expression and characterization of recombinant PRV gB protein. A, SDS-PAGE analysis of gB protein. Lane M, protein marker. Lane 1, purified gB protein. B, Western blot analysis of purified gB protein. Lane 2, purified gB protein
Determination of cut-off value for the TRFIC strip
Swine gB-antibody negative serum samples were analyzed using the newly developed TRFIC strip. The results showed that the mean FI value was 2605.6 arbitrary units (a.u.), with a standard deviation (SD) of 1580.2 (Fig. 3). According to the critical value determination criterion based on the mean FI value ± 2SD of the negative sera, the cutoff value for this test strip method was established as 5766 a.u. Serum samples with an FI value ≥ 5766 a.u. were classified as positive, whereas those with an FI value < 5766 a.u. were classified as negative.
Fig. 3.
Determination of the cut-off value for the established TRFIC strip
Limit of detection (LOD) of the TRFIC strip
The LOD of the TRFIC strip was presented in Fig. 4. An anti-PRV positive sample was initially diluted at a ratio of 1:200, followed by serial 2-fold dilutions, resulting in a final dilution titer of up to 1:204,800. As illustrated in Fig. 4A, a visible T line was observed up to a dilution of 1:102,400. At the dilution of 1:204,800, the T line could no longer be discerned visually. Therefore, the LOD of the TRFIC strip was determined to be 1:102,400. Simultaneously, the FI value of the T lines was quantitatively analyzed using a fluorescence immunoassay analyzer. As shown in Fig. 4B, the fluorescence signal gradually decreased with increasing dilution and reached levels comparable to those of the negative control at a dilution of 1:204,800.
Fig. 4.
Limited of detection (lOD) of the TRFIC strip test. A, The tested results were observed by a UV lamp. B, The intensities of the T lines and C lines fluorescence were collected by fluorescence immunity analyzer
Cross-reactivity of the TRFIC strip
The cross-reactivity of the TRFIC strip is shown in Fig. 5. The T line and C line were clearly visible only when PRV gB-antibody positive sera were detected. In contrast, no visible T line was observed when testing PRV gB-antibody negative sera or sera positive for antibodies against PRRSV, CSFV, PEDV, or FMDV. These results indicate that the TRFIC strip does not exhibit cross-reactivity with antibodies against other viruses, demonstrating high specificity for the detection of PRV antibodies.
Fig. 5.
Cross-reactivity of the TRFIC strip test. 1, PRV gB antibody positive serum. 2, PRV gB antibody negative serum. 3 and 4, CSFV antibody positive sera. 5 and 6, FMDV antibody positive sera. 7 and 8, PRRSV antibody positive sera. 9 and 10, PEDV antibody positive sera
Comparative detection by multiple methods
Simultaneous testing of swine serum samples was performed using the developed TRFIC strip and the IDEXX PRV gB Ab ELISA. The FI value of the T lines was quantitatively measured, and the results indicated that positive and negative sera could be effectively differentiated based on the defined cut-off value. As presented in Table 1, the analysis revealed a positive coincidence rate of 96.8% (92/95) and a negative coincidence rate of 94.2% (49/52) between the TRFIC strip and the ELISA kit. Consequently, the overall coincidence rate between the TRFIC strip developed in this study and the ELISA kit was 95.9%, which was slightly higher than that of the previously established QDs-FIC strip (92.6%).
Table 1.
Coincidence rate of three detection methods
| TRFIC strip | QDs-FIC strip | ||||
|---|---|---|---|---|---|
| Positive | Negative | Positive | Negative | ||
| IDEXX ELISA | Positive | 92 | 3 | 88 | 8 |
| Negative | 3 | 49 | 3 | 50 | |
| Coincidence rate | 95.9% (141/147) | 92.6% (138/149) | |||
Table 1. Analysis of clinical serum samples in TRFIC strip and comparison with the results obtained by the IDEXX ELISA and LFIC strip.
Discussion
Most PRV eradication programs depend on the application of vaccines. Monitoring antibody production in vaccinated swine populations and evaluating the efficacy of immune protection are essential components of these programs. The advancement of antibody detection technologies is significantly affected by the structural characteristics, purity, and relative proportions of antigens. The PRV gB gene encodes a highly conserved glycoprotein, which constitutes one of the major structural proteins of the viral envelope. The gB protein contains multiple antigenic epitopes that are capable of eliciting protective immune responses in susceptible hosts. Therefore, gB represents a promising antigenic candidate for the development of both vaccines and diagnostic assays [26]. In this study, the gB protein was selected as the diagnostic antigen for the development of a gB-specific antibody detection assay, thereby ensuring that the obtained results are closely correlated with neutralizing antibody titers.
Antigens play a critical role in determining the accuracy and sensitivity of diagnostic detection systems. Protein expression and purification represent essential procedures in biochemical research. The choice of expression system significantly influences the structural integrity and functional properties of the target protein [27]. Proteins that retain structural features closer to their native viral counterparts demonstrate enhanced reactivity with antibodies, thereby improving the sensitivity and reliability of diagnostic assays. Escherichia coli expression system is widely employed as a protein expression platform due to its advantages of high expression yield, low cost, and rapid production cycle. However, this system presents several limitations, including improper disulfide bond formation, poor solubility, inability to assemble into higher-order nanoparticle structures, absence of post-translational glycosylation modifications, and potential endotoxin contamination [28]. In this study, the gB protein was expressed using an insect cell-based expression system. This system possesses advanced capabilities in translation and post-translational modification, such as glycosylation, phosphorylation, acylation, and signal peptide processing, enabling the production of recombinant proteins that closely resemble their natural counterparts in terms of antigenicity, immunogenicity, and biological activity [29]. Based on the gB recombinant protein, a rapid antibody detection strip was developed, capable of sensitively and specifically measuring gB antibody levels in animal sera. By analyzing both gB-negative and gB-positive serum samples, a reference standard was established, allowing for more accurate assessment of antibody responses in vaccinated pig herds and reflecting the protective immunity induced by vaccination. This assay provides technical support for the formulation and evaluation of clinical vaccine immunization strategies.
Immunochromatographic test strips offer the advantage of rapid, on-site detection without the need for specialized instruments or equipment for result interpretation. However, with ongoing technological advancements, there is a higher demand for rapid detection. Ultra-sensitive detection and digitalization have become the development trends of immunochromatographic assays. To meet growing market demands, numerous researchers and industry players have conducted digital analysis studies on test strips, primarily through grayscale scanning for quantitative detection [30, 31]. Although many immunochromatographic analyzers are currently available for digitizing test strip detection results, most rely on converting the color density of the C and test T lines into optical density values. However, as the color density signal originates from only the top 10 μm layer of the NC membrane, approximately 90% of the signal may be lost due to optical phenomena such as refraction and scattering, thereby compromising detection accuracy [32]. Time-resolved fluorescence nanoparticles (TRFN) exhibit a substantial Stokes shift (> 150 nm), with fluorescence lifetimes that are 5–6 orders of magnitude longer than those of background autofluorescence. By implementing time-delayed detection, non-specific fluorescence interference can be effectively minimized [23]. This characteristic offers significant advantages in enhancing the sensitivity, stability, and analytical precision of rapid diagnostic assays. Consequently, TRFN technology has been widely adopted in nano-biosensor applications, enabling ultrasensitive detection of antigens, antibodies, and nucleic acids [33]. Immunochromatographic test strips incorporating time-resolved fluorescent microspheres not only allow visual interpretation under a portable ultraviolet light source but also support precise quantitative analysis via instrumentation, thereby facilitating digital result acquisition. This capability is particularly beneficial for data-driven management and large-scale epidemiological analysis in modern swine production systems. In this study, the fluorescence intensity of negative samples was consistently below 5766 a.u. Under ultraviolet illumination, no visible red fluorescence band was observed at the T line. Therefore, in the absence of a fluorescence immunoassay analyzer, the test strips developed in this study can still be visually interpreted under UV light. A positive result is indicated when distinct red fluorescence bands appear simultaneously at both the C and T lines, conversely, a negative result is indicated when only a clear red fluorescence band appears at the C line.
Conclusions
To facilitate the rapid and convenient assessment of the immune response elicited by pseudorabies virus (PRV) vaccines in swine, a time-resolved fluorescence immunochromatographic (TRFIC) strip was developed for the serological detection of PRV gB antibodies. The assay demonstrated a strong correlation with established reference methods and exhibited no cross-reactivity with antibodies against other porcine pathogens. Due to its high specificity, sensitivity, and operational convenience, the TRFIC strip is well-suited for on-site detection of PRV gB antibodies and can serve as a valuable tool for monitoring the immune status of PRV in animal herds.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We would like to thank all participants for their help in the research.
Author contributions
SY, Y Sun and YX: Conceptualization, methodology, writing-original draft preparation. SY, Y Sun and LF: writing-review and editing. Y Li, Y Shang and SC: formal analysis, data analysis. SY, Y Liu and Y Shang: project administration. All authors contributed to the article and approved the submitted version.
Funding
This study was supported by the Henan Provincial Scientific and Technological Research Project (No. 252102110064), Intergovernmental international scientific and technological innovation cooperation projects of National Key Research and Development Program (2024YFE0199100), and the Independent Innovation Project of Henan Academy of Agricultural Sciences (No. 2025ZC153).
Data availability
All data supporting our findings is contained within the manuscript.
Declarations
Ethics approval and consent to participate
The study was approved by the Ethics and Animal Welfare Committee of Henan Academy of Agricultural Sciences (Approval No. LLSC41024037). The experiments were conducted in accordance with the local legislation and institutional requirements.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Suzhen Yang, Yaning Sun and Yunrui Xing contributed equally to this work.
Contributor Information
Suzhen Yang, Email: 49549173@qq.com.
Yunchao Liu, Email: yunchaoliu2012@163.com.
References
- 1.Zheng H, Fu P, Chen H, Wang Z. Pseudorabies virus: from pathogenesis to prevention strategies. Viruses-Basel. 2022;14(8):1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Ye C, Zhang Q, Tian Z, Zheng H, Zhao K, Liu F, Guo J, Tong W, Jiang C, Wang S. Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence: evidence for the existence of two major genotypes. Virology. 2015;483:32–43. [DOI] [PubMed] [Google Scholar]
- 3.Guo Z, Xu H, Zhang S, Kang H, Li C, Sun Q, Zhao J, Li J, Zhou G, Wang Q. Improved detection sensitivity of anti-PRV variant antibodies through Preparation of anti-gB and anti-gE monoclonal antibodies and development of blocking ELISAs. Int J Biol Macromol. 2024;260(1):129425. [DOI] [PubMed] [Google Scholar]
- 4.Ai J, Weng S, Cheng Q, Cui P, Li YJ, Wu H, Zhu Y, Xu B, Zhang W. Human endophthalmitis caused by pseudorabies virus infection, china, 2017. Emerg Infect Dis. 2018;24(6):1087–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Wang Y, Nian H, Li Z, Wang W, Wang X, Cui Y. Human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: A case report. Int J Infect Dis. 2019;89:51–4. [DOI] [PubMed] [Google Scholar]
- 6.Yang H, Han H, Wang H, Cui Y, Liu H, Ding S. A case of human viral encephalitis caused by pseudorabies virus infection in China. Front Neurol. 2019;10:534. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bo Z, Li X. A review of pseudorabies virus variants: genomics, vaccination, transmission, and zoonotic potential. Viruses-Basel. 2022;14(5):1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol R. 2005;69(3):462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Nie Z, Zhu S, Wu L, Sun R, Shu J, He Y, Feng H. Progress on innate immune evasion and live attenuated vaccine of pseudorabies virus. Front Microbiol. 2023;14:1138016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Wang H, Qiao Y, Cai B, Tan J, Na L, Wang Y, Lu H, Tang Y. Genome editing of pseudorabies virus in the CRISPR/Cas9 era: a mini-review. Front Vet Sci. 2023;10(1):6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Talha SM, Salminen T, Swaminathan S, Soukka T, Pettersson K, Khanna N. A highly sensitive and specific time resolved fluorometric Bridge assay for antibodies to HIV-1 and– 2. J Virol Methods. 2011;173(1):24–30. [DOI] [PubMed] [Google Scholar]
- 12.Li X, Luo P, Tang S, Beier RC, Wu X, Yang L, Li Y, Xiao X. Development of an immunochromatographic strip test for rapid detection of melamine in Raw milk, milk products and animal feed. J Agr Food Chem. 2011;59(11):6064–70. [DOI] [PubMed] [Google Scholar]
- 13.Li X, Shen J, Wang Q, Gao S, Pei X, Jiang H, Wen K. Multi-residue fluorescent microspheres immunochromatographic assay for simultaneous determination of macrolides in Raw milk. Anal Bioanal Chem. 2015;407(30):9125–33. [DOI] [PubMed] [Google Scholar]
- 14.Rong Y, Wang F, Li X, Liang X, Zhou Y, Zhang D, Liu J, Zeng H, Wang J, Shi Y. Correlation of the ratio of igm/igg concentration to days after symptom onset (IgM/T or IgG/T) with disease severity and outcome in non-critical COVID-19 patients. Am J Transl Res. 2021;13(3):1197–208. [PMC free article] [PubMed] [Google Scholar]
- 15.Le N, Zhang M, Kim K. Quantum Dots and their interaction with biological systems. Int J Mol Sci. 2022;23(18):10763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Huang Z, Wen J, Ma G, Liu Y, Tan H. Time-resolved fluorescence immunoassay based on glucose oxidase-encapsulated metal-organic framework for amplified detection of foodborne pathogen. Anal Chim Acta. 2024;1287:342111. [DOI] [PubMed] [Google Scholar]
- 17.Simonyan TR, Protasova EA, Mamontova AV, Shakhov AM, Lukyanov KA, Maksimov EG, Bogdanov AM. A single fluorescent protein-based indicator with a time-resolved fluorescence readout for precise pH measurements in the alkaline range. Int J Mol Sci. 2022;23(21):12907. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Wu Q, Hang C, Li T, Wang B, Qin Y, Wang Y, Zhou X, Xia P, Sheng H, Huang P. Establishment of time-resolved fluoroimmunoassay of IgG4 based on magnetic microspheres. J Clin Lab Anal. 2021;35(8):23874. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wang Y, Yu Z, Ning Z, Li M, Li W, Zhong Y, Chen H, Zhang X, Tang X, Cheng X. Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of GFAP in serum. Microchim Acta. 2024;191(6):325. [DOI] [PubMed] [Google Scholar]
- 20.Liu Z, Chen J, Zhao S, Pang Y, Shen X, Lei H, Li X. Immunochromatographic assays based on three kinds of nanoparticles for the rapid and highly sensitive detection of Tylosin and Tilmicosin in eggs. Microchim Acta. 2021;189(1):42. [DOI] [PubMed] [Google Scholar]
- 21.Yang Q, Zhu J, Ma F, Li P, Zhang L, Zhang W, Ding X, Zhang Q. Quantitative determination of major capsaicinoids in serum by ELISA and time-resolved fluorescent immunoassay based on monoclonal antibodies. Biosens Bioelectron. 2016;81:229–35. [DOI] [PubMed] [Google Scholar]
- 22.Wei Q, Bai Y, Song Y, Liu Y, Zhang G. Generation and immunogenicity analysis of Recombinant classical swine fever virus glycoprotein E2 and Erns expressed in baculovirus expression system. Virol J. 2021;18(1):44. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Chen J, Chen L, Zhang Y, Xiang S, Zhang R, Shen Y, Liao J, Xie H, Yang J. Development of a time-resolved fluorescent microsphere test strip for rapid, on-site, and sensitive detection of picoxystrobin in vegetables. Foods. 2024;13(3):14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Paweska JT, van Vuren PJ, Kemp A, Buss P, Bengis RG, Gakuya F, Breiman RF, Njenga MK, Swanepoel R. Recombinant nucleocapsid-based ELISA for detection of IgG antibody to rift Valley fever virus in African Buffalo. Vet Microbiol. 2008;127(1–2):21–8. [DOI] [PubMed] [Google Scholar]
- 25.Ji C, Wei Y, Wang J, Zeng Y, Wang H. Development of a dual fluorescent microsphere immunological assay for detection of pseudorabies virus gE and gB IgG antibodies. Viruses-Basel. 2020;12(9):912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Li X, Yang F, Hu X, Tan F, Qi J, Peng R, Wang M, Chai Y, Hao L, Deng J, et al. Two classes of protective antibodies against pseudorabies virus variant glycoprotein B: implications for vaccine design. Plos Pathog. 2017;13(12):1006777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Schütz A, Bernhard F, Berrow N, Buyel JF, Ferreira-Da-Silva F, Haustraete J, Heuvel JVD, Hoffmann JE, Marco AD, Peleg Y. A concise guide to choosing suitable gene expression systems for Recombinant protein production. Star Protoc. 2023;4(4):102572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Jia B, Jeon CO. High-throughput Recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biology. 2016;6(8):160196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Jarvis DL. Baculovirus-insect cell expression systems. Methods Enzymol. 2009;463:191–222. [DOI] [PubMed] [Google Scholar]
- 30.Wang Y, Wang T, Wang M, Wang J, Xu Z, Zhang H. Photothermal card reader assay using the commercial colloidal gold test strip for the rapid quantitative detection of food hazards. Microchim Acta. 2022;189(3):112. [DOI] [PubMed] [Google Scholar]
- 31.Zheng Q, Wu H, Jiang H, Yang J, Gao Y. Development of a smartphone-based fluorescent immunochromatographic assay strip reader. Sensors-Basel. 2020;20(16):4521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Huang Z, Hu S, Xiong Y, Wei H, Xu H, Duan H, Lai W. Application and development of superparamagnetic nanoparticles in sample pretreatment and immunochromatographic assay. Trac-Trend Anal Chem. 2019;114:151–70. [Google Scholar]
- 33.Li M, Wang H, Sun J, Ji J, Ye Y, Lu X, Zhang Y, Sun X. Rapid, on-site, and sensitive detection of aflatoxin M1 in milk products by using time-resolved fluorescence microsphere test strip. Food Control. 2021;121:107616. [Google Scholar]
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Supplementary Materials
Data Availability Statement
All data supporting our findings is contained within the manuscript.