Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

A fluorescence polarization immunoassay(FPIA) for the determination of salinomycin(SAL) was developed by using anti-SAL monoclonal antibodies(mAb).Fluorescein labeled SAL(tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography(TLC).The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit(LOD) of 0.08 ng/mL.No significant cross-reactivities were observed with other drugs but 67.6%...     

量子点标记荧光免疫分析测定雌三醇

采用包被雌三醇完全抗原竞争免疫分析模式,以量子点标记羊抗兔抗体为荧光探针,建立了一种测定雌三醇的量子点标记的荧光免疫分析新方法.方法测定雌三醇的线性范围为0.01～10 000μg/L;检出限为0.007 5μg/L.此法用于尿液样品的分析,并利用高效液相色谱法进行对照,测定结果一致。     

Fluorescence quenching immunoassay performed in an ionic liquid

We describe the first example of immunoanalysis performed within an ionic liquid with minimal deleterious effect; our results bode well for the development of second-generation biosensors, particularly in applications involving poorly water soluble analytes including pesticides, phospholipids, and illicit drugs.     

Enzyme immunoassay of angiotensin II in human plasma.

We established a highly sensitive double-antibody enzyme immunoassay (EIA) for angiotensin (ANG) II. For competitive reactions, the ANG II-antibody was incubated with ANG II standard (or sample) and beta-D-galactosidase-labeled ANG III (delayed addition). Free and antibody-bound labeled antigen were separated using an anti-rabbit immunoglobulin G (IgG) coated immunoplate. The enzyme activity on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 72 fmol/well of ANG II. Using the present EIA, ANG II-like immunoreactivity (-LI) in human plasma was determined. The level of ANG II-LI in human plasma from 10 healthy volunteers was 33.3 +/- 10.4 pmol/l.     

Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine

A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer, has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and 5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min. The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation (r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for 70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for white and rosé wines, which are known to contain less interfering compounds such as polyphenols.     

Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue

Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL–DTAF) in the optimized FPIA with antibody IgY₁₀₀ demonstrated an IC₅₀ value of 12 ng mL^?1 in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 μg kg^?1) were higher than 79 % with coefficients of variation (CVs) lower than 12 %. The limit of detection (LOD) of the FPIA in swine tissue was 26 μg kg^?1 and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.

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Graphical Abstract FPIA based on IgY for rapid detection of Valnemulin in swine tissue

     

Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat

Analytical and Bioanalytical Chemistry - Fluorescence polarization immunoassays (FPIAs) for thiabendazole and tetraconazole were first developed. Tracers for FPIAs of thiabendazole and...     

Fluorescence signal transduction mechanism for immunoassay based on zinc ion release from ZnS nanocrystals

In this work, a fluorescence signal transduction mechanism based on cation release from ZnS nanocrystals was developed for sandwich immunoassay. In this mechanism, ZnS nanocrystals as labels in immunoassay are dissolved by acid to release zinc ions. After pH adjustment of the dissolving solution using a basic solution, zinc-ion sensitive fluorescence indicator Fluozin-3 is added to bind with the released zinc ions for sensitive fluorescence measurement. Using mouse IgG as a model analyte, the immunoassay adopting this signal transduction mechanism demonstrates a low detection limit around 1 pM and a detection range with two orders of magnitude (1 pM to 0.5 nM).     

非标记铕配合物荧光免疫分析法研究: 测定血清中的金属硫蛋白

本文报告一种新型非同位素免疫分析法, 非标记稀土铕配合物荧光免疫分析。它仍以免疫反应为基础, 所不同的是无须制备标记物。较标记免疫分析法简便、快速、在所建立的方法中, 金属硫蛋白(MT)的量与Eu-β-二酮配合物的荧光强度成负相关。测定范围为0-1.3μg/mL, 测定血清中MT的回收率为95%-100%, 相对标准偏差<10%。     

Fluorescence immunoassay based on alkaline phosphatase-induced in situ generation of fluorescent non-conjugated polymer dots

Alkaline phosphatase (ALP) activity assay is not only significant to the clinical diagnosis of some related disease, but also momentous to the construction of ALP-based enzyme-linked immunosorbent assay (ELISA). Herein, for the first time, we have discovered that ascorbic acid (AA) can specially react with N-methylethylenediamine (N-MEDA) to generate fluorescent non-conjugated polymer dots (NCPDs) under mild conditions. On the basis of the AA-responsive emission and ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to AA, we have exploited a fluorometric ALP activity assay with high sensitivity and selectivity. Furthermore, by means of conventional ALP-based ELISA platform, a conceptual fluorescent ELISA has been constructed and applied in the potential clinical diagnosis, during which cardiac troponin I (cTnI), a well-established biomarker of acute myocardial infarction, has been chosen as the model target. We envision that such original fluorescent NCPDs generation-enabled ELISA could become a versatile tool in biochemical sensing and medical diagnosis in the future.     

Washing-free chemiluminescence immunoassay for rapid detection of cardiac troponin Ⅰ in whole blood samples

Chemiluminescence immunoassay(CLⅠA) has always been a great challenge in detecting cardiac troponin Ⅰ(c Tn Ⅰ) in whole blood samples without centrifugation because of the interference of red blood cells and low sensitivity. Ⅰn this study, the antigens and erythrocytes in the blood were captured by the antibodies immobilized on the magnetic particles, recognized by another biotinconjugated c Tn Ⅰ antibody and detected by streptavidin/acridine aster-conjugated polychloromethylstyrene microspheres(...     

Upconverting nanophosphors as reporters in a highly sensitive heterogeneous immunoassay for cardiac troponin I

Photon upconverting nanophosphors (UCNPs) have a unique capability to produce anti-Stokes emission at visible wavelengths via sequential multiphoton absorption upon infrared excitation. Since the anti-Stokes emission can be easily spectrally resolved from the Stokes' shifted autofluorescence, the upconversion luminescence (UCL) is a highly attractive reporter technology for optical biosensors and biomolecular binding assays – potentially enabling unprecedented sensitivity in separation-based solid-phase immunoassays.     

Novel fluorogenic substrates containing bimane system for microdetermination of angiotensin I converting enzyme

As a sensitive fluorometric assay for the activity of angiotensin converting enzyme, bimane-peptides containing tryptophan, i.e., 1,7-dioxo-2,5,6-trimethyl-1H,7H-pyrazolo [1,2-a]pyrazol-3-yl-methylthiomethylcarbonyl-glycyl (or L-phenylalanyl)-L-tryptophyl-L-leucine (or L-proline), were synthesized and shown to be potent fluorogenic substrates for the micro-determination of angiotensin I converting enzyme activity.     

Fluorescence immunoassay of α-1-fetoprotein with hemin as a mimetic enzyme labelling reagent

A novel mimetic enzyme immunoassay to determine α-1-fetoprotein (AFP) in solution was developed. Hemin, a horseradish peroxidase substitute, was used as a labelling reagent to catalyze the reaction of p-hydroxyphenylacetic (HPA) and hydrogen peroxide in alkaline media. In the competitive immunoassay, monoclonal anti-AFP antibody was coated on a 96-well plate (polystyrene) and a constant amount of hemin-labelled AFP and a known volume of test solution were added. Non-labelled and hemin-labelled AFP compete for binding to the plate-bound antibody. After the immunoreaction, the immunochemically adsorbed hemin-AFP conjugate moiety was determined by measuring the fluorescence produced in a solution containing HPA and hydrogen peroxide. The calibration graph for AFP was linear over the range 0 ∼ 380 ng/ well with a detection limit of 1.0 ng/well. The method has been applied to determine the AFP in human blood serum with satisfactory results. Received: 21 January 1998 / Revised: 1 April 1998 / Accepted: 4 April 1998     

Chemilumiescence immunoassay for oxazepam

For the sensitive assay descibed, antiserum against oxazepam-3-hemisuccinate/bovine serum albumin is raised in rabbits. The chemiluminogenic N-(4-aminobutyl)-N-ethylisoluminol conjugate of oxazepam-3-hemisuccinate is used as tracer. The sample or standard is incubated with a suitable antiserum and chemiluminogenic tracer for 60 min. After separation (dextrancoated charcoal) of bound and free tracer, the chemiluminescence in the bound fraction is measured. As little as 0.9 pg of oxazepam could be oxazepam could be detected. In urine samples, down to 40 ng ml^?1 could easily be determined after HPLC separation.