Immunochromatographic Test Strip Assay Research Articles

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

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.

Smartphone-Based Dual-Channel Immunochromatographic Test Strip with Polymer Quantum Dot Labels for Simultaneous Detection of Cypermethrin and 3-Phenoxybenzoic Acid.

Currently, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) are the primary methods used to detect pesticides and their metabolites for biomonitoring of exposure. Although GC-MS and LC-MS can provide accurate and sensitive measurements, these techniques are not suitable for point-of-care or in-field biomonitoring applications. The objective of this work is to develop a smartphone-based dual-channel immunochromatographic test strip (ICTS) for on-site biomonitoring of exposure to cypermethrin by simultaneous detection of cypermethrin and its metabolite, 3-phenoxybenzoic acid (3-PBA). Polymer carbon dots (PCDs) with ultrahigh fluorescent brightness were synthesized and used as a signal amplifier in ICTS assay. Cypermethrin (a representative pyrethroid pesticide) and its major metabolite 3-PBA were simultaneously detected to provide more comprehensive analysis of cypermethrin exposure. After competitive immunoreactions between the target sample and the coating antigens preloaded on the test line, the tracer antibody (PCD-conjugated antibody) was quantitatively captured on the test lines. The captured PCDs were inversely proportional to the amount of the target compound in the sample. The red fluorescence on the test line was then recorded using a smartphone-based device capable of conducting image analysis and recording. Under optimal conditions, the sensor showed excellent linear responses for detecting cypermethrin and 3-PBA ranging from 1 to 100 ng/mL and from 0.1 to 100 ng/mL, respectively, and the limits of detection were calculated to be ∼0.35 ng/mL for cypermethrin and ∼0.04 ng/mL for 3-PBA. The results demonstrate that the ICTS device is promising for accurate point-of-care biomonitoring of pesticide exposure.

Highly sensitive detection of Cronobacter sakazakii based on immunochromatography coupled with surface-enhanced Raman scattering

The presence of Cronobacter sakazakii must be controlled in infant powder plants, because it may cause infectiousdisease in infants, with high mortality. Testing for C.sakazakiiin powdered infant formula should be performed before delivery, and it requires rapid and specific detection methods. In this study, we established a surface-enhanced Raman scattering (SERS) immunochromatographic test strip for the quantitativedetermination of C.sakazakiiin powdered infant formula. Monoclonalantibodies for C. sakazakii were labeled with p-aminothiophenol-bound colloidal gold nanoparticles. Color change in the test line indicated the presence of C. sakazakii. A highly sensitive and quantitative test method was developed based on the Raman signal produced by the p-aminothiophenol bonding on gold nanoparticles. The SERS immunochromatographic test strip assay required a short analysis time (12 min) and exhibited a linearityrangefrom 102 to 107 cfu/mL. The limit of detection was 201 cfu/mL without preculture. The SERS immunochromatographic test strip assay is a promising tool for the simple and rapid quantitativeanalysis of C.sakazakiiand other pathogenic bacteria.

Rapid and sensitive detection of ochratoxin A in rice flour using a fluorescent microsphere immunochromatographic test strip assay

ABSTRACT In this study, a high sensitivity and specificity monoclonal antibody (mAb) was developed based on the hydroxy and carboxy-termini of ochratoxin A (OTA). A fluorescent microsphere immunochromatographic test strip (FM-ICTS) was assembled for the rapid detection the OTA in rice flour. Some relevant parameters were optimized including the best concentration of coating antigen and optimal diluent buffer composition. Under optimal conditions, the strip assay had a visual limit of detection (LOD) of 2 ng/mL and a cut-off value of 16 ng/mL for OTA in rice flour samples. By means of a strip scan reader, we obtained a quantitative measurement for OTA with an LOD of 1.27 ng/mL. No significant difference in the strip test results was seen when compared to an ic-ELISA assay or LC–MS/MS. This also shows, therefore, that the FM-ICTS has potential application for rapid and accurate quantitative detection in the market place.

Development of an immunochromatographic strip assay based on a monoclonal antibody for detection of cimaterol

ABSTRACT An anti-cimaterol (cima) monoclonal antibody, 2H8, was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic test strip assay for the detection of cima, a newly described β-agonist. The half-maximal inhibitory concentration of the antibody was 1.56 ng/mL, and no cross-reactivity with other β-adrenergic agonists was observed. The antibody’s limit of detection was 0.416 ng/mL, and its linear range of detection was 0.416–5.868 ng/mL. The recovery rates of cima in swine urine ranged from 86% to 92.5%, and the test strip had a cut-off value of 10 ng/mL. Both ic-ELISA and the developed strip assay are effective when detecting cima in swine urine.

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.

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

A rapid lateral flow fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MAb 1D3 for AFM1 was 23 ng/L, and the cross-reactivity (CR) of the MAb with aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 was 18%, 9%, <0.15%, 43% and <0.3%, respectively. The MAb was covalently conjugated with carboxylate-modified FMs, and the FMs were used as the label in a competitive immunochromatography assay. Using a moderate hapten-to-protein coupling ratio (˜2) in the coating antigen resulted in improved immunoassay sensitivity. Under optimal conditions, the IC50 value of the assay was 36.3 ng/L with the limit of detection (LOD) of 4.4 ng/L in milk samples using a fluorescence reader, and the recoveries of AFM1 in spiked milk samples ranged from 76.6 to 110.8% with coefficient of variation (CV) of 4–14.7%. The whole procedure could be completed within 30 min. The results demonstrated that the FM-ICTS assay is easy to conduct, rapid, highly sensitive and specific for the detection of AFM1 residues in milk.

Synthesis of carboxyl-capped and bright YVO4:Eu,Bi nanoparticles and their applications in immunochromatographic test strip assay

Carboxyl-capped YVO4:Eu,Bi nanoparticles with average diameter of ∼10nm were synthesized via a copolymer of phosphono and carboxylic acid mediated hydrothermal method. Under a 350nm ultraviolet light excitation, the YVO4:Eu,Bi NPs exhibit sharp and bright red emission peaked at 615nm and with highest quantum yield of ∼43%. Furthermore, the nanoparticles show good water/buffer stability and feasible bioconjugation benefiting from the carboxylic groups on their surface. Based on these kind optical and surface properties of the YVO4:Eu,Bi nanoparticles, an immunochromatographic test strip assay for quantitative determination of human IgG was achieved. This protocol can be extended to other rare-earth nanoparticles with the purpose of developing bioprobes for desired applications.

The effects of size and synthesis methods of gold nanoparticle-conjugated MαHIgG4 for use in an immunochromatographic strip test to detect brugian filariasis

This study describes the properties of colloidal gold nanoparticles (AuNPs) with sizes of 20, 30 and 40 nm, which were synthesized using citrate reduction or seeding-growth methods. Likewise, the conjugation of these AuNPs to mouse anti-human IgG4 (MαHIgG4) was evaluated for an immunochromatographic (ICG) strip test to detect brugian filariasis. The morphology of the AuNPs was studied based on the degree of ellipticity (G) of the transmission electron microscopy images. The AuNPs produced using the seeding-growth method showed lower ellipticity (G ≤ 1.11) as compared with the AuNPs synthesized using the citrate reduction method (G ≤ 1.18). Zetasizer analysis showed that the AuNPs that were synthesized using the seeding-growth method were almost monodispersed with a lower polydispersity index (PDI; PDI≤0.079), as compared with the AuNPs synthesized using the citrate reduction method (PDI≤0.177). UV–visible spectroscopic analysis showed a red-shift of the absorbance spectra after the reaction with MαHIgG4, which indicated that the AuNPs were successfully conjugated. The optimum concentration of the BmR1 recombinant antigen that was immobilized on the surface of the ICG strip on the test line was 1.0 mg ml−1. When used with the ICG test strip assay and brugian filariasis serum samples, the conjugated AuNPs–MαHIgG4 synthesized using the seeding-growth method had faster detection times, as compared with the AuNPs synthesized using the citrate reduction method. The 30 nm AuNPs–MαHIgG4, with an optical density of 4 from the seeding-growth method, demonstrated the best performance for labelling ICG strips because it displayed the best sensitivity and the highest specificity when tested with serum samples from brugian filariasis patients and controls.

An integrated electrochemical device based on immunochromatographic test strip and enzyme labels for sensitive detection of disease-related biomarkers

A novel electrochemical biosensing device that integrates an immunochromatographic test strip and a screen-printed electrode (SPE) connected to a portable electrochemical analyzer was presented for rapid, sensitive, and quantitative detection of disease-related biomarker in human blood samples. The principle of the sensor is based on sandwich immunoreactions between a biomarker and a pair of its antibodies on the test strip, followed by highly sensitive square-wave voltammetry (SWV) detection. Horseradish peroxidase (HRP) was used as a signal reporter for electrochemical readout. Hepatitis B surface antigen (HBsAg) was employed as a model protein biomarker to demonstrate the analytical performance of the sensor in this study. Some critical parameters governing the performance of the sensor were investigated in detail. Under optimal conditions, this sensor was capable of detecting a minimum of 0.3ngmL−1 (S/N=3) HBsAg with a wide linear concentration range from 1 to 500ngmL−1. The sensor was further utilized to detect HBsAg spiked in human plasma with an average recovery of 91.3%. In comparison, a colorimetric immunochromatographic test strip assay (ITSA) was also conducted. The result shows that the SWV detection in the electrochemical sensor is much more sensitive for the quantitative determination of HBsAg than the colorimetric detection, indicating that such a sensor is a promising platform for rapid and sensitive point-of-care testing/screening of disease-related biomarkers in a large population.

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10(2) CFU/ml, which was significantly higher than the detection limit (10(7) CFU/ml) of the commercial immunochromatographic test strip assay.

Quantum Dot-Based Immunochromatographic Fluorescent Biosensor for Biomonitoring Trichloropyridinol, a Biomarker of Exposure to Chlorpyrifos

A novel and portable fluorescent sensor that integrates an immunochromatographic test strip assay (ITSA) with a quantum dot (QD) label and a test strip reader was described in this study for simple, rapid, and sensitive biomonitoring of an organophosphorus pesticide metabolite. The principle of this sensor is based on a competitive immunoreaction that was performed on an immunochromatographic test strip, where analytes compete with competitors (QD-conjugated analogs) to bind to antibodies on a test zone. Captured QDs serve as signal vehicles for fluorescent readout. In this work, 3,5,6-trichloropyridinol (TCP) is used as a model analyte to demonstrate the performance of the immunosensor. QD-TCP conjugates were synthesized and characterized with X-ray photoelectron spectroscopy (XPS) and fluorescence spectroscopy. Some parameters (e.g., the amount of QD-modified TCP and immunoreaction time) that govern sensitivity and reproducibility of ITSA were optimized. Under optimal conditions, the sensor has a wide dynamic range and is capable of detecting a minimum 1.0 ng/mL TCP standard analyte in 15 min. The sensor has been successfully applied for detection of TCP spiked in rat plasma with average recovery of 102.0%. Results demonstrate that this sensor provides a rapid, clinically accurate, and quantitative tool for TCP detection and shows great promise for in-field and point-of-care (POC) quantitative testing and screening for metabolite biomarkers, e.g., TCP, for humans exposed to pesticides.