Colloidal Gold Immunochromatographic Assay for Rapid On-Site Detection of Tetracycline in Seawater

Recently increasing concerns from the scientists and public have been paid for seawater pollution due to tetracycline(TC)overuse in maricultural area.However,there are few methods or instruments that can be used for specific and rapid detection of this antibiotic in seawater.In this study,the colloidal gold immunochromatographic assay(CG-ICA)was used to achieve this goal.A commercialized monoclonal antibody against TC(anti-TC mAb)was selected because of its higher sensitivity(half-maximal inhibitory concentration of 2.38μgL^(-1)).The prepared CG particles(average diameter of 20 nm)were used to label anti-TC mAb at pH 8.0.The conjugate pad was formed by spraying the CG-labeled anti-TC mAb on a glass fibre membrane followed by proper dryness.The test pad was made by immobilizing artificial antigen and anti-mouse mAb in the test line and the control line,respectively,in a nitrocellulose membrane.The test strip,assembled with sample pad,conjugate pad,test pad and absorbent pad,could be used to detect TC during seawater sample flowing through these components in turn.The results could be observed by the naked eye in 10min.The visible limit of detection(vLOD)was 20μgL^(-1) for TC in seawater.The CG-ICA test results were in good agreement with those of liquid chromatography-tandem mass spectrometry(LC-MS/MS).The assay also showed that,oxytetracycline(OTC)and chlortetracycline(CTC),as the structural analogues of TC,did not interfere with TC determination.Furthermore,the TC concentration given by test strip could not be affected by the fluctuation of temperature(10℃–30℃),pH(7–9)and salinity(0–40)of seawater.Therefore,CG-ICA is a suitable tool for rapid,on-site,and semi-quantitative detection of TC in seawater.

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.

Effects of 105 traditional Chinese medicines on the detection ofβ-agonists in medicine extracts and swine urine based on colloidal gold immunochromatographic assay

Colloidal gold immunochromatographic assay(CGIA)is commonly used for the on-site detection ofβ-agonists that are sometimes used illegally as feed additives in swine diets.However,few studies have evaluated the causes of false-positive results that sometimes occur when applying CGIA in agricultural settings.In this study,we investigated if this false-positive phenomenon is related to the addition of certain traditional Chinese medicines(TCMs)to swine feed.We established and verified an extraction method for TCMs,and then applied CGIA to detectβ-agonists in the extracts of 105 TCMs and in the urine of swine dosed with TCMs,respectively.Liquid chromatography-tandem mass spectrometry was used to validate the results of the urine samples tested positive forβ-agonists using CGIA.The results were also verified using TCMs and colloidal gold test strips produced by different manufacturers.The extracts of Citri Reticulatae Pericarpium Viride,Citri Reticulatae Pericarpium,Magnoliae Officinalis Cortex,Chaenomelis Fructus,and Rhodiolae Crenulatae Radix Et Rhizoma were tested positive forβ-agonists.Meanwhile,the addition of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium to swine feed resulted in false-positive results forβ-agonists in swine urine.The results provide a new way to explain false-positive CGIA results and provide valuable information for livestock feeding programs.

Universal probe with oriented antibody to improve the immunochromatographic assay of lead ions in Procambarus clarkii

Objectives:Based on the information from the random inspection of foods by the China Food and Drug Administration in 2022,the contamin-ation levels of lead ions are high in many edible products.Traditional methods of detecting lead ions cannot meet the requirements of on-site analysis of food due to the need for large equipment.The immunochromatographic assay(iCA)is an effective,rapid,on-site analytical technique for determining lead ions in foods.However,the performance of ICA based on the traditional probe(AuNP-mAb)is limited by ignoring the influ-ence of theantibody orientation.Materials and Methods:In this study,we developed an efficient technology for constructing a universal probe(AuNP-PrA-mAb)based on the oriented immobilization of antibody.The performance of ICA was largely improved due to specific binding of the Fc region of the antibody with recombinant protein A(PrA)on the surface of a gold nanoparticle(AuNP).The ICA based on a universal probe was applied for the qualitative and quantitative detection of lead ions in Procambarus clarki within 30 min.Meanwhile,a simple and fast pretreatment method based on dilute acid extraction was developed forpretreating thePclarkii containing leadions.Results:The visual limit of detection and the scanning limit of quantization of the developed iCA strip for lead ions were O.5 ng/mL and 0.28 ng/mL,respectively.The sensitivity of ICA based on universal probe was 10-fold higher than that of the ICA using traditional probe.Furthermore,the detection results had no obvious difference between the ICA and ICP-MS with t-test statistical method.Conclusions:The developed ICA based on a universal probe presented broad application prospects in detecting contaminants in foods.

Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine

A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentra- tion （IC50） value of 4.65 ng/ml and the IC20value of 0.21 ng/ml in phosphate buffered saline （PBS）. At optimum con- ditions, an indirect competitive enzyme-linked immunosorbent assay （ELISA） and a colloidal gold-based immuno- chromatographic assay （CGIA） were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/rnl were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum de- tection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples

A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry （LC-MS/MS） and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.

Rapid and sensitive determination of difenoconazole in cucumber and pear samples using an immunochromatographic assay

Difenoconazole is triazole fungicide,widely used to prevent the growth and reproduction of fungi and to control fungal diseases in fruits and vegetables.In this work,we prepared a hapten of difenoconazole and produced a highly-sensitive anti difenoconazole-monoclonal antibody(mAb)with an IC50 of 0.64 ng/mL.Using the mAb,an immunochromatographic assay(ICA)was developed to analyze difenoconazole residues in pear and cucumber samples.The ICA exhibited LOD values of 2.60 and 2.30 ng/g for difenoconazole in pear and cucumber samples,respectively.The ICA showed average recoveries of difenoconazole ranging from 94.3%±3.6%–105.4%±1.6%,and CVs of 1.5–4.2%in cucumber and pear samples.When used to measure difenoconazole in samples,the ICA results were compatible with those detected by LC/MSMS.The results of this study support the idea that an ICA is a practical and effective tool for high-throughput and rapid analysis of difenoconazole residues in vegetables and fruits.

An immunochromatographic assay for rapid and simultaneous detection of levonorgestrel and methylprednisolone in water samples

Synthetic contraceptive levonorgestrel （LNG） and glucocorticoid methylprednisolone （MP） residues are eventually discarded to environmental water system and function as environmental hormones, displaying potential risk to humans and ecosystems, thus there is an urgent need for fast, sensitive and simultaneous detection of these compounds in water samples. In this study, a competitive immunochromatographic assay （ICA） using colloidal gold-labeled polyclonal antibodies as probes for rapid and simultaneous detection of LNG and MP in water samples was developed. The visual detection limits of LNG and MP in water samples were 10 ng/mL. The detection process could be completed within 10 min. There was no cross-reactivity of the ICA with other seven compounds. The strips could be stored at 4 ℃ for 10 weeks without significant loss of activity. The assay is a suitable tool for rapid and semi- quantitative detection of LNG and MP in water samples on site.

The Fluorescence Immunochromatographic Strips for Natamycin and Its Application in Food Safety Detection

A fluorescence immunochromatographic strip was developed in this study for natamycin detection in food. The results showed that the best amount of labeled antibody was 10 μg, for every 50 μl of fluorescent microspheres with a 2.5%(w/v) concentration. This labeled antibody was diluted for 10 times, and the diluted solution was dispensed into conjugate pad at the amount of 3 μl/cm. The concentrations of natamycin labeled BSA for test line and goat anti-mouse IgG for control line were 2.0 and 1 mg/ml, respectively, which performed best. With the best conditions, the limit of detection was 1 ng/ml, the linearity ranged from 2 to 100 ng/ml, the recovery was about 80% to 120%, and the CV was below 23%.

A gold-based multiplex immunochromatographic sensor for detecting paclitaxel

Paclitaxel(PTX),methotrexate(MTX),and 5-fluorouracil(5-FU)are commonly-used small molecule anti-tumor drugs for breast cancer.Unfortunately,drug resistance occurs with long-term treatment or excessive use,and the high concentrations of PTX,MTX,and 5-FU in patients may lead to side effects with dose-limiting toxicities,such as myelosuppression,hepatotoxicity,and peripheral neuropathy.Therefore,concentration monitoring of PTX,MTX,and 5-FU in clinical treatment is very important.We prepared highly sensitive and specific monoclonal antibodies using several haptens,and established a multiple paper-based sensor utilizing optical signals of gold nanoparticles,which simultaneously detected PTX,MTX,and 5-FU in human plasma or urine with 10 min.A portable scanner for quantitative detection in plasma and urine samples was used,and the calculated limit of detection(cLOD)values were 0.002 and 0.142μg/mL for PTX,0.187 and 0.976 ng/mL for MTX,and 0.057 and 0.128μg/mL for 5-FU,respectively.In the recovery test,the recovery rates and the coefficient of variation were 85.84%–108.81%and 1.23%–8.80%,respectively,indicating the reliability and accuracy of the multiple gold immunochromatographic strip(GIS).In addition,for the determination of collected clinical plasma samples,the correlation between the test data of the multiple GIS and liquid chromatography–tandem mass spectrometry(LC–MS/MS)was good.Therefore,the developed multiple GIS could be applied to the rapid detection of PTX,MTX,and 5-FU in different clinical samples.

A multi-colloidal gold immunochromatography assay for rapid and simultaneous detection of 13 herbicides

Herbicide residues in agricultural products can have adverse effects on the environment and human health,therefore,there is an urgent need to establish a sensitive,rapid,and wide-ranging detection method.In this study,haptens of phenylurea herbicides(PUs)and sulfonylurea herbicides(SUs)were analyzed and designed based on computational simulation techniques,and two high-performance broad-spectrum monoclonal antibodies against PUs and SUs were prepared.On this basis,a multi-colloidal gold immunochromatography assay(multi-CGIA)was developed to simultaneously detect 13 herbicides in wheat.The visual limit of detection(vLOD)for PUs including diuron,chlortoluron,neburon,chlorbromuron,and linuron was 1-2μg/kg.The vLOD for SUs including metsulfuron methyl,ethametsulfuron-methyl,sulfometuron-methyl,tribenuron methyl,cinosulfuron,triasulfuron,chlorimuron-ethyl,and chlorsulfuron was 2-10μg/kg.The results of real sample determination indicated that the multi-CGIA is accurate,stable,and reliable,and adaptable to on-site preliminary screening of actual samples.

Rapid Detection of Serum Procalcitonin by Immunochromatograghy Technology Based on Freeze-dried Up-conversion Nanoparticles/Antibody Conjugates

Procalcitonin （PCT） is a sensitive and specific biomarker for sepsis diagnosis and widely used as a biomarker to improve the diagnosis of bacterial infections and to guide the antibiotic therapy. In our work, an improved up-converting nanoparticle （UCP） technology based on the immunochromatographic assay （UPT-ICA） was developed for rapid and quantitative detection of PCT. In order to further improve the accuracy, sensitivity and stability of the assay on the basis of our previous study, the UCP coupling with rnonoclonal antibody of PCT （UCP-Abl） was freeze-dried under certain conditions. And the detections of PCT levels with UCP-Abl conjugates before and after freeze-drying were evaluated. The results show that, compared to the UCP-Abl conjugates without freeze-drying, the detection sensitivity of freeze-dried UCP-Abl is slightly improved, having a lower immunochromatogragh background and better stability. This improved method can provide a rapid, accurate, and relatively easy way for the clinical detection of PCT.