Quantum dot-based fluorescence-linked immunosorbent assay for the rapid detection of lomefloxacin in animal-derived foods.

Lomefloxacin (LMF), a third-generation fluoroquinolone antibacterial agent, is often used to treat bacterial and mycoplasma infections. However, due to its prolonged half-life and slow metabolism, it is prone to residues in animal-derived foods, posing a potential food safety risk. Therefore, it is particularly urgent and important to establish a method for detecting lomefloxacin. In this study, direct and indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) based on quantum dots (QDs) was established for the detection of LMF. As for dc-FLISA, the half-maximal inhibitory concentration (IC50) and limit of detection (LOD) were 0.84 ng/mL, 0.04 ng/mL, respectively, the detection ranges from 0.08 to 9.11 ng/mL. The IC50 and LOD of ic-FLISA were 0.43 ng/mL and 0.03 ng/mL, respectively, meanwhile the detection ranges from 0.05 to 3.49 ng/mL. The recoveries of dc-FLISA and ic-FLISA in animal-derived foods (milk, fish, chicken, and honey), ranged from 95.8% to 105.2% and from 96.3% to 103.4%, respectively, with the coefficients of variation less than 8%. These results suggest that the dc-FLISA and ic-FLISA methods, which are based on QD labelling, are highly sensitive and cost-effective, and can be effectively used to detect LMF in animal-derived foods.

Development and characterization of monoclonal antibodies against p37 protein of African swine fever virus.

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines.

Thiolated chitosan encapsulation constituted mucoadhesive nanovaccine confers broad protection against divergent influenza A viruses.

Influenza A virus (IAV) poses a significant threat to human and animal health, necessitating the development of universal influenza vaccines that can effectively activate mucosal immunity. Intranasal immunization has attracted significant attention due to its capacity to induce triple immune responses, including mucosal secretory IgA. However, inducing mucosal immunity through vaccination is challenging due to the self-cleansing nature of the mucosal surface. Thiolated chitosan (TCS) were explored for mucosal vaccine delivery, capitalizing on biocompatibility and bioadhesive properties of chitosan, with thiol modification enhancing mucoadhesive capability. The focus was on developing a universal nanovaccine by utilizing TCS-encapsulated virus-like particles displaying conserved B-cell and T-cell epitopes from M2e and NP proteins of IAV. The optimal conditions for nanoparticle formation were investigated by adjusting the thiol groups content of TCS and the amount of sodium tripolyphosphate. The nanovaccine induced robust immune responses and provided complete protection against IAVs from different species following intranasal immunization. The broad protective effect of nanovaccines can be attributed to the synergistic effect of antibodies and T cells. This study developed a universal intranasal nanovaccine and demonstrated the potential of TCS in the development of mucosal vaccines for respiratory infectious diseases.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies.

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200-350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251-305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262-279. Amino acid 262-279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262-273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay for rapid detection of VZV.

BACKGROUND: Varicella zoster virus (VZV) is the pathogen of varicella and herpes zoster, it is necessary to develop a rapid, sensitive and specific detection method for the prevention and control of related diseases. METHODS: We inserted the gB protein extracellular region gene (gB-ex, 1-2208 bp) of VZV into lentivirus vector, and then obtained the recombinant gB protein through mammalian expression system. BALB/c mice were immunized multiple times with purified gB protein as immunogen. Then four strains of high affinity monoclonal antibodies targeting gB protein were prepared by cell fusion technique. Monoclonal antibodies 5G4 and HRP-4E9 were selected as capture and detection antibodies respectively, and a double-antibody sandwich ELISA method was established for detection. RESULTS: The detection limit of the DAS-ELISA was 156 PFU/mL, and there was no cross-reaction with Herpes simplex virus-1/Herpes simplex virus-2/Pseudorabies virus. The coefficients of variation of intra-assay and inter-assay repeatability were less than 5%. CONCLUSIONS: In this study, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established for the detection of VZV. The assay has good sensitivity, specificity and repeatability, which provides strong technical support and product guarantee for the rapid clinical detection of VZV.

AP2/EREBP Pathway Plays an Important Role in Chaling Wild Rice Tolerance to Cold Stress.

Cold stress is the main factor limiting rice production and distribution. Chaling wild rice can survive in cold winters. AP2/EREBP is a known transcription factor family associated with abiotic stress. We identified the members of the AP2/EREBP transcription factor family in rice, maize, and Arabidopsis, and conducted collinearity analysis and gene family analysis. We used Affymetrix array technology to analyze the expression of AP2/EREBP family genes in Chaling wild rice and cultivated rice cultivar Pei'ai64S, which is sensitive to cold. According to the GeneChip results, the expression levels of AP2/EREBP genes in Chaling wild rice were different from those in Pei'ai64S; and the increase rate of 36 AP2/EREBP genes in Chaling wild rice was higher than that in Pei'ai64S. Meanwhile, the MYC elements in cultivated rice and Chaling wild rice for the Os01g49830, Os03g08470, and Os03g64260 genes had different promoter sequences, resulting in the high expression of these genes in Chaling wild rice under low-temperature conditions. Furthermore, we analyzed the upstream and downstream genes of the AP2/EREBP transcription factor family and studied the conservation of these genes. We found that the upstream transcription factors were more conserved, indicating that these upstream transcription factors may be more important in regulating cold stress. Meanwhile, we found the expression of AP2/EREBP pathway genes was significantly increased in recombinant inbred lines from Nipponbare crossing with Chaling wild rice, These results suggest that the AP2/EREBP signaling pathway plays an important role in Chaling wild rice tolerance to cold stress.

Design, Synthesis and Biological Evaluation of Novel PEG-Rakicidin B1 Hybrid as Clostridium difficile (CD) Targeted Anti-Bacterial Agent.

Rakicidin B1 was isolated and purified from the culture broth of a marine Streptomyces sp. as a potent anti-cancer agent, and lately the compound and its derivatives have firstly been found to possess anti-Clostridium difficile (CD) activity but with high cytotoxicity. Herein, following our previous discovery on anti-CD activity of Rakicidin B1, structure modification was performed at the OH position of Rakicidin B1 and a new Rakicidin B1-PEG hybrids FIMP2 was facilely designed and synthesized by conjugating the PEG2000 with the scaffolds of Rakicidin B1 via the linkage of carbamate. The cytotoxicity of the FIMP2 was first evaluated against three different cancer cell lines, including HCT-8 cells, PANC-1, and Caco-2, with IC50 values at 0.519 µM, 0.815 µM, and 0.586 µM, respectively. Obviously, as compared with a positive control group treated with Rakicidin B1, the IC50 value of FIMP2 increased by nearly 91-fold, 50-fold, and 67-fold, suggesting that the PEGylation strategy significantly reduced the cytotoxicity of FIMP2. Thus, this preliminary result may be beneficial to increase its safety index (SI) value due to the decreased cytotoxicity of FIMP2. In addition, this decreased cytotoxicity of FIMP2 was further confirmed based on a zebrafish screening model in vivo. Thereafter, the anti-CD activity of FIMP2 was evaluated in vivo, and its efficacy to treat CDI was found to be better than that of vancomycin. The mortality and recurrence rate of FIMP2 is not as low compared with that of vancomycin; these results demonstrated that compound FIMP2 is a new, promising anti-CD agent with significant efficacy against CD recurrence with low cytotoxicity towards bodies.

Development of a Gold Nanoparticle-Based Immunochromatographic Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is an important swine infectious pathogen that seriously threatens the global swine industry. PCV2 Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. In this study, a gold nanoparticle-based immunochromatographic strip with high sensitivity and specificity was developed which could be used for rapid detection of PCV2 virions or Cap protein in research. The visual detection limit of the strip was 103.18 50% tissue culture infective does (TCID50)/mL for PCV2, and 2.03 µg/mL for PCV2 Cap protein. No cross-reactivity was observed with the PCV1 and PCV3 Cap proteins and other common swine pathogens such as porcine reproductive and respiratory syndrome virus, classical swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine parvovirus, and swine influenza virus. The repeatability of the strip was good. The stability of the strip was perfect for 12 months in a dry state at room temperature. Visual results could be obtained within 5 min by simply inserting the strip into the diluted sample. The strip is a time-saving, labor-saving, and reliable tool for testing of PCV2 virions or Cap protein in research. The idea of this study might open a new perspective for the application of the strip. IMPORTANCE Porcine circovirus type 2 (PCV2) Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. Although many methods can be used to identify PCV2 or PCV2 Cap protein in vaccine research, they usually require high workload and time. The developed strip can specifically detect PCV2 virions or Cap protein, and visual qualitative results can be obtained within 5 min by simply diluting the sample and inserting the strip into the sample. The final value of the strip is providing a simple and time-saving method for real-time monitoring of PCV2 antigen in vaccine research with reliable results, such as the different stages of PCV2 Cap protein expression and purification, as well as the different stages of PCV2 reproduction and purification.

Rapid detection of varicella-zoster virus based on an immunochromatographic strip.

Varicella-zoster virus (VZV) is a highly infectious DNA virus that can cause varicella (chickenpox) and herpes zoster (HZ). A simple, sensitive and specific detection method is desirable for the VZV infection. In this study, VZV gE protein, expressed in CHO cells, was used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). For the first time, we developed a colloidal gold-based immunochromatographic strip for rapid detection of VZV using a pair of mAbs against gE protein. The limit of detection (LOD) of the strip was 30 ng mL-1 of purified VZV gE antigen, and it could specifically test VZV without cross-reactivity with Enterovirus 71 (EV-71), Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2). The coincidence rate between the strip and commercial real-time PCR diagnostic kit was 100% using vesicle as the clinical sample. Our strip provided a technical support for rapid and specific detection of VZV.

One-Step Transformations from ACQ Luminogens to DSEgens via the Boc Protection Process.

Dual-state emission luminogens (DSEgens), as a new type of luminescent materials that can effectively emit light in solution and solid state, have attracted tremendous attention due to their potential application in chemical sensing, biological imaging, organic electronic devices, etc. In this study, two new rofecoxib derivatives ROIN and ROIN-B have been synthesized, and their photophysical properties are fully investigated by experimental studies and theoretical calculations. The key intermediate ROIN, resulting from one-step conjugation of rofecoxib with an indole unit, shows the classical aggregation-caused quenching (ACQ) effect. Meanwhile, by introducing a tert-butoxycarbonyl (Boc) group on the basis of ROIN without enlarging the π conjugation system, ROIN-B was successfully developed with an obvious DSE property. In addition, both fluorescent behaviors and their transformation from ACQ to DSE were elucidated clearly by going through the analysis of their single X-ray data. Moreover, the target ROIN-B, as a new DSEgens, also displays reversible mechanofluorochromism and lipid droplet-specific imaging ability in HeLa cells. Taken together, this work proposes a precise molecular design strategy to afford a new DSEgens, which may provide guidance for the future exploration of new DSEgens.

Label-free electrochemical immunosensor based on staphylococcal protein a and AgNPs-rGO-Nf for sensitive detection of virginiamycin M1.

Virginiamycin (VIR), a feed additive, is used to promote pig and poultry growth. However, it is hazardous to human health. This work described a label-free electrochemical immunosensor based on silver nanoparticles-reduced graphene oxide (AgNPs-rGO) nanocomposites and staphylococcal protein A (SPA) for the first time to directly detect the residual marker VIR M1. Good catalytic currents for oxygen reduction reaction were apparently obtained after the modification of nanocomposites on gold electrode. Nanocomposites were characterized using UV-Vis, X-ray diffraction (XRD) patterns, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). SPA was targeted to immobilize VIR M1 monoclonal antibody (mAb) by binding to Fc region of antibody. The proposed immunosensor showed a wide linear range from 0.25 ng mL-1 to 100 ng mL-1, providing detection limit (LOD) of 0.18 ng mL-1 of VIR M1. Recovery rates ranged from 92.27% to 98.84%, and relative standard deviation (RSD) was not above 6.6%, indicating the immunosensor could detect VIR M1 in actual samples with high accuracy. The sensor showed good selectivity, reproducibility and stability and could be considered as a potential tool for detection of VIR M1 in feed and animal derived food.

Facile Transformation from Rofecoxib to a New Near-Infrared Lipid Droplet Fluorescent Probe and Its Investigations on AIE Property, Solvatochromism and Mechanochromism.

Lipid-related cancers cause a large number of deaths worldwide. Therefore, development of highly efficient Lipid droplets (LDs) fluorescent imaging probes will be beneficial to our understanding of lipid-related cancers by allowing us to track the metabolic process of LDs. In this work, a LDs-specific NIR (λmax = 698 nm) probe, namely BY1, was rationally designed and synthesized via a one-step reaction by integrating triphenylamine (electron-donor group) unit into the structure of rofecoxib. This integration strategy enabled the target BY1 to form a strong Donor-Acceptor (D-A) system and endowed BY1 with obvious aggregation-induced emission (AIE) effect. Meanwhile, BY1 also showed observable solvent effect and reversible mechanochromatic luminescent property, which could be interpreted clearly via density functional theory (DFT) calculations, differential scanning calorimetry (DSC), powder X-ray diffraction (XPRD), and single crystal X-ray data analysis. More importantly, BY1 exhibited highly specific fluorescent imaging ability (Pearson's correlation = 0.97) towards lipid droplets in living HeLa cells with low cytotoxicity. These results demonstrated that BY1 is a new promising fluorescent probe for lipid droplets imaging, and it might be beneficial to facilitate biological research of lipid-related cancers.

Electrochemical immunosensor for ultrasensitive detection of human papillomaviruse type 16 L1 protein based on Ag@AuNPs-GO/SPA.

Human papillomaviruse type 16 (HPV16) is a high-risk serotype. As the main protective antigen protein, L1 protein is also the target protein for diagnosis. A simple label free electrochemical immunosensor (ECIS) was fabricated for ultrasensitive detection of HPV16 L1 protein in this work. Quasi-spherical Ag@Au core-shell nanoparticles on graphene oxide (Ag@AuNPs-GO) was developed as current response amplifier and characterized by UV-Vis Spectroscopy, Transmission Electron Microscopy and energy dispersive X-ray spectroscopy. Staphylococcal protein A was decorated on the modified electrode and utilized to immobilized the Fc portion of the monoclonal antibody specific for HPV16 L1 protein. Cyclic Voltammetry, Differential Pulse Voltammetry and Electrochemical Impedance Spectroscopy were used to verify the electrochemical performance and interfacial kinetic property. The increased concentration of HPV16 L1 protein led to slow electron transport and linearly decreased differential pulse voltammetry peak current with a detection limit of 0.002 ng mL-1 and a wide linear relationship in the range of 0.005-400 ng mL-1at a regression coefficient (R2) of 0.9948. Furthermore, this ECIS demonstrated acceptable accuracy with good reproducibility, stability and selectivity, suggesting a promising immunological strategy for HPV typing and early screening.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Expression of extracellular domain of ASFV CD2v protein in mammalian cells and identification of B cell epitopes.

African swine fever virus (ASFV), a highly pathogenic large DNA virus, is the cause of African swine fever worldwide. The ASFV virulence gene EP402R encodes CD2v, a structural protein that plays an important role in the ASFV infection process. In this study, a CHO-S cell line stably expressing the extracellular region of CD2v was generated and secretory CD2v(sCD2v)was purified from the cell culture supernatant. The purified glycosylated sCD2v protein possessed high immunoreactivity and immunogenicity. In addition, we found that glycosylation had a decisive effect on the immune reactivity of CD2v. Then sCD2v was used to generate five CD2v-specific monoclonal antibodies. The reactivity of all monoclonal antibodies with CD2v protein was confirmed by Western blot and indirect immunofluorescence assay (IFA). Interestingly, mAb 8D5 reactivity with sCD2v depended on sCD2v glycosylation status. Subsequent B cell epitope mapping experiments conducted using a series of overlapping synthetic peptides of the CD2v extracellular domain led to identification of mAb B cell epitopes of 128TCKKNNGTNT137 for mAb 4B11 and 148VKYTNESILE157 for mAbs 5H4 and 5F7. Due to their well-defined epitopes, these three mAbs will likely serve as valuable tools for use in ASFV CD2v structure-function studies, diagnostic assays, and prophylactic methodologies to control ASFV transmission.

Identification of a novel B-cell epitope of the African swine fever virus p34 protein and development of an indirect ELISA for the detection of serum antibodies.

African swine fever (ASF) is a viral disease caused by the African swine fever virus that can be highly transmitted and lethal in domestic pigs. In the absence of a vaccine, effective diagnosis is critical for minimizing the virus's spread. In recent years, with the decline of African swine fever virus (ASFV) virulence, antibody detection has become an important means of detection. ASFV nucleocapsid protein p34 is a mature hydrolytic product of pp220, which is highly conserved and has a high content in the structural protein of the virus. Prokaryotic cells were chosen to generate highly active and high-yield p34 protein, which was then used as an antigen for producing mouse monoclonal antibodies. The B-cell epitope 202QKELDKLQT210, which was highly conserved and found on the surface of the p34 protein, was first identified by an anti-p34 monoclonal antibody utilizing the peptide scanning technique and visualized in helix. This supported the viability of p34 protein detection even further. In addition, we established an indirect ELISA assay based on p34 to detect ASFV antibodies. The coincidence rate of this method with commercially available kits was shown to be 97.83%. Sensitivity analysis revealed that it could be detected in serum dilution as low as 1:6400, and there was no cross-reaction with other prevalent porcine epidemic diseases classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus 2 (PCV2). In summary, the established ELISA method and anti-P34 monoclonal antibody have demonstrated that the p34 protein has a promising application prospect for the detection of African swine fever antibodies.

Serological survey of SARS-CoV-2 in companion animals in China.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can be transmitted from human to companion animals. The national wide serological surveillance against SARS-CoV-2 was conducted among pet animals, mainly in cats and dogs, 1 year after the first outbreak of COVID-19 in China. All sera were tested for SARS-CoV-2 IgG antibodies using an indirect enzyme linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of spike protein. This late survey takes advantage of the short duration of the serological response in these animals to track recent episode of transmission. A total of 20,592 blood samples were obtained from 25 provinces across 7 geographical regions. The overall seroprevalence of SARS-CoV-2 infections in cats was 0.015% (2/13397; 95% confidence intervals (CI): 0.0, 0.1). The virus infections in cats were only detected in Central (Hubei, 0.375%) and Eastern China (Zhejiang, 0.087%) with a seroprevalence estimated at 0.090 and 0.020%, respectively. In dogs, the seroprevalence of SARS-CoV-2 infections was 0.014% (1/7159; 95% CI: 0.0, 0.1) in the entire nation, seropositive samples were limited to Beijing (0.070%) of Northern China with a prevalence of 0.054%. No seropositive cases were discovered in other geographic regions, nor in other companion animals analyzed in this study. These data reveal the circulation of SARS-CoV-2 in companion animals, although transmission of the virus to domestic cats and dogs is low in China, continuous monitoring is helpful for the better understand of the virus transmission status and the effect on animals.

Establishment of an Immunological Method for Detection of Bluetongue Virus by Fluorescence-Linked Immunosorbent Assay.

Bluetongue (BT) is a severe noncontagious infectious disease that occurs in sheep and wild ruminants but occasionally also in cattle and camels. The worldwide BT pandemic has had a significant impact on global livestock production. Rapid detection helps prevent outbreaks of bluetongue disease. Fluorescence-linked immunosorbent assay (FLISA) labeled with quantum dots (QDs) is typically used for detection due to its high sensitivity. There has been no reported detection of BT virus (BTV) using QD-based fluorescence immunoassays. In this study, monoclonal antibodies (MAbs) against BT were prepared by immunizing BALB/c mice with recombinant VP7 protein. Two MAbs with high sensitivity and specificity were selected as the detection antibody (2F11) and capture antibody (11B7). Then, the detection antibody was coupled with QDs to prepare QD-MAb fluorescence probes. Fluorescence-linked immunosorbent assay is highly specific, detecting only VP7 protein/BTV, and did not show any nonspecific reactions with other reoviruses. The detection limit of VP7 protein was 3.91 ng/mL using fluorescence-linked immunosorbent assay, with a coefficient of variation (CV) of less than 15%. The establishment of rapid, sensitive direct FLISA has potential for bluetongue virus detection and control of BT vaccine quality. IMPORTANCE Bluetongue virus causes the severe infectious disease BT. BTV has many serotypes, and there is no cross-protection among different serotypes. BT is listed as a notifiable animal infectious disease by the World Organisation for Animal Health (OIE) and occurs throughout the world, causing significant economic losses. The establishment of a fast and effective detection method is the key to controlling and preventing this disease. Current methods for detecting BTV mainly include reverse transcription-PCR (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and immunochromatographic strips that are based on antigen-antibody recognition. Immunoassays are most commonly used because of their low cost, high specificity, and fast analysis, making them particularly useful for routine monitoring. These conventional detection strategies for BTV have some drawbacks. Recently, FLISA has been drawing attention due to its sensitivity, which is higher than traditional immunoassays. Fluorescence-linked immunosorbent assays (FLISA) using fluorescent materials as labels overcome ELISA's disadvantage of being time-consuming.

Modification of nanoscale polymeric adsorbent for the preparative separation and purification of tacrolimus from fermentation broth of Streptomyces tsukubaensis.

Tacrolimus is an important immunosuppressant produced by microbial fermentation. In this study, a modified nanoscale polymeric adsorbent, Ag+-exchanged resin, was prepared and studied for the preparative separation and purification of tacrolimus from fermentation broth of Streptomyces tsukubaensis. The performance and absorption characteristics of the modified nanoscale polymeric adsorbent namely Ag-NPS was evaluated. Notably, Ag-NPS resin displayed the pronounced separation capacities for tacrolimus and its equilibrium adsorption data was well-fitted to the Langmuir isotherm. Moreover, the dynamic adsorption and desorption tests was carried out to obtain the operational parameters for further purification of tacrolimus. Finally, tacrolimus and the two major impurities, ascomycin and dihydrotacrolimus, were separated well in the scale-up purification process. The purity and recovery of tacrolimus was recorded to be 99.12±0.25% and 90.41±2.05%. In conclusion, this method displayed a high potential for separation and purification of tacrolimus and other unsaturated bioactive compounds in high yield from the fermentation broth.

Analysis of virginiamycin M1 in swine feed, muscle and liver samples by quantum dots-based fluorescent immunochromatographic assay.

Based on a highly sensitive and specific monoclonal antibody (mAb) against virginiamycin M1 (VIR M1), a quantum dots-based fluorescent immunochromatographic assay (QDs-ICA) for quick and sensitive analysis of VIR M1 was established for the first time. The mAb showed a half-maximal inhibitory concentration (IC50) of 0.5 ng/mL and cross-reactivity (CR) values below 0.1% for other three analogues when used in enzyme-linked immunosorbent assay (ELISA). The mAb was conjugated to ZnCdSe/ZnS (core/shell) QDs with maximum emission wavelength of 610 nm (orange-red) which was selected as fluorescent probe to increase QDs-ICA sensitivity. The cut-off value of QDs-ICA was 12.5 ng/mL. QDs-ICA showed a linear range from 0.7 to 14.5 ng/mL with a limit of quantification of 0.7 ng/mL. Compared with existing methods for the analysis of VIR M1, the QDs-ICA exhibited higher sensitivity. For analysis of VIR M1 concentrations spiked into swine feed, muscle and liver samples, recovery rates ranged from 94.0% to 111.6% with the highest coefficient of variation (CV) of 6.7% for intra-assay, and for inter-assay ranged from 94.7% to 107.6% with the highest CV of 9.4%. In conclusion, the QDs-ICA could be a potential method for analyzing VIR M1 in animal feed and animal-derived food.