Computerized analysis of haptens for the ultrasensitive and specific detection of Pyriftalid.

Pyriftalid (Pyr) is one of the most commonly used herbicides and due to its widespread and improper use, it has led to serious pollution of groundwater, soil and other ecosystems, threatening human health. A rapid method to detect Pyr was urgently needed. A high specific monoclonal antibody (mAb) against Pyr with IC50 values of 4.7 ng/mL was obtained by mAb screening technique and method with enhanced matrix effect. The study firstly proposed colloidal gold immunochromatographic test strips (CGIA) for Pyr, which enables rapid qualitative and quantitative determination of a large number of samples anytime and anywhere, so as to effectively monitor Pyr in environment and grain samples. Based on the properties of the desired Pyr antibody, the hapten Pyr-hapten-4 with high structural similarity to Pyr molecule, similar electrostatic potential distribution, and the ability to expose Pyr functional groups was screened out from five different Pyr haptens, which was consistent with mouse antiserum test. The CGIA quickly analyze the Pyr content in positive samples such as water samples, soil samples, paddy samples, brown rice samples within 10 min, the LOD for Pyr by CGIA as low as 1.84 ng/g, the v LOD value as low as 6 ng/g, and the extinction value as low as 25 ng/g. The content of positive samples detected by CGIA was consistent with the quantitative results of LC-MS/MS, the relative accuracy was within the range of 97-103 %. The recovery rate range for Pyr by CGIA was 92.0-99.7 %, and the coefficient of variation was between 1.30-8.56 %. It indicated Pyr-targeted CGIA test strip was an efficient and fast detection method to detect real environment and food samples.

Establishment and application of a gold nanoparticle-based immunochromatographic test strip for the detection of avian leukosis virus P27 antigen in egg white samples.

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.

An immunochromatographic strip sensor for rapid and sensitive detection of candesartan, olmesartan medoxomil, and irbesartan in herbal beverages.

Sartans, as a class of antihypertensive drugs, pose a threat to human health when illegally added to herbal beverages. It is crucial to detect sartans in herbal beverages. We have developed a highly sensitive monoclonal antibody against candesartan (CAN), olmesartan medoxomil (OLM), and irbesartan (IRB), with 50% inhibitory concentrations (IC50) that were obtained via indirect enzyme-linked immunosorbent assay (ic-ELISA) as 0.178 ng mL-1, 0.185 ng mL-1, and 0.262 ng mL-1 against CAN, OLM, and IRB, respectively. Based on this monoclonal antibody, we developed a rapid screening method for CAN, OLM, and IRB in herbal beverage samples using an immunochromatographic assay (ICA) strip. Test for 15 minutes after simple and rapid sample pre-treatment and the results of this method can be obtained through naked eye observation. The detection limits (LODs) of the ICA strip for CAN, OLM, and IRB in herbal beverage samples are lower than 0.15 ng mL-1, and the results of the ICA strip and ic-ELISA are consistent in spiked samples and recovery experiments. Therefore, this method can quickly, efficiently, and reliably achieve high-throughput on-site rapid detection of illegally added CAN, OLM, and IRB in herbal beverages.

Nonalcoholic Fatty Liver Disease Development in Male Mice upon Exposure to Flubendiamide.

Flubendiamide (FLU), a widely used diamide insecticide, has been observed to potentiate adipogenesis in 3T3-L1 preadipocytes in vitro. Whether exposure to FLU disrupts hepatic lipid homeostasis in mammals and induces visceral obesity, however, remains unclear. The aim of this study was to assess the effects of FLU when administered orally to male C57BL/6J mice under normal diet (ND) and high-fat diet (HFD) conditions. FLU accumulated at higher levels in the tissues of the HFD group than those of the ND group, indicating that an HFD contributed to the accumulation of lipophilic pesticides in vivo. Notably, FLU (logP = 4.14) is highly lipophilic and easily accumulates in fat. Exposure to FLU had opposing effects on the lipid metabolism of the liver in the ND and HFD groups. Liver triacylglycerol levels in the ND group were reduced, while those in the HFD group were increased, resulting in more severe hepatic steatosis. More lipid accumulation was also observed in HepG2 cells exposed to FLU. Changes in hepatic lipid deposition in vivo occurred as the enhanced transcriptional regulation of the genes involved in lipid uptake, de novo lipogenesis, and fatty acid ß-oxidation (FAO). Moreover, an excessive increase in FAO caused oxidative stress, which in turn exacerbated the inflammation of the liver. This study revealed the disruptive effect of FLU exposure on hepatic lipid homeostasis, which may facilitate the triggering of nonalcoholic fatty liver disease in HFD-fed mice.

Development of an ic-ELISA and immunochromatographic assay strip for the rapid detection of chloridazon in oranges and celery.

Chloridazon (CLZ) is a selective herbicide used in the control of annual broadleaf weeds. The misuse or abuse of CLZ may result in the accumulation of CLZ in crops and water, which can pose a risk to human health. In this study, a hapten of CLZ with three carbon spacer arms was designed and a highly sensitive and specific antibody against CLZ was prepared with a half-maximal inhibitory concentration of 0.630 ng mL-1 and a linear range of 0.181-2.195 ng mL-1.Based on this antibody, we developed an immunochromatographic assay (ICA) strip for the detection of CLZ in oranges and celery. Under optimized conditions, the visual limit of detection was 2 ng mL-1 and 10 ng mL-1 in oranges and celery, respectively, and the cut-off value was 50 ng mL-1. In CLZ-spiked samples and the recovery test, the results of the ICA strip were consistent with those of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Therefore, the ICA strip developed in our study represents an efficient and reliable method for the rapid screening of CLZ in oranges and celery.

Immunological strip sensor for the rapid determination of niacin in dietary supplements and foods.

Herein, four haptens of niacin (Vitamin B3, VB3) were designed, and after a series of experiments, it was concluded that hapten D had the best immune effect. To avoid false positives in the detection of real samples, a monoclonal antibody (mAb) against VB3 was prepared by a matrix effect-enhanced mAb screening method. The concentration of the inhibition rate reaching 50% (IC50) was 603.41 ng mL-1 and the limit of detection (LOD) using an indirect enzyme-linked immunosorbent assay (ic-ELISA) was 54.89 ng mL-1. A lateral flow immunochromatographic assay (LFIA) based on gold nanoparticles was established to detect the concentration of VB3 in compound vitamin B tablets and infant formulas, with a visual LOD of 5 µg mL-1. Using a handheld reader, the quantitative LOD was calculated to be 0.60 µg mL-1. The contents of the compound vitamin B tablets and infant formulas were also verified by liquid chromatography. Therefore, the LFIA developed in this study can be applied to the specific identification and rapid detection of niacin in nutritional dietary supplements, thus meeting the market's demand for efficient niacin detection methods.

Gold nanoparticle-based immunochromatographic assay for rapid detection of imazalil.

Imazalil (IMZ) is a commonly used fungicide for controlling fungus in agriculture, leaving residual IMZ in crops that could be hazardous to human health. In this work, we designed IMZ haptens for mice immunization and prepared sensitive monoclonal antibody (mAb) against IMZ. The subtype of anti-IMZ mAb is IgG2a. It possessed a half inhibition concentration (IC50) of 0.95 ng mL-1 and showed no cross-reactivity against other chemicals in ic-ELISA. Taking advantage of the mAb, we developed a gold nanoparticle-based immunochromatographic assay (GICA) for the rapid detection of IMZ in grapes and tomatoes. The assay gave a visual limit of detection (vLOD) of 25 ng g-1 and cut-off value of 500 ng g-1 in both samples. According to the calibration curves, the calculated LOD were 4.12 ng g-1 and 4.70 ng g-1 in grapes and tomatoes, respectively. The recovery rates of IMZ ranged from 84.7% to 104.4% with variation coefficients (CVs) of 5.7-11.8% in spiked samples, indicating a potent practicability of the GICA. The whole GICA process took 30 min. Therefore, the developed assay can be used for on-site detection and quantitation of IMZ in grape and tomato samples.

Monoclonal antibody production and development of immunochromatographic strip assays for screening of the herbicide bispyribac-sodium in rice.

Bispyribac-sodium (BIS) is a new broad-spectrum and efficient herbicide, which is widely used for the control of weeds in rice. To protect the human body from the threat of BIS exposure, it is essential to establish a sensitive and simple detection method. In this work, a high-affinity monoclonal antibody against BIS was produced for the first time, and a colloidal gold immunochromatographic strip assay (ICSA) was developed to screen for BIS in rice samples. The visual limit of detection and the calculated limit of detection of the ICSA were 0.2 µg kg-1 and 0.018 µg kg-1, respectively, which could be accurately obtained within 8 min. The average recoveries of BIS ranged from 90.0% to 109.0% in tests, with CVs ranging from 4.0% to 8.9% for rice samples. Therefore, our ICSA would be a good option for the sensitive and rapid detection of BIS in rice samples.

Immunochromatographic test strip for quantitative and rapid detection of tolfenpyrad in food samples.

In the study, a hapten was designed to preserve the molecular structure of tolfenpyrad while introducing a carboxyl group and was coupled with a carrier protein to synthesize an immunogen and coating antigen. A monoclonal antibody was fabricated against tolfenpyrad and its performance was assessed by indirect competitive enzyme-linked immunosorbent assay. Finally, we developed a colloidal gold nanoparticle immunochromatographic test strip (CGN-ICTS) for the detection of tolfenpyrad in kale, Chinese cabbage, and eggplant samples. The results shows that CGN-ICTS was sensitive, with calculated detection limits of 0.49 ng/g for kale and Chinese cabbage and 0.99 ng/g for eggplant. Subsequently, CGN-ICTS and LC-MS were used to analyze the tolfenpyrad-spiked samples. The recovery rate of the CGN-ICTS for kale samples was 97.1-103.0%, for Chinese cabbage samples was 93.7-103.4%, and for eggplant samples was 92.7-105.7%. Recovery rates were similar between CGN-ICTS and LC-MS. Therefore, CGN-ICTS can be used to quickly screen tolfenpyrad residues in foods.

Lateral flow immunoassay based on gold nanoparticles for rapid and sensitive detection of zoxamide in grape, tomato and cucumber samples.

In the study, we discovered zoxamide hapten (ZOX-hapten) by introducing a carboxyl extension chain, combined it with protein to make a complete antigen to immunize mice, and generated a monoclonal antibody (mAb) against ZOX. To identify ZOX residues in grape, tomato, and cucumber samples, we used our anti-ZOX mAb to develop a lateral flow immunoassay (LFIA) strip. In grape, tomato, and cucumber samples, the calculated detection limit of the LFIA strip in grape, tomato and cucumber samples was 3.44, 4.78 and 3.53 ng/g, respectively. Using the LFIA strip, the recovery rate from grape samples was 96.4-106.8%, and that from tomato samples was 98.4-107.5%, while the recovery from cucumber samples was 99.4-111.3%. These results showed that our LFIA strip could be expected to achieve rapid screening of ZOX residues in fruits and vegetables.

A Rapid Immunochromatographic Method Based on Gold Nanoparticles for the Determination of Imidacloprid on Fruits and Vegetables.

Imidacloprid (IMP) is toxic and a potential carcinogen that is most widely used as an insecticide for pest control and seed treatment. It is important to produce a rapid and sensitive assay for on-site monitoring. We have developed a novel lateral flow assay (LFA) using a sensitive monoclonal antibody (mAb) for monitoring IMP residues on fruits and vegetables. The 50% inhibition concentration result that was found when using the ELISA method was 0.247 ng mL-1, with the cut-off limits using the LFA method the result was 10 ng mL-1 (0.01 M PBS), and in the samples it was 20 ng mL-1 (with a recovery rate of 96-104.7% for Chinese cabbage, cowpea, apple, and pear samples, respectively). All of the results can be determined within seven minutes. The proposed LFA method is a valid, quick, and stable assay for the on-site detection of IMP in large numbers of samples.

Immunochromatographic assay for the analysis of methomyl in cabbage and tomato.

In this study, a hapten of methomyl was designed and used to produce monoclonal antibodies (mAbs) against methomyl. Based on these mAbs, we developed an enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) strip for the determination of methomyl residues. Results from the ELISA showed that mAb 1D10 exhibited higher affinity with an affinity constant of 2.76 × 1010 L/mol and higher sensitivity with a limit of detection (LOD) was 8.12 ng/mL. After optimizing the ICA, a visible limit of detection (vLOD) was found to be 100 ng/g and the cut-off value was 500 ng/g for methomyl in cabbage and tomato. The calculated LODs were 3.2 ng/g and 5.4 ng/g in cabbage and tomato, respectively. Moreover, results from the ICA were consistent with those of the ELISA in our recovery assay using spiked samples. Hence, the ICA method has a bright future and great prospects for the detection of methomyl in food samples.

Integrated analysis identifies the IL6/JAK/STAT signaling pathway and the estrogen response pathway associated with the pathogenesis of intracranial aneurysms.

Objective: We intended to identify the potential key biomarker and pathways that correlated with infiltrating immune cells during the pathogenesis of intracranial aneurysms (IA), to develop a diagnostic model, and to predict therapeutic drugs. Methods: Three datasets containing intracranial aneurysm tissue samples and normal artery control samples from Gene Expression Omnibus (GEO) were included. Gene-set variation analysis(GSVA) and gene set enrichment analysis (GSEA) were conducted to find the significant differentially expressed pathways in IA formation. The least absolute shrinkage and selection operator (LASSO) regression and the multivariate logistic regression analysis were performed to identify the characteristic genes in the IL6/JAK/STAT signaling pathway (ISP) and the estrogen response pathway (ERP). A diagnostic model was constructed. xCell was used to identify immune cell types in IA pathogenesis. We used the weighted gene co-expression network analysis (WGCNA) algorithm to explore the correlations between the key modules and the four traits. Potential therapeutic drugs were investigated in Enrichr and Drugbank database. Results: The ISP is significant positively correlated with IA onset. The biological function of the ISP is positively correlated with that of the ERP, and is significantly associated with immune cells activities. CSF2RB, FAS, IL6, PTPN1, STAT2, TGFB1 of the ISP gene set and ALDH3A2, COX6C, IGSF1, KRT18, MICB, NPY1R of the ERP gene set were proved to be the characteristic genes. The STAT2 gene can be the potential biomarker of IA onset. The immune score of IA samples was significantly higher than the controls. The STAT2 gene expression is associated with infiltration of immune cells. The WGCNA results were consistent with our finds. Acetaminophen can be a potential therapeutic drug for IA targeting STAT2. Conclusions: We identified that the ISP was one of the most significant positively correlated pathways in IA onset, and it was activated in this process concordant with the ERP and immune responses. Except for beneficial effects, complex and multiple roles of estrogen may be involved in IA formation. STAT2 could be a potential biomarker and a promising therapeutic target of IA pathogenesis.

Rapid and sensitive detection of flubendiamide in grapes and tomatoes using a colloidal gold immunochromatography assay.

In this study, we developed a monoclonal antibody (mAb)-based colloidal gold immunochromatography assay (CG-ICA) strip for the rapid and sensitive detection of flubendiamide (FBD) in food samples. Our anti-FBD mAb 2B1 was highly specific and had a half-maximal inhibitory concentration (IC50) of 1.55 ng/mL under optimal conditions. It belongs to the IgG2a isotype and has a Kaff value of 1.52 × 109 mol/L. The strip provided a visual detection limit of 50 ng/g for both grapes and tomatoes, with a cut-off value of 1,000 ng/g and these qualitative results were observed within 15 min. For quantitative analysis, the calculated detection limits were 6 ng/g and 5 ng/g in grapes and tomatoes, respectively. The average recoveries of FBD ranged from 88.8% to 111.8% in grapes and 94.5% to 110.6% in tomatoes. The proposed strip assay is highly practical for screening FDB in food samples.

Bioinformatics analysis reveals potential biomarkers associated with the occurrence of intracranial aneurysms.

To better understand the molecular mechanisms of intracranial aneurysm (IA) pathogenesis, we used gene coexpression networks to identify hub genes and functional pathways associated with IA onset. Two Gene Expression Omnibus (GEO) datasets encompassing intracranial aneurysm tissue samples and cerebral artery control samples were included. To discover functional pathways and potential biomarkers, weighted gene coexpression network analysis was employed. Next, single-gene gene set enrichment analysis was employed to investigate the putative biological roles of the chosen genes. We also used receiver operating characteristic analysis to confirm the diagnostic results. Finally, we used a rat model to confirm the hub genes in the module of interest. The module of interest, which was designated the green module and included 115 hub genes, was the key module that was most strongly and negatively associated with IA formation. According to gene set variation analysis results, 15 immune-related pathways were significantly activated in the IA group, whereas 7 metabolic pathways were suppressed. In two GEO datasets, SLC2A12 could distinguish IAs from control samples. Twenty-nine hub genes in the green module might be biomarkers for the occurrence of cerebral aneurysms. SLC2A12 expression was significantly downregulated in both human and rat IA tissue. In the present study, we identified 115 hub genes related to the pathogenesis of IA onset and deduced their potential roles in various molecular pathways; this new information may contribute to the diagnosis and treatment of IAs. By external validation, the SLC2A12 gene may play an important role. The molecular function of SLC2A12 in the process of IA occurrence can be further studied in a rat model.

Gold immunochromatographic strip assay for the detection of triamcinolone acetonide and budesonide in milk.

A monoclonal antibody against triamcinolone acetonide (TCA) and budesonide (BUD) was prepared using a hapten that was generated by introducing a carboxyl group into the structure of TCA. Based on the prepared monoclonal antibody, a gold nanoparticle-based lateral-flow immunoassay (GLFA) was developed with the ability to screen TCA and BUD in milk. The visible limits of detection of the GLFA for the analysis of TCA and BUD were 0.1 and 0.5 ng/mL with a cutoff value of 5 and 10 ng/mL, respectively, in milk. Average recoveries of TCA and BUD in milk were 92.0-102.2% and 96.0-98.8% with a good correlation between the results from the GLFA and LC-MS/MS analysis. These results demonstrated that the GLFA method for the rapid detection of TCA and BUD in milk samples is reliable and sensitive.

Ultrasensitive detection of four organic arsenic compounds at the same time using a five-link cardboard-based assay.

In order to effectively control the excessive use of organic arsenic reagents in livestock and poultry products, there is an urgent need to develop a method for rapid detection of multiple organic arsenic reagents. In this study, two haptens were designed and derivatized around the structural formula of roxarsone, and a highly-sensitive group-selective mAb 3F2 was prepared, which can simultaneously detect roxarsone, 4-aminophenylarsonic acid, 2-aminophenylarsonic acid and phenylarsonic acid. We further developed a colloidal gold immunochromatographic test strip (ICS) and prepared a five-link card that can simultaneously detect four organic arsenics in chicken and pork samples. Its quantitative detection limits (LOQ) for the four compounds in chicken and pork samples were 0.06 and 0.32 ng/mL, 0.11 and 0.29 ng/mL, 0.34 and 0.99 ng/mL, and 0.88 and 1.5 ng/mL, respectively. This multi-ICS detection provides a powerful tool for the on-site detection and rapid screening of organic arsenic reagents in actual samples.

A monoclonal antibody-based colloidal gold immunochromatographic strip for the analysis of novobiocin in beef and chicken.

In this study, a monoclonal antibody (mAb) 1G5 against novobiocin with high sensitivity and specificity was prepared from a newly-designed hapten. According to the results of an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), the 50%-inhibitory concentration of the anti-novobiocin mAb was 6.9 ng/mL and the cross-reactivity was less than 0.1% to its analogues. Furthermore, a rapid colloidal gold immunochromatographic assay (ICA) was successfully developed for the determination of novobiocin in spiked samples. Two calibration curves were established respectively, for beef and chicken samples. The ICA results showed a visual colorimetric value of 50 ng/mL and a cut-off value of 300 ng/mL in beef samples. The ICA results of chicken samples were almost the same as that of beef. When quantitative detection was performed using a strip reader, the detection ranges for quantitative analysis in beef and chicken were 23.7-287.5 and 19.7-263.8 µg/kg respectively. Recoveries were between 82.7 and 95.3% for beef samples with the coefficient of variation (CV) ranging from 2.5 to 5.1%. Recoveries were in the range of 89.6-105.5% with the CV ranging from 2.9% to 6.3% for chicken samples. Importantly, these results from the ICA were highly consistent with the results obtained by LC-MS/MS. Therefore, this ICA could be used as an alternative means for the rapid determination of NOV in a large number of beef and chicken samples.

Gold-based strip sensor for the rapid and sensitive detection of butralin in tomatoes and peppers.

Butralin is a widely used dinitroaniline herbicide. Butralin residues in vegetables or fruits represent a threat to human health. In this study, we developed a rapid and sensitive gold-based lateral flow immunoassay (LFIA) for butralin detection in tomato and green pepper samples based on a screened monoclonal antibody (mAb) against butralin. The mAb possessed a half-maximal inhibitory concentration (IC50) of 12.7 ng/mL, with no cross-reactivity toward other dinitroaniline herbicides. The established LFIA strip had a visible limit of detection (LOD) of 50 ng/g and a cut-off value of 2000 ng/g in tomato and green pepper samples. According to the calibration curves for quantitative analysis, the calculated LODs of the LFIA strip were 4.7 ng/g and 4.3 ng/g in tomato and green pepper, respectively. The results were obtained within 10 min. The average recoveries ranged between 95.4% and 109.6% with a coefficient of variation (CV) of 4.3% to 7.1% in tomato samples and between 94.8% and 109.1% with a CV of 3.9% to 6.1% in green pepper samples. These data suggested that our proposed LFIA is a sensitive, specific, and reliable method for the rapid detection of butralin in real samples.

Secretory expression and purification of recombinant PLA2R epitopes for the detection of anti-PLA2R autoantibody in serum.

Most patients with idiopathic membranous nephropathy (IMN) have autoimmune antibodies specifically against the M-type phospholipase A2 receptor (PLA2R). PLA2R acts as a significant biomarker contributing to the clinical diagnosis of IMN. Herein, we performed the secretory expression of the exocellular domain and immune-dominant regions of PLA2R by using mammalian cells. Using ELISA, we confirmed that the purified His-tagged PLA2R variant of CysRC1C2C7, which contained CysRC1C2 (aa 21-510) and CTLD7 (aa 1097-1246), possesses the strongest binding affinity toward serum anti-PLA2R in IMN patients. The signal peptide of interleukin-2 for secretion was selected, and parameters for transient expression were optimized to achieve the highest titer of CysRC1C2C7. Stepwise purification of CysRC1C2C7 using anion-exchange chromatography and gel filtration apparently increased its capability of anti-PLA2R recognition or interaction in ELISA. Under optimal conditions of expression and purification, the yield of CysRC1C2C7 in monomer form was ∼14.1 mg L-1, with a recovery rate of ∼77%. This recombinant PLA2R variant had decent potential for serological analysis of anti-PLA2R in IMN patients.