Immunochromatographic Strip Assay Research Articles

Hapten synthesis and a colloidal gold immunochromatographic strip assay to detect nitrofen and bifenox in fruits.

In this study, we synthesized two haptens similar in structure to nitrofen (NIT), and screened out five monoclonal antibodies with the ability to recognize NIT and bifenox (BIF) by competitive ELISA, with the lowest IC50 values of 0.87 ng mL-1 and 0.86 ng mL-1, respectively. The antibody 5G7 was selected to be combined with colloidal gold to establish a lateral flow immunochromatographic assay strip. This method was shown to qualitatively and quantitatively detect the residues of NIT and BIF in fruit samples. The visual limits of detection for qualitative detection were 5 μg kg-1 and 10 μg kg-1 for NIT and BIF, respectively. The calculated limits of detection for quantitative detection were 0.75 μg kg-1, 1.77 μg kg-1 and 2.55 μg kg-1 respectively, for nitrofen in orange, apple and grapes, and 3.54 μg kg-1, 4.96 μg kg-1 and 5.26 μg kg-1, respectively, for bifenox. Thus the strip assay could be used for rapid analysis of fruit samples.

Highly resistant and sensitive colorimetric immunochromatographic assay for sibutramine (SBT) illegally adulterated into diet food based on PDA/AuNP labelling.

A gold nanoparticle (AuNP) based immunochromatographic assay strip is a valuable tool for monitoring chemicals in foods. However, the sensitive ICA strip for SBT is rarely reported due to the fact that monoclonal antibodies (mAbs) against SBT with high affinity are commercially unavailable. Herein, a monoclonal antibody against SBT was prepared through a designed hapten with a carboxyl end-capped space arm. The obtained mAb showed high affinity for SBT and N-desmethylsibutramine, a metabolite of SBT. Furthermore, a series of core-shell NPs, polydopamine (PDA) coated AuNPs (PDA/AuNPs) with controlled shell thickness and packing density were synthesized. The obtained PDA/AuNP-mAb conjugate demonstrated high tolerance to salt and good stability in a wide pH range, which is beneficial for improving the matrix interference common in ICA. As a result, PDA/AuNP-based ICA could quantify SBT in the range of 3.39-69.60 ng mL-1 with a limit of detection (LOD) of 0.98 ng mL-1. This novel ICA improved the sensitivity of the traditional AuNP-based ICA by nearly 12 times. Method validation was conducted with spiked samples of slimming food and human serum and compared with HPLC-MS/MS. Consistent results indicated that high sensitivity, accuracy, and reliability of the PDA/AuNP-based ICA approach were achieved. To the best of our knowledge, this study reported the most sensitive immunoassay for SBT thus far.

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

AbstractObjectivesBased on the information from the random inspection of foods by the China Food and Drug Administration in 2022, the contamination 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 influence of the antibody orientation.Materials and MethodsIn 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 clarkii within 30 min. Meanwhile, a simple and fast pretreatment method based on dilute acid extraction was developed for pretreating the P. clarkii containing lead ions.ResultsThe visual limit of detection and the scanning limit of quantization of the developed ICA strip for lead ions were 0.5 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.ConclusionsThe developed ICA based on a universal probe presented broad application prospects in detecting contaminants in foods.

Ratiometric fluorescent immunochromatography for simultaneously detection of two nitrofuran metabolites in seafoods

Competitive immunochromatographic assay (ICA) has been widely used in food safety on-site monitoring. However, traditional competitive ICA relies on the change of single-signal intensity to discriminate the results, leading to limited sensitivity and accuracy of naked-eye visual inspection. In this study, we developed a sensitive ratiometric fluorescent immunochromatographic assay (RFICA) judging results based on the color changes of fluorescence. Two different color quantum dot nanobeads (QBs) were introduced as signal labels for rapid and accurate detection of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) in seafoods. Under the optimal conditions, the visual detection limit for 2-NPAOZ and 2-NPSEM reached up to 1 and 0.5 ng/mL, which were 5- and 14-fold lower than that of traditional single-signal response fluorescent ICA strips. In addition, the RFICA strips were applied to real samples with acceptable recoveries of 81.33 ∼ 99.23 %. These results demonstrate that the developed RFICA platform possesses considerable potential in the on-site detection.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China

BackgroundThe existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host’s immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed.ResultsThe truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%.ConclusionsThis study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

An Immunochromatographic Assay for the Rapid and Qualitative Detection of Mercury in Rice.

Mercury is a major pollutant in food crops. In this study, we synthesized an anti-mercury monoclonal antibody (mAb; IC50 was 0.606 ng mL−1) with high sensitivity and specificity and different immunogens and coating antigens and developed an immuno-chromatographic assay (ICA) for the detection of mercury in rice. The ICA strip had a visible detection limit of 20 ng g−1 and a cut-off value of 500 ng g−1 in rice. The performance of the ICA strip was consistent with that of ICP-MS and ic-ELISA. The recoveries of mercury in rice ranged from 94.5% to 113.7% with ic-ELISA and from 93.6% to 116.45% with ICP-MS. Qualitative analysis by ICA can be obtained with the naked eye. The ICA strip is an effective and practical method for the rapid and high-throughput determination of mercury in rice.

Development of an immunochromatographic assay for the rapid screening of pendimethalin in potato and apple

Pendimethalin (PDM), a dinitroaniline group herbicide, is extensively used for controlling weeds in amounts of agricultural crops. In this work, Hapten-1 and Hapten-2 were synthesized for the preparation of an anti-PDM monoclonal antibody (mAb), which was used to establish an indirect competitive ELISA. Based on the anti-PDM mAb, we developed an immunochromatographic assay (ICA) for detection of PDM in potato and apple. Under optimal conditions, the ICA exhibited a cut-off value of 200 ng/g and the visible limit of detection was 10 ng/g for apple and potato samples in qualitative detection. Analysis of quantitative detection suggested that the limit of detection were 5.4 and 3.2 ng/g in apple and potato samples, respectively. Moreover, the results obtained from the developed ICA strip were shown to coincide with those of LC–MS/MS in the analysis of apple and potato samples. In addition, the ICA exhibited excellent performance with regard to sensitivity, speed, and portability in contrast to previous determination methods for PDM. Hence, the proposed ICA strip showed superior prospects in PDM residue determination.

Improving the sensitivity of lateral flow immunoassay for Salmonella typhimurium detection via flow-rate regulation

Application of the traditional immunochromatographic assay (ICGA) has been limited by its poor sensitivity. The objective of this study was to increase the sensitivity of the traditional ICGA. A dual-mode ICGA (D-M ICGA) was developed by combining a nanozyme-assisted signal-amplification strategy with a magnetic-nanoparticle-based flow-speed-control strategy. Salmonella typhimurium can be detected simultaneously based on color and magnetic signals in the detection area of the D-M ICGA strip. The calculated limits of detection of 50 cfu·mL−1 and 75 cfu·mL−1 in the color and magnetic modes, respectively, were approximately 1000 times lower than those of the traditional ICGA. The selectivity and practical applicability of the D-M ICGA were also confirmed in this study. The results prove that the D-M ICGA is an assay that could be used for Salmonella typhimurium detection and can be easily adapted to detect other pathogenic bacteria.

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.

Carboxylated fluorescent microsphere based immunochromatographic test strip enabled sensitive and quantitative on-site detection for florfenicol in eggs

Florfenicol (FF), used popularly in prevention and treatment of virus infections in livestock and poultry, has widely been found in eggs and harmful to human health. In this work, a sensitive and quantitative on-site detecting solution, monoclonal antibody-based carboxylated fluorescent microsphere immunochromatographic test strip assay (FM-ICTS), is design and applied for FF detection. The proposed method can sensitively detect FF in low detection limit of 0.030 ng/g and quantitatively measure its concentration from 0.1 ng/mL to 8.1 ng/mL (R2 = 0.9991) with high repeatability (CV<8.0 %). In addition, the established FM-ICTS method exhibited high measurement accuracy in FF samples as compared with HPLC-MS analysis and demonstrated satisfied recoveries (99.1–101.3 %). More importantly, the quantitative FF test strip demonstrate ultra-high stability, which presents approximately equivalent detection ability to the fresh one after stored at 4 °C for more than one year or stored at 37 °C for 60 days. Therefore, the proposed method is a promising solution for rapidly and sensitively quantitative determination of FF in eggs.

Ultrasensitive multichannel immunochromatographic assay for rapid detection of foodborne bacteria based on two-dimensional film-like SERS labels

Rapid and sensitive detection of multiple foodborne bacteria without DNA amplification is still challenging. Here, we proposed an immunochromatographic assay (ICA) with multiplex analysis ability and high sensitivity for direct detection of bacteria in real food samples, based on an improved surface-enhanced Raman scattering (SERS) sensing strategy. Multifunctional Au shell-coated graphene oxide nanosheets (GO@Au) were fabricated and for the first time introduced into the ICA system as a two-dimensional film-like SERS label, which possessed huge surface area, excellent stability, and superior SERS activity. Different from the conventional spherical nanotags, the antibody-conjugated GO@Au nanosheet effectively and rapidly adhered to bacterial cells, improved the dispersibility of bacteria-nanolabel complexes on the ICA strips, and provided numerous stable hotspots for SERS signal enhancement. The combination of GO@Au labels and the ICA system achieved the multiplex and ultrasensitive determination of three major foodborne pathogens, namely, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium, in a single test, with low detection limits (8, 10, and 10 cells/mL) and short detection time (20 min). The proposed biosensor demonstrated high stability and good accuracy in various food samples and is thus a promising tool for the rapid identification of bacteria.

Dengue fever as a reemerging disease in upper Egypt: Diagnosis, vector surveillance and genetic diversity using RT-LAMP assay.

BackgroundThe recent increase in dengue virus (DENV) outbreaks and the absence of an effective vaccine have highlighted the importance of developing rapid and effective diagnostic surveillance tests and mosquito-based screening programs. To establish effective control measures for preventing future DENV transmission, the present study was established to identify the main mosquito vector involved in the dengue fever (DF) outbreak in Upper Egypt in 2016 and detect the diversity of dengue virus serotypes circulating in both humans and vectors.MethodsWe investigated the prevalence of DENV infection and circulating serotypes in the sera of 51 humans clinically suspected of DF and 1800 field-collected Aedes aegypti adult female mosquitoes grouped into 36 pooled samples. Both DENV non-structural protein (NS1) immunochromatographic strip assay and loop-mediated isothermal amplification (LAMP) were used for screening.ResultsOverall, the rate of DENV infection in both human sera and pooled mosquito homogenate was 33.3%, as revealed by rapid dipstick immunochromatographic analysis. However, higher detection rates were observed with RT-LAMP assay of 60.8% and 44.4% for humans and vector mosquitoes, respectively. DENV-1 was the most prevalent serotype in both populations. A combination of two, three, or even four circulating serotypes was found in 87.5% of total positive pooled mosquito samples and 83.87% of DENV-positive human sera.ConclusionThe study reinforces the evidence of the reemergence of Aedes aegypti in Upper Egypt, inducing an outbreak of DENV. Mosquito-based surveillance of DENV infection is important to elucidate the viral activity rate and define serotype diversity to understand the virus dynamics in the reinfested area. Up to our knowledge, this is the first report of serotyping of DENV infection in an outbreak in Egypt using RT-LAMP assay.

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for detection of 17β-estradiol in sheep sera

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for detection of 17β-estradiol in sheep sera

Development of an Immunochromatographic Strip for Rapid Detection of Mink Enteritis Virus.

Although mink enteritis virus (MEV) is an acute, virulent, and highly contagious pathogen in minks, there is currently a lack of a quick diagnostic method. By conjugating colloidal gold nanoparticles with the MEV-specific monoclonal antibody, monoclonal antibody (MAb) 14, we developed a single-step competitive immunochromatographic strip (ICS) assay for simple determination of MEV. The optimal concentrations of the colloidal gold-coupled MAb 14 (coating antibody), the capture protein (MEV VP2 protein), and the goat anti-mouse antibody were 1.0, 0.8, and 1.0 mg/ml, respectively. The limit of detection was approximately 512 hemagglutination units/100 μl of MEV B strain. Other common viruses of mink were tested to evaluate the specificity of the ICS, and the results showed no cross-reactivity for other pathogens. In comparison with the Anigen Rapid canine parvovirus (CPV) Ag Test Kit (BioNote, Korea) in testing 289 samples, the percentage of agreement and relative sensitivity and specificity of the MEV ICS assay were 94.1, 93.2, and 97.1%, respectively. The ICS test was found to be a sufficiently sensitive and specific detection method for the convenient and rapid detection of MEV.

Synthesis of quantum dot encoded multicolour nanobeads for the ultrasensitive and multiplex immunochromatographic detection of neonicotinoid insecticides

In this study, a novel fluorescent immunochromatographic assay (ICA) strip based on quantum dot encoded multicolour nanobead (QB) was developed to detect three neonicotinoid insecticides in the vegetable samples simultaneously. The results showed that three QBs with red-, yellow- and green-emitting were successfully synthesized by embedding different ratios of two kinds of hydrophobic CdSe/ZnS QDs (λem = 565 nm and λem = 620 nm) into the polymer matrix. The obtained three QBs were further used to label the monoclonal antibodies (mAbs) specific to imidacloprid (IMI), clothianidin (CLT) and thiamethoxam (TMX). The resultant QB@mAbs nanoprobes, including QBred@IMI mAbs, QByellow@CLT mAbs and QBgreen@TMX mAbs, presented excellent monodispersity with distinguishable emission colours. Furthermore, the fabricated QB-ICA strip based on QB@mAbs nanoprobes achieved a more sensitive simultaneous detection for three neonicotinoids with the detection limits of 14.58 ng/mL for IMI, 0.27 ng/mL for CLT, and 0.36 ng/mL for TMX, respectively. This was better than most of the conventional based enzyme-linked immunosorbent assay (ELISA). Moreover, the QB-based ICA strips also exhibited good stability and specificity for the multiplex detection of IMI, CLT, and TMX. In the spiked Chinese cabbage samples, the recovery of IMI, CLT, and TMX ranged from 72.08% to 98.70%, with RSD values ranging from 3.60% to 13.09%. In brief, the great performance of the developed QB-ICA approach demonstrates its potential for simultaneous and quantitative neonicotinoids detection in agricultural products.

Difunctional immunochromatographic assay based on magnetic quantum dot for ultrasensitive and simultaneous detection of multiple mycotoxins in foods

There is a strong need for the development of a sensitive biosensor for real-time monitoring mycotoxins contamination in real food samples. Here, we reported a multiplexed immunochromatographic assay (ICA) using functionalized magnetic-quantum dot (QD) as capture/detection difunctional immunoprobes that can simultaneously and sensitively detect aflatoxin B1 (AFB1), ochratoxin A (OTA), and fumonisin B1 (FB1) in food. The antibodies-modified multilayer magnetic-QD nanobeads (MagQBD) allowed effective capture of three target mycotoxins from complex solution and simultaneously detect them on one ICA strip through fluorescence signal reading. The detection sensitivity of MagQBD-ICA was markedly improved due to the signal amplification of the multilayer QD shell and target enrichment. With capture/detection strategy, multiplexed detection of AFB1, OTA, and FB1 was conducted, resulting in low detection limits of 0.42, 11.48, and 4.21 pg mL–1, respectively. The excellent accuracy, specificity, and stability of the method were further confirmed by testing actual samples including rice, peanut, corn and juices. Results suggested the great potential of MagQBD-ICA for practical detection of mycotoxins-contaminated food.

An immunochromatographic assay for the rapid detection of oxadixyl in cucumber, tomato and wine samples

Oxadixyl is a systemic phenylamide fungicide, used to prevent and control disease caused by phytophthora and downy mildew in a variety of cash crops. In this study, a hapten of oxadixyl was designed and used to prepare a highly specific and sensitive anti-oxadixyl monoclonal antibody (mAb) with a 50% inhibition concentration of 3.68 ng/mL. An immunochromatographic assay (ICA) was developed for screening of oxadixyl residues in cucumber, tomato and wine. The ICA strip provided a visible limit of detection (vLOD) of 1 ng/g and a cut-off value of 20 ng/g in cucumbers and tomatoes and displayed a vLOD of 5 ng/mL and a cut-off value of 100 ng/mL in wine. The calculated limit of detection (cLOD) values of the ICA were 1.45 and 0.55 ng/g for cucumbers and tomatoes and 2.11 ng/mL for wine. The ICA gave recovery rates of 95.4–106.0% for cucumber samples, 96.4–99.5% for tomato samples and 93.0–97.4% for wine samples. Moreover, the test results provided by the ICA were highly consistent with those of LC/MS. These results and data indicated that the ICA was an effective and practical method for rapid and high-throughput detection of oxadixyl residues in cucumber, tomato, wine and other food samples.

Immunological quantitative detection of dicofol in medicinal materials.

In this study, a highly specific and sensitive monoclonal antibody against dicofol (DIC) was prepared and used to create a colloidal gold immunochromatographic strip (ICS) for the quantitative analysis of DIC in liquorice and Angelica sinensis samples. We found that for both the liquorice and Angelica sinensis samples, the cut-off value for the ICS was 500 ng g-1, respectively, and using a colloidal gold test strip reader, the half-maximal inhibitory concentrations (IC50) were found to be 35 ng g-1 and 88 ng g-1, the limits of detection were 2 ng g-1 and 3 ng g-1, and the linear detection scopes (IC20-IC80) were from 6 to 201 ng g-1 and from 10 to 819 ng g-1, respectively. In the recovery test, we found that recoveries were within the range of 90.6% to 97.2% and the coefficients of variation were less than 8.6% in the intra-assay and inter-assay for the liquorice samples and the recoveries ranged from 90.8% to 97.3% and the coefficients of variation were less than 9.1% in the intra-assay and inter-assay for the Angelica sinensis samples. The established ICS assay could be used for the rapid quantitative screening of chemicals in Chinese traditional medicinal materials.

Gold-based immunochromatographic strip assay for detecting dimethomorph in vegetables

Gold-based immunochromatographic strip assay for detecting dimethomorph in vegetables.

A colloidal gold immunochromatographic strip assay for the rapid detection of Shigella in milk and meat products

Anti-Shigella mAb was produced using IpaC and an immunochromatographic strip was developed to detect different serotypes of Shigella in food.