Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements

A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg⁻¹. The detection capability (CCβ) of the assay was determined to be ≤0.85 mg kg⁻¹ for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg⁻¹. The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.     

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 μg l⁻¹ for carbaryl and TCP, respectively. The sensitivity attained (I₅₀ value) was around 30 μg l⁻¹ for both compounds. Linear working ranges were 15-53 μg l⁻¹ for carbaryl and 13-83 μg l⁻¹ for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.     

A spectrophotometric procedure for DNA assay with a micro-sequential injection lab-on-valve meso-fluidic system

A micro-sequential injection spectrophotometric procedure for DNA assay was developed based on the employment of a lab-on-valve (LOV) meso-fluidic analytical system. A small amount of crystal violet solution (10 μl) was de-colored inside the flow cell of the LOV at the presence of 5 μl λ-DNA/HindIII within a certain pH range, and the absorbance decrease of crystal violet solution at 591 nm was measured via optical fibers and was employed as the basis of quantification. A uni-variant approach was adopted for the optimization of experimental parameters, including buffer pH, concentration and volume of crystal violet solution, reaction time and sample/reagent loading flow rates. A linear calibration graph was obtained within 0.2-6.0 μg ml⁻¹, along with a detection limit of 0.07 μg ml⁻¹. The procedure was applied for the determination of λ-DNA/HindIII in synthetic samples in comparison with a documented procedure.     

Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O-methylguanine-DNA methyltransferase

O⁶-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120 ng mL⁻¹ with a detection limit of 0.5 ng mL⁻¹. By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.     

Development of a disposable amperometric immunosensor for the detection of ecstasy and its analogues using screen-printed electrodes

An amperometric immunosensor for the specific and simple detection of 3,4-methylenedioxyamphetamine (MDA) and its analogues, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) in saliva and urine was developed. A direct competitive assay in which free analyte and horseradish peroxidase labelled species were simultaneously added to an immobilised polyclonal antibody was employed. Both MDA and MDMA could be labelled with the enzyme and the use of an MDMA-HRP tracer greatly enhanced the sensitivity of the assay. Amperometric detection was performed at +100 mV versus Ag/AgCl, using tetramethylbenzidine (TMB)/H₂O₂ as substrate. The antibody, raised specifically against the methylenedioxy moiety of an MDA-BSA immunogen allowed highly specific detection of these analogues with negligible cross-reactivity towards any other amphetamine related compounds. Total assay time was 45 min and the standard curve using MDA could be evaluated within the range 0.61-400 ng ml⁻¹ with corresponding limit of detection (LOD) of 0.36 and 0.042 ng ml⁻¹ for saliva and urine, respectively. The cross-reactivity pattern of the analytes was determined and showed the order of sensitivity increased with increased alkyl chain length (MDA < MDMA < MDEA). The overall performance of the sensor, working range, precision and sensitivity demonstrate its usefulness for rapid and direct measurement of methylenedioxy analogues of ecstasy in saliva and urine. The sensor has better specificity than any previous method for ecstasy, with greater sensitivity than ELISA methods, is less expensive/assay with an “easier to use” format than previous methods. The detection works in saliva or urine, eliminating requirement of blood sampling, with improved precision.     

Determination of trace amounts of bromide by flow injection/stopped-flow detection technique using kinetic-spectrophotometric method

A simple, sensitive and selective method for the determination of bromide in seawater by using a flow injection/stopped-flow detection technique was examined. The detection system was developed for a new kinetic-spectrophotometric determination of bromide in the presence of chloride matrix without any extraction and/or separation. The detection was based on the kinetic effect of bromide on the oxidation of methylene blue (MB) with hydrogen peroxide in a strongly acidic solution. Large amounts of chloride could enhance the sensitivity of the method as an activator. The decolorisation of the blue color of MB was used for the spectrophotometric determination of bromide at 746 nm. A stopped-flow approach was used to improve the sensitivity of the measurement and provide good linearity of the calibration over the range of 0-3.2 μg ml⁻¹ of bromide. The relative standard deviation was 0.74% for the determination of 2.4 μg ml⁻¹ bromide (n = 5). The detection limit (3σ) was 0.1 μg ml⁻¹ with a sampling frequency of 12 h⁻¹. The influence of potential interfering ions was studied. The proposed method was applied to the determination of bromide in seawater samples and provided satisfactory results.     

Direct chiral resolution and its application to the determination of fungicide benalaxyl in soil and water by high-performance liquid chromatography

A simple chiral high-performance liquid chromatography (HPLC) method with ultraviolet (UV) and circular dichroism (CD) detection was developed and validated for measuring benalaxyl enantiomers using (R,R) Whelk-O 1 column. The effects of mobile phase composition and column temperature on the entioseparation were investigated. A CD detector was used to determine the elution order of the enantiomers. Excellent resolution was easily obtained using n-hexane-polar organic alcohols mobile phase. The chiral recognition mechanism was also discussed. Based on the developed chiral HPLC method, enantioselective analysis methods for this fungicide in environment matrix (soil and water) were developed and validated. Good linearities were obtained over the concentration range of 0.25-25 mg L⁻¹ for both enantiomers. Liquid-liquid extraction and solid phase extraction (SPE) were used for the enrichment and cleanup of soil and water samples. Recoveries for the two enantiomers were 79-91% at 0.02, 0.04 and 0.2 mg kg⁻¹ levels from soil, and 89-101% at 0.0025, 0.01 and 0.05 mg L⁻¹ levels from water. Run-to-run and day-to-day assay precisions were below 10% for both enantiomers at concentrations of 0.5, 1 and 5 mg L⁻¹. Individual detection limits of the two enantiomers were both 2 ng. Limits of detection (LOD) were 0.004 mg kg⁻¹ in soil and 0.001 mg L⁻¹ in water.     

A novel, selective, and rapid fluorimetric method for the simultaneous analysis of coproporphyrin and uroporphyrin in urine

A simple and rapid spectrofluorimetric method is described for the determination of the closely overlapping mixture of coproporphyrin (CP) and uroporphyrin (UP) in urine samples. Matrix Isopotential Synchronous Fluorescence Spectrometry (MISFS) was applied to improve the spectral resolution for the severely overlapped spectra of the urinary porphyrins. First-order derivative technique eliminates the background interference of each component on the other. Using these two techniques together, selectivity was improved, while maintaining a high sensitivity, and time-consuming separation processes and multiple scanning processes were avoided. The limits of detection were 0.15 nmol L⁻¹ and 0.1 nmol L⁻¹ for CP and UP, respectively. The concentrations of CP and UP were determined from the peak amplitudes of the Derivative Matrix Isopotential Synchronous Fluorescence (DMISF) spectra, at their detection points where the interference was suppressed. Porphyrins excretion in urine samples, collected from normal subjects, was studied. A comparison between the new method and the anion-exchange chromatographic method of Martinez and Mills was established using Bland-Altman method and the results indicate that these two methods are in a good agreement with each other.     

Screening and determination of melamine residues in tissue and body fluid samples

Melamine is a chemical product that was sporadically mixed into animal feeds to boost protein content. Excessive melamine in animal feed can induce renal failure and even death in animals. The residue of melamine in edible animal products also threatens human health. Currently, there is no real-time and high throughput method to detect residual melamine in animal tissues. Successful development of such methods is very important for fast and on-site screening of melamine residue in animal tissues to eliminate the potential threat to human health. Here we demonstrate the detection of residual melamine from swine and chicken tissues and body fluids using indirect competitive enzyme-linked immunosorbent assay (ELISA) method. A detection sensitivity of 0.5 μg mL⁻¹ and a limit of detection of 0.05 μg mL⁻¹ were achieved with this method. A gas chromatography-mass spectrometry (GC-MS) method was also developed to act as a confirmatory and quantitative procedure for the ELISA results. The limits of quantitation (LOQ) of were 0.01 μg g⁻¹ and 0.005 μg mL⁻¹ for tissues and body fluids, respectively. The two methods showed good agreement (r² > 0.992). The method developed was performed on samples of tissues from chickens fed with melamine-spiked feed.     

Development of a sensitive detection method of cancer biomarkers in human serum (75%) using a quartz crystal microbalance sensor and nanoparticles amplification system

A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 μg mL⁻¹ BSA, 0.5 M NaCl, 500 μg mL⁻¹ dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL⁻¹ PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL⁻¹ was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples.     

Highly-sensitive label-free electrochemical carcinoembryonic antigen immunosensor based on a novel Au nanoparticles–graphene–chitosan nanocomposite cryogel electrode

For the first time, a simple and highly sensitive label-free electrochemical carcinoembryonic antigen (CEA) immunosensor based on a cryogel electrode has been developed and tested. The as-prepared nanocomposite combined the advantages of the graphene, AuNPs and chitosan (AuNPs–GP–CS) together with the ease of preparing a cryogel coupled to a silver deposition, to act as a redox mediator, on a Au electrode. Under the optimal conditions, the decrease of the cyclic voltammetry (CV) silver peak current was proportional to the CEA concentration over a range of from 1.0 × 10⁻⁶ to 1.0 ng mL⁻¹ with a detection limit of 2.0 × 10⁻⁷ ng mL⁻¹. This AuNPs–GP–CS cryogel electrode gave a 1.7 times higher sensitivity and 25 times lower detection limit than the non-cryogel electrode. Moreover, the proposed electrochemical immunosensor exhibited good selectivity, reproducibility and stability. When applied to analyse clinical serum samples, the data determined by the developed immunosensor were in agreement with those obtained by the current hospital analysis system (enzyme linked fluorescent assay) (P > 0.05), to indicate that the immunosensor would be potentially useful for clinical diagnostics.     

Development of fast Fourier transform continuous cyclic voltammetry at Au microelectrode in flowing solutions as a novel method for sub-nanomolar monitoring of lidocaine in injection and biological fluids

In this work a novel method for the fast monitoring of lidocaine in flow-injection systems has been developed. The fast Fourier transform continuous cyclic voltammetry (FFTCV) at gold microelectrode in flowing solution system was used for determination of lidocaine in its pharmaceutical formulation. The presented technique was very simple, precise, accurate, time saving and economical, compared with all of the previously reported methods. The recommended technique demonstrated some advantages over other reported methods. Firstly, there was no need for the oxygen removal from the test solution. Secondly, a picomolar detection limit was achieved, and additionally, the method was fast enough for the determination of any such compound, in a wide variety of chromatographic methods. The method was linear across the concentration range of 240-1.1 × 10⁵ pg mL⁻¹ (r = 0.996) with a limit of detection and quantitation 117.3 and 240 pg mL⁻¹, respectively. As a conclusion this system offers the requisite accuracy, sensitivity, precision and selectivity to assay lidocaine in injections.     

Alternative method for measurement of albumin/creatinine ratio using spectrophotometric sequential injection analysis

A simple, automatic and practical system for successive determination of albumin and creatinine has been developed by combining sequential injection analysis (SIA) and highly sensitive dye-binding assays. Albumin detection was based on the increase in the absorbance due to complex formation between albumin and eosin Y in acidic media. The absorbance of the complex was monitored at 547 nm. For the creatinine assay, the concentration of creatinine was measured by reaction with alkaline picrate to form a colored product which absorbs at 500 nm. The influences of experimental variables such as effects of pH, reagent concentration, standard/sample volume and interferences were investigated. Under optimal conditions, the automated method showed linearity up to 20 mg L⁻¹ for albumin and 100 mg L⁻¹ for creatinine. The 3σ detection limits were 0.6 and 3.5 mg L⁻¹ for albumin and creatinine, respectively, and the relative standard deviations (n = 10) were 2.49% for 20 mg L⁻¹ albumin, and 3.14% for 20 mg L⁻¹ creatinine. Application of the proposed method to the direct analysis of urinary samples yielded results which agreed with those obtained from the Bradford protein assay and a creatinine enzymatic assay according to a paired t-test. The results obtained should be a step towards developing a fully automated and reliable analytical system for clinical research, which requires direct determination of albumin and creatinine and/or its ratios.     

Electrochemiluminescence biosensor for the assay of small molecule and protein based on bifunctional aptamer and chemiluminescent functionalized gold nanoparticles

An electrochemiluminescence (ECL) biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs) was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of N-(aminobutyl)-N-(ethylisoluminol) enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of 5.0 × 10⁻¹²–5.0 × 10⁻⁹ M with a detection limit of 2.2 × 10⁻¹² M. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer–target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from 5 × 10⁻¹⁴ M to 5 × 10⁻¹⁰ M with a detection limit of 1.2 × 10⁻¹⁴ M. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The sensitivity of the present biosensor is superior to most available aptasensors for adenosine and thrombin. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples.     

Frequency-selective absorbance detection: Refractive index and turbidity compensation with dual-wavelength measurement

A frequency-selective absorbance detection approach and its applications are described. First, a digital signal processor-lock-in amplifier (DSP-LIA)-based absorbance detector was evaluated. Compared to a simple operational amplifier (TL082CP)-based detector, the DSP-LIA-based detector showed lower noise levels, but the relative advantage was reduced under very low photocurrent levels (down to few nA). A 7 cm pathlength flow cell with this commercial LIA-based detector exhibited excellent Beer's law linearity (r² = 0.9999) and a noise level of 7 micro absorbance units (μAU). The limit of detection (LOD, S/N = 3) for methyl orange (MO) was 7 nM with this detector. Finally, as a more affordable alternative to an LIA, a balanced demodulator integrated circuit chip was used to fabricate a dual wavelength-frequency-selective LED-based absorbance detector. This device successfully compensated refractive index (RI) effect and turbidity effect in test flow systems. The LOD for MO with this system was 8 nM.     

Nanosilver-doped DNA polyion complex membrane for electrochemical immunoassay of carcinoembryonic antigen using nanogold-labeled secondary antibodies

A simple and sensitive electrochemical immunoassay protocol was developed for the detection of carcinoembryonic antigen (CEA) using nanosilver-doped DNA polyion complex membrane (PIC) as sensing interface. To construct such an immunosensor, double-stranded DNA was initially assembled onto the surface of thionine/Nafion-modified screen-printed carbon electrode to adsorb silver ions with positive charges, then silver ions were reduced to nanosilver particles with the aid of NaBH₄, and then anti-CEA antibodies were immobilized on the nanosilver surface. Gold nanoparticles conjugated with horseradish peroxidase-labeled anti-CEA were employed as signal antibodies for the detection of CEA with a sandwich-type assay format. Under optimal conditions, the immunosensor exhibited a dynamic range of 0.03-32 ng mL⁻¹ with a low detection limit of 10 pg mL⁻¹ CEA. Intra- and inter-assay imprecision (CVs) were <9.5% and 6.5%, respectively. The response could remain 90.1% of the original current at 30th day. 50 real samples were evaluated using the immunosensor and the enzyme-linked immunosorbent assay, respectively, and received in accordance with those two methods.     

Quantification of biogenic amines by microchip electrophoresis with chemiluminescence detection

A highly sensitive microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of biogenic amines including agmatine (Agm), epinephrine (E), dopamine (DA), tyramine, and histamine in human urine samples. To achieve a high assay sensitivity, the targeted analytes were pre-column labeled by a CL tagging reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). ABEI-tagged biogenic amines after MCE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing CL emission. Since no CL reagent was added to the running buffer, the background of the CL detection was extremely low, resulting in a significant improvement in detection sensitivity. Detection limits (S/N = 3) were in the range from 5.9 × 10⁻⁸ to 7.7 × 10⁻⁸ M for the biogenic amines tested, which were at least 10 times lower than those of the MCE–CL methods previously reported. Separation of a urine sample on a 7 cm glass/poly(dimethylsiloxane) (PDMS) microchip channel was completed within 3 min. Analysis of human urine samples found that the levels of Agm, E and DA were in the ranges of 2.61 × 10⁻⁷ to 4.30 × 10⁻⁷ M, 0.81 × 10⁻⁷ to 1.12 × 10⁻⁷ M, and 8.76 × 10⁻⁷ to 11.21 × 10⁻⁷ M (n = 4), respectively.     

Fast Fourier transformation with continuous cyclic voltammetry at an Au microelectrode for the determination of morphine in a flow injection system

For the fast morphine monitoring in flow injection systems a highly sensitive method is being introduced in this work. The fast Fourier transformation with continuous cyclic voltammetry (FFTCV) in a flowing solution as a detection system was applied for the prompt morphine monitoring. Here it should be stressed that this technique is simple, precise, accurate, time saving and economical. This research includes the observation of the effects of various parameters on the sensitivity of the detection system. Eventually, it was concluded that the best condition was obtained within the pH value of 2, scan rate value of 40 V s⁻¹, accumulation potential of 400 mV and accumulation time of 0.6 s.In detail, the noteworthy advantages which this method illustrates in comparison with other reported methods are the following; no necessity for the oxygen removal from the test solution, a sub-nano molar detection limit and the fast determination of any such compound in a wide variety of chromatographic methods.The method proved to be linear over the concentration range of 285-305,300 pg mL⁻¹ (r = 0.999) with a detection limit and a quantitation limit of 95.5 and 285 pg mL⁻¹, respectively. Consequently, the method illustrates the requisite accuracy, sensitivity, precision and selectivity to assay morphine in its tablets and biological fluids.     

Benzimidazole carbamate residues in milk: Detection by Surface Plasmon Resonance-biosensor, using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for extraction

A surface plasmon resonance (SPR) biosensor screening assay was developed and validated to detect 11 benzimidazole carbamate (BZT) veterinary drug residues in milk. The polyclonal antibody used was raised in sheep against a methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein conjugate. A sample preparation procedure was developed using a modified QuEChERS method. BZT residues were extracted from milk using liquid extraction/partition with a dispersive solid phase extraction clean-up step. The assay was validated in accordance with the performance criteria described in 2002/657/EC. The limit of detection of the assay was calculated from the analysis of 20 known negative milk samples to be 2.7 μg kg⁻¹. The detection capability (CCβ) of the assay was determined to be 5 μg kg⁻¹ for 11 benzimidazole residues and the mean recovery of analytes was in the range 81-116%. A comparison was made between the SPR-biosensor and UPLC-MS/MS analyses of milk samples (n = 26) taken from cows treated different benzimidazole products, demonstrating the SPR-biosensor assay to be fit for purpose.     

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I₅₀ of the assay ranged from 0.64 to 20.98 μg mL⁻¹ for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I₅₀ and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL⁻¹, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.