A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I₅₀ of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.     

Development of an indirect competitive immunoassay for parathion in vegetables

An indirect competitive immunoassay for the insecticide parathion has been optimized and characterized. This assay is based on a monoclonal antibody (2H₉) produced from an immunogen, a bovine thyroglobulin (BTG) conjugate wherein the reduced form of parathion was multiply bound to the carrier protein via diazo bonds. Assay was performed in the parathion-HSA coated (0.25 μg/ml) ELISA format in which antibody was diluted 1:2000. Several physicochemical factors (pH, ionic strength, BSA concentrations and organic solvent) that influence assay performance were studied and optimized. Finally, the assay was applied to the analysis of parathion in spiked vegetable samples. The sensitivity, estimated as the IC₅₀ value, was 360 ng/ml, with a practical working range between 47 and 6000 ng/ml, a limit of detection of 26 ng/ml, and inter-assay and intra-assay variations less than 10%. The average recovery of parathion added to potato, celery and Chinese cabbage were 173 ± 34%, 108 ± 15% and 98 ± 6%, respectively.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle,liver and egg

A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC₅₀ value was 1.32 ± 0.18 ng/ml. The limit of detection was 0.14 ± 0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50–600 ng/g were 84.6–85.5%, 88.2–89.1%, and 84.4–89.3%, respectively. The coefficient variations (CVs) were 5.1–9.3%, 3.7–11.3%, and 4.7–9.8%, respectively. Linear regression analysis showed good correlation, with r² values 0.9909 for chicken liver and 0.9916 for chicken muscle.     

Development of an enzyme-linked immunoassay for the detection of gentamicin in swine tissues

We developed an enzyme-linked immunoassay that provides rapid and sensitive detection of gentamicin in swine tissues. Rabbit was immunized with gentamicin-BSA conjugate and antiserum was collected after the fifth immunization. After optimizing the concentration of immunoreagents, competitive indirect ELISA (ciELISA) gave an IC₅₀ value of 0.98 ng/ml, while competitive direct ELISA (cdELISA) exhibited lower IC₅₀ value of 0.92 ng/ml, thus cdELISA was further optimized under various pH values and ionic strengths of assay buffer, different coating methods and incubation time. The optimized ELISA can be completed within 45 min and it showed negligible cross-reactivity with other aminoglycosides. The recoveries of gentamicin from spiked swine tissues at levels of 25–200 μg/kg ranged from 64.7% to 101.2% with CVs of 4.5–12.1%, and the detection limits were 6.2 μg/kg in muscle, 3.6 μg/kg in liver and 2.7 μg/kg in kidney, respectively.     

A highly sensitive method for simultaneous determination of ultra trace levels of copper and cadmium in food and water samples with luminol as a chelating agent by adsorptive stripping voltammetry

In the present study a selective method is presented for the simultaneous determination of copper and cadmium in food samples by adsorptive stripping voltammetry. In preliminary studies, it has been proven that the copper and cadmium react with 3-aminophthalhydrazide (luminol), giving rise to the formation of these complexes. These complexes have adsorptive characteristics on hanging mercury drop electrode (HMDE) and can be reduced in a reduction step. In this study the optimum reaction parameters and conditions studies are investigated. The calibration graphs were linear in the concentration range of 0.5–105.0 and 0.8–70.0 ng/ml for copper and cadmium, respectively. The limit of detection of the method was 0.04 ng/ml for Cu²⁺ and 0.02 ng/ml for Cd²⁺. The interference of some common ions was studied and it was concluded that application of this method for the determination of copper (II) and cadmium in food and water samples led to satisfactory results.     

Simultaneous determination of ultra trace amounts of lead and cadmium in food samples by adsorptive stripping voltammetry

A selective and sensitive method for simultaneous determination of lead and cadmium by adsorptive differential pulse cathodic stripping voltammetry is presented. The method is based on adsorptive accumulation of the complexes of Pb (II) and Cd (II) ions with 2-mercaptobenzothiazole onto hanging mercury drop electrode (HMDE), followed by the reduction of the adsorbed species by differential pulse cathodic stripping voltammetry. Optimal conditions were obtained at pH 8.0, 2-mercaptobenzothiazole concentration of 1.0 × 10⁻⁴ M, the accumulation potential of −0.4 V (vs. Ag/AgCl), the accumulation time of 160 s, and the scan rate of 100 mV/s. Under optimised conditions, linear calibration curves were established for the concentration of Pb (II) and Cd (II) in the range of 0.5–70 and 0.2–30 ng/ml, respectively, with detection limit of 0.017 ng/ml for Pb (II) and 0.01 ng/ml for Cd (II). The procedure was successfully applied to the simultaneous determination of both ions in food samples (rice, soya and sugar).     

Simultaneous detection of sulfamethazine,streptomycin, and tylosin in milk by microplate-array based SMM–FIA

This paper presents an approach to simultaneously detect sulfamethazine, streptomycin, and tylosin in milk by indirect competitive multianalyte Fluorescence immunoassay (FIA). Microscope glass slides modified with agarose were used for the preparation of small molecule microarrays (SMMs). Bovine serum albumin (BSA) conjugates of the haptens were immobilized on glass slides. The system consists of four glass slides containing 96 wells formed by an enclosing hydrophobic mask, which precisely matches a standard microplate. All liquid handling and sample processing were fully automated as 96-wells ELISA format. Monoclonal antibodies against sulfamethazine, streptomycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with Cy5 generating fluorescence, which was scanned with chip scanner. The detection limits for three analytes were 3.26 ng/ml (sulfamethazine), 2.01 ng/ml (streptomycin), and 6.37 ng/ml (tylosin), being far below the respective MRLs. The system proved to be the first SMM–FIA platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of safety in the food industry.     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

Simultaneous determination of four phenolic components in citrus honey by high performance liquid chromatography using electrochemical detection

A sensitive and accurate method for simultaneous separation and determination of four phenolic compounds, including caffeic acid, p-coumaric acid, ferulic acid, and hesperetin in Chinese citrus honey by high performance liquid chromatography using electrochemical detection (HPLC-ECD) has been established. Chromatographic separation was performed using a reversed phase column and methanol/4% (v/v) aqueous acetic acid as the mobile phase. The detection and quantification limits of the four compounds with ECD were 6–14 times greater than those obtained with diode-array detection (DAD). All calibration curves of the four phenolic compounds showed good linearity (r ? 0.9994) within the test ranges, 1.10–66 μg/ml, 0.35–70 μg/ml, 0.16–16 μg/ml and 0.03–10 μg/ml, respectively. The recoveries ranged from 98.9% to 100.3%. The extraction process was very simple, because of the dissolution of honey only involving water. Taken together, the application of ECD in honey determination leads to a significant improvement in the quantification of phenolic compounds, whereby paying the way for the establishment of a better quality control of citrus honey.     

Double-antibody based immunoassay for the detection of β-casein in bovine milk samples

The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1–10.0 μg mL⁻¹. The detection limit was 0.04 μg mL⁻¹. In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.     

Cloud point extraction coupled with derivative of carbofuran as a preconcentration step prior to HPLC

Carbofuran can be hydrolysed to from 2,3-dihydro-2,2-dimethyl-7-benzofuranol (BF). BF is coupled with 4-aminoantipyrene (AP) in presence of potassium ferricyanide (K₃Fe(CN)₆) to generate red coloured derivative(BFAP)having λmax 530 nm. Cloud point extraction (CPE) methodology and using surfactant Triton X-100 as extractant was applied as a preconcentration step prior to HPLC, the surfactant-rich phase containing BFAP was then analysed by HPLC in visible region. The high back ground absorbance of Triton X-100 in UV region was completely avoided. A new visible detection method with high-performance liquid chromatography (HPLC) has been developed for the determination of carbofuran. Using this method, we found that carbofuran residues could be determined with recoveries ranging from 80.4% to 84.5%, relative standard deviations in the range of 2.51–3.26% for three fortified rice levels, and the limit of detection as 5.0 × 10⁻⁴ cJ/cK.     

Validation of HPLC method for determination of tetracycline residues in chicken meat and liver

High-performance liquid chromatographic method with diode-array detection (HPLC–DAD) was optimised and validated for determination of tetracyclines (TCs) residue in chicken meat and liver through evaluating each step of various methods. The principle steps involved ultrasonic-assisted extraction of TCs from chicken samples by 2 ml of 20% trichloroacetic acid and citrate buffer (pH 4) which gave a clearer supernatant and high recovery, followed by centrifugation and purification on SPE (Strata C18-E cartridge) using 10 ml of 0.01 M methanolic oxalic acid for TCs elution. Separation was on reversed-phase column (Nuclosil 100 C18, 25 cm × 4.6 mm ID, 5 μ) by multisteps gradient elution which provided a better chromatographic peak resolution and the late eluting peaks were as sharp as those eluting earlier. Monitoring was at 351 nm which gave a higher detector response factor. Validity study of the method revealed that all obtained calibration curves showed good linearity (r² > 0.999) over the range of 50–5000 ng. Sensitivity was found to be 1.44, 1.90, 0.95 and 1.23 ng for OTC, TC, CTC and DC, respectively. Accuracy was in the range of 71.88–92.44.3% and 68.88–84.84% for meat and liver, respectively. Precision was lower than 10% in all cases indicating that the method can be used as a validated method. Limit of detection (LOD) was found to be 4.4, 5, 13 and 10 ng for OTC, TC, CTC and DC, respectively. The corresponding values of limit of quantitation (LOQ) were 10, 13, 27 and 22 ng.     

Ultra trace quantification of chromium(VI) in food and water samples by highly sensitive catalytic adsorptive stripping voltammetry with rubeanic acid

A simple and highly selective and sensitive catalytic adsorptive stripping voltammetric procedure for determination of ultra trace levels of chromium(VI) on hanging mercury drop electrode is reported. The method is based on the adsorptive preconcentration of the Cr(III)–dithiooxamide (rubeanic acid) complex and the utilization of the catalytic reaction in the presence of nitrate. At optimized conditions the calibration graph is linear from 0.01 to 6 ng/ml and detection limit is 0.002 ng/ml for accumulation time of 50 s. The interference of some common ions was studied and this method has been applied to the determination of chromium in food and waste water samples with satisfactory results.     

Quantitative analysis of acrylamide in tea by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An effective sample preparation procedure was optimized and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantitative analysis of acrylamide in tea. [¹³C₃]-acrylamide was used as internal standard. Acrylamide was extracted at 25 °C for 20 min by 10 ml water followed by 10 ml acetonitrile, and then 4 g of magnesium sulfate and 0.5 g of sodium chloride were added to the above mixture under stirring thoroughly. In order to increase the response of acrylamide, 9 ml acetonitrile layer was taken and concentrated to 0.5 ml. Solid-phase extraction with an Oasis MCX cartridge was carried out for clean-up. The limit of detection (LOD) and limit of quantification (LOQ) were 1 and 5 ng/ml, respectively. The recovery efficiency of the extraction procedure ranged between 74% and 79%. The levels of acrylamide in 30 tea samples were less than 100 ng/g. Black, oolong, white and yellow tea samples had quite low acrylamide contents (<20 ng/g). Higher acrylamide levels occurred in baked, roasted, and one sun-dried green tea samples (46–94 ng/g).     

Lead and cadmium concentrations in goat,cow, sheep,and buffalo milks from different regions of Iran

In total, 137 goat, cow, sheep, and buffalo milk samples were collected in different regions of Iran and analysed to determine concentrations of lead and cadmium by a graphite furnace atomic absorption spectrometric method. The mean recovery of the analytical method was 96.3% and 104% for cadmium and lead, respectively. The mean lead and cadmium contents obtained from 137 samples were 1.93 ± 1.48 (range: 0.18–6.11 ng/ml) and 9.51 ± 4.93 ng/ml (range: 1.84 ng/ml–30.50 ng/ml), respectively. Lead concentration in 8.1% of sheep and 1.9% of cow milk samples was higher than the newly established Codex standard. The mean concentrations of cadmium and lead in animals aged ?3 years (n = 80; 1.40 ± 1.05 ng/ml and 7.91 ± 3.60 ng/ml, respectively) were lower than in animals aged >3 years (n = 58; 2.69 ± 1.67 ng/ml and 11.8 ± 5.71 ng/ml, respectively).     

Development of monoclonal antibodies and a competitive ELISA detection method for glycinin,an allergen in soybean

Soybean glycinin is a major food allergen causing anaphylaxis. A sensitive detection method for glycinin is needed to evaluate soybean allergies in food and feed products. In the present study, monoclonal antibodies (Mabs) against glycinin were prepared using purified glycinin as the immunogen. The generated Mabs, named 3B2 and 4B2, were identified as being IgG_2b and IgG_2a iso-types respectively, and exhibited high specificity to glycinin. Then we developed a competitive ELISA based on Mab 4B2 to measure glycinin which showed an IC₅₀ value of 1.7 ng/mL with a detection limit of 0.3 ng/mL, and the linear portion of the curve was 0.3–11.2 ng/mL. Recovery tests indicated that the competitive ELISA based on Mab 4B2 gave reliable reproducibility. The produced Mab 4B2 and the developed ELISA could provide a valuable tool for sensitive determination of glycinin and for future studies conducted to reveal the mechanism of how glycinin functions in anaphylaxis.     

A non-destructive method to monitor changes in a troponin T peptide in beef drip with a monoclonal antibody

To simplify the monitoring of postmortem beef aging, we established a system to detect a troponin T (TnT) peptide fragment in bovine muscle drip (natural exudates) with an original monoclonal antibody. The antibody was raised against a synthetic peptide APPPPAEVPEVHEEVH corresponding to the N-terminal region of bovine fast-type TnT. In a competitive enzyme-linked immunosorbent assay (ELISA), our antibody detected the standard peptide dose-dependently. According to the monitoring examination with a competitive ELISA during 22 days postmortem, the concentration of the peptide in both the drip and trichloroacetic acid extracts from the longissimus muscle (n = 4) significantly increased in parallel, up to 10 nmol/ml and 16.4 nmol/g at day 14 postmortem, respectively. These events were accompanied by an increase in the conventional 30 kDa fragment in western blot analysis and a decrease in the Warner–Bratzler shear force value of the beef from 5.0 to 2.4 N/cm². The peptide detection system using drips with the antibody has advantages applicable to a non-destructive, simple, quick, and on-site monitoring method, such as immunochromatography.     

Tocopherols in espresso coffee: Analytical method development and validation

The present paper reports the development and validation of an analytical micro-method for tocopherols quantification in espresso coffee by normal-phase HPLC with fluorescence detection. The proposed method consists in a liquid–liquid extraction with n-hexane:ethyl acetate, followed by a clean-up with dimethylformamide to eliminate co-eluting interferences. The method showed good intra- and inter-day precisions (coefficient of variation < 6.5%), good accuracies (98 ± 6%), and high correlation coefficients (r > 0.999) for standards subjected to the entire procedure. Only α- and β-tocopherols were identified in the brews. The detection and quantification limits were 0.5 and 1.4 ng/ml, for α-tocopherol, and 0.4 and 1.1 ng/ml, for β-tocopherol, respectively. A mean total tocopherol content (α + β) of 3.5 ± 0.9 μg in commercial espresso coffee blends (30 ml) was detected. The proposed method requires low solvent consumption and proved to be sensitive, precise and accurate.     

Determination of ultra trace levels of copper in food samples by a highly sensitive adsorptive stripping voltammetric method

A new method is presented, for the determination of copper, based on adsorptive stripping voltammetry of the complex of copper with thiosemicarbazide at a hanging mercury drop electrode (HMDE). The most suitable operating conditions and parameters, such as pH, accumulation potential, deposition time, ligand concentration and scan rate, were selected. The calibration graph for copper (II) was linear over the concentration range 0.01–90.0 ng/ml; the detection limit of the method was 0.007 ng/ml. The interferences of some common ions were studied and the method was found suitable for the determination of copper (II) in rice, tea, tomato, blood and water samples. Moreover, with the use of the proposed method, there is a considerable improvement in the detection limit, the linear dynamic range and the deposition time, compared with the methods other than adsorptive stripping voltammetry for the determination of copper.     

Analysis of residual oxytetracycline in fresh milk using polymer reversed-phase column

A simple and rapid reversed phase high performance liquid chromatograph (HPLC) method for analysis of oxytetracycline (OTC) was developed and applied in the determination of the antibiotic in fresh milk sample. Isocratic elution was performed with acetic acid:water (pH 4.5):acetonitrile (4:68:28), using a polymer reversed-phase (PLRP) column and UV detection at 354 nm wavelength. The average recoveries of OTC spiked milk at 0.1, 0.2, 0.5 and 100 ng/mL were in excess of 92% with intraday and interday precision between 0.8% and 6.6% respectively. A good linearity was established between the range 100–1000 ng/mL with r² = 0.9995. The limit of detection and quantitation were 30 and 100 ng/mL respectively. The method demonstrated successful application for analysis of 100 milk samples. Two samples out of 70 from livestock keepers tested OTC positive while none of the 30 samples from milk centers tested positive.