Competitive Time-resolved Fluoroimmunoassay Research Articles

Biotin-labelled peptidomimetic for competitive time-resolved fluoroimmunoassay of benzothiostrobin.

In recent years, more and more functional peptide ligands have been identified from phage display libraries and served the immunoassay of small molecules. After the identification, the phage particle instead limits further application of peptide ligands, so it is of great significance to explore the peptide ligand as an independent detection reagent. In this work, the identified peptidomimetic of benzothiostrobin was synthesized and labelled with biotin, which wascombined with Eu3+-labelled streptavidin to develop the peptide-based time-resolved fluoroimmunoassay (P-TRFIA). Under the optimal conditions, the half-maximum inhibitory concentration (IC50) of proposed P-TRFIA is 3.63ngmL-1, which is similar to the TRFIA using phage-borne peptidomimetic and Eu3+-labelled anti-phage antibody (IC50: 4.55ngmL-1), also more sensitive than previously reported immunoassays for benzothiostrobin. In addition, the proposed P-TRFIA shows excellent specificity and accuracy for analysis of spiked samples, and its detection results shows good consistency with high-performance liquid chromatography for the detection of environment and agro-products samples with unknown benzothiostrobin concentrations.

Establishment of an ultrasensitive indirect competitive time-resolved fluoroimmunoassay for vancomycin determination

ABSTRACT Vancomycin (Van) is a common antibiotic that pollutes food, feed and ecological environment. In the present study, an anti-Van polyclonal antibodies (pAbs) and Eu3+-labeled goat anti-rabbit IgG monoclonal antibody based indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for ultrasensitive determination of Van, and its half-maximum inhibition concentrations (IC50) was 13.74 ng/L and the limit of detection (LOD) was 1.07 ng/L. The IC-TRFIA showed strong cross-reactivity (CRs) of 36.2% for norvancomycin, and less than 0.1% for polymyxin B, kanamycin and ampicillin. It was found that the IC-TRFIA for Van spiked in different milk powder samples determination were with good accuracy, stability and repeatability, and its recoveries are 83.0–97.9%, also with its coefficient of variations (CVs) are 3.0–12.9% in intra-assay and inter-assay. The results showed that the established IC-TRFIA was promising for ultrasensitive determination of Van in food samples. Highlights Prepared high-quality Van-KLH/BSA conjugates. Obtained high activity and purify anti-Van polyclonal antibodies. Prepared high-quality Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. Firstly established an IC-TRFIA for Van based on the anti-Van polyclonal antibodies and Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. The IC-TRFIA can be used for ultrasensitive determination of Van in milk powder samples.

Rapid and sensitive determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone by biotin–streptavidin-amplified time-resolved fluoroimmunoassay

A competitive time-resolved fluoroimmunoassay (TRFIA) using a biotin–streptavidin system was developed for the detection and quantification of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in aquatic tissues. AMOZ-bovine serum albumin was coated to a solid phase and then competed with a free nitrophenyl derivative of AMOZ (2-NP-AMOZ) in standards or samples for binding with biotin-anti-AMOZ-ovalbumin polyantibody. Later, the complex was recognized by europium-labelled streptavidin, which was used as a novel tracer to amplify the signal, and it also reacted faster than europium-labelled second antibody in traditional TRFIA. After optimizing the reaction conditions, the method showed high sensitivity and specification to 2-NP-AMOZ with a half maximal inhibitory concentration of 0.190 μg/l and a sensitivity of 0.019 μg/l with a working range from 0.025 to 10 μg/l. The data from fish and shrimp samples indicated that the limit of detection was 0.021 μg/kg, and the recovery rates were 84.1–107.0% and 80.9–98.4%, respectively, with an average RSD below 10%. High correlation rates were observed in TRFIA, high performance liquid chromatography and universal enzyme-linked immunosorbent assay methods. The experiment above confirms that biotin–streptavidin-amplified TRFIA could be an ultrasensitive and accurate tool for screening large numbers of aquatic products for the determination of AMOZ residues.

Development of a Competitive Time-Resolved Fluoroimmunoassay for Paclitaxel.

Paclitaxel (Tax) is a diterpene alkaloid isolated from Taxus species and has proved clinically effective in treating a number of malignancies. Current quantitative analytical methods for Tax such as high-performance liquid chromatography (HPLC) often involve complicated sample preparation procedures with low recovery rates. To establish a rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) for measuring Tax in Taxus materials with convenient sample preparation and a high recovery rate. Rabbit anti-mouse IgG was coated onto a 96-well microplate, which was then incubated with standard solutions of Tax and anti-Tax monoclonal antibody 3A3. A Eu3+ -labelled conjugate of Tax and human serum albumin was used as the tracer. The luminescent system was enhanced with a solution containing 2-naphthoyltrifluoroacetone. The established TRFIA showed a linear response within the Tax concentration range of 3.2 to 80ng/mL, with a limit of detection of 1.4ng/mL. The intra- and inter-assay coefficient of variation of the assay was 9.6% and 9.7%, respectively, with an average recovery rate from spiked samples of 108.5%. Tax contents in Taxus samples were determined using both the established TRFIA system and a previously established enzyme-linked immunosorbent (ELISA), and the results of two assays were well correlated. This TRFIA system shows a high sensitivity, precision and accuracy for detection of Tax. This assay, which is convenient and less time-consuming, allows rapid analysis of Tax and provides another option for Tax measurement for quality control of Taxus materials and products. Copyright © 2017 John Wiley & Sons, Ltd.

Time-resolved immunoassay based on magnetic particles for the detection of diethyl phthalate in environmental water samples

Diethyl phthalate (DEP) is an extensively used phthalic acid diester (PAEs) with estrogenic activity and the potential for carcinogenic and teratogenic effects. To monitor trace DEP in environmental waters, a sensitive direct competitive time-resolved fluoroimmunoassay based on magnetic particles (MPs) as solid support was established. For the assay system, the anti-DEP antibody was oriented on the surface of the MPs using goat anti-rabbit antibody as linkers, and DEP-OVA was labeled using Eu3+. Several physicochemical factors that potentially influence the assay performance of the proposed method were investigated in detail, including concentration of MPs, dilution of DEP-OVA-Eu3+ and incubation time. Under the optimized conditions, the method showed: (i) low limit of detection (LOD) of 5.92ng/L; (ii) satisfactory accuracy (recoveries, 91.97–134.54%) with good reproducibility (inter-CV, 4.17–9.17%; intra-CV, 7.41–14.72%). All of which indicated that the newly established method had much higher efficiency and great potential for use in environmental water analysis for DEP. In addition, the proposed immunoassay was applied for investigation of DEP in aquatic environments at Zhenjiang City. Our results showed that DEP was detected at the concentration of 2.98–65.18ng/mL in river samples and 46.95–306.19ng/mL in wastewater treatment plants (WWTPs), which showed rather high concentrations compared with reported data. Our study provides background data important for risk assessment and contamination control of DEP in the aquatic environment of this area.

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08–6.44 ng mL−1 and the medium inhibition of control (IC50) was 0.73 ng mL−1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%–96.6% and with a coefficient of variation (CV) among 2.0%–8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.

Highly efficient detection of paclobutrazol in environmental water and soil samples by time-resolved fluoroimmunoassay

A fast and ultrasensitive indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed for the analysis of paclobutrazol in environmental water and soil samples. Paclobutrazol hapten was synthesized and conjugated to bovine serum albumin (BSA) for producing polyclonal antibodies. Under optimal conditions, the 50% inhibitory concentration (IC50 value) and limit of detection (LOD, IC20 value) were 1.09μgL−1 and 0.067μgL−1, respectively. The LOD of TRFIA was improved 30-fold compared to the already reported ELISA. There was almost no cross-reactivity of the antibody with the other structural analogues of triazole compounds, indicating that the antibody had high specificity. The average recoveries from spiked samples were in the range from 80.2% to 104.7% with a relative standard deviation of 1.0–9.5%. The TRFIA results for the real samples were in good agreement with that obtained by high-performance liquid chromatography analyses. The results indicate that the established TRFIA has potential application for screening paclobutrazol in environmental samples.

Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix).

The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu3+-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 μg/mL for standard SSa sample with a detection limit of 0.006 μg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.

Development of a time-resolved fluoroimmunoassay for salmon insulin-like growth factor binding protein-1b

In salmon plasma/serum, three major insulin-like growth factor binding proteins (IGFBPs) are consistently detected at 22-, 28- and 41-kDa. The 22-kDa form has been identified as IGFBP-1b and shown to increase under catabolic conditions. We developed a competitive time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1b. Purified salmon IGFBP-1b was used for biotin-labeling, assay standard and antiserum production. The TR-FIA did not cross-react with the 41-kDa form (IGFBP-2b) but showed 3% cross-reactivity with the 28-kDa form (IGFBP-1a). It measured IGFBP-1b levels as low as 0.4ng/ml, and ED80 and ED20 were 0.9 and 24.6ng/ml, respectively. There appears to be little interference by IGF-I. Using the TR-FIA, serum IGFBP-1b levels were measured in individually-tagged underyearling masu salmon fed or fasted for 5weeks, or fasted for 3weeks followed by refeeding for 2weeks. Fasting for 3weeks significantly increased circulating IGFBP-1b levels, while it returned to the basal levels after prolonged fasting for additional 2weeks. Serum IGFBP-1b level negatively correlated with body weight, condition factor, specific growth rate and serum IGF-I level. During parr–smolt transformation of masu salmon, average circulating IGFBP-1b levels were the highest in May. There was a positive correlation between serum IGFBP-1b and IGF-I, which is in contrast to that in the fasting/feeding experiment. IGFBP-1b also showed a positive relationship with gill Na+, K+-ATPase activity. These results suggest that the relationship between circulating IGFBP-1b and IGF-I during smoltification differs from that during fasting and IGFBP-1b may play a role in the development of hypoosmoregulatory ability.

A new sensitive method for the detection of chloramphenicol in food using time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TRFIA) technique was developed to detect chloramphenicol (CAP) contamination in food. By using CAP-ovalbumin, anti-CAP antibodies, and europium-labelled goat anti-rabbit antibodies, an indirect, competitive method for CAP-TRFIA was established. The sensitivity was high, with a detection limit of 0.008 μg/L (8 ppt) for indirect competitive TRFIA formats. Testing showed that the sensitivity of the technique was 2.67 μg/kg in honey, prawn, and chicken muscle tissues, and 0.32 μg/L in milk. The detection range was between 0.008 and 100 μg/L: within this, the intra- and inter-batch coefficients of variation of the CAP-TRFIA method were 6.8 and 13.5 %, respectively. The study suggested that CAP-TRFIA was a simple, sensitive, and cost-effective method of screening large quantities of samples and had good prospects for further application.

Sensitive time-resolved fluoroimmunoassay for quantitative determination of clothianidin in agricultural samples

Europium (Eu(3+))-labeled antibody was used as a fluorescent label to develop a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for determination of clothianidin residues in agricultural samples. Toward this goal, the Eu(3+)-labeled polyclonal antibody and goat anti-rabbit antibody were prepared for developing and evaluating direct competitive TRFIA (dc-TRFIA) and indirect competitive TRFIA (ic-TRFIA). Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) of clothianidin were 9.20 and 0.0909μg/L for the dc-TRFIA and 2.07 and 0.0220μg/L for the ic-TRFIA, respectively. The ic-TRFIA has no obvious cross-reactivity with the analogues of clothianidin except for dinotefuran. The average recoveries of clothianidin from spiked water, soil, cabbage, and rice samples were estimated to range from 74.1 to 115.9%, with relative standard deviations of 3.3 to 11.7%. The results of TRFIA for the blind samples were largely consistent with gas chromatography (R (2) = 0.9902). The optimized ic-TRFIA might become a sensitive and satisfactory analytical method for the quantitative monitoring of clothianidin residues in agricultural samples.

Time-resolved fluoroimmunoassay for quantitative determination of thiacloprid in agricultural samples

A fast and sensitive indirect competitive time-resolved fluoroimmunoassay (TRFIA) for thiacloprid is developed. Europium-labeled goat anti-rabbit antibody was used as the tracer. Under optimal conditions, the TRFIA showed a 50% inhibition concentration (IC50) value of 2 μg L−1 with a limit of detection (LOD, IC10) of 1.9 ng L−1. The recoveries from agricultural samples, including water, soil, pear and tomato, were in the range from 83.4% to 121% with a relative standard deviation of 2.40–11.3%. The TRFIA results for real soil samples were confirmed by high performance liquid chromatography with a high correlation coefficient of 0.9854. This analytical method demonstrates good sensitivity and stability, and its potential for practical applications.

Increased plasma concentrations of soy isoflavones by increased doses of soy isoflavones are irrelevant to lipid peroxidation and aberrant crypt foci formation in colonic cancer model rats

We have investigated whether soy isoflavones supplemented to diets increase plasma concentrations of soy isoflavones and equol, and whether increased plasma concentration of soy isoflavones suppresses tissue levels of lipid peroxides and aberrant crypt foci (ACF) formation in colon cancer model rats. Sprague Dawley male rats were injected with 1,2‐dimethylthydrazine (DMH) and given one of AIN93‐based experimental diets (0, 10, 50, 150, or 500 mg isoflavones per kg diet) for 12 weeks. Thiobarbituric acid reactive substances (TBARS) of plasma and homogenates of liver and kidneys were determined using TBARS assay kit. Plasma concentrations of genistein, daidzein and equol were determined using kits by competitive time‐resolved fluoroimmunoassay technique. Plasma concentrations of genistein (R2=0.521 by genistein intake, p&lt;0.001), daidzein (R2=0.623 by daidzein intake, p&lt;0.001) and equol (R2=0.528 by daidzein intake, p&lt;0.001) were increased dose‐dependently. However, the numbers of ACF decreased significantly (p&lt;0.01) in 10 or 50 mg isoflavones‐fed groups as compared with the control group, and there was no significant correlation between the number of ACF and plasma concentration of genistein, daidzein or equol. Plasma TBARS level was not influenced by either dose or plasma level of soy isoflavones. Hepatic levels of TBARS were significantly lower in 10 and 50 mg isoflavones‐fed groups as compared with the control group. The level of kidney TBARS was significantly lower in only 50 mg isoflavones‐fed group as compared with the control group. Therefore, the results indicate that optimal level of soy isoflavones seemed to be most effective in suppressing ACF formation and lipid peroxidation in DMH‐induced colon cancer model rats.

Time-Resolved Fluoroimmunoassay for 19-Nortestosterone Residues in Aquaculture Tissues

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of 19-nortestosterone (17beta-NT) residues in aquaculture tissues. The limit of detection (LOD) was determined to be 0.08 ng g-1 and the limit of quantification (LOQ) was less than 0.8 ng g-1. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS). This proposed technique could be applied to routine residue analysis.

Rapid Determination of Time-Resolved Fluoroimmunoassay for Medroxyprogesterone Acetate Residues in Pork Tissues and Comparison with Liquid Chromatography and Tandem Mass Spectrometry

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of medroxyprogesterone acetate (MPA) residues in pork tissues. The limits of detection (LOD) was determined to be 0.06 ng g-1 and the limits of quantification (LOQ) was less than 0.8 ng g-1. The intra-assay variations were below 10% and the interassay variations ranged between 9.7 and 12.7%. The mean recoveries established at six concentration levels varied from 87.3 to 108.3%. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred pork tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.

Comparison of the determination of chloramphenicol residues in aquaculture tissues by time-resolved fluoroimmunoassay and with liquid chromatography and tandem mass spectrometry

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of chloramphenicol (CAP) residues in aquaculture tissues based on monoclonal antibodies. The limits of detection (LOD) were determined to be 0.04 ng g−1 and the limits of quantification (LOQ) were less than 0.15 ng g−1. The intra-assay variations were below 10% and the interassay variations ranged between 9.7 and 13.3%. The mean recoveries established at six concentration levels varied from 83.7–109.6% and the coefficient of variation was from 8.3–11.5%. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.

RETRACTED: Rapid time-resolved fluoroimmunoassay for diethylstilbestrol residues in chicken liver

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of diethylstilbestrol (DES) residues in chicken liver. Prior to analysis, the residues were extracted from chicken liver with acetonitrile. The assay could be used in a quantitative or qualitative mode. The limit of detection (LOD) was determined to be 0.05 ngg(-1), and the limit of quantification (LOQ) was less than 0.18 ngg(-1). The intraassay variations were less than 10%, and the interassay variations were from 9.8 to 12.7%. The mean recoveries established at six concentration levels varied from 84.3 to 109.6%, and the coefficients of variation were from 8.3 to 12.4%. The results obtained by the TR-FIA and enzyme-linked immunosorbent assay (ELISA) showed a good correlation. The established TR-FIA was validated for the determination of incurred chicken liver and was confirmed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). This proposed technique could be applied to routine residue analysis.

Determination of ochratoxin A by polyclonal antibodies based sensitive time-resolved fluoroimmunoassay

Using indirect competitive time-resolved fluoroimmunoassay (TRFIA), a rapid, highly selective and extremely sensitive method has been established for the determination of ochratoxin A (OA). Tests can be performed in a 96-well microplate using the toxin-specific polyclonal antibodies, obtained from rabbits immunized with ochratoxin A-keyhole limpet's hemocyanin (OA-KLH). In indirect TRFIA format, ochratoxin A-bovine serum albumin conjugate (OA-BSA) is coated onto the microtitre plate and incubated with standard toxin (samples) and anti-OA antibody. A goat anti-rabbit IgG Eu(3+) conjugate is used to enable the detection. The suitability of the assay for quantification of OA is also studied and samples are determined by OA-TRFIA using autoDELFIA1235 system. The results show that the polyclonal antibodies can be used at a dilution exceeding 1:8,000 and the OA detection limit is 0.02 microg/l for indirect competitive TRFIA formats. The 80, 50, and 20% inhibition binding effect dose (ED80, ED50, ED20) of OA were 0.195, 1.018, and 5.314 microg/l, respectively. The assay ranges from 0.02 to 400 microg/l. The cross reactivity with ochratoxin B is 5.6% and antibodies do not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OA-TRFIA are 2.6 and 5.2%, respectively. The mean recoveries from the OA-free cereals spiked with 1-200 microg of OA/kg of cereals sample were 95.8%. Both OA-TRFIA and OA-ELISA tests are applied for the quantitative measurement of OA in the same cereals, and the coefficient of correlation is 0.912. The results show that the novel TRFIA method can be applied to detect the OA contamination in cereals. It provides very high sensitivity and optimal range and will be useful to screen OA contamination easily, simply, and economically when the number of samples is large.

Rapid time-resolved fluoroimmunoassay for the screening of monensin residues in eggs

Polyether ionophores, such as monensin, are commonly used as feed additives in the poultry production. However, ionophore residues in foodstuffs may have adverse health effects on humans. In order to effectively implement residue control programmes in the European Union (Council Directive 96/23/EC), rapid analysis methods are required. A competitive time-resolved fluoroimmunoassay was developed for the determination of monensin residues in eggs. Prior to analysis, the residues were extracted from eggs with acetonitrile according to a simple protocol. The assay, which could be utilised in a quantitative or qualitative mode, was validated in compliance with Commission Decision 2002/657/EC. The detection capability of the assay was determined to be less than 2 μg kg −1. The intra-assay variations ( n = 6) were below 10% and the interassay variations ( n = 18) ranged between 10.3 and 13.9%. The mean recoveries ( n = 6), established at three concentration levels, varied from 88.0 to 102.1%.

Synthesis of novel europium-labeled estradiol derivatives for time-resolved fluoroimmunoassays.

The O-(5-carboxypentyl)-, O-(4-aminobutyl)-, O-(6-aminohexyl)oximes of 2- and 4-formylestradiol as well as the 4-carboxyethylthioether derivative of estradiol were synthesized. These estradiol derivatives were characterized using IR-, 1H-, and 13C NMR spectroscopy. All synthesized estradiol derivatives were labeled with europium chelates. These "tracers" were purified and tested in a competitive time-resolved fluoroimmunoassay with monoclonal antibody (SSI 57-2) raised against the 6-O-(carboxymethyl)oxime-bovine serum albumin derivative of estradiol. All the tested europium-labeled estradiol-4-derivatives were bound by the antibody, whereas tracers linked via position 2 were not recognized by this antibody. It was observed that tracers conjugated via C-4 gave more sensitive standard curves than tracers conjugated via C-6. Especially, the estradiol-4-thioether derivative was found to be highly useful in time-resolved fluoroimmunoassays of estradiol while using this antibody.