Development and evaluation of a rapid lateral flow immunochromatographic strip assay for screening 19-nortestosterone

The lateral flow strip test for 19-nortestosterone is one kind of immunochromatographic assay. Nitrocellulose membrane was separately immobilized with goat anti-rabbit IgG (control line) and 19-NT-OVA conjugate (test line). Anti-19-NT polyclonal antibody labeled with colloidal gold particles acted as the detector reagent. The assay is qualitatively, not quantitatively, judged with positive or negative result. We tested the sensitivity of the strip using spiked swine urine, and each specimen was independently measured by LC/MS/MS. The sensitivity, measure by eye, was determined to be 200 ng/mL. The assay time was less than 15 min, and so suitable for on-site rapid test.     

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.

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.     

Development of a competitive liposome-based lateral flow assay for the rapid detection of the allergenic peanut protein Ara h1

A competitive lateral flow assay for detecting the major peanut allergen, Ara h1, has been developed. The detector reagents are Ara h1-tagged liposomes, and the capture reagents are anti-Ara h1 polyclonal antibodies. Two types of rabbit polyclonal antibodies were raised either against the entire Ara h1 molecules (anti-Ara h1 Ab) or against an immunodominant epitope on Ara h1 (anti-peptide Ab). All of them reacted specifically with Ara h1 in Western Blot against crude peanut proteins. Moreover, the anti-Ara h1 Ab was chosen for this assay development because of its highest immunoactivity to Ara h1-tagged liposomes in the lateral flow assay. The calculated limit of detection (LOD) of this assay is 0.45 g mL^–1 of Ara h1 with a dynamic range between 0.1 and 10 g mL^–1 of Ara h1 in buffer. Additionally, the visually determined detection range is from 1 to 10 g mL^–1 of Ara h1 in buffer. Results using this assay can be obtained within 30 min without the need of sophisticated equipment or techniques; therefore, this lateral flow assay has the potential to be a cost-effective, fast, simple, and sensitive method for on-site screening of peanut allergens.     

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.     

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.     

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.     

Development of an immunochromatographic assay for rapid detection of 1-Aminohydantoin in urine specimens

A rapid immunochromatographic assay was developed and validated for detection of 1-aminohydantoin (AHD) in urine specimens. Colloidal gold-labeled polyclonal antibody specific to AHD derivative was used as the marker; based on the competitive reactivity theory, the metabolite of nitrofurantoin after derivatization with benzaldehyde would compete with carboxyphenyl AHD derivative-conjugated ovalbumin. The test strip could efficaciously detect the novel analyte with a visual detection limit of 10 ng mL(-1) and high specificity. The reliability of the assay was determined by testing 80 standard samples comparing with enzyme-linked immunosorbent assay. The semi-quantitative detection was accomplished in less than 15 min with low cost, especially for requirements of rapid and simple screening. This is the first publication of an immunochromatographic assay for detection of nitrofuran residues.     

A competitive immunochromatographic assay for testosterone based on electrochemical detection

An immunochromatographic assay using nitrocellulose membrane was combined with electrochemical detection using an electrode chip in order to quantitatively detect testosterone as a model analyte. The electrode chip consisted of a gold working electrode, a counter electrode and a pseudo-reference electrode, all fabricated on the bottom of a 3.2 mm × 3.2 mm well. Competitive immunoreactions on the membrane were initiated by flowing a solution containing testosterone and horseradish peroxidase (HRP)-labeled testosterone (a competitor) over the membrane. Prepared membrane was placed in a solution containing ferrocenemethanol (FcOH) and H₂O₂ in the well of the electrode chip, and the enzyme reaction was detected by amperometry. Labeled HRP captured on the membrane catalyzed the oxidation of FcOH to the oxidized form FcOH⁺, which was reduced electrochemically by the electrode chip. The electrochemical response of the reduction current decreased with increasing concentration of testosterone over the range 1–625 ng/ml.     

A liposome-based assay for quantitation of detergents

The amount of enzyme released from liposomes exposed to detergents varies with the amount of detergent present. This fact makes it possible to quantitate the detergent. Multilamellar liposomes containing entrapped peroxidase were adsorbed to paper discs and then exposed to solutions containing detergents. The assay procedure proved useful for assaying detergents down to their critical micelle concentration (CMC), and for an induced leakage of ionic detergents also below their CMCs.     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin

A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 μg mL⁻¹of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL⁻¹ to 2 μg mL⁻¹), obtainable in an easy and timely manner.     

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.     

胶体金免疫层析法检测罂粟碱的研究

建立了准确、快速,简便的检测食品中罂粟碱的胶体金免疫层析技术(GICA)。采用柠檬酸三钠还原法制备胶体金颗粒,标记罂粟碱(Papavarine)抗体。方法检出限为0.2μg/mL,正确检出率约为97%,特异性强,具有推广应用价值。     

胶体金免疫层析法快速检测三聚氰胺

采用戊二醛法制备三聚氰胺-BSA偶联抗原,胶体金标记三聚氰胺单克隆抗体,建立了检测三聚氰胺的胶体金免疫层析试验。结果显示,该试验具有良好的敏感性,胶体金免疫层析试验对鲜奶、奶粉和饲料样品中三聚氰胺的最低检测量分别为1.0、2.0和2.5μg/g,该法适合现场快速检测三聚氰胺。     

Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol

A novel immunochromatographic assay (ICA) was proposed for rapid and multiple assay of β₂-agonists, by utilizing ractopamine (RAC) and salbutamol (SAL) as the models. Owing to the introduction of chemiluminescent (CL) approach, the proposed protocol shows much higher sensitivity. In this work, the described ICA was based on a competitive format, and horseradish peroxidase-tagged antibodies were used as highly sensitive CL probes. Quantitative analysis of β₂-agonists was achieved by recording the CL signals of the probes captured on the two test zones of the nitrocellulose membrane. Under the optimum conditions, RAC and SAL could be detected within the linear ranges of 0.50–40 and 0.10–50 ng mL⁻¹, with the detection limits of 0.20 and 0.040 ng mL⁻¹ (S/N = 3), respectively. The whole process for multianalyte immunoassay of RAC and SAL can be completed within 20 min. Furthermore, the test strip was validated with spiked swine urine samples and the results showed that this method was reliable in measuring β₂-agonists in swine urine. This CL-based multianalyte test strip shows a series of advantages such as high sensitivity, ideal selectivity, simple manipulation, high assay efficiency and low cost. Thus, it opens up new pathway for rapid screening and field analysis, and shows a promising prospect in food safety.     

Antigen detection based on background fluorescence quenching immunochromatographic assay

Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100 ng mL⁻¹ of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1 ng mL⁻¹. Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r = 0.98, p < 0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases.     

Development of a one-step immunochromatographic strip test for the rapid detection of nevirapine (NVP), a commonly used antiretroviral drug for the treatment of HIV/AIDS

Currently, high-performance liquid chromatographic (HPLC) methods are mainly used to measure antiretroviral plasma concentrations in HIV-infected patients. Although the utility of routine therapeutic drug monitoring (TDM) as an additional tool to optimize long-term antiretroviral therapy is unclear, if TDM is to be widely used, the availability of simple, cheap and reliable methods for the measurement of antiretroviral drug levels are needed, particularly in resource-limited settings. In this study, an immunochromatograhic (IC) strip test to detect the presence of nevirapine (NVP) in body fluids has been developed. Antiserum to NVP was first raised in rabbits by immunization against NVP chemically conjugated with bovine serum albumin, and subsequently validated by Western immunoblotting and competitive indirect ELISA. The partially purified anti-NVP antibodies were conjugated with colloidal gold particles. The conjugation of the colloidal gold and polyclonal antibodies was monitored by UV-vis spectroscopy, while transmission electron microscopy images were used to characterize the particle size and shape of the conjugates. The resulting colloidal gold conjugates were used for the production of an IC strip test to detect nevirapine in human plasma. Preliminary assessment suggests no-cross reactivity of the NVP polyclonal antibodies but assessment of plasma samples from HIV-infected patients receiving HAART needs to be conducted. This assay could potentially be used for drug monitoring as part of the clinical care of HIV infected patients.     

Nano-gold capillary immunochromatographic assay for parvalbumin

A novel non-instrumental bioanalysis based on colloidal-gold immunochromatography in a modified glass capillary was developed and named capillary immunochromatographic assay (CICA). In this report, glass capillary was proposed as a support in immunochromatographic assay because of its excellent characteristics. Goat anti-rabbit IgG and parvalbumin (PV) were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively. The CICA was constructed, and main variables for the performance were optimized. Using an important allergen of fish products (parvalbumin, PV) as the target, the analytical efficiency of the developed technique was investigated and the visual detection limit (VDL) and semi-quantitative limit of detection (LOD) were estimated to be 70 ng mL^?1 and 40 ng mL^?1, respectively. The coefficient of variation (CV) for the intra-assay and inter-assay was calculated for the PV concentration of 50 ng mL^?1, and the entire operation, including sample preparation, was consistently performed in 30 min. The developed technique was implemented and validated with different foodstuffs, including Scophthalmus maximus (Linnaeus), surimi products, and livestock, confirming sufficient accuracy and precision of results and verifying the method to be efficacious. These results enabled us to propose CICA as a new and promising technique for simple, rapid, and on-site screening of PV in biological samples. Graphical abstract

The scheme of the CICA system: (a) the control zone and test zone on the capillary, (b) negative results and (c) positive results