Immunochromatographic Strip Test Research Articles

Rapid detection of aldicarb in cucumber with an immunochromatographic test strip

ABSTRACTA rapid, simple, and sensitive immunochromatographic strip test has been developed to detect aldicarb (ALD) in 0.01 M phosphate buffer solution (PBS) (pH 7.4) and cucumber samples. The test was developed with a sensitive monoclonal antibody specific for ALD, generated by immunizing BALB/c mice with a well-characterized ALD – keyhole limpet hemocyanin conjugate, produced in our laboratory. Under the optimized condition, the cut-off limits of test strip for ALD were found to be 25 ng/mL in 0.01 M PBS (pH 7.4) and 100 ng/mL in cucumber samples, and the highest standard relative deviation was 9.05%. Both results were obtained within 5 min. The data indicate that the method is sensitive, rapid, and specific, so this immunochromatographic test strip has utility for the rapid high-throughput screening of the pesticide, especially in food samples.

Multicolor immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin B1 and fumonisins

The authors describe the preparation of desert rose-like gold nanoparticles (DR-GNPs) with a plasmon resonance band at 620 nm which gives them a blue color. They have a hydrodynamic diameter of ∼72 nm and were prepared by a seeding growth approach. The DR-GNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. These nonspherical GNPs were used as a label for the antibody in an immunochromatographic strip test (ICST). Despite their particular shape and the higher surface area compared to spherical gold nanoparticles, the DR-GNPs are useful blue labels for the GNP-based strip test. A multicolor ICST for aflatoxin B1 and fumonisins is described that employs both blue DR-GNPs and red spherical GNPs. It allows for simultaneous rapid determination of the two mycotoxins in maize flour, with visual cut-off levels of 2 μg⋅kg-1 for aflatoxin B1 and of 1000 μg⋅kg-1 for fumonisins.

Integrated immunochromatographic strip with glucometer readout for rapid quantification of phosphorylated proteins

A new technology to quantify phospho-p5315 by converting its content to the amount of glucose which is detectable by a glucometer was developed. An immunochromatographic test strip (ITS) was used as a disposable platform, where primary antibody (Ab1)-modified Fe3O4 magnetic nanoparticles (Fe3O4-Ab1) were settled on the test zone to capture both the target phospho-p5315 and the detection antibody (Ab2)-glucose encapsulating liposome (GEL) conjugate. The measurement was based on the release and subsequent detection of glucose from Ab2-GEL using a glucose meter (GM). The amount of glucose is proportional to the phospho-p5315 concentration from 0.1 to 50 ng mL−1, the limit of detection is 50 pg mL−1 (3S/N). The high sensitivity was a result of the huge number of glucose encapsulated in the liposome. Taking the advantage of low cost, widespread availability and portability of the test trip, together with the personal GM, the presented approach can be easily used to detect other disease biomarkers in medical diagnostics and environmental monitoring.

Hollow Au-Ag Nanoparticles Labeled Immunochromatography Strip for Highly Sensitive Detection of Clenbuterol

The probe materials play a significant role in improving the detection efficiency and sensitivity of lateral-flow immunochromatographic test strip (ICTS). Unlike conventional ICTS assay usually uses single-component, solid gold nanoparticles as labeled probes, in our present study, a bimetallic, hollow Au-Ag nanoparticles (NPs) labeled ICTS was successfully developed for the detection of clenbuterol (CLE). The hollow Au-Ag NPs with different Au/Ag mole ratio and tunable size were synthesized by varying the volume ratio of [HAuCl4]:[Ag NPs] via the galvanic replacement reaction. The surface of hollow Ag-Au NPs was functionalized with 11-mercaptoundecanoic acid (MUA) for further covalently bonded with anti-CLE monoclonal antibody. Overall size of the Au-Ag NPs, size of the holes within individual NPs and also Au/Ag mole ratio have been systematically optimized to amplify both the visual inspection signals and the quantitative data. The sensitivity of optimized hollow Au-Ag NPs probes has been achieved even as low as 2 ppb in a short time (within 15 min), which is superior over the detection performance of conventional test strip using Au NPs. The optimized hollow Au-Ag NPs labeled test strip can be used as an ideal candidate for the rapid screening of CLE in food samples.

Development of a quantum dot-based immunochromatography test strip for rapid screening of oxytetracycline and 4-epi-oxytetracycline in edible animal tissues

ABSTRACTA rapid and sensitive immunochromatographic test strip (ICTS) was developed in a competitive format with the quantum dot-conjugated monoclonal antibody specifically to determine the residues of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC). Using an ICTS reader, the 50% inhibition concentration (IC50) was found to be 3.41 ± 0.29 ng ml–1 of OTC in phosphate-buffered saline samples and the detection limit was 0.44 ± 0.05 ng ml–1. The visual cut-off level of the ICTS is 25 ng ml–1. The recoveries from tissue samples spiked with OTC of 50–600 μg kg–1 were 74.2–107.2%, with coefficients of variation below 15%. The method was used to analyse incurred tissue samples, and there was good correlation (R2 = 0.999) between the ICTS and high-performance liquid chromatography. These results indicate that ICTS is suitable for the rapid and quantitative detection of OTC and 4-epi-OTC residues in edible animal tissues.

Development of an indirect competitive immunochromatographic strip test for rapid detection and determination of anticancer drug, harringtonine.

Harringtonine (HT) is a natural compound, which is mainly produced by the genus Cephalotaxus, and has been clinically utilized in China for the treatment of acute leukemia and lymphoma. However, the amounts of HT in the Cephalotaxus species are very small; therefore, plant tissue cultures have been focused upon to enhance HT production. Qualitative/quantitative methods for HT detection are required to screen superior cell lines. We developed a one-step indirect competitive immunochromatographic assay (ICA) using colloidal gold nanoparticles conjugated with highly specific monoclonal antibodies against HT (MAb 1D2) for simple, rapid, and sensitive detection of HT in plant samples. This ICA can be completed in 15min after dipping the strip into analytes with a limit of detection of ∼313ng/mL. In developed ICA, fiber pad which is usually used for conventional ICA, was not used to shorten the time for preparing chromatographic strip, resulting in a decrease in the volume of valuable analytes (20μL). Considering simplicity, rapidity, and sensitivity of the developed ICA, this study could be applied to a fieldwork study for finding new natural resources containing HT.

Using fluorescence immunochromatographic test strips based on quantum dots for the rapid and sensitive determination of microcystin-LR.

A novel immunosensor for the detection of microcystin-LR (MC-LR) was constructed with use of immunochromatographic test strips (ICTS). Quantum dots were chosen to be the fluorescent labels for the immune sensor in ICTS because of their excellent optical and electronic properties with a relatively narrow emission spectrum. The detection sensitivity of the ICTS was related to the concentration of the fluorescent probe and the amount of the MC-LR standards. Under optimal conditions, with MC-LR as the target, the ICTS sensor had a linear range from 0.25 to 5μg/L, with a correlation coefficient of 0.9901 and a detection limit of 0.1μg/L. Furthermore, the repeatability of the ICTS was good, and the coefficient of variation was 10%. The ICTS immunosensor allows the reliable detection of MC-LR in water, and has potential in simple, sensitive detection applications. Graphical Abstract A novel method was developed to detect MC-LR using QDs based immunochromatographic test strip.

Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.

CdTe/CdS quantum dot-labeled fluorescent immunochromatography test strips for rapid detection of Escherichia coli O157:H7

Immunochromatography test strips have been widely used to detect foodborne pathogens due to their high sensitivity, stability and specificity, and the selection of the label is an important factor.

An Immunochromatographic Test Strip to Detect Ochratoxin A and Zearalenone Simultaneously.

Lateral flow immunochromatographic assays are simple, useful tools for the detection of many different targets. In this format, a liquid sample containing target of interest moves along a strip of polymeric material adhered with various lines where molecules have been attached that excert specific interactions with the target. Here, we describe a colloidal gold-based immunochromatographic test strip for the simultaneous detection of ochratoxin (OTA) and zearalenone (ZEN) in corn and other cereals qualitatively and quantitatively.

Optimizations needed for lateral flow assay for rapid detection of pathogenic E. coli.

Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 105 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.

Fluorescence quenching-based signal amplification on immunochromatography test strips for dual-mode sensing of two biomarkers of breast cancer.

Recently, immunochromatography test strips (ICTS) have been fully developed for point-of-care testing (POCT). However, the intrinsic limitations including non-quantitative detection of colloidal gold ICTS and low sensitivity of fluorescence ICTS (FICTS) significantly restrict their further application in clinical diagnosis. Taking advantages of rapid colorimetric qualitative detection and fluorescence quantitation, we designed a kind of sensitive and dual-mode magnetic FICTS (mFICTS) based on PLGA@Fe3O4 super-paramagnetic nanosphere (SPMN) probes quenching multiplex fluorescer on the test line through sandwich immunoreactions. Owing to the large number of Fe3O4 nanoparticles (about 47) encapsulated in one SPMN, about 2680 Cy5 molecules were quenched by one SPMN on the test line such that to significantly improve the analytical sensitivity as well as the detection of whole blood samples via magnetic separation. Moreover, the aggregation of black SPMN on the test line enabled a quick naked-eye screening in 3 min. For high accuracy breast cancer diagnosis, combined determination of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA153) was performed on one mFICTS with the limits of detection of about 0.06 ng mL-1 and 0.09 U mL-1, respectively. Then, more than 50 clinical serum samples were investigated for high-resolution screening by mFICTS, and the results were coincident with those obtained by electrochemiluminescence immunoassay (ECLIA). Thus, the designed mFICTS is suitable for point-of-care diagnostics.

Identification of B-cell Epitope of Leishmania donovani and its application in diagnosis of visceral leishmaniasis

Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) is 97–100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (P1P2) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient’s sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16–96.27)% and specificity 100%; Sp Cl95% (84.56–100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-P1P2 antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-P1P2 antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.

High sensitivity immunochromatographic strip test (ICP11 strip test) for white spot syndrome virus detection using monoclonal antibodies specific to ICP11 non-structural protein

Two monoclonal antibodies (MAbs) specific to different epitopes on ICP11, a non-structural protein of white spot syndrome virus (WSSV), were employed to develop a highly sensitive immunochromatographic strip test (ICP11 strip test). One MAb (WI-11) was conjugated to colloidal gold to bind to ICP11 and another MAb (WI-16) was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. A downstream control line (C) of goat anti-mouse immunoglobulin G (GAM) antibody was used to capture the free colloidal gold conjugated MAb to validate the test performance. Homogenate of shrimp sample in application buffer could be applied directly to the application well and the test result was obtained within 15min. The sensitivity of the kit is 40 times more sensitive than the detection limit of previously reported strip test using MAbs specific to VP28, and approximately 400 times more sensitive than that of Shrimple®, but approximately 50 times less sensitive than that of one-step PCR. For experimentally WSSV-infected shrimps, the ICP11 strip could detect WSSV as early as 12–18h post infection. Due to its high specificity, simplicity and no requirement on sophisticated equipment or specialized skills, the strip test could be adopted to surveillance of early WSSV infection at the pond site.

Immunochromatographic detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli with cross-detection of cholera toxin

Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG. This test strip detected not only LT in the culture supernatant of LT gene-positive strains, but also cholera toxin in the culture supernatant of Vibrio cholerae. These results indicate that this test strip is suitable for the diagnosis of both enterotoxigenic E. coli and V. cholerae infection.

Use of quantum dot beads-labeled monoclonal antibody to improve the sensitivity of a quantitative and simultaneous immunochromatographic assay for neuron specific enolase and carcinoembryonic antigen

Detection of multiplex tumor markers was of great importance for cancer diagnosis. Immunochromatographic test strip (ICTS) was the most frequently-used point-of-care detection means. Herein, a convenient and fast method for simultaneous quantitative detection of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) was developed based on ICTS using quantum dot beads (QBs) as marking material. Good monodispersity, high colloidal stability and carboxyl-modified (COOH–) QBs were used. For this method, two test lines were applied to the NC membrane for simultaneous analysis of CEA and NSE respectively. The ideal limit of CEA and NSE detection was 0.0378ng/mL and 0.0426ng/mL with scarcely any cross-reactivity. Moreover, the fluorescent signal intensity of the nitrocellulose membrane could be easily read out in the cooperation of the “Handing” system without professional operators. The possible clinical utilization of this platform was demonstrated by detecting 100 clinic human serums. The result showed that the platform had sensitivity of 99% and 97% for CEA and NSE, while the specificity was 97% and 100% respectively. Our results indicated that the QBs based ICTS not only owning the ability of sensitive and specific simultaneous detection of CEA and NSE, but also showing the potential in developing this ICTS into a routine part of early lung cancer diagnosis.

An Integrated Electrochemical Immunochromatographic Test Strip Based on the Amplification of Gold Nanoparticles for Quantitative Detection of Alpha-Fetoprotein

An Integrated Electrochemical Immunochromatographic Test Strip Based on the Amplification of Gold Nanoparticles for Quantitative Detection of Alpha-Fetoprotein

Multiplexed immunochromatographic test strip for time-resolved chemiluminescent detection of pesticide residues using a bifunctional antibody

A novel bifunctional antibody (BfAb) that could recognize methyl parathion and imidacloprid simultaneously was prepared by a hybrid hybridomas technique. Using the BfAb as the sole recognition reagent, a multiplexed immunochromatographic test strip based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of pesticide residues. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were used as the CL probes to label the haptens of methyl parathion and imidacloprid, respectively. After the labeled haptens competed with methyl parathion and imidacloprid to bind with the BfAb immobilized on the test strip, the two CL reactions catalyzed by the enzymes were triggered simultaneously by coreactants injection. Due to the distinct CL kinetics characteristics of HRP and ALP, the signals for methyl parathion and imidacloprid detections were collected at 2.5s and 300s, respectively. The linear ranges for methyl parathion and imidacloprid were both 0.1–250ngmL−1, with detection limits of 0.058ngmL−1 (S/N=3). The whole assay process could be accomplished within 22min. The detection results for spiked traditional Chinese medicine samples demonstrated its application potential. The proposed method provided a low-cost, facile and rapid tool for multiplexed screening of pesticide residues using single antibody.

A sensitive point-of-care testing chip utilizing superabsorbent polymer for the early diagnosis of infectious disease

We developed a highly sensitive point-of-care testing (POCT) chip using an antigen-antibody complex reaction for the early diagnosis of infectious disease. The central concept of the chip was to balance the sensitivity enhancement with appropriate safety measures for infectious risk during POCT. The chip consisted of a sheet of glass fiber sealed in a polymethylmethacrylate microfluidic channel. Antibody-immobilized microbeads were accumulated in a transparent location in the channel that served as the test line, trapping the target antigen bound to fluorescently labeled antibodies and allowing diagnosis by a portable fluorescence scanner. In this work, we applied a superabsorbent polymer to drive a stable and continuous fluidic flow for both the sample and a rinsing solution without an external power supply. The generated flow enabled thorough bound/free separation of the antigen-antibody interactions and significantly improved the signal-to-noise ratio and the reproducibility. Moreover, the polymer had the advantage of retaining the potentially infectious sample within the chip, contributing to its safety for practical use. An influenza test using a virus protein exhibited a sensitivity enhancement of more than a thousand-fold compared with commercial immunochromatographic test strips.

Reverse Fluorescence Enhancement and Colorimetric Bimodal Signal Readout Immunochromatography Test Strip for Ultrasensitive Large-Scale Screening and Postoperative Monitoring.

Ultrasensitive and quantitative fast screening of cancer biomarkers by immunochromatography test strip (ICTS) is still challenging in clinic. The gold nanoparticles (NPs) based ICTS with colorimetric readout enables a quick spectrum screening but suffers from nonquantitative performance; although ICTS with fluorescence readout (FICTS) allows quantitative detection, its sensitivity still deserves more efforts and attentions. In this work, by taking advantages of colorimetric ICTS and FICTS, we described a reverse fluorescence enhancement ICTS (rFICTS) with bimodal signal readout for ultrasensitive and quantitative fast screening of carcinoembryonic antigen (CEA). In the presence of target, gold NPs aggregation in T line induced colorimetric readout, allowing on-the-spot spectrum screening in 10 min by naked eye. Meanwhile, the reverse fluorescence enhancement signal enabled more accurately quantitative detection with better sensitivity (5.89 pg/mL for CEA), which is more than 2 orders of magnitude lower than that of the conventional FICTS. The accuracy and stability of the rFICTS were investigated with more than 100 clinical serum samples for large-scale screening. Furthermore, this rFICTS also realized postoperative monitoring by detecting CEA in a patient with colon cancer and comparing with CT imaging diagnosis. These results indicated this rFICTS is particularly suitable for point-of-care (POC) diagnostics in both resource-rich and resource-limited settings.