Search Results (116)

The Akabane virus (AKAV) is a significant member of the Orthobunyavirus genus, with its envelope glycoprotein Gc, focusing on its molecular structural features, immunoregulatory mechanisms, and application value in pathogen diagnosis and vaccine design. As a key structural protein of AKAV, Gc mediates virus adsorption and neutralizing antibody recognition through the N-terminal highly variable region (HVR), while the C-terminal conserved region (CR) dominates the membrane fusion process, and its glycosylation modification has a significant regulatory effect on protein function. In clinical diagnostics, serological assays based on Gc proteins (e.g., ELISA, immunochromatographic test strips) have been standardized; in vaccine development, the neutralizing epitope of Gc proteins has become a core target for subunit vaccine design. Follow-up studies were deeply needed to analyze the structure-function interaction mechanism of Gc proteins to provide theoretical support for the construction of a new type of AKAV prevention and control system. Full article

Nipah virus (NiV) and Hendra virus (HeV), which both belong to the genus henipavirus, are zoonotic pathogens that cause severe systemic, neurological, and/or respiratory disease in humans and a variety of mammals. Therefore, monitoring viral prevalence in natural reservoirs and rapidly diagnosing cases of henipavirus infection are critical to limiting the spread of these viruses. Current laboratory methods for detecting NiV and HeV include virus isolation, reverse transcription quantitative real-time PCR (RT-qPCR), and antigen detection via an enzyme-linked immunosorbent assay (ELISA), all of which require highly trained personnel and specialized equipment. Here, we describe the development of a point-of-care customized immunochromatographic lateral flow (ILF) assay that uses recombinant human ephrin B2 as a capture ligand on the test line and a NiV-specific monoclonal antibody (mAb) on the conjugate pad to detect NiV and HeV. The ILF assay detects NiV and HeV with a diagnostic specificity of 94.4% and has no cross-reactivity with other viruses. This rapid test may be suitable for field testing and in countries with limited laboratory resources. Full article

Canine distemper, a fatal and highly transmissible disease caused by the canine distemper virus (CDV), poses a major threat to the companion animal industry. An urgent need exists for a rapid, specific, and simple method for the detection of this disease in order to improve its prevention and control. In this research, two monoclonal antibodies (mAbs), 1D3E9 and 1H9B7, were prepared, both of which specifically recognize the nucleoprotein (N protein) of CDV, and an immunochromatographic assay for CDV detection was subsequently developed using these mAbs. The results showed that both mAbs belong to the IgG1 subclass with kappa light chains. 1D3E9 was found to recognize the linear epitope ⁴¹⁰AGPKQSQITFLH⁴²¹, while 1H9B7 targeted the epitope ⁴⁵⁰HFNDERFPGH⁴⁵⁹. The test strips exhibited high specificity and good stability for up to two months when stored at 4, 25, and 37 °C. The assay exhibited a sensitivity of 10^2.39 TCID₅₀/0.1 mL. When compared with RT-PCR for detecting CDV in clinical samples, the concordance rate was 91.67%. Thus, this method shows great potential for facilitating rapid on-site detection of CDV and could be highly beneficial from the viewpoint of disease surveillance and control. Full article

The development of rapid, sensitive and cost-effective lateral flow assays is crucial for the detection of mycotoxins, ideally at the point-of-care level. This study presents the design and optimization of a competitive lateral flow assay based on gold nanoparticles (AuNPs) for the detection of zearalenone in food samples. Beginning with the synthesis and functionalization of gold nanoparticles, it proceeds to compare the immobilization of antibodies using chemical conjugation and physical adsorption binding strategies, upon optimizing parameters including the pH, antibody concentration and blocking conditions to enhance the stability of the prepared bioconjugates. The bioconjugates are characterized using UV–visible absorption spectroscopy and dynamic light scattering to monitor changes in the spectra and hydrodynamic size of AuNPs upon the addition of antibodies. The assessment of these bioconjugates is based on their ability to bind and manifest a color, developed due to nanoparticle binding with the test zone on the strip with the toxin–protein conjugate. The lateral flow immunochromatographic assay (LFIA) strips are then prepared by dispensing a control line (IgG) and test line (toxin–protein conjugate) on a nitrocellulose membrane using a lateral flow strip dispenser. The sensitivity of the LFIA strips is evaluated after standardizing the conditions by varying the concentration of zearalenone in the spiked samples and optimizing the running buffer solution. The limit of detection and limit of quantification under optimized conditions are determined to be 0.7 ng/mL and 2.37 ng for zearalenone-spiked samples. Furthermore, the mean pixel intensity and RGB values are plotted against the concentration of zearalenone, which can be used in a colorimetric smartphone-based application for the quantification of the amount of mycotoxin in the sample. Full article

Toxoplasma gondii is a globally significant zoonotic pathogen responsible for severe parasitic diseases in humans and animals. This study aimed to design, develop, and evaluate a novel immunochromatographic test (ICT) using a recombinant MIC2-MIC3 fusion protein (rMIC2-MIC3) for detecting specific antibodies against T. gondii. The ICT demonstrated exceptional sensitivity, capable of detecting T. gondii-specific antibodies in sera diluted up to 1:8. Specificity evaluation confirmed no cross-reactivity with antibodies against other parasites, such as Neospora caninum, Cryptosporidium suis, Eimeria tenella, and Sarcocystis tenella. Stability tests revealed the test strips maintained full functionality after 12 weeks of storage at 24 °C. The coincidence rate of the colloidal gold test strips prepared in this study with a commercial ELISA kit was 94.59%. Comparisons with advanced serodiagnostic tools, such as chimeric antigen-based ELISAs and recombinant protein diagnostics, further highlighted its robustness and applicability. These findings underscore the potential of the rMIC2-MIC3-based ICT as a reliable, economical, and accessible diagnostic tool for toxoplasmosis in veterinary and human medicine. Full article

Procymidone (PCM) is an effective, low-toxicity fungicide commonly used to control plant diseases in grains, vegetables, and fruits. Its usage has significantly increased in recent years, resulting in higher residues in vegetables. This study developed a sensitive and rapid immunoassay method utilizing a gold- and fluorescence-labeled monoclonal antibody (mAb) for detecting PCM residues in vegetable samples. Under optimal conditions, the fluorescent microsphere-labeled monoclonal antibody immunochromatographic strips achieved a limit of detection (LOD) of 1.67 ng/mL, with a visual LOD of 50 ng/mL. Intra-batch accuracy ranged from 94.98% to 103.82%, with a coefficient of variation (CV) of 1.97% to 8.26%. Inter-batch accuracy ranged from 96.16% to 102.51%, with a CV of 4.62% to 8.91%. The visual detection range of the gold nanoparticle-labeled monoclonal antibody immunochromatographic strips was 50 to 200 ng/g. The method demonstrated excellent performance in actual vegetable samples, confirming its applicability across various matrices. This dual-method approach enables rapid screening of negative samples with gold test strips, followed by accurate quantitative analysis of positive samples using fluorescent test strips, thereby enhancing efficiency and addressing diverse detection needs. Consequently, this method holds significant market potential for practical applications. Full article

E. coli O157:H7 contamination in food and the environment poses a serious threat to human health. Rapid and sensitive identification of foodborne pathogens remains challenging. Here, we prepared tungsten disulfide (WS₂)–Au nanocomposites coupled with the Raman signal molecule 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and antibodies to replace the conventional colloidal gold nanoparticles and applied SERS-active nanotags in the SERS-ICA method for highly sensitive detection of E. coli O157:H7. The large surface area and numerous effective SERS hotspots of WS₂-Au nanotags provide superior SERS signals. Under optimized conditions, this ICA achieves the quantitative detection of E. coli O157:H7 in a broad linear range of 8 × 10²–8 × 10⁷ CFU/mL and at a low detection limit of 175 CFU/mL. In addition, the test strip indicates high specificity for E. coli O157:H7 identification, favorable reproducibility, and shows good accuracy in the detection of actual food samples, such as milk and pork. The proposed assay can be used for rapid qualitative and quantitative detection of E. coli O157:H7 and has great potential for field application. Full article

Actinobacillus pleuropneumoniae (APP) is a bacterial pathogen causing porcine pleuropneumonia, causing great economic loss to the global pig industry. Although natural apxIV contributes to the prevention and control of porcine pleuropneumonia, its isolation poses a great challenge, and recombinant soluble apxIV proteins tend to carry large molecular weight tags. The traditional serologic methods tend not to accurately detect the apxIV-partially deleted vaccine (GDV). In this study, we screened the soluble protein apxIVA N2 (756 bp) from six apxIV-truncated proteins and applied it to the enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic strip for detecting the samples vaccinated with APP GDV. The results indicate that N2 was close to the natural apxIV protein in terms of structure and function as it only contained a single His (0.86 kDa) tag and a single S (2 kDa) tag. Among the six candidate proteins, N2 exhibited the best performance in distinguishing APP-infected samples from those vaccinated with the APP GDV. Both ELISA and colloidal gold immunochromatographic strips based on this protein exhibited an excellent performance in detecting and distinguishing wild-strain-infected samples from those vaccinated with the subunit vaccine or the GDV. In addition, three monoclonal antibodies against different antigenic epitopes were identified using these truncated proteins. Our studies are of great significance for further research on APP, the differential diagnosis of wild strains and vaccine strains, and pig control breeding, exhibiting a broad application prospect in the on-site diagnosis of APP, particularly in remote areas lacking detection instruments and professionals. Full article

Bacterial fruit blotch (BFB) caused by Paracidovorax citrulli is a severe threat to melon, watermelon, and other cucurbit crop production worldwide. The long-term adaptation of the pathogen to environmental conditions has resulted in substantial genetic diversity. In this study, we used P. citrulli strains from two groups as immunogens to obtain antibodies that were used to generate A.C1 and A.C2 colloidal gold immunochromatographic single test strips, which specifically identified group I and group II P. citrulli strains, respectively. We combined the A.C1 and A.C2 single test strips in a dual-channel plastic cartridge to construct a dual-channel colloidal gold immunochromatographic test strip able to distinguish between P. citrulli strains from two distinct groups. Test strip sensitivity reached 10⁶ CFU/mL under ideal conditions. Moreover, it was relatively stable, with no cross-reactions with strains of closely related genera. The dual-channel test strip developed in this study may provide farmers with a useful tool for managing BFB through the prompt implementation of quarantine procedures in the field. Full article

Porcine circovirus type 2 (PCV2) is the main and primary causative agent of Postweaning Multisystemic Wasting Syndrome (PMWS). To date, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are the most commonly diagnostic methods for detecting PCV2 antigens. However, these methods require specialized equipment and technical expertise and are suitable for laboratory use only. This study aims to develop an immunochromatographic strip and a magnetic chemiluminescence immunoassay for the detection of PCV2 antigens. The recombinant protein was constructed using a prokaryotic expression system, and the polyclonal antibody was obtained by animal experiments. Polystyrene microspheres are used as solid phase carriers to covalently bind to the amino groups of proteins to form immunoprobes. Monodisperse beads are covalently bound to antigens or antibodies as solid phases to bind antibodies or antigens in the liquid phase in a superior manner, thereby capturing and separating antigens and antibodies in the liquid phase. The immunochromatographic strip is qualitative detection method, this method can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 10^2.89TCID₅₀/0.1 mL, 10^3.19TCID₅₀/0.1 mL, and 10^3.49TCID₅₀/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The results of testing PEDV (CV777 strain), PRV (HB2000 strain), CSFV (WH-09 strain), PRRS (JXA1-R strain), PPV (WH-1 strain), and ASFV (SD strain) were negative. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866 (Kappa > 0.81). On the premise of ensuring sensitivity, the most important feature of the immunochromatographic strip is that this method can save time when testing; results can be obtained within 5 to 10 min. Magnetic chemiluminescence immunoassay is quantitative detection method; this method can detect PCV2 Cap proteins in swine serum, the linear range of this method was 0.25 ng/mL to 32 ng/mL and R² of the standard curve was 0.9993. The limit of detection (LOD) is 0.051 ng/mL and the limit of quantitation (LOQ) is 0.068 ng/mL. The agreement between the magnetic chemiluminescence immunoassay and the ELISA kit (test PCV2 Cap proteins) was 97.14% (68/70). This method took less than 30 min to achieve results, which is less than the ELISA kit. The results of this study show that immunochromatographic strip and magnetic chemiluminescence immunoassay for PCV2 antigens had great sensitivity and specificity, which lays the foundation for PCV2 clinical detection. Full article

In this study, a novel rapid immunochromatographic (IC) test for African swine fever virus (ASFV) antibodies is presented. An immunochromatographic test (IC) is a detection technique that combines membrane chromatography with immunolabeling. This approach saves time for antibody preparation, resulting in a shorter production cycle. p54 is an important structural protein of African swine fever, and an ideal protein for serotype diagnosis. Gold nanoparticles are attached to the ASFV p54-Fc fusion protein, and the ASFV-specific antigen p54 and Staphylococcus aureus protein A (SPA) are labeled on a nitrocellulose membrane, at positions T and C, respectively. We developed a SPA double sandwich IC test strip, and assessed its feasibility using ASFV p54 and p54-Fc fusion proteins as antigens. ASFV p54 and p54-Fc fusion proteins were expressed and purified. A sandwich cross-flow detection method for p54, which is the primary structural protein of ASFV, was established, using colloidal gold conjugation. Our method can detect ASFV antibodies in field serum samples in about 15 min using a portable colloidal gold detector, demonstrating high specificity and sensitivity (1:320), and the coincidence rate was 98% using a commercial ELISA kit. The dilution of the serum sample can be determined by substituting the absorbance (T-line) interpreted by portable devices into the calibration curve function formula of an African swine fever virus standard serum. In summary, our method is rapid, cost-effective, precise, and highly selective. Additionally, it introduces a new approach for constructing IC test strips using SPA protein without antibody preparation, making it a reliable on-site antibody test for ASFV. Full article

The livestock industry uses ofloxacin, an antibiotic, to prevent several animal diseases; however, the overdose of ofloxacin used in animal farming treatments may appear in food products and cause some adverse human health effects. Hence, there is an immediate need to develop a method suitable for on site large-scale detection of ofloxacin residues in animal-derived foods. This study aimed to prepare a monoclonal antibody with high sensitivity and affinity for ofloxacin by re-synthesizing the ofloxacin hapten and synthesizing the corresponding complete antigen. The IC₅₀ of the enzyme-linked immunosorbent assay (ic-ELISA) was 0.13 ng/mL, and the detection limit was 0.033 ng/mL. The visual detection limit of the established colloidal gold immunochromatographic test strip, for the visual detection of actual samples, was 1 ng/g. In summary, this work establishes a rapid detection method of ofloxacin residues on the basis of colloidal gold immunochromatography that is suitable for actual detection. Full article

In this study, we developed a colloidal gold immunochromatographic strip (CGIS) method that used the matrix-matched calibration curves of contamination ratio models to quantitatively determine the total aflatoxin in five herbal medicines. This approach addresses issues related to false results and poor accuracy associated with conventional methods. The CGIS was analyzed using a Vertu touch reader, and the matrix-matched calibration was established based on the absorbance ratios of the T and C lines, as well as the logarithmic values of the total aflatoxin concentrations. The total aflatoxins could be accurately and digitally detected from 2.5 to 40 μg/kg, and the LOD of total aflatoxins was 1 μg/kg in the five herbal medicines. The recovery rates from the spiked samples ranged from 65.1% to 98.6%, and the RSD was less than 16.9%. A total of 229 samples were analyzed by both CGIS and HPLC-FLD, with agreement ranging from 78.4% to 132.6% (Arecae semen), 82.6% to 133.0% (Nelumbinis semen), 79.9% to 117.9% (Coicis semen), 78.1% to 119.0% (Platycladi semen), and 76.1% to 123.0% (Ziziphi spinosae semen). This process for the discrimination of the CGIS results was established to assess if samples met the requirement of aflatoxin limits, which could save approximately 75% in time and reduce the workload of retesting by a designated confirmatory reference method to less than 10%. This study demonstrated that the application of matrix-matched calibration curves based on contamination ratio models to CGIS can effectively enhance the rapid quantitative determination capability of total aflatoxins in herbal medicine matrices. Full article

Gatifloxacin (GAT), an antibiotic belonging to the fluoroquinolone (FQ) class, is a toxicant that may contaminate food products. In this study, a method of ultrasensitive immunochromatographic detection of GAT was developed for the first time. An indirect format of the lateral flow immunoassay (LFIA) was performed. GAT-specific monoclonal antibodies and labeled anti-species antibodies were used in the LFIA. Bimetallic core@shell Au@Ag nanoparticles (Au@Ag NPs) were synthesized as a new label. Peroxidase-mimic properties of Au@Ag NPs allowed for the catalytic enhancement of the signal on test strips, increasing the assay sensitivity. A mechanism of Au@Ag NPs-mediated catalysis was deduced. Signal amplification was achieved through the oxidative etching of Au@Ag NPs by hydrogen peroxide. This resulted in the formation of gold nanoparticles and Ag⁺ ions, which catalyzed the oxidation of the peroxidase substrate. Such “chemical enhancement” allowed for reaching the instrumental limit of detection (LOD, calculated by Three Sigma approach) and cutoff of 0.8 and 20 pg/mL, respectively. The enhanced assay procedure can be completed in 21 min. The enhanced LFIA was tested for GAT detection in raw meat samples, and the recoveries from meat were 78.1–114.8%. This method can be recommended as a promising instrument for the sensitive detection of various toxicants. Full article

Toxoplasma gondii (T. gondii) is an important zoonotic pathogen which induces both acute and chronic toxoplasmosis. Timely diagnosis of T. gondii is crucial for effective disease management. Here, we present a pioneering approach using europium (III)-chelated nanoparticles (EuNPs) in a rapid lateral flow immunochromatographic test strip (ICTS) for detecting T. gondii antibodies in serum samples. By conjugating EuNPs with Staphylococcus aureus protein A, we efficiently captured T. gondii-specific antibodies, which bound to T. gondii antigens on the test line (T-line), generating a distinct fluorescent signal. Employing this novel method, we conducted an extensive epidemiological investigation of T. gondii infections among dogs and cats in Shanghai, China. This innovative ICTS allows for rapid results within 25 min, which include a qualitative result through naked-eye observation under an ultraviolet lamp and a quantitative one derived using a strip reader. With a detection limit of 1:6400 for dog positive serum and no cross-reactivity with other canine and feline pathogens, the EuNPs-ICTS demonstrated excellent consistency with standard enzyme-linked immunosorbent assay results for dogs (κ = 0.91) and cats (κ = 0.92). In addition, 20.38% of 996 dog serum samples and 14.18% of 416 cat serum samples revealed T. gondii antibodies, highlighting the efficacy of this approach. Our study presents a rapid, sensitive, specific, and reproducible EuNPs-ICTS, serving as a promising tool for on-the-spot diagnosis of T. gondii infections in dogs and cats. Full article