A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples.

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC50 and LOD of the ELISA for ZEN were 0.37 ng mL-1 and 0.04 ng mL-1, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB1, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, ß-zearalanol, zearalanone, α-zearalenol, ß-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R2 = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.

An Extremely Highly Sensitive ELISA in pg mL-1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples.

In this study, an extremely highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly produced monoclonal antibody (mAb) for the detection of ochratoxin A (OTA) in food samples was developed. OTA-Bovine serum albumin (BSA) conjugate was prepared and used as the immunogen for the production of the mAb. Among four hybridoma clones (8B10, 5C2, 9B7, and 5E11), the antibody from 8B10 displayed the highest affinity recognition for OTA. Based on the mAb (8B10), the IC50 and LOD of the ELISA for OTA were 34.8 pg mL-1 and 1.5 pg mL-1, respectively, which was 1.53~147 times lower than those in published ELISAs, indicating the ultra-sensitivity of our assay. There was no cross-reactivity of the mAb with the other four mycotoxins (AFB1, ZEN, DON, and T-2). Due to the high similarity in molecular structures among OTA, ochratoxin B (OTB), and ochratoxin C (OTC), the CR values of the mAb with OTB and OTC were 96.67% and 22.02%, respectively. Taking this advantage, the ELISA may be able to evaluate total ochratoxin levels in food samples. The recoveries of the ELISA for OTA in spiked samples (corn, wheat, and feed) were 96.5-110.8%, 89.5-94.4%, and 91.8-113.3%; and the RSDs were 5.2-13.6%, 8.2-13.0%, and 7.7-13.7% (n = 3), respectively. The spiked food samples (corn) were measured by ELISA and HPLC-FLD simultaneously. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation of y = 0.918x - 0.034 (R2 = 0.985, n = 5) was obtained. These results demonstrated that the newly produced mAb-based ELISA was a feasible and ultra-sensitive analytical method for the detection of OTA in food samples.

A SERS-based lateral flow immunochromatographic assay using Raman reporter mediated-gap AuNR@Au nanoparticles as the substrate for the detection of enrofloxacin in food samples.

A lateral flow immunochromatographic assay (LFIA) based on surface-enhanced Raman scattering (SERS) for sensitive and specific detection of antibiotic enrofloxacin (ENR) in food samples was developed. 1,4-benzenedithiol (BDT) was selected as the Raman reporter, and the BDT mediated-gap AuNR@Au nanoparticles (NPs) were synthesized, characterized and used as the substrate in SERS-LFIA due to the existence of the anisotropic gold nanorods (AuNRs) and the nano-gap with the high SERS enhancement. AuNRs were prepared, then covered by monolayer BDT. Under reduction condition and in presence of HAuCl4, the reduced gold was deposited and grown on AuNRs to form AuNRBDT@Au NPs. As the two thiol groups on para-positions in BDT were respectively linked to AuNR (core) and Au (shell), the gap size inside the NPs was uniform. The immunoprobe (e.g. AuNRBDT@Au-Ab) was obtained by immobilizing Ab against ENR on the surface of AuNRBDT@Au NPs. The performance of SERS-LFIA was similar to that in colloidal gold based-LFIA, and the entire assay time was within 15 min. After LFIA procedures, the specific SERS intensity of BDT at 1560 cm-1 on the test line was measured for the quantitative detection of ENR. The IC50 and limit of detection (LOD) of the LFIA for ENR were 59 pg mL-1 and 0.12 pg mL-1 (e.g. 71 pg g-1 and 0.14 pg g-1 in real sample), respectively. There was no cross-reactivity (CR) of the LFIA with other five antibiotics. The recoveries of ENR from spiked food samples were in range of 89.2%-102.4% with the relative standard deviation (RSD) of 1.70%-6.38%. It was proven that the proposed method was able to simply and rapidly detect ENR in food samples with high sensitivity, specificity, accuracy and precision. The platform can be also an alternative platform for the detection of other target analytes using corresponding Abs.

Protein-enriched extracts from housefly (Musca domestica) maggots alleviates atherosclerosis in apolipoprotein E-deficient mice by promoting bile acid production and consequent cholesterol consumption.

Currently, atherosclerosis control is important to prevent future heart attacks or strokes. Protein-enriched extract (PE) from housefly maggots (Musca domestica) can inhibit the development of atherosclerosis partially through its antioxidant effects. Whether PE exerts other anti-atherosclerosis functions remains unclear. Here, PE was found to simultaneously promote cholesterol metabolism effects in apolipoprotein E knockout (ApoE-/- ) mice. Bile acid synthesis plays a key role in regulating cholesterol homeostasis in atherosclerosis. Whether PE alleviates atherosclerosis by promoting bile acid production and consequent cholesterol consumption was further explored. First, 8-week-old male ApoE-/- mice were recruited and fed on a cholesterol-enriched diet. After 8 weeks, these mice were divided into three groups and received gavage administration of PE, simvastatin, and saline for another 8 weeks. Atherosclerosis severity was then assessed. Real-time quantitative polymerase chain reaction and western blot were employed to determine the expression of hepatic ATP-binding cassette transporter A1 (ABCA1), liver X receptor α (LXRα), and peroxisome proliferator-activated receptor-γ (PPAR-γ). Serum levels of high-density lipoprotein-cholesterol (HDL), low-density lipoprotein-cholesterol (LDL), and total cholesterol (TC) were determined by enzyme-linked immunoassay. Results revealed that PE reversed the formation of atherosclerotic lesion; increased the expression of PPAR-γ, LXRα, and ABCA1; increased the amount of bile flow and total bile acid; reduced the serum level of LDL and TC; and increased the level of HDL. In conclusion, enhancement on bile acid production and consequent cholesterol consumption may partially contribute to the anti-atherosclerotic effects of PE. The reversal of PPARγ-LXRα-ABCA1 signaling pathway may be involved in this process.

Synergistic Effect of Combined Sub-Tenon Triamcinolone and Intravitreal Anti-VEGF Therapy for Uveitic Macular Edema.

Purpose: To investigate effects of intravitreal anti-VEGF in combination therapy with sub-Tenon triamcinolone acetonide (STA) injection for uveitic macular edema (UME). Design: A single-center, retrospective cohort study. Methods: The medical records were obtained for 65 eyes of 65 patients with UME. Of which, 32 eyes received combined anti-VEGF with STA injection, and 33 eyes received 40 mg of STA injection alone. The primary outcome was the reduction of central macular thickness (CMT) measured with optical coherence tomography (OCT). Resolution rate of clinical UME and changes of best corrected visual acuity (BCVA) over 24 weeks were secondary outcomes. Results: There was a significantly greater reduction of CMT with the combination treatment than with STA alone at 1-week (ß = -157.9, P < 0.001) and 1-month (ß = -53.1, P = 0.019) after injection. The cumulative incidence of macular edema resolution of all eyes was 87.7%, with 90.6% (29/32) in the combined group and 84.8% (28/33) in the STA group, respectively. More incidence of UME resolution was observed in the combined group than the STA group after 1 week (71.9% vs 15.2%, P < 0.001) and 4 weeks (84.4% vs 54.5%, P = 0.009), respectively. BCVA was better for the combination treatment than STA alone at 1-week (ß = -0.085, P = 0.070) and 1-month (ß = -0.108, P = 0.019) after injection, respectively. Increased intraocular pressure (>25 mmHg) was observed in 4 eyes (12.5%) in the combined group and 5 eyes (15.2%) in the STA group, respectively. Conclusion: Combined intravitreal anti-VEGF and STA is superior to STA alone for reduction of UME and visual restoration. Addition of anti-VEGF did not increase risk for steroid-induced elevation of intraocular pressure over 6 months.

Evaluation of microvasculature alterations in convalescent Vogt-Koyanagi-Harada disease using optical coherence tomography angiography.

PURPOSE: To evaluate the microvasculature alterations in convalescent Vogt-Koyanagi-Harada (VKH) disease using optical coherence tomography angiography (OCTA), and to explore the association between microvasculature and the presence of sunset glow fundus (SGF). METHODS: A cross-sectional study was conducted with 28 VKH patients at convalescent stage and 25 healthy individuals. Both eyes of each participant were enrolled. The VKH patients were classified into two subgroups based on the existence of SGF. OCTA images (3 × 3 mm) were assessed for the data of superficial capillaris plexus (SCP), deep capillaris plexus (DCP), choriocapillaris, and foveal avascular zone (FAZ). RESULTS: Compared with healthy control eyes and eyes without SGF, the vessel densities of the SCP and DCP decreased significantly in most regions of eyes with SGF (p < 0.0167). No significant difference of vascular perfusion was found between eyes without SGF and control eyes (p > 0.05). VKH patients with SGF had slightly increased FAZ area (p = 0.067) and decreased choroid flow area (p = 0.427) than those in the control group. CONCLUSION: Convalescent VKH patients with SGF showed decreased macular capillary perfusion. OCTA could serve as a sensitive tool to assess the microvasculature alterations of VKH disease.

Long noncoding RNA LINC00982 upregulates CTSF expression to inhibit gastric cancer progression via the transcription factor HEY1.

Upregulating the expression of long noncoding RNA LINC00982 controlled cell proliferation in gastric cancer, but the regulatory molecular mechanisms are yet to be expounded. We here aimed to elaborate how LINC00982 regulated the malignancy of gastric cancer cells. RT-qPCR and Western blot analysis were used to detect the expression of LINC00982 and cathepsin F (CTSF) in gastric cancer tissues and cells. Modulatory effect of LINC00982 on gastric cancer cells was assessed by CCK-8, colony formation, Transwell migration, and invasion assays. The relationship between LINC00982, YRPW motif 1 (HEY1), and CTSF was examined by RNA-binding protein immunoprecipitation, luciferase assay, and chromatin immunoprecipitation, and their interaction in the regulation of gastric cancer cellular functions was analyzed by performing gain-of-function and rescue assays. The nude mouse model of tumor formation was developed to examine the effects of LINC00982 on tumorigenesis. LINC00982 was lowly expressed in gastric cancer tissues, whereas its overexpression impaired the proliferative, migratory, and invasive properties of gastric cancer cells. Furthermore, LINC00982 could bind to transcription factor HEY1 and inhibited its expression. Through blocking the binding of HEY1 to CTSF promoter, LINC00982 promoted the expression of CTSF. Overexpression of HEY1 or inhibition of CTSF could reverse the antitumor effects of LINC00982 on gastric cancer, which were further demonstrated in vivo. All these taken together, LINC00982 acted as a tumor suppressor in gastric cancer, which is therefore suggested to be a potential antitumor target for gastric cancer.NEW & NOTEWORTHY We identified LINC00982 as a promising antitumor target for the treatment of patients with gastric cancer. We also determined a regulatory network involved in the pathophysiology of gastric cancer wherein LINC00982 could bind to HEY1 to impair its binding to cathepsin F (CTSF) promoter and hence promote CTSF expression, which aids in better understanding of molecular mechanisms related to gastric tumorigenesis.