Search Results (73)

Brucellosis is a zoonotic disease causing significant economic losses, and necessitating standardized diagnostic kits for effective surveillance. The objectives of this study are to investigate brucellosis serosurveillance in ruminants, and to evaluate three serological tests—Rose Bengal Plate Tests (RBPTs), Serum Tube Agglutination Tests (STATs), and Enzyme-Linked Immunosorbent Assays (ELISAs)—each from three manufacturers. A total of 5797 serum samples (5260 sheep, 83 bovines, and 454 spotted deer) were initially screened with the RBPT, and 54 RBPT-positive selected samples were subsequently analyzed by the STAT and ELISA. RBPT screening showed significant variability: kit A (0.86%) showed significantly lower positivity than kits B and C (1.45% and 1.43%, p < 0.05). Among 54 RBPT-positive samples, STAT positivity from manufacturers A, B, and C was 61.11%, 70.37%, and 66.67%, respectively, with no significant differences (p > 0.017 in pairwise comparisons). All ELISAs consistently outperformed STATs. Kit B (competitive ELISA, 90.74%) had significantly higher positivity than kits A (indirect ELISA, 77.78%) and C (competitive ELISA, 74.07%) (p < 0.017 in pairwise comparisons). Key implications: Results from different manufacturers are not directly comparable, and concurrent use of different techniques on the same sample can yield discrepant outcomes. This underscores the urgent need for diagnostic kit standardization in brucellosis prevalence studies. Full article

Mycoplasma bovis (M. bovis) is a major pathogen in cattle, primarily responsible for bovine respiratory disease complex (BRDC). Characterized by high infectivity and morbidity, M. bovis spreads rapidly within herds and is challenging to control and eradicate, underscoring the need for early and accurate diagnosis. To address this, we developed a competitive enzyme-linked immunosorbent assay (cELISA) for detecting M. bovis-specific antibodies in bovine sera. The assay uses recombinant MbovP579 protein (1 μg/mL) as the coating antigen and an HRP-conjugated 1A2 monoclonal antibody against MbovP579 (mAb, 0.35 μg/mL) as the competitor. With a cut-off value of 40.69% (percentage inhibition, PI), the cELISA demonstrated apparent sensitivity (90%; 95% CI: 78.64–95.65%) and specificity (96.15%; 95% CI: 90.53–98.49%) using the known background positive and negative sera. In addition, it yielded 100% positivity (9/9) against sera collected from 21 days post-immunization. The assay showed no cross-reactivity with other Mycoplasma species or two common bovine bacterial pathogens, confirming its high specificity. The intra- and inter-assay coefficients of variation (CVs) were below 10%. In clinical evaluations, this cELISA generated a 55.57% positivity rate for field samples (n = 1069) and exhibited 90.46% overall agreement (967/1069; 95% CI: 88.54–92.15%) with the commercial indirect ELISA kit, demonstrating strong diagnostic consistency. Discrepant specimens were further analyzed by a metabolic inhibition test (MIT), which suggested improved specificity, although further validation is required of this cELISA. Full article

Isofenphos-methyl (IFP), a highly toxic and persistent organophosphate pesticide (OP), is widely used for soil pest control in crops but poses severe risks to ecological safety and human health because of its environmental accumulation and bioaccumulation. Herein, a sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for rapid IFP detection in vegetables. A novel IFP hapten was rationally designed and synthesized via computer-aided molecular simulation, and its structure was validated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR). High-specificity anti-IFP monoclonal antibodies (mAbs) with strong anti-matrix interference were prepared for the first time using a matrix effect-enhanced screening strategy. The optimized ic-ELISA showed high sensitivity, with an IC₅₀ of 6.087 ng/mL and a detection range of 1.165–30.490 ng/mL, and no cross-reactivity with other common OPs. Spiked recovery experiments in celery and chili pepper matrices yielded recoveries of 81.87–97.95% (RSD < 5.44%), with highly consistent LC–MS/MS results. The method exhibited a weak positive matrix effect in vegetable matrices, eliminating complex pretreatment and enabling rapid onsite detection. Full article

Food safety requires real-time monitoring of mycotoxins in food, as food products contaminated with these toxins poses major threat to human health. In this study, we proposed a remote-controlled microfluidic platform (RCMP) integrated with chemiluminescent/colorimetric detection system for rapid, cost-effective and real-monitoring of multiple mycotoxins in real samples based on the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). The RCMP enabled sensitive and automatic detection of deoxynivalenol (DON), zearalenone (ZEA), and fumonisin B1 (FB1) in the range of 4–128 ng/mL, 1–32 ng/mL, and 0.5–16 ng/mL, respectively. The limits of detection (LOD) were 2.881 ng/mL for DON, 0.702 ng/mL for ZEA, and 0.470 ng/mL for FB1. In further validation, satisfactory recoveries between 93.57% to 108.47% with the relative standard deviations (RSDs) of 6.92–11.39% were obtained in beer samples. Overall, RCMP provides an automatic, high-throughput and cost-effective method for detection of DON, ZEA, and FB1 and can be confidently applied for monitoring in beer samples. Full article

The contamination of traditional Chinese medicines (TCMs) with neonicotinoid pesticides, notably acetamiprid (ACE), poses a significant challenge to product safety. Conventional detection methods are often hampered by operational complexity, prolonged analysis times, and dependence on sophisticated instrumentation, rendering them impractical for rapid on-site screening. To address these limitations, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the efficient quantification of ACE residue in TCM matrices. A monoclonal antibody-based ic-ELISA was developed through the synthesis of an ACE antigen. Critical assay parameters—including coated antigen concentration, antibody dilution ratio, and blocking buffer composition—were systematically optimized. The validated protocol was subsequently applied to ACE detection in five representative TCMs. The sensitivity (IC₅₀), limit of detection (IC₁₅), and detection range (IC₂₀-IC₈₀) of the developed ic-ELISA for ACE were 13.61 ng/mL, 0.50 ng/mL, and 1.00–150.99 ng/mL, respectively. The ic-ELISA demonstrated good stability and specificity, with cross-reactivity for ACE analogs all below 1.5%. Additionally, the ic-ELISA for ACE achieved recoveries of 86.87–104.80% in spiked TCM samples (Lonicerae Japonicae Flos, Lycii Fructus, Bulbus Lilii, Citri Reticulatae Pericarpium, and Jasminum sambae Flos), with relative standard deviations (RSDs) of 3.33–12.05%. The recovery rate of ic-ELISA was verified to be in good consistency with that of high-performance liquid chromatography (86.09–102.10%), indicating that ic-ELISA has acceptable accuracy and precision. This approach is simple and sensitive, making it suitable for the rapid quantitative detection of ACE residues in TCM products. It also provides technical references for the development of ic-ELISA for other small-molecule contaminants. Full article

Due to the severe hazard of aflatoxins (AFs) to humans, it is of great significance to detect the key aflatoxins, aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁), in food and feed in simple, rapid, and semi-quantitative ways. The hybridoma clone 3A1 was prepared in this study, and anti-AFB₁ monoclonal antibody (mAb) with high specificity and affinity (9.38 × 10⁸ L/mol) from 3A1 was purified. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) demonstrated that the linear detection range for AFB₁ was 0.029–1.526 ng/mL with a limits of determination (LOD) of 0.023 ng/mL. A latex microsphere-based immunochromatographic test strip (LM-ICTS) was constructed based on 3A1, which showed that the strip could detect AFB₁ (LOD: lower than 1.79 ng/mL) and AFG₁ (LOD: lower than 8.08 ng/mL), and the linear detection ranges for AFB₁ and AFG₁ are 1.79–48.46 ng/mL and 8.08–107.40 ng/mL, respectively. The average recoveries of intra-assay and inter-assay for peanuts were (98.4 ± 4.7)% and (92.6 ± 7.6)%, and the average coefficient of variation (CVs) were 4.38% and 8.15%, respectively. For sunflower seeds, the intra-assay and inter-assay recoveries were (94.4 ± 7.2)% and (89.2 ± 4.3)%, and the average CVs were 6.6% and 4.9%, respectively. In summary, the developed LM-ICTS exhibited excellent sensitivity and specificity, which provided a rapidly stable on-site detection choice for AFB₁ and AFG₁ to contaminated agricultural samples, including grain and feed. Full article

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe lymphoproliferative vascular disease of cattle that is caused by ovine gammaherpesvirus 2 (OvGHV2), which is a Macavirus within the Gammaherpesvirinae subfamily. SA-MCF occurs worldwide in several mammalian hosts. Alternatively, alcelaphine gammaherpesvirus 1 (AlGHV1) is a Macavirus that causes wildebeest-associated malignant catarrhal fever (MCF), which principally occurs in cattle from Africa. Previous serological assays to evaluate the presence of MCF in mammals used a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA). This CI-ELISA is based on the 15A antigenic epitope that is common to all Macavirus associated with the development of MCF in their respective hosts. This study evaluated an indirect MCF-specific ELISA assay based on the AlGHV1 C500 strain to detect antibodies against OvGHV2 in 43 closed dairy cattle farms from Southern Brazil. These farms are located in a region where subclinical infections by OvGHV2 have been detected in free-ranging wild boars (Sus scrofa). Sheep or goats were not reared at these farms or within the proximity of these farms. Risk factors associated with seropositivity to OvGHV2 were evaluated, while the possible participation of subclinically infected wild boars in the dissemination of OvGHV2 was estimated using spatial analysis. Sera from 29 dairy cows from 16 farms demonstrated sample/positive (S/P) values considered positive with this MCF-specific ELISA (cutoff S/P, 0.063). The S/P values for the positive dairy cows varied between 0.0633 and 0.2510 (mean, 0.0998; standard deviation, 0.0476). At least one cow was seropositive in 16/43 (37.2%) of these farms, with seropositivity identified in 29/367 (7.9%) of dairy cows maintained at these farms. Additionally, dairy cows raised within the intensive system had a more than threefold higher chance of being seropositive to OvGHV2 relative to those reared within the semi-intensive system. Furthermore, the spatial evaluation revealed that cows on dairy farms within a 50 km radius of the home range of subclinically infected wild boars had an increased risk of being seropositive to this assay. These findings demonstrated that the AlGHV1 C500-specific MCF ELISA can be efficiently used to monitor the occurrence of OvGHV2 in cattle. In addition, the occurrence of subclinically infected free-ranging wild boars within a radius of 50 km from susceptible cattle may be a possible risk factor for the occurrence of OvGHV2-related infections in these animals from Southern Brazil. These initial results are fundamental to understanding the epidemiology of OvGHV2-associated infections and clinical SA-MCF in mammals in Brazil. Full article

Benzo[a]pyrene (B[a]P) is a hazardous polycyclic aromatic hydrocarbon that accumulates in several environmental matrices as a result of incomplete combustion. Its presence, carcinogenic properties, and tendency for bioaccumulation provide significant risks to human health and the environment. The objective of this study is to create an immunoassay for the detection of benzo[a]pyrene utilizing immunoglobulin Y antibodies. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was utilized to develop a speedy, straightforward, sensitive, and economical approach for detecting B[a]P residues. Following the immunization of hens with the hapten pyrenebutyric acid-bovine serum albumin (PyBA-BSA), the IgY antibody extracted from egg yolk was utilized to identify B[a]P residues. To evaluate antibody specificity, six PAH derivatives—PyBA, B[a]P, Chrysene, Benzo[b]fluoranthene, Benzo[a]anthracene, and Benzo[k]fluoranthene—were examined in the experiment to compete for binding with PyBA. The findings indicate that the antibody had considerable affinity for Chrysene (1.15%), Benzo[b]fluoranthene (311.32%), Benzo[k]fluoranthene (10.62%), Benzo[a]anthracene (22.82%), and PyBA (9.55%). Nonetheless, its affinity for B[a]P remained at 100%. The recovery range for grilled pork samples spiked with B[a]P doses of 10.00–0.1 μg/mL was 74.99% to 143.11%. This study utilized a polyclonal antibody, employing the IgY antibody for the inaugural development of an immunoassay to detect benzo[a]pyrene. The ELISA had a higher IC₅₀ value compared to the other immunoassays; however, it yielded good results. This immunoassay signifies a substantial progression in environmental analytical chemistry, offering a cost-effective and accessible technique for the detection of B[a]P to protect human health and the environment. Full article

The target of traditional immunological detection methods for milk allergens is usually the whole β-lactoglobulin molecule. However, thermal processes and hydrolysis can destroy the epitope of β-lactoglobulin and interfere with its accurate detection and labeling in prepackaged foods, posing a health risk to milk-allergic patients. There currently remains a need to excavate and locate recognition sites for β-lactoglobulin in thermally processed and hydrolyzed products. Therefore, a stable epitope of β-lactoglobulin (CAQKKIIAEKTKIPAVFKIDA) was selected as the ideal recognition site, and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using an antibody against this stable β-lactoglobulin epitope in order to improve the detection of β-lactoglobulin in thermally processed and hydrolyzed foods in this study. The stable epitope of β-lactoglobulin was selected using a molecular dynamics simulation, and the binding ability of anti-stable epitope antibodies was characterized using indirect ELISA and indirect competitive ELISA. The limit of detection (LOD) and limit of quantitation (LOQ) of the established ELISA were 0.25 and 1.07 mg·kg⁻¹, respectively. Furthermore, the developed ELISA only showed cross-reactivity to goat milk among 23 common foods, therefore exhibiting high specificity to bovine β-lactoglobulin. In addition, the developed ELISA was able to effectively detect β-lactoglobulin residue in processed commercial foods and hydrolyzed formula milk powder. Our findings provide a novel strategy for accurately detecting milk allergens based on stable epitope recognition in thermally processed and hydrolyzed foods. Full article

Prolonged consumption of animal-derived foods containing high levels of lincomycin (LIN) residues can adversely impact human health. Therefore, it is essential to develop specific antibodies and immunoassay methods for LIN. This study utilized computational chemistry to predict the efficacy of LIN haptens prior to chemical synthesis, with subsequent confirmation obtained through an immunization experiment. A hybridoma cell line named LIN/1B11 was established, which is specific to LIN. The optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method exhibited high specificity for detecting LIN residues, with an IC50 value of 0.57 ± 0.03 µg/kg. The method effectively detected LIN residues in pork and milk samples, achieving a limit of detection (LOD) ranging from 0.81 to 1.20 µg/kg and a limit of quantification (LOQ) ranging from 2.09 to 2.29 µg/kg, with recovery rates between 81.9% and 108.8%. This study offers a valuable tool for identifying LIN residues in animal-derived food products. Furthermore, the efficient hapten prediction method presented herein improves antibody preparation efficiency and provides a simple method for researchers in screening haptens. Full article

Lomefloxacin (LOM), an antibiotic crucial for preventing various animal diseases in animal husbandry, can pose serious health risks when found in excessive amounts in meat products. The development of highly specific and sensitive colloidal gold immunochromatographic test strips is essential for the accurate detection of this class of antibiotics. Our study utilized a monoclonal antibody (mAb) assay and immunochromatographic strips to detect lomefloxacin residues in meat products. The results showed minimal cross-reactivity with other structural analogs, with a maximum half inhibitory concentration (IC₅₀) of 0.93 ng/mL and a linear range of 0.38 to 2.3 ng/mL for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). The recovery of LOM was 80% to 120%, with an average coefficient of variation below 5%. The immunochromatographic strip test results showed a visual detection limit of 2.5 ng/g, meeting the market requirements for the test. This study highlights the significance of specific and sensitive testing methods for detecting lomefloxacin, ensuring consumers’ safety and health. Full article

Chlorphenamine maleate is a prohibited additive found in herbal teas and health foods. Excessive intake of this substance can result in adverse health effects. In this study, two novel haptens, PEM and bepotastine (PB1), mimicking chlorphenamine maleate structure were designed and synthesized based on molecular simulation for developing two corresponding polyclonal antibodies (PEM-Ab and PB1-Ab), respectively. Afterward, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to quickly and accurately detect chlorphenamine maleate in herbal teas using PB1-Ab, which has a high sensitivity and specificity. For chlorphenamine maleate, the half-maximal inhibitory concentration (IC₅₀) and limit of detection (LOD) of PB1-Ab under ideal circumstances were found to be 1.18 µg/L and 0.07 µg/L, respectively. Besides, an environmentally friendly sample pre-treatment strategy was employed that allowed easy and effective elimination of complex matrices. The ic-ELISA method observed the average recovery rate from 87.7% to 94.0% with the variance coefficient (CV) ranging from 2.2% to 9.4%. Additionally, the identification of 25 commercially available herbal teas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) further confirmed the validity of our detection. The results of the two methods are consistent. Overall, the proposed ic-ELISA could be an ultrasensitive and reliable method for chlorphenamine maleate adulterated in foods or exposure to the environment. Full article

Gallic acid (GA) is closely related to the quality of herbal medicines and other agricultural products. In order to facilitate the rapid detection of GA, we developed a monoclonal antibody-based ic-ELISA method. Antigens with and without connecting arms were prepared. It was found that the introduction of connecting arms (linear carbon chain) was beneficial for immune response. By utilizing hybridoma technology, a specific mAb (anti-GA-M702) was screened and identified, which exhibited a 1:40,500 antibody titer and IgG2b antibody subtype. The ic-ELISA assay was established based on anti-GA-M702. The optimal working concentrations of the encapsulated antigen and antibody were 0.5 µg/mL and 0.67 µg/mL, respectively. The ic-ELISA method showed a linear detection range of 297.17–2426.61 ng/mL for GA with a sensitivity of 849.18 ng/mL. It displayed a good applicability for the determination of GA in Galla chinensis. In conclusion, the ic-ELISA method provides an efficient approach to the rapid detection of GA in products. Full article

Methylene blue (MB) is a chemical dye which is used as an alternative drug for malachite green. In this study, based on computational chemistry, a novel hapten (TM) of MB was designed by comparing the conformational and electronic properties of MB. TM was synthesized and further conjugated with bovine serum albumin (BSA) and Ovalbumin (OVA). Then, the polyclonal antibodies were obtained by immunization with the immunogen of TM-BSA. Under optimal conditions, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for the determination of MB, with an IC₅₀ value of 41.5 μg L⁻¹ and displaying a cross reaction of 78.2~88.9% with three different MB metabolins. The limit of detection (LOD) and the limit of quantitation (LOQ) for MB were validated to be 4.8 µg/kg and 6.0 µg/kg, respectively. Spiking experiments showed recoveries between 82.3% and 84.3%, with a relative standard deviation (RSD) of ≤6%. The results showed the generated polyclonal antibodies from the new hapten TM were reliable and could be used for detecting MB in fishery productions. Full article

Bovine neosporosis is an infection caused by the protozoan parasite Neospora caninum and has substantial veterinary hazards. Neosporosis cannot be controlled by vaccination or chemotherapy. Thus, accurate diagnosis followed by isolation and culling of infected animals is regarded as the most efficient method of control. In vivo diagnosis often relies on serologic testing of the animals, and milk represents a non-invasive and easy-to-collect sample matrix. However, indirect enzyme-linked immunosorbent assay (ELISA) specifically designed for antibody detection in milk are sometimes not easily available and it is tempting to use ELISA kits that are originally designed for use in serum in milk samples instead. Herein, we evaluated a widely used commercial ELISA (ID Screen^® Neospora caninum competition Multispecies ELISA (ID. Vet, Grabels, France)), developed for detection of N. caninum antibodies in serum samples, for its performance on milk samples. Milk samples from dairy ruminants (cows, buffaloes, sheep, and goats; n = 149) were tested in parallel with the serum ELISA and a commercial milk ELISA as a standard test (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France). The detected prevalence values were 28.2% (42/149), 17.4% (26/149), and 17.4% (26/149) using milk ELISA, serum ELISA, and both ELISAs, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for the serum ELISA used with milk samples were 61.9%, 100%, 100%, and 87%, respectively. The agreement and kappa value between the two ELISAs were 89.3% and 0.70, respectively, suggesting substantial agreement. High values of Pearson correlation coefficient (0.904, p ≥ 0.0001) and area under the receiver operating characteristic (ROC) curve (0.789, p ≥ 0.0001) demonstrated the high diagnostic performance of the serum ELISA in milk samples. Also, a Bland–Altman Plot and histogram describing the frequency of distribution of ELISA optical densities confirmed the high agreement of both serum and milk ELISAs. The current results revealed the high specificity but moderate sensitivity of the serum ELISA used for milk samples compared with the milk ELISA. However, the excellent positive predictive value of the serum ELISA makes it an alternative option in case of the unavailability of milk ELISAs. With this study, we provided additional evidence that a widely used serum ELISA test kit may also be used for the detection of N. caninum antibodies in milk samples. Full article