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.

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.

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.

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.

Ultrasensitive immunochromatographic strip assay for the detection of diminazene.

We prepared monoclonal antibodies (mAbs) against diminazene and used them in the development of a gold nanoparticle-based lateral-flow test (GNT) strip and indirect competitive enzyme-linked immunosorbent assay for the detection of diminazene in beef and beef liver samples. MAbs, which belong to the IgG2a subclass, had a half maximal inhibitory concentration of 0.04 ng mL-1. Based on cross-reactivity, mAb against diminazene was highly specific. The visual limit of detection (vLOD) of the GNT strip was 0.1 µg kg-1, and the cut-off value was 1 µg kg-1 in beef samples. The vLOD of the GNT assay was 0.1 µg kg-1, and the cut-off value was 2 µg kg-1 in beef liver samples. The average recoveries of diminazene ranged from 97.5% to 103.7% when using ic-ELISA. The accuracy of the developed GNT strip was confirmed by comparing the results with the ic-ELISA results. We developed a reliable GNT strip assay for the rapid detection of diminazene in beef and beef liver samples.

Development of immunochromatographic strips for the detection of dicofol.

In this study, a monoclonal antibody (mAb) against dicofol was developed to prepare immunochromatographic strips (ICAs) for the detection of dicofol residues in fruit and vegetables. The mAb exhibited high affinity and high sensitivity, with an affinity constant of 2.96 × 1010 and a limit of detection of 3.142 ng mL-1. A cross reactivity test revealed that the mAb also had good specificity for dicofol. This ICA method gave a visible limit of detection of 50 ng g-1, and a cut-off value of 500 ng g-1 for the detection of dicofol in both apple and cucumber with the naked eye. Importantly, the results here are consistent with results obtained using liquid chromatography mass spectrometry and ic-ELISAs indicating that this ICA method is reliable and practical when used for the detection of dicofol in fruit and vegetables.

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.

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.

Immunochromatographic strip for rapid and sensitive detection of bupirimate residues in peach, orange, and carrot.

Bupirimate (BPM) is a high-efficiency and low-toxicity fungicide used to combat powdery mildew in crops. To mitigate potential health risks to consumers resulting from improper BPM usage, we prepared a monoclonal antibody against BPM based on novel hapten synthesis, which has high sensitivity and strong specificity, and then successfully designed a colloidal gold-based immunochromatographic (ICG) strip. The newly designed ICG strip was then employed for detecting BPM residues in peach, orange, and carrot. The results show that for the peach, orange, and carrot samples, the calculated detection limits of the ICG strip are 9.36, 0.79, and 0.57 ng/g, respectively, and that it is resistant to the matrix effect and meets the maximum residue limit requirements of European Commission for BPM. Therefore, this developed ICG strip is expected to enable swift detection of BPM residues on the spot.

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.

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 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.

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 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.

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.

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.

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.

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.

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.

Ultrasensitive detection of phenolphthalein in slimming products by gold-based immunochromatographic paper.

An anti-phenolphthalein monoclonal antibody (mAb) was prepared based on the N,N'-Carbonyldiimidazole (CDI) method through phenolphthalein conjugated with proteins. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold-based immunochromatographic assay (ICA) methods were used to determine phenolphthalein in slimming products. A standard curve was established, and the IC50 and limit of detection of ic-ELISA were 0.95 and 0.10 ng/mL with a linear detection range of 0.27-3.37 ng/mL. The developed ICA was used to detect phenolphthalein in tablets, capsules, and slimming tea samples with visual limit of detection values of 10 µg/kg, and cut-off values of 200 µg/kg. The results indicated that these two methods could be used to quickly detect phenolphthalein in slimming products.