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.     

High-Performance Liquid Chromatographic Determination of Trace Quantities of Azinphos Methyl and Azinphos Methyl Oxon in Various Water Sources by Direct Injection and Trace Enrichment

Abstract

A high-performance liquid chromatographic (HPLC) method is described for the determination of trace amounts of the organophosphate insecticide, azinphos methyl and its degradation product, azinphos methyl oxon, by direct injection and by trace enrichment. The compounds were analyzed on a uBondapak C₁₈ column with UY detection at 224 nm. The mobile phase for the analysis was acetonitrile-water (50:50) at a flow rate of 1.3 ml/min. Ten minutes were required for the chromatographic analysis. Water from three sources, public water supply, stream, and ocean was analyzed for azinphos methyl and azinphos methyl oxon at concentrations as low as 11.9 and 11.3 ppb, respectively, without a clean-up, concentration or derivatization step. Azinphos methyl was quantitated at 0.29 ppb and azinphos methly oxon at 0.29 ppb by employing a concentration step involving a C₁₈ Sep-Pak cartridge. The coefficients of variation for all determinations ranged from 0.77 to 9.06%. Peak heights were used for quanti-tation. Several other pesticides have been shown not to interfere with the analysis of either compound.     

Use of a nitrate-form anion exchange resin for the determination of traces of gold in copper and cadmium by neutron activation analysis

A method is described for the determination of traces of gold in copper and cadmium by neutron activation analysis, using anion exchange resin as a preconcentration agent: gold was separated from large amount of copper or cadmium with Cl-form Dowex 1X8 AG, 100–200 mesh, resin. To reduce the interfering activities, the resin was irradiated in NO ₃ ⁻ -form and washed with dilute hydrochloric acid after irradiation.¹⁹⁸Au in the resin was then counted with a Ge(Li) or NaI(Tl) detector. The chemical yields were more than 99%. The concentration factors of gold for copper and cadmium samples were 1.1×10⁸ and 2.7×10⁶, respectively. The analytical results of gold in 99.99% copper and 99.999% cadmium were 65 and 0.15 ppb, respectively. The blank was 0.05 ng Au per 200 mg of wet resin.     

Characterization of s-triazine antibodies and comparison of enzyme immunoassay and biotin-avidin enzyme immunoassay for the determination of s-triazine

Triazines have been used widely as herbicides and known to cause environmental contamination. For developing ELISA of s-triazines, six kinds of s-triazine derivatives (one from simazine, one from atrazine and four from cyanuric chloride) which contained a C3- or C6-carboxylic acid group bridge were prepared. These derivatives were conjugated to bovine serum albumin (BSA) for the use of immunogens and to KLH for the use of coating ligands on the microtiter plate wells. Polyclonal antibodies produced from rabbit or sheep using atrazine-BSA (1b-BSA) and simazine-BSA (1a-BSA) immunogens. These antibodies were characterized and biotinylated for the use of enzyme immunoassay (EIA). We evaluated EIA and biotin-streptavidin mediated EIA in terms of the sensitivities and specificities with these antibodies. The results in both assay systems showed that coating ligand synthesized from atrazine derivative was better than that from simazine derivative. Comparing binding ability between C3-carboxylic acid and C6-carboxylic acid spacers of N-alkyl group in s-triazine ring, C3-carboxylate-KLH (2c-KLH) derived from atrazine showed better sensitivity for the both assay systems. The detection limit was found to be 0.01 ppb in biotin-streptavidin mediated EIA (B-Av EIA). Comparing IC₉₀ values of EIA (0.5 ppb) and B-Av EIA (0.05 ppb), B-Av EIA was able to detect one order lower concentration range of atrazine than EIA.     

Monitoring of Atmospheric Sulfur Dioxide by Fluorescence Monitoring System

A fluorescence monitoring system was used to measure gaseous sulfur dioxide at Taoyuan Agricultural Farm. A gas-diluting system and gas chromatograph equipped with a sulfur chemiluminescence detector used to audit the accuracy of diluted sulfur dioxide are described. The accuracy was ?8% and ?2%, respectively, for the preparation of 10 and 50 ppb SO₂ standard gas. The pattern of variation of SO₂ concentration at Taoyuan Agricultural Farm is reported. In summer at Taoyuan Agricultural Farm, the concentration of SO₂ varied with a pattern of night concentration (15-20 ppb) greater than that in the afternoon (0–2 ppb). Computer-analyzed wind trajectories in Taiwan for this pattern are reported.     

Comparison of Electrochemical and Spectro-Photometric Detection in Hrp-Based Elisa for Tobacco Mosaic Virus

ABSTRACT

An electrochemical method was compared with a spectrophotometric method using a horseradish peroxidase (HRP)-based indirect enzyme-linked immunosorbent assay (ELISA) with direct antigen coating (DAC) technique for the determination of tobacco mosaic virus (TMV). In substrates for a spectrophotometric HRP-based ELISA method, was selected as the substrate for the electrochemical method as well as the spectrophotometric method. 2, 3-Diaminophenazine is the product of the oxidation of OPD with H₂O₂ catalyzed by HRP in the selected conditions. It is electroactive and has a sensitive voltammetric response at -0.93 V (vs. Ag/AgCl) in alkaline solution. The detection limit of free HRP, by second-order derivative linear-sweep voltammetry, is 1.1 mU/L. This method can be further used to detect TMV antigen. The peak current is linear with TMV concentration in the range of 0.25-5.0 ng/mL. The detection limit for TMV is 0.25 ng/mL and the highest dilution ratio detected for the infected leaf sap is 1:102400. Compared with the classical OPD ELISA spectrophotometric method, the detection limits of the electrochemical method for the clarified TMV and the infected leaf sap detection are about ten and four times lower, respectively.     

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.     

Photothermal Deflection Spectroscopy for Ultra Trace Analysis of Cobalt and Copper Ions

Abstract

Photothermal deflection spectroscopy (PDS) is applied for the absolute determination at room temperature of ultra trace concentration of cobalt and copper ions in aqueous solution. The samples have been prepared by percolating 2 liters of solution prepared from Merck Titrisol Standards in 200 mg Merck-I cationic exchange resin. With the present experimental conditions the detection limit is 20 ppb and 200 ppb for copper and cobalt ions respectively within an overall accuracy of 5%. The response of the spectrometer has been calibrated using ICP technique.     

A readily-prepared and efficient solid-supported scavenger for molybdenum alkoxides and a structurally characterized model complex

A very effective solid support for the removal of molybdenum-based metathesis catalysts can be prepared by placing a salicylimine on a polystyrene support. The resin is produced by treating Merrifield’s resin with H₂NBuⁿ, 4-chloromethylsalicylaldehyde, and p-toluidine in succession. The final resin had an identifiable CN stretch in the IR and a resonance assigned to the HCN hydrogen was found by MAS ¹H NMR of the swelled resin. Solutions of Mo[C(H)CMe₂Ph](NAr)(OBu^t_F6)₂ (Mo_F6) and Mo[C(H)CMe₂Ph](NAr)(OAd)₂ (Mo_Ad), where Ar = 2,6-diisopropylphenyl and Ad = 1-adamantyl, were treated with the scavenger, which reduced the remaining molybdenum concentration as examined by ICP-MS to 30-50 ppb. Catalyst was also scavenged from ring-closing metathesis of diethyl diallylmalonate by Mo_F6; the decrease in molybdenum concentration on addition of scavenger followed first order kinetics with initial and final concentrations of 54 000 and 15 ppb, respectively. We also prepared a model system where a soluble salicylimine (H-DIB) was reacted with Mo[C(H)CMe₂Ph](NAr)(OAd)₂ to produce Mo[C(H)CMe₂Ph](NAr)(DIB)₂, which was structurally characterized.     

Indirect Trace Determination of Ethylenediminetetraacetic Acid (Edta) in Water by Potentiometric Stripping Analysis

Abstract

An analytical method for indirect trace determination of ethylenediaminetetraacetic acid (EDTA) in water, by potentiometric stripping analysis is described. Excess Bi(III) was added to form a 1:1 complex with EDTA at pH 2.3. The uncomplexed Bi(III) was then deposited on a glassy carbon electrode at a potential of ?0.40 V vs. SCE and subsequently stripped potentiometrically using potassium dichromate as oxidant. The stripping time of uncomplexed Bi(III) was recorded. The Concentration of EDTA in the sample was determined from the concentration of added Bi(III) and the potentiometrically stripped Bi(III) at ?0.4 V by the standard addition method. The relative standard deviation for EDTA concentration of 95 ppb and 4.5 ppb was 1.9% and 2.6%, respectively. The detection limit was about 1 ppb EDTA for a deposition time of 3 minutes.     

Preparation and Characterization of Olaquindox Polyclonal Antibody

本研究先将喹乙醇琥珀酸单酯化,使其转变为带有羧基的衍生物。合成产物经重结晶纯化,得率为52.47%。合成产物进行结构鉴定,合成了具有喹乙醇分子结构特征的喹乙醇半抗原。采用活化酯法将半抗原与牛血清白蛋白（BSA）进行偶联,采用混合酸酐法将半抗原与卵清蛋白（OVA）进行偶联,分别制备免疫原和包被抗原。紫外扫描与红外图谱分析结果表明,半抗原与载体蛋白偶联成功,与BSA、OVA的结合比分别为3.8:1和5.0:1。以OLA-BSA作为免疫原免疫4只新西兰大白兔,获得了较高效价的抗血清,以OLA-OVA作为包被抗原,间接ELISA法测定各兔的效价,分别为1:6400;1:1600;1:12800;1:6400。间接ELISA法喹乙醇的抑制中浓度IC50为743.3 ng/mL,最低检测限IC20为5.71 ng/mL。从电泳图谱分析可以看出,经纯化得到了纯度较高的喹乙醇多克隆抗体,为喹乙醇免疫分析方法的进一步研制和开发奠定基础。     

Absorptiometric Determination of Acetylsalicylic Acid in Aqueous Ethanolic Solution

Abstract

Acetylsalicylic acid (ASA) was found to be quite soluble and quite stable in a 50/50% volume/volume water–ethanol solution. In this solution, a well-defined spectrum with a maximum absorbance, λ_max, of 276 nm of ASA was observed, and a molar absorptivity, ε, of 1.1 × 10³ M-cm was determined. These characteristic physical properties were used to determine ASA concentration in different brands of aspirin. The λ_max and the ε of salicylic acid were also determined. The characteristic values determined were 300 nm and 3.83 × 10³ M-cm, respectively. In this solution and using the method developed here, the concentrations of salicylic acid and ASA in a given aspirin formulation can be determined simultaneously.     

High Performance Liquid Chromatographic Determination of Neomycin in Milk Using a Hisep Column

Abstract

A high performance liquid chromatographic (HPLC) method for the determination of neomycin in milk is described. Milk is passed directly through an amberlite CG-50 ion exchange resin column, and the neomycin which is retained on the column is derivatized with ortho-phthalaldehyde (OPA) reagent. The derivatized neomycin is eluted from the column with potassium borate buffer/methanol and analyzed by HPLC. A HISEP HPLC column with fluororoetric detection was used. Recoveries ranged from 94 to 102% in samples fortified between 0.1 to 5ppra levels. The detection limit is 50ppb.     

Development of an Enzyme Immunoassay for the Determination of the Herbicide Metsulfuron-Methyl Based on Chicken Egg Yolk Antibodies

Abstract

The development of an indirect competitive enzyme immunoassay for the sulfonylurea herbicide metsulfuron-methyl (MSM) is described. In contrast to traditional antibody generation in mammals, this extremely sensitive method is based on chicken egg yolk antibodies (IgY). They were raised in laying hens using an MSM-derivative-BSA hapten as immunogen. With a 1:10000 dilution of the antibody solution and a coating antigen (MSM-derivative-KLH) concentration of 10 μg L^?1 the IC₅₀ value achieved for the target analyte was 0.4 μg L^?1. The least detectable dose was established at 13 ng L^?1. Cross-reactivity was tested with 5 structurally related compounds, where only sulfometuron showed a significant binding. The ELISA was tested with spiked tap and surface water samples. This paper, for the first time, demonstrates the production of high-affinity IgY antibodies for a herbicide compound.     

Preparation and adhesive properties of colored Ti/Zr-based conversion coating on aluminum foil for lithium battery packaging

In this study, an aluminum (Al) foil used for lithium ion battery packaging film was treated with titanium (Ti)/zirconium (Zr) solution containing hexafluorotitanic acid and hexafluorozirconic acid using tannic acid as a colorant and metavanadate as an accelerator, respectively, and a golden conversion coating was successfully deposited on the surface of Al foil. The morphology and composition of the coating were characterized by scanning electron microscopy, energy dispersive spectroscopy, and X-ray photoelectron spectroscopy, and the hydrophilicity was assessed by contact angle measurement. The effect of Ti/Zr treatment on the adhesion properties of Al foil was evaluated by T-peeling test and compared with that of traditional chromate-phosphate treatment. The results show that tannic acid contributes to the formation of the golden coating, and metavanadate accelerates the formation of the conversion coating. The results also indicate that the Ti/Zr-based conversion coating is mainly composed of Al₂O₃, Al (OH)₃, AlF₃, TiO₂/ZrO₂, NH₄VO₃, and V₂O₅. The Ti/Zr treatment cannot only improve the heat-sealing strength, but also the T-peeling strength by approximately 12 times compared with that of untreated Al foil. Thus, Ti/Zr treatment has the potential to replace the traditional chromate conversion treatment.     

Resin Bead Detection and Spectrophotometric Determination of Ascorbic Acid in Pharmaceutical Preparations Using m-Dinitrobenzene

Abstract

A colorimetric reaction has been studied for the detection and determination of ascorbic acid in samples at the microgram level. the method is simple, rapid, and sensitive, the ascorbic acid was detected using resin beads and determined spectrophotometrically using m-dinitrobenzene in formaldehyde. the detection limit was 10μg. Beer's law is obeyed in the concentration range of 2 – 50μg/ml of ascorbic acid.     

Simultaneous Detection of Ascorbic Acid and Hydrogen Peroxide by Flow‐Injection Analysis with a Thin Layer Dual‐Electrode Detector

Based on preliminary voltammetric investigations at unmodified and modified electrodes in aqueous solutions buffered at different pHs, a profitable thin layer dual‐electrode detector was developed for the selective and simultaneous determination of ascorbic acid (AA) and hydrogen peroxide present in the same samples. It consists of a small thickness (0.1 mm) cell, used as a detector for a flow injection system, in which a glassy carbon (GC) and a polyeugenol coated Pt electrode are encased. The GC electrode is selective for AA, thanks to the potential applied (0.3 V vs. Ag/AgCl,Cl^?_sat), while H₂O₂ alone can be oxidized at the Pt electrode (0.75 V), thanks to the size selectivity and electrostatic screen properties displayed by the polyeugenol coating layer which prevents oxidation of ascorbate anions. Repeatable sharp peaks (±4.5 %) were detected for both analytes over wide linear ranges (0.005–1.000 mM) and detection limits of about 10^?6 M (176 ppb) and 5×10^?7 M (16 ppb) were inferred for AA and H₂O₂ respectively. This flow injection approach was applied to the analysis of some orange‐taste soft drinks, used as prototype real samples, spiked with controlled amounts of both analytes, thus inferring good recoveries which pointed out that deviations never exceeding 5 % affected the relevant accuracy.     

A Piezoelectric Crystal Detector for Acetoin

Abstract

A piezoelectric quartz crystal coated with tetrabutyphosphoniun chloride was found to be a good detector for acetoin. The coating is sensitive in the ppb range and completely reversible. The response curve is linear over a concentration range of 8-120 ppb. The detector can be tuned to give a satisfactory response in 30 seconds.     

Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLC and HPLC

Abstract

High performance thin layer chromatographic (HPTLC) and high performance liquid chromatographic (HPLC) methods were developed for the simultaneous determination of Tinidazole and Furazolidone in suspension.

In the HPTLC method the separation of Tinidazole and Furazolidone was carried out on silica gel 60F₂₅₄ HPTLC glass plate using chloroform:methanol:ammonia (9:1:0.1 v/v) as a mobile phase. Rf values obtained were 0.63 and 0.79 for Furazolidone and Tinidazole respectively. Densitometric evaluation was done at 335 nm. Linearity was obtained within the concentration range 10–50 μg/ml and 3.5–17.5 μg/ml for Tinidazole and Furazolidone respectively.

The second method is based on high performance liquid chromatography on a reversed phase column (μ Bondapak C₁₈) using a mobile phase comprised of water: acetonitrile: triethylamine (80:20:0.1 v/v) adjusted to pH = 3.0 with dil. phosphoric acid. Retention times were 5.24 and 7.82 min for Tinidazole and Furazolidone respectively at a flow rate of 1.5 ml/min. Detection was done at 335 nm. Linearity was obtained within the concentration range 30–180 μg/ml and 10.5–63 μg/ml for Tinidazole and Furazolidone resp.     

Electrochemical Oxidation Behavior of Nitrogen Dioxide for Gas Detection Using Boron Doped Diamond Electrodes

The electrochemical oxidation reaction of nitrogen dioxide (NO₂) using boron doped diamond (BDD) electrodes is presented. Cyclic voltammetry of NO₂ in a 0.1 M KClO₄ solution exhibits oxidation peaks at +1.1 V and +1.5 V (vs. Ag/AgCl) which are attributable to oxidation of HONO and NO₂⁻, respectively. Moreover, the pH and scan rate dependences were investigated to study the oxidation mechanism. A linear calibration curve was observed in the concentration range of ∼1 to 5 mM (R²=0.99) with a detection limit of 11.1 ppb (S/B=3) for HONO and 58.6 ppb (S/B=3) for NO₂⁻. In addition, the analytical performance was compared with those using glassy carbon, platinum and stainless steel as the working electrode.