Production of Antibodies and Development of Specific Polarization Fluoroimmunoassay for Acetochlor

Specific polyclonal antibodies towards acetochlor (2-chloro- N -(ethoxymethyl)- N -(2-ethyl-6-methylphenyl)acetamide) were obtained from rabbits immunized against a 3-mercaptopropionic acid derivative of acetochlor, covalently attached to bovine serum albumin. A polarization fluoroimmuoassay (PFIA) based on these antibodies was developed and optimized to detect acetochlor in water samples. The optimized PFIA had a detection limit of 9 µg/L, linear working range from 50 to 5500 µg/L and within-assay coefficient of variation less than 4%. Cross-reactivity studies demonstrated that these antibodies are capable of specific detection of acetochlor amongst structurally related chloroacetanilide herbicides. Assay cross-reactivity values were: alachlor 0%, metolachlor 2.4%, propachlor 0%, butachlor 0.2% and dimethachlor 0.5%. Five organic solvents commonly used in sample extraction were evaluated for their effect on acetochlor PFIA performance, and methanol and ethanol were found to be compatible with the assay up to 10% v/v.     

Ultrasound‐assisted extraction and solid‐phase extraction for the simultaneous determination of five amide herbicides in fish samples by gas chromatography with electron capture detection

An efficient sample extraction and clean‐up method was developed for simultaneous determination of five amide herbicides (alachlor, acetochlor, propisochlor, metazachlor, and butachlor) in fish samples. The protocol consisted of ultrasound‐assisted solvent extraction and solid‐phase extraction clean‐up. In detail, aliquots of homogenized fish flesh were thoroughly mixed with 20 mL of n‐hexane and then extracted with ultrasonication for 40 min. Each sample was centrifuged and the supernatant was collected for the subsequent clean‐up. For the sample preparation, the above supernatant was processed with a C₁₈ column with 3 mL of dichloromethane/n‐hexane (1:1, v/v) as the eluant. Then the samples were analyzed by gas chromatography with electron capture detection. The correlation coefficients of the five calibration curves were 0.9976–0.9998 (n = 3). The limits of detection (S/N = 3, n = 11) and limits of quantification (S/N = 10, n = 11) were 0.19–0.42 and 0.63–1.39 μg/kg, respectively. The recoveries of this method were 71.2–92.6% with good precision (<4.7% relative standard deviations, n = 6). The developed method was successfully applied to monitor the five amide herbicides in fish samples collected from different cities.     

Monoclonal antibody-based enzyme-linked immunosorbent assay for the insecticide imidacloprid

An enzyme-linked immunosorbent assay (ELISA) was developed for the neonicotinoid insecticide imidacloprid, 1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine using monoclonal antibodies (MAb). Three MAbs, designated as E6A6, E6F3 and H7F7, were raised from mice immunized with an imidacloprid hapten-ovalbumin conjugate. These MAbs performed similarly in indirect competition ELISA (icELISA), so one, E6F3, was selected for detailed study. The equilibrium constants (K_d) and association and dissociation rate constants (k_on, k_off) for five neonicotinoids and one imidacloprid metabolite to E6F3 were determined by kinetic exclusion fluoroimmunoassay (KinExA). Affinities (1/K_d) of E6F3 for acetamiprid and clothianidin were similar, but 50-fold weaker than that of imidacloprid. MAb E6F3 had no measurable affinity for the other neonicotinoids. The icELISA can tolerate up to 15% (v/v) acetone or 20% (v/v) methanol. Assay sensitivity was similar at pH 4-9, 1-10-fold concentration of PBS with or without 0.05% Tween 20, and incubation times of 30-180 min. The half-maximal inhibition and the limit of detection were approximately 0.8 and 0.1 μg/l of imidacloprid in icELISA, and 0.3 and 0.03 μg/l in direct competition ELISA (dcELISA), respectively. Analysis of imidacloprid-fortified water and cucumber samples by the icELISA showed average recoveries from 70 to 120%.     

Development of Polarization Fluoroimmunoassay for the Detection of s-Triazine Herbicides

Abstract

A rapid, non-isotopic polarization fluoroimmunoassay (PFIA) for the monitoring of the simazine (striazine herbicide) level in water was developed. Polyclonal antiserum was raised in rabbits by immunization with simazine – Keyhole Limpet Haemocyanin conjugate. Sensitivity of the PFIA with the use of heterologous tracer with the shortest bridge between antigen and fluorescein proved to be the highest. All analytical criteria for PFIA were satisfied. The detection limit of simazine (3 ng/ml in 50 μl of sample) was comparable to that for liquid or gas chromatography method. The detection limit of ELISA using the same antiserum and conjugate derivative of atrazine with horseradish peroxidase was 0.1 ng/mL of simazine. The cross-reactivity for PFIA with widely used s-triazine herbicides: atrazine, propazine, terbuthylazine was 100%, 32% and 20%, respectively. The cross-reactivity for PFIA with some metabolites of s-triazines and other herbicides was negligible.     

Thin Layer Chromatographic Method for Rapid Quantification and Identification of Trimethoprim and Sulfamethoxazole in Pharmaceutical Dosage Forms

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of sulfamethoxazole (SMA) and trimethoprim (TMP) present in combined dosage forms were described. SMA and TMP were extracted with 90% acqueous methanol and the interfering and related contaminants were removed by thin layer chromatography (TLC) or high performance TLC (HPTLC) on silicagel plates using chloroform : isopropanol : diethylamine :: 10 : 6 : 1 (v/v) as mobile phase. Assay was done at the respective absorption maxima of the drugs by in situ densitometry and by spectroscopy after extracting the drugs from TLC plates with 90% acqueous ethanol. Results obtained by both the methods agreed well with those obtained by the method prescribed by the United States Pharmacopoeia XXI edition. Total time required for HPTLC and densitometric assay of 32 samples using 4 standards was 30 min. Probable source of errors in densitometric studies and their rectification was discussed.     

Monoclonal Antibody-Based Immunoassay for the Detection of Maduramicin in Chicken Tissues

Abstract

This paper reports on the preparation of a monoclonal antibody (Mab) against maduramicin (MD) and the development of a simple and sensitive ELISA for MD in chicken tissues. MD was conjugated to bovine serum albumin for immunogens and ovalbumin for coating antigens by mixed anhydride (MA) and active ester (AE) methods. Hybridoma cells were generated using spleen cells from a mouse immunized with MD-BSA conjugate. After screening with ELISA, the Mab with high anti-interference ability and high affinity was selected, and it exhibited negligible cross-reactivity with other usual-used polyether antibiotics. After optimization, the developed ELISA showed an IC₅₀ value of 2.12 ± 0.46 ng/ml (n = 20). Chicken muscle and liver samples were extracted with methanol-8% NaCl solution (4:1) and then directly diluted with PBS-10% methanol for analysis. The recoveries of MD from spiked chicken tissues at levels of 40–480 µg/kg ranged from 81.3% to 91.3% with variation coefficients of 5.2–12.1%, and the detection limits were 6.5 µg/kg in muscle and 9.2 µg/kg in liver, respectively (n = 20).     

Competitive enzyme-linked immunosorbent assay for the determination of catecholamine, dopamine in serum

A competitive enzyme-linked immunosorbent assay for dopamine (DA) has been optimized and characterized. DA is sensitive to oxygen and light according to a function of the pH on the DA oxidation process. The phenolic groups in DA are readily oxidisable to a quinoid form and thus, free DA deteriorates in alkaline media. Thus, effect of factors such as pH, enzyme-label with substrate, ionic strength and reaction time was considered on performance of ELISA. Assay was performed with 5 μg mL⁻¹ of BSA-DA and 1/7500 dilution of anti-DA antibody. A dose-response curve was constructed, and a limit of detection and a dynamic range for DA were accomplished to 1.0 × 10⁻⁹ M (0.19 μg L⁻¹) and five orders (3.2 × 10⁻⁸ M to 3.2 × 10⁻³ M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown good agreement with correlation coefficient (R² = 0.9947): Abs. (in serum) = 0.6128 × Abs. (in buffer) + 0.2926. The cross-reactivity was examined with the structurally similar compounds. And the results demonstrated that epinephrine and 3-methoxytyramine showed cross-reactivity (18.9% each), whereas 3,4-dihydroxyphenylacetic acid and homovanillic acid showed low cross-reactivity (<1%). And percent recoveries of DA in serum were quite satisfactory. This provides usefulness of the present assay to monitor DA in serum.     

Development and validation of a highly sensitive ELISA for the determination of pharmaceutical indomethacin in water samples

The development and validation of a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of pharmaceutical indomethacin in water samples was presented. The immunogen and coating antigen were prepared by covalently linking indomethacin to bovine serum albumin and ovalbumin by anhydride ester method. Two rabbits were immunized by standard immunization processes and the superior antibody was characterized in terms of sensitivity, specificity, precision, accuracy and stability. Under optimal experimental conditions, the standard curve was constructed in the concentration range of 0.01-10 ng/mL. For 10 consecutive standard curves run in 2 weeks, IC₅₀ value (the concentration of analyte producing 50% of inhibition) were found within 0.10-0.25 ng/mL, and the detection limit (DL) at a signal-to-noise ratio of 3 (S/N = 3) was about 0.01 ng/mL. The antiserum recognized acemetacin, a precursor of indomethacin with 92.3% cross-reactivity, while the cross-reactivity values of antiserum with other tested compounds were very low. From the spiking experiments, the recoveries were found within 98-123%. The ELISA was applied for the determination of indomethacin in different water samples and the results were confirmed by conventional HPLC. The correlation coefficient of 0.988 was obtained, demonstrating a good correlation of ELISA with HPLC.     

Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Herbicide Propanil

Although the extensively used postemergence herbicide propanil itself is of low acute toxicity in mammals, it raises environmental concerns due to its effect on aquatic organisms and other adverse impacts. Therefore, in order to obtain a rapid analytical method for this pesticide, an indirect enzyme-linked immunosorbent assay (ELISA) has been developed. Antibodies obtained against a conjugate of 3,4-dichloroaniline coupled to succinylated proteins were tested in hapten-homologous and heterologous indirect ELISA formats using various N -(dichlorophenyl)-succinamic acid derivatives conjugated to carrier proteins as coating antigens. Titers in ELISA were found to be significantly affected by the type and quantity of coating antigen. One of the optimized systems using N -(2,4-dichlorophenyl)-succinamic acid and N -(3,5-dichlorophenyl)-succinamic acid conjugated to ovalbumin allowed serum dilution of 1 : 10,000 and IC 50 values of 2.2 and 2.7 ng/mL for propanil, respectively. The limit of detection (LOD) of the immunoassays is 0.2 ng/mL. Other optimized ELISA systems based on different dichloroaniline-based coating antigens also offered similar sensitivities. The ELISA systems appeared to tolerate methanol and ethanol upto 5% concentration. For confirmatory purposes, the ELISA protocol was compared with a highly sensitive gas chromatographic method coupled with mass spectrometric detection (GC-MS). Spiked propanil content was detected both by ELISA and GC-MS in methanolic rice extract. Detection sensitivities of the two analytical systems appeared to closely correlate with each other in the range of 10-90 ng/mL (0.02-0.18 µg/g), indicating the utility of the immunoanalytical method in detecting propanil content in rice, the main commodity propanil is being applied on.     

Preparation of Anti-Cadmium-EDTA Complex Polyclonal Antibody and Its Application for Determination of Cadmium in Aqueous Solution

Abstract

A polyclonal antibody that can recognize cadmium-ethylenediaminetetraacetic acid (CD-EDTA) complex was prepared via the injection of New Zealand white rabbits with Cd-1-(4-isothiocyanatobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid–BSA (Cd-ITCBE-BSA). The polyclonal antibody displayed high levels of affinity for Cd-1-(4-isothiocyanatobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid-OVA (Cd-ITCBE-OVA) with favorable titer of 1.28 × 10⁶. A simple, reliable, and economical indirect competitive immunoassay based on this polyclonal antibody was developed and validated for detection of cadmium. Assay optimization was performed with respect to chelator concentration, ionic strength, blocking solution, pH, and reaction time. The detection limit of the assay was 0.21 µg L^?1, and the effective linear range was from 10^?1 to 10³µg L^?1. The coefficient of variation (CV) of intra- and interassay were 1.0–8.2% and 2.3–6.9%, respectively. Results yielded low cross-reactivity of the assay to other tested metals such as Pb²⁺, Ni²⁺, Mg²⁺, Ca²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Co²⁺, Cr³⁺, and Fe³⁺, except Hg²⁺, which showed a cross-reactivity of 7.4%. Spike recoveries of ultrapure water, tap water, and samples of the Yangtze River were 85.5–116.3%. These results show that this assay is suitable for quantitative detection of cadmium at trace levels in water samples.     

Reliable enzyme immunoassay detection for chlorothalonil: fundamental evaluation for residue analysis and validation with gas chromatography

This work describes the analytical performance of the enzyme-linked immunosorbent assay (ELISA) for the fungicide chlorothalonil to effectively exploit as a simple and rapid detection system for pesticide residue on the scenes of the agricultural production and distribution. This ELISA represents the satisfactory analytical characteristics (I50 value, 0.34 ng/g; limit of detection, 0.052 ng/g) to detect chlorothalonil at the regulatory values or thereabout in a sample. Noticeable cross-reactivities were shown with two fungicides, fthalide (58.8%) and pentachloronitrobenzene (quintozene) (20.0%), and some non-agrochemicals such as tetrachloroterephthalonitrile (96.8%) and tetrachlorophthalonitrile (68.3%). The influence of three organic solvents (methanol, acetone, and acetonitrile) used as extractants for chlorothalonil residue was evaluated, with the result that methanol was the most suitable solvent for the ELISA, and the final concentration in the well could be up to 5% (v/v) without any negative influence on the ELISA. It has been possible to directly analyze chlorothalonil residue only by giving dilution of each sample extract with water prior to the ELISA analysis. The average recovery values from the spiked samples by the ELISA were between 101.7 and 113.6% with the average coefficients of variation between 2.6 and 5.9%. Although the results obtained from the ELISA correlated well with those from the reference GC/MS methods for all agricultural samples (r>0.98), the linear function inclined to the ELISA results because of loss during complex sample preparations for GC/MS analysis. Nevertheless, the results demonstrated that the proposed ELISA is a reliable, cost-effective, and rapid quantitative method for chlorothalonil residue.     

Semi-Preparative High Performance Liquid Chromatographic Separation of Carbon-14 Labeled Avermectin B1a from a Mixture of Avermectins

Abstract

A semi-preparative high performance liquid chromatographic method has been developed to separate carbon-14 labeled avermectin B_1a from a fermentation mixture of carbon-14 labeled avermectins, i. e., avermectins A₁a, A₁b, A₂a, A₂b, B₁a, B₁b, B₂a, and B₂b. Two HPLC systems were employed for the separation: I. A Whatman M20, Partisil 10, normal phase column and a solvent system of 10% ethanol in isooctane (v/v), and II. A Whatman M20, Partisil 10, ODS-3, reverse phase column and a solvent system of acetonitrile/methanol/water (56:18:26, v/v/v); the flow rate was 18 ml/ min. Avermectin separations were monitored using ultraviolet detection (254 nm). Further analyses of avermectin B₁a were done using analytical HPLC and TLC/radioassay to check compound purity and identity.     

Quantitative Analysis of Metoclopramide in Tablet Formulations by Hplc

Abstract

A rapid, specific and reliable high performance liquid chromatographic assay of Metoclopramide in tablets has been developed. Reversed-phase chromatography was conducted using a mobile phase of acetate buffer and acetonitrile, (75% v/v) and detection at 273nm. The recovery and coefficient of variation from six placebo tablets containing 10 mg of Metoclopramide were 100.5% and 0.889% respectively. Relicate regression analyses of three standard plots in the concentration range of 0.5 – 7 mcg/mL obtained on three different days gave a correlation coefficient > 0.996 and the coefficient of variation of the slopes < 1%. The assay was precise within day and between days as indicated by ANOVA test. The recoveries from 10 replicate tablets of three different commercial Metoclopramide brands was 98.7, 100.8 and 100.3% of the label amount and their coefficients of variation were 1.16, 1.76 and 1.6%.     

An Enzyme Immunoassay for the Determination of Methabenzthiazuron

Abstract

A sensitive enzyme immunoassay is described for the determination of the urea herbicide methabenzthiazuron. The assay is carried out with polyclonal antibodies, which were raised in rabbits by immunization with a methabenzthiazuron-BSA conjugate containing five methabenzthiazuron residues per molecule. The ELISA was optimized on microtiter plates with a peroxidase-methabenzthiazuron tracer. The middle of the test (50% B/B₀) was found at 1.0 μg/l. The lower detection limit of methabenzthiazuron is c. 0.05 μg/l. Samples can be measured up to 10 μg/l methabenzthiazuron (upper detection limit). The assay does not require concentration or clean-up steps for drinking or ground water samples. Validation experiments showed a good accuracy and precision. Work with monoclonal antibodies is in progress.     

Determination of Part-Per-Billion Levels of Formaldehyde in Aqueous Solution by Ion Chromatography with Post-Column Derivatization

Abstract

A method based on the use of an ion-exchange column, post-column derivatization with 2,4-pentane-dione (acetylacetone), and photometric detection for the determination of low levels of formaldehyde in aqueous solution is described. The lower limit of detection is 5 μg/L (ppb) and response is linear up to at least 10 mg/L. Reproducibility at the 1 mg/L level is 0.6% RSD. Determination of formaldehyde concentration in several aldehyde systems is demonstrated.     

A Rapid and Inexpensive Thin Layer Chromatographic Method for Quantitative Analysis of Bleomycin Complex

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of different components of bleomycin injections has been described. The bleomycin components were separated by reversed phase thin layer chromatography on silanized silicagel plates using a mixture of aqueous ammonium nitrate (2.5%) : methanol :: 7 : 3 (v/v) as mobile phase. Assay was done at the absorption maxima of the components (291 nm) by in situ densitometry or by spectroscopy after extracting the drugs from the adsorbent with the mobile phase. Results obtained by both the methods agreed well with each other and with those obtained by an official HPLC method. The densitometric method described was highly suitable for routine quality control of bleomycines as a large number of samples could be analysed within a short time (68 samples/analyst/day).     

Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum

A rapid and cost-effective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in serum has been optimized and validated. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassays. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3′-end on aptamer and at neutravidin plate. Various factors such as incubation temperature, divalent ion – Mg²⁺ ion and treatment of serum solution were evaluated for the performance of ELAA. The aptamer was incubated for 1 h at 4 °C in the assay buffer containing 5 mM Mg²⁺ ion, and serum was diluted (1:9, serum:assay buffer) and filtrated through a 3 kDa dialysis membrane to extract the proteins present in the serum. Assay was performed with 0.01 μg mL⁻¹ of aptamer and 1.205 × 10⁻⁷ M DA-HRP conjugate using the optimized method. A dose–response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 1.0 × 10⁻¹² M and four orders (1.0 × 10⁻⁷ M to 5.0 × 10⁻¹¹ M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown a good agreement with the correlation coefficient (R² = 0.9872): Abs. (in serum) = 0.9612 × Abs. (in buffer) − 0.0556. The cross-reactivity evaluation demonstrated that norepinephrine showed some cross-reactivity (3.68%) whereas 3-methoxytyramine, epinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid showed almost no cross-reactivity (<1%). Percent recoveries of DA in serum were quite satisfactory (∼95%). This paper describes usefulness of the aptamer assay in monitoring DA in human serum.     

Deoxynivalenol and its conjugates in beer: a critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry

Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen^® DON (R-Biopharm), Veratox 5/5 DON^® (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant^® DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile-water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep™# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.     

Separation of Hydroxybenzoic Acid Isomers by Capillary Zone Electrophoresis

Abstract

Capillary zone electrophoresis is a highly efficient analytical technique that has been shown to be particularly useful for the analysis of isomers. Using a cathodic injection and anodic detection scheme, ortho-, meta- and para-hydroxybenzoic acids were separated in a fused-silica capillary column with a phosphate buffer at pH 10.3 (25.0 mmol/1 phosphate + 0.20 mmol/1 cetyltrimethylammonium bromide (CTAB) + 10% (v/v) 1-propanol with an applied voltage of 10 KV followed by direct UV detection. The use of CTAB as electroosmotic flow modifier allows the rapid separation of the three isomers by reversing the direction of electro-osmotic flow. The influence of pH, CTAB concentration and organic solvents on the migration behaviour of the solutes were also studied.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.