Quantum Dot Nanobeads as Multicolor Labels for Simultaneous Multiplex Immunochromatographic Detection of Four Nitrofuran Metabolites in Aquatic Products

A multicolor immunochromatographic assay platform based on quantum dot nanobeads (QBs) for the rapid and simultaneous detection of nitrofuran metabolites in different aquatic products is documented. These metabolites include 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), and 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ). QBs with emission colors of red, yellow, green, and orange were employed and functionalized with the corresponding antibodies to each analyte to develop a multicolor channel. The visual detection limits (cutoff values) of our method for AOZ, AHD, SEM, and AMOZ reached up to 50 ng/mL, which were 2, 20, 20, and 20 times lower than those of traditional colloidal gold test strips, respectively. The test strip is capable of detection within 10 min in real samples while still achieving good stability and specificity. These results demonstrate that the developed multicolor immunochromatographic assay platform is a promising technique for multiplex, highly sensitive, and on-site detection of nitrofuran metabolites.     

Colloidal gold-based immunochromatographic assay for detection of diethylstilbestrol residues

One-step membrane-based competitive colloidal gold-based immunoassays in immunochromatographic formats for the rapid detection of diethylstilbestrol (DES) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and DES hapten-ovalubumin conjugate (test line). Anti-DES polyclonal antibody labeled with colloidal gold particles was first incubated with DES. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for immunochromatographic of 0.5 microg/kg for detecting DES standard solution, and the limit of detection was 5 microg/kg for detecting the DES spiked in swine pork and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.     

Lateral flow immunoassay of human troponin-I

an immunochromatographic assay for a rapid determination of troponin-I, in which gold nanoparticles were used as visual tags, was developed. The optimum analysis conditions were determined. The detection limit of the assay was 1 ng/mL, and the coefficient of variation did not exceed 10%.     

Rapid determination of chloramphenicol residues in aquaculture tissues by immunochromatographic assay.

An immunochromatographic assay was developed to detect chloramphenicol (CAP) residues in aquaculture tissues. The limit of detection (LOD) was 10 ng g(-1) for detecting CAP spiked in the aquaculture tissues. The results were confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and indicated that there was a good agreement between the two methods. The linear regression equation was y = 1.19x + 0.539 with R(2) = 0.978. The assay time for test was less than 5 min and the method is suitable for rapid testing on-site.     

Gold nanoparticle-based immunochromatographic assay for the detection of 7-aminoclonazepam in urine

An analytical system of immunochromatographic assay based on gold nanoparticles was developed for the detection of 7-aminoclonazepam (7-ACLZ) in human urine. The qualitative assay was based on the competitive immunoassay using anti-7-ACLZ polyclonal antibody (PcAb) and a detector reagent that contains colloidal gold particles coated with anti-7-ACLZ PcAb. Nitrocellulose membrane was separately immobilised with goat anti-rabbit IgG (control line) and 7-ACLZ-OVA conjugate (test line). The sensitivity of the strip was tested for detecting 7-ACLZ spiked in urine and each specimen was independently measured by liquid chromatography tandem mass spectrometry. Good correlation was showed by the recovery results. The limit of detection for the strip test in urine was 100 ng mL^?1. The assay can be applied to the rapid detection of 7-ACLZ with the short testing time.     

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 semiquantitative detection of LNG and MP in water samples on site.     

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 monoclonal antibody of PCT (UCP‐Ab1) was freeze‐dried under certain conditions. And the detections of PCT levels with UCP‐Ab1 conjugates before and after freeze‐drying were evaluated. The results show that, compared to the UCP‐Ab1 conjugates without freeze‐drying, the detection sensitivity of freeze‐dried UCP‐Ab1 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.     

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 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay.     

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.      

Immunochromatographic rapid analysis of human heart-type fatty acid-binding protein for acute myocardial infarction diagnosis

A method of immunochromatographic assay for rapid detection of human heart-type fatty acid-binding protein (h-FABP) as an early marker of acute myocardial infarction was developed. Gold nanoparticles were used as a visual label. The optimum conditions for assay were determined. The limit of detection in the assay was 1.5 ng/mL, and the variation coefficient did not exceed 8%. Using the developed test system, the clinical diagnosis of acute myocardial infarction (N = 10) was confirmed, and the results of testing the serum of healthy individuals (N = 25) were negative.     

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos

An immunochromatographic assay (ICA) based on competitive antigen-coated format using colloidal gold as the label was developed for the detection of the organophosphorus insecticide chlorpyrifos. The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with chlorpyrifos Hapten-OVA conjugate (test line) and anti-mouse IgG (control line). Based on the fact that the competition is between the migrating analyte in the sample and the analyte hapten immobilized on the test strip for the binding sites of the antibody-colloidal gold (Ab-CG) conjugate migrating on the test strip, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. This criterion was utilized for the confirmation of appropriateness of a nitrocellulose (NC) membrane for chlorpyrifos ICA. The detection limit of the ICA for chlorpyrifos standard and chlorpyrifos spiked into agricultural samples were 10 and 50 ng mL(-1), respectively. The assay time for the ICA test was less than 10 min, suitable for rapid on-site testing of chlorpyrifos.     

Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ngmL(-1) with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.     

孔雀石绿的磁性免疫层析快速检测方法研究

开发了一种适用于现场快速检测孔雀石绿(MG)的免疫层析试纸条,在超顺磁性纳米微球上偶联MG单克隆抗体作为检测探针,分别将孔雀石绿完全抗原(MG-B SA)和羊抗鼠IgG喷涂于NC膜的T线和C线.结果 发现,T线最佳喷涂量为0.25 mg/mL,抗体最佳偶联量为20 μg,构建的试纸条可在25 min内实现养殖用水及鱼肉...     

Enzyme-linked immunosorbent assay and immunochromatographic strip for rapid detection of atrazine in water samples

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic (ICG) strip for the determination of the herbicide atrazine in water samples. The ELISA had a half-maximum inhibition concentration (IC₅₀) of 0.12 ng mL^?1 and a limit of detection (LOD, calculated as the IC₁₅ value) of 0.01 ng mL^?1. The average of recoveries for all spiked water samples was 96.5%. There was a good correlation between the data determined by this ELISA and those obtained by high performance liquid chromatography (HPLC) (r ² ?=?0.996). The visual LOD of the ICG strip assay was 2 ng mL^?1. The assay process only took 10 min, and no sample pretreatment was required. Its high specificity, sensitivity and fast detection made the strip well suited for on-site screening of atrazine in water samples. Both the ELISA and the ICG strip assay are useful for rapid analysis of a large number of water samples at low cost.

Production of monoclonal antibody and development of enzyme-linked immunosorbent assay for kanamycin in biological matrices

Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin-bovine gamma-globulin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in two hybridoma cell lines. Fifty per cent inhibition concentrations (IC50) for the MAbs were 2 and 5 ng ml-1. One MAb (IC50 = 2 ng ml-1) was named #22 and was used to develop quantitative assays for kanamycin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 0.2 ng ml-1 and the standard deviations were 0.2-4.4% for intra-assay and 0.6-4.7% for inter-assay, respectively. The detection limits using peroxidase were 4 ppb in cattle milk, cattle plasma, cattle urine, swine plasma, swine urine and chicken plasma. Using the MAb #22 produced, a rapid test kit based on an immunochromatographic method was developed. The detection limits using the kit were 50 ppb in cattle milk, cattle plasma, cattle urine and chicken plasma.     

A one-step immunochromatographic assay for detecting ginsenosides Rb1 and Rg1

An immunochromatographic strip test has been developed for detecting ginsenosides Rb1 (G-Rb1) and Rg1 (G-Rg1). This qualitative assay system is useful as a rapid screening method for detecting G-Rb1 and G-Rg1 in plants and plant preparations. Our assay is a competitive immunoassay that uses anti-G-Rb1 and anti-G-Rg1 monoclonal antibodies (MAbs) and a detection reagent that contains colloidal gold particles coated with anti-G-Rb1 and anti-G-Rg1 MAbs. Detection limits are 2 g mL^–1 for both G-Rb1 and G-Rg1.     

Rapid, quantitative and sensitive immunochromatographic assay based on stripping voltammetric detection of a metal ion label

A novel, sensitive immunochromatographic electrochemical biosensor (IEB) which combines an immunochromatographic strip technique with an electrochemical detection technique has been demonstrated. The IEB takes advantages of the speed and low-cost of the conventional immunochromatographic test kits and high-sensitivity of stripping voltammetry. Bismuth ions (Bi(3+)) have been coupled with the antibody through the bifunctional chelating agent diethylenetriamine pentaacetic acid (DTPA). After immunoreactions, Bi(3+) was released and quantified by anodic stripping voltammetry at a built-in single-use screen-printed electrode. As an example for the applications of such novel device, the detection of human chorionic gonadotronphin (HCG) in a specimen was performed. This biosensor provides a more user-friendly, rapid, clinically accurate, less expensive immunoassay for such analysis in specimens than currently available test kits.     

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-ELISAwas 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.     

EZ Gluten for the qualitative detection of gluten in foods, beverages, and environmental surfaces

The EZ Gluten assay is a rapid immunochromatographic screening method for qualitative detection of gluten in raw and cooked foods and beverages and on environmental surfaces. This AOAC Performance Tested Method study evaluated the EZ Gluten assay as an effective method for the detection of gluten in four selected matrixes: rice flour, cooked dough, beer, and dog food. In addition, the method was evaluated for its effectiveness in detecting gluten contamination of > or =1 microg/2 in.2 (25 cm2) stainless steel surface area. The EZ Gluten demonstrated 100% specificity [probability of detection (POD) 0.00, confidence interval (CI) 0.00-0.01] and 99% sensitivity (POD 0.99, CI 0.97-0.995) at the 10 ppm level for all four matrixes, and 100% specificity (POD 0.00, CI 0.00-0.11) and sensitivity (POD 1.00, CI 0.886-1.00) at the 1 microg level on the stainless steel surface. Independent laboratory testing confirmed the internal validation results in one matrix and on the stainless steel surface. Lot-to-lot, stability, and robustness studies provided evidence that the EZ Gluten is a rugged, consistent method for the detection of gluten at levels as low as 10 ppm.     

A novel immunochromatographic assay based on a time-resolved chemiluminescence strategy for the multiplexed detection of ractopamine and clenbuterol

A novel multiplexed immunochromatographic assay (ICA) based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of β-agonists, by utilizing ractopamine (RAC) and clenbuterol (CLE) as the models. Different from conventional multiplexed ICA methods which usually require two or more test lines, this strategy was developed for detection of two β-agonists by using only one test line on the nitrocellulose membrane. In this study, horseradish peroxidase and alkaline phosphatase were used as the signal probes to label RAC antibody and CLE antibody, respectively. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by injecting the coreactants, then the signals for RAC and CLE detections were recorded at 3 s and 300 s after coreactants injection, respectively. Owing to the utilization of CL detection, this protocol showed ideal sensitivity for quantitation. Under the optimal conditions, the detection limits for RAC and CLE were 0.17 ng mL⁻¹ and 0.067 ng mL⁻¹ (S/N = 3), respectively. The whole assay process can be accomplished within 20 min without complicated sample pretreatment. The proposed method was successfully applied for the detection of RAC and CLE in spiked swine urine. It opens up a new pathway for designing a low cost, time-efficiency and multiplexed strategy for rapid screening and field assay.