Fast purification of trace vitellogenin from Chinese rare minnow using protein A-immobilized antibody

Vitellogenin (Vtg) is a highly responsive biomarker for environmental exposure to various estrogenically active compounds. Here we present a simple, fast, mild, and stable immobilization of anti-Vtg antibody, and demonstrate its powerful applications for preconcentration and purification of fish Vtg proteins, allowing for the monitoring and screening of environmental exposure to estrogenically active compounds. In this immobilization method, rabbit antiserum containing a specific polyclonal antibody against Vtg was directly immobilized on an antibody-binding Staphylococcal protein A matrix (SpA) without the need for prior purification. Under the unique elution conditions, the Vtg protein can be eluted out alone without any leaked specific antibody. The developed method was further used to purify Vtg from whole-body homogenate of Chinese rare minnow. Compared with previous purification methods, the isolated Vtg fraction by this method displays higher purity and well-preserved structure integrity. Moreover, our method is eight times faster. The simple one-step protein A-based specific antibody immobilization and its associated elution strategy may be extended to a number of antibodies for various application purposes, highlighting the paramount advantages over traditional immunoprecipitation and covalent immobilization of antibodies, and suggesting a wide range of promising applications in environmental monitoring and proteome analysis.     

A simple and sensitive high‐performance liquid chromatography–electrochemical detection assay for the quantitative determination of monoamines and respective metabolites in six discrete brain regions of mice

A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4‐dihydroxybenzlyamine hydro‐bromide in mouse brain homogenate using high‐performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C₁₈ column. The method was accurate over the linear range of 0.300–30 ng/mL (r = 0.999) for dopamine and 0.300–15 ng/mL (r = 0.999) for norepinephrine, 3,4‐dihydroxybenzlyamine hydro‐bromide, homovanillic acid and 5‐hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625–30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.     

Monoclonal antibody-based enzyme-linked immunosorbent assays for the detection of the organophosphorus insecticide diazinon

Four haptens of the organophosphorus (OP) insecticide diazinon were synthesized to develop enzyme-linked immunosorbent assays (ELISAs) for this pesticide. One of them was conjugated to KLH to be used as the immunogen for production of monoclonal antibodies. By using the antibodies and a coating antigen, an indirect competitive ELISA was developed, which showed an IC₅₀ of 4.0 ng/mL with a detection limit of 0.7 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 6.0 ng/mL with a detection limit of 0.9 ng/mL. The antibodies in both assays showed negligible cross-reactivity with metabolites of diazinon and other OP pesticides. Recovery of diazinon from fortified lettuce and rice samples was satisfactory except at the fortified concentration of 100 ppb.     

Determination of levofloxacin (the Levorotatory Stereoisomer of Ofloxacin) in milk by an indirect enzyme-linked immunosorbent assay

In this work we obtained polyclonal antibodies for levofloxacin. We synthesized conjugates of levofloxacin with cationized BSA for immunization and with ovalbumin for development of an ELISA for the detection of levofloxacin. The method is characterized by a range of detectable concentrations of 0.03 ng/mL to 0.41 ng /mL and the limit of detectable concentrations is 0.01 ng/mL. We tested the 28 fluoroquinolones for cross reactivity and only ofloxacin (145%), marbofloxacin (82%), ofloxacin in its dextrorotatory form (68%), rufloxacin (67%), and garenoxacin (24%) had cross reactivity. The optimized ELISA technique allowed the detection of levofloxacin in milk from 0.33 ng/mL to 3.34 ng/mL. The recoveries were in the range of 89.5–102% with a relative standard deviation of 3%. We tested 45 real samples of milk purchased in local stores; for 5 of them the results were positive (near 1 ng/mL).     

Comparsion of an immunochromatographic strip with ELISA for simultaneous detection of thiamphenicol,florfenicol and chloramphenicol in food samples

Rapid and sensitive indirect competitive enzyme‐linked immunosorbent assays (ic‐ELISA) and gold nanoparticle immunochromatographic strip tests were developed to detect thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in milk and honey samples. The generic monoclonal antibody for TAP, FF and CAP was prepared based on a hapten [D ‐threo‐1‐(4‐aminophenyl)‐2‐ dichloroacetylamino‐1,3‐propanediol], and the haptenwas linked to a carrier protein using the diazotization method. After the optimization of several parameters (coating, pH, sodium chloride content and methanol content), the ic‐ELISA was established. The quantitative working range for TAP was 0.11–1.36 ng/mL, with an IC₅₀ of 0.39 ng/mL. The optimized ELISA showed cross‐reactivity to CAP (300%) and FF (15.6%), with IC₅₀ values of 0.13 and 2.5 ng/mL, respectively. The analytical recovery of TAP, FF and CAP in milk and honey samples in the ic‐ELISA ranged from 81.2 to 112.9%. Based on this monoclonal antibody, a rapid and sensitive immunochromatographic test strip was also developed. This strip had a detection limit of 1 ng/mL for TAP, FF and CAP in milk and honey samples. Moreover, the test was completed within 10 min. Our results showed that the proposed ic‐ELISA and immunochromatographic test strip method are highly useful screening tools for TAP, FF and CAP detection in milk and honey samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of haptens of organophosphorus pesticides and development of immunoassays for fenitrothion

A simple synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (aminocarboxylic acid) attached at the pesticide thiophosphate group was developed. While the previous synthetic approach for this type of haptens requires seven steps, the present method involves only two steps. Using this method, five haptens of fenitrothion were synthesized and two of them were conjugated to proteins to be used as immunogens for production of polyclonal antibodies. Using the antibodies and a coating antigen, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for fenitrothion was developed, which showed an IC₅₀ of 3.7 ng/mL with a detection limit of 0.5 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 5.6 ng/mL with a detection limit of 0.4 ng/mL. The antibodies in both assays showed negligible cross-reactivity with other OP pesticides except with the insecticide parathion-methyl only in the direct ELISA. Recoveries of fenitrothion from fortified lettuce and rice samples ranged 84-116 and 100-121%, respectively.     

Development of an Ultra-sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Diethylstilbestrol in Seafood

An ultra-sensitive indirect competitive chemiluminescence enzyme immunoassay was developed for screening diethylstilbestrol in fish and shrimp samples. The concentration of diethylstilbestrol that caused 50% inhibition of the binding enzyme marker (IC₅₀) was 0.32 ng/mL and the limit of detection was 0.0068 ng/mL; the linear range was from 0.028 ng/mL to 3.60 ng/mL. The assay showed cross-reactivity of 7.1% and 2.8% with dienestrol and hexoestrol, respectively, but negligible cross-reactivity with estradiol, estrone, ethinyloestradiol, and progestin. The recovery from spiked fish and shrimp samples varied from 68.5% to 92.5%, and the mean coefficients of variation within groups and between groups were 6.2% and 8.0%, respectively. Our results indicated that the assay is a simple, sensitive, specific, and accurate method for screening fish and shrimp samples for diethylstilbestrol.     

Simultaneous quantitation of testosterone,estradiol, ethinyl estradiol,and 11‐ketotestosterone in fathead minnow fish plasma by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

In the present work, for the first time, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry (LC/APPI‐MS/MS) method has been developed and validated for the simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11‐ketotestosterone in fathead minnow fish plasma using no more than 10 µL of plasma. Compounds present in plasma were directly derivatized with dansyl chloride and 25 µL of the derivatized mixture was injected into the LC/APPI‐MS/MS system. The gradient chromatographic elution was achieved on an Agilent Zorbax SB‐C18 analytical column (2.1 mm × 50 mm, 1.8 µm particle size) with mobile phases consisting of acetonitrile, water and acetic acid. The flow rate was 0.5 to 0.7 mL/min and the total run time was 11.5 min. The lower limits of quantitation for testosterone, estradiol, ethinyl estradiol, and 11‐ketotestosterone and were 1, 1, 1, and 2.5 ng/mL, respectively. Intra‐batch precision was less than 19.4% and inter‐batch precision was less than 11.7% for all four analytes. Accuracy was within 83.5–115.4% of nominal concentrations. This method is used for quantitation of sex steroid levels in fathead minnow tested in endocrine disruptor screening experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

高效液相色谱法与酶联免疫试剂盒测定海水中痕量氯霉素

详细报道了海水样品中氯霉素的高效液相色谱检测方法及酶联免疫试剂盒检测方法。应用高效液相色谱法测定海水中氯霉素,方法的线性范围为5～50ng,检出限为0．064ng,回收率为96．2％-118．4％,相对标准偏差小于8．9％。酶联免疫试剂盒测定海水中的氯霉素背景值偏高,在加标浓度为10ng／mL时回收率为101．0％,相对标准偏差为8．32％。     

Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study

A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.     

芯片液相色谱-质谱法高灵敏度检测水产品中微囊藻毒素

建立了一种芯片液相色谱-高分辨质谱测定3种微囊藻毒素（MC-RR、MC-YR和MC-LR）的分析方法.在最优条件下,3种藻毒素在16 min内实现基线分离,并选择电喷雾源正离子模式进行质谱测定.方法在2.5~2 000 ng/mL范围内线性良好,线性相关系数大于0.996 5,检测限介于0.5~2.0 ng/mL之间.鱼肉和虾肉样品经固相萃取法净化处理后未测得上述毒素残留,加标回收率为82.8%~120.3%.方法具有样品用量少、定性快速准确、灵敏度高等优点,适合用于水产品等生物样品中藻毒素的痕量监测.     

Determination of Meldonium in human urine by HPLC with tandem mass spectrometric detection

A procedure is proposed for determining Meldonium in human urine, including sample preparation to analysis and analyte determination by HPLC with tandem mass spectrometric detection. For sample preparation, the procedure of “dilute-and-shoot” was used. The lower limit of the analytical range is 10 ng/mL; the limit of detection is 7.5 ng/mL; and the linearity range is 10–250 ng/mL. The proposed procedure is tested on real samples obtained from volunteers. A possibility of the direct analysis of urine samples after dilution is demonstrated; the limit of detection is 20 ng/mL. The high sensitivity of the procedure ensures its use for the determination of Meldonium in clinical diagnosis and doping control.     

Analytical evaluation of enzyme-linked immunosorbent assay for neonicotinoid dinotefuran for potential application to quick and simple screening method in rice samples

The analytical performance of a kit-based enzyme-linked immunosorbent assay (ELISA) for the determination of a neonicotinoid insecticide dinotefuran residue in rice samples is addressed. The sensitivity (I₅₀ value) was 5.4 ng/mL, with the limit of detection, 0.6 ng/mL and the dynamic range from 1.0 to 30 ng/mL. The ELISA showed substantially high specificity toward dinotefuran besides clothianidin (184%). For rice samples, dinotefuran was extracted with methanol and the extracts were directly determined with the ELISA because of no significant matrix interference. Good recoveries were observed and ranged from 92.5% to 113.2% with coefficients of variation below 10%. The results obtained with the ELISA correlated well with those by the HPLC method for rice samples (r > 0.98). These findings strongly indicate that the evaluated and validated ELISA has a potential utility in a quick, simple, and reliable residue analysis, especially a screening method before shipment contributing to food safety.     

Optimization of large volume injection-programmable temperature vaporization-gas chromatography-mass spectrometry analysis for the determination of estrogenic compounds in environmental samples

Large volume injection-programmable temperature vaporization-gas chromatography-mass spectrometry (LVI-PTV-GC-MS) was optimized for the determination of estrone (E1), 17β-estradiol (E2), 17α-ethynyl estradiol (EE2), mestranol (MeEE2) and estriol (E3) for their determination in environmental samples (estuarine water, wastewater, fish bile and fish homogenate) after derivatization with 25 μL (BSTFA+1% TMCS) and 125 μL of pyridine. Experimental designs such as Plackett-Burman (PBD) and central composite designs (CCDs) were used to optimize the LVI-PTV variables (cryo-focusing temperature, vent time, vent flow, vent pressure, injection volume, purge flow to split vent, splitless time and injection speed). Optimized conditions were as follows: 45 μL of n-hexane extract are injected at 60°C and 6 μL/s with a vent flow and a vent pressure of 50 mL/min and 7.7 psi, respectively, during 5 min; then the split valve is closed for 1.5 min and afterwards the injector is cleaned at 100 mL/min before the next injection. The method was applied to the determination of estrogenic compounds in environmental samples such as estuarine water, wastewater, and fish homogenate and bile. Limits of detection (0.04-0.15 ng/L for water samples, 0.04-0.67 ng/g for fish bile and 0.1-7.5 ng for fish homogenate) obtained were approx. ten times lower than those obtained by means of a common split/splitless inlet.     

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.     

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.     

Determination of 4- n -nonylphenol in water by enzyme immunoassay

A range of monoclonal antibody-based competitive immunoassays in the format of microtitre plate ELISA and dipstick tests for quantitative and semi-quantitative detection of 4- n -nonylphenol in water was developed. A simple visual dipstick test was based on changing of spot colour from green to brown in the presence of 4- n -nonylphenol at concentrations within the range 10-100 ng mL(-1). Two different detection systems were used for quantitative immunoassay. Application of enhanced chemiluminescence (ECL) resulted in an increase of the sensitivity of ELISA when compared to conventional colorimetric detection. Thus a detection limit of 0.06 ng mL(-1 )of 4- n -nonylphenol was achieved with IC(50) 2.0 ng mL(-1). The tests developed were applied to natural and spiked water samples.     

Label-free immunosensor for monitoring vitellogenin as a biomarker for exogenous oestrogen compounds in amphibian species

A label-free, optical waveguide lightmode spectroscopy based immunosensor was developed for frog (Bombina orientalis) vitellogenin (Vtg) determination in biological samples as a biomarker for exogenous oestrogen compounds. Antibody against Vtg was produced in rabbits immunised with purified lipovitellin (Lpv), a precursor of Vtg, from the homogenised ovary of oriental fire-bellied toads (B. orientalis). The purified protein and Lpv/Vtg-specific serum were applied in both competitive and direct immunoassay formats using optical waveguide lightmode spectroscopy immunosensor. When measuring Vtg in direct manner, the Lpv antibody (1.76 µg mL^?1) was immobilised on the sensor surface, and the linear measuring range for Vtg was 0.1–10 µg mL^?1. During the competitive measurement, 100 ng mL^?1 Lpv was applied for the immobilisation. The linear measuring range for Vtg was 0.5–50 ng mL^?1. We studied the relative substrate specificity of the antibody, and it was concluded that the method is suitable for the sensitive and selective determination of Vtg levels in toads. Heart, liver and gonad samples from male animals were spiked with Vtg and were analysed using the newly developed method, and female toads and spawn samples were tested and compared to the calibration curve obtained by the spiked samples.     

UPLC–TOF–MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction

The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC–TOF–MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5–1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.     

Stressed Kinetics of Nanoemulsion Formulation Encapsulated Ropinirole with a Validated Ultra High Performance Liquid Chromatography–Synapt Mass Spectrometry (UPLC‐MS/MS ESI‐Q‐TOF)

A highly sensitive ultra high pressure liquid chromatography (UHPLC‐MSMS) method for estimation of ropinirole in rat brain homogenate and plasma has been validated. The method was successfully used for the degradation kinetics in different stress condition and regulated temperature. The chromatographic separation was achieved using isocratic mobile phase, consisting of acetonitrile–2mM ammoniumacetate (28:72 v/v; 0.25 mL min^?1). The mass spectrometer was operated in synapt mass spectrometry mode via positive electrospray ionization using the transitions m/z 260 → m/z 261 for ropinirole, and m/z 324.39 → m/z 262.161 as a parent ion of escitalopram (IS). The assay for ropinirole was linear over the range of 0.5–100 ng mL^?1 (r²; 0.999). The intra‐ and inter day precisions were less than 11.2% in terms of relative standard deviation (R.S.D.), and the accuracy was within ±6.4% in terms of relative error (RE). The mean extraction‐efficiency of QC samples (MQC, 8 ng/mL) was ≥80%. The lower limit of quantification (LLOQ) was 0.049 ng/mL where as lower limit of detection (LLOD) was 0.016 ng/mL. All the peaks of degradation were well resolved. The degradation kinetics of ropinirole, showed highest stability (t_1/2 256.66/h; t_0.9, 39.11/h) in acidic medium, lower stability in alkaline environment (t_1/2, 103.43/h; t_0.9, 15.76/h) and highly susceptible in oxidative environment (t_1/2, 21.58/h; t_0.9, 3.28/h). The applicability of this assay was demonstrated and successfully applied for pharmacokinetic profiling of ropinirole in Wister rat brain homogenate after intranasal administration.