A rapid and highly sensitive portable chemiluminescent immunosensor of carcinoembryonic antigen based on immunomagnetic separation in human serum

To detect a biomarker for lung cancer, carcinoembryonic antigen (CEA), a highly sensitive, selective, rapid and portable immunosensor based on immunomagnetic separation and chemiluminescence immunoassay was introduced. A sandwich scheme assay has been utilized with horseradish peroxidase (HRP) labeled anti-CEA antibody and immunomagnetic beads (IMBs). The presence of target protein CEA caused the formation of the sandwich structures (IMBs-CEA-HRP labeled antibody). IMBs were applied to capture CEA and immobilize CEA through the external magnetic field. The HRP at the surface of the antibody catalytically oxidized the luminescence substrate to generate optical signals which were detected by a portable home-made luminometer and which were directly proportional to the concentration of CEA in the samples. The signals were dependent on CEA concentrations in a linear range from 0 to 50 ng mL⁻¹. The limit of detection (LOD) of this method was as low as 5.0 pg mL⁻¹ (S/N = 3). The novel immunosensor was highly sensitive with an assay time of <35 min. The intra- and inter-assay coefficients of variation were <10%. The anti-CEA antibody can be bound to the bead efficiently with a conjugation rate of 73%. IMBs could be stored in 4 °C protecting from light for 2 months without obvious reduction of biological activity. Human reference sera mixed with various concentrations of CEA were tested with the proposed method and commercial enzyme-linked immunosorbent assay (ELISA) kit, and a good linear relationship was obtained. This proposed technique demonstrated an excellent performance for quantifying CEA and was expected to be used for clinical testing.     

Chemiluminescence determination of citrate and pyruvate and their mixtures by the stopped-flow mixing technique

A novel kinetic chemiluminescent method using the stopped-flow mixing technique has been investigated for the rapid and sensitive determination of citrate and pyruvate. The method is based on a tris(2,2′-bipyridiyl)ruthenium(III) (Ru(bpy)₃³⁺) chemiluminescence (CL) reaction. Ru(bpy)₃³⁺ was generated in the mixing chamber by oxidising tris(2,2′-bipyridyl)ruthenium(II) with cerium(IV). After selecting the best operating parameters, calibration graphs were obtained over the concentration ranges 0.38-38 μg ml⁻¹ and 8.7-1300 ng ml⁻¹ for citrate and pyruvate, respectively. The limits of detection were 0.1 μg ml⁻¹ for citrate and 0.3 ng ml⁻¹ for pyruvate. Based on the differential rate of the chemiluminescent reaction corresponding to citrate and pyruvate, a very simple kinetic procedure was developed for the simultaneous determination of both compounds. Mixtures of citrate and pyruvate in ratios between 15:1 and 1.5:1 were satisfactorily resolved. The proposed method was successfully applied to the determination of citrate in pharmaceutical formulations, pyruvate in animal blood serum and both compounds in human urine.     

A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL⁻¹ for CEA, with a detection limit of 0.3 ng mL⁻¹. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.     

Determination of umbilical cord and maternal plasma concentrations of fentanyl by using novel spectrophotometric and chemiluminescence enzyme immunoassays

Two novel enzyme immunoassays of fentanyl have been developed using Horseradish Peroxidase (HRP) as an enzyme, 3,3′,5,5′tetramethylbenzidine (TMB) and luminol as its colorimetric and its chemiluminescence substrate, respectively. A fentanyl polyclonal antibody was used as a capture antibody for fentanyl and fentanyl-bovine serum albumin (BSA) conjugate. The latter was first biotinylated and then bound by streptavidin labeled with HRP, resulting in the development of two novel competitive immunoassays. The detection limits were 0.045 and 0.0048 ng ml⁻¹ for spectrophotometric and chemiluminescence detection, respectively, and were much lower than existing HRP-fentanyl based kits. Intra- and inter-assay CVs were 2.6-4.5 and 5.4-11.2%, respectively, at concentrations of 0.050-5.000 ng ml⁻¹ for the colorimetric assay, whilst for the chemiluminescent assay intra- and inter-assay CVs were 3.7-6.2 and 6.2-12.3%, respectively, for the linear range of the assay at concentrations of 0.008-0.800 ng ml⁻¹. The methods in this study were developed in order to measure maternal and neonatal fentanyl plasma samples during cesarean section, after the application of a novel subarachnoid analgesia technique. The 28 maternal and neonatal plasma samples were measured by both assays, providing data for subarachnoid administration of fentanyl that had never been presented before.     

An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles

A quick and reproducible electrochemical-based immunosensor technique, using magnetic core/shell particles that are coated with self-assembled multilayer of nanogold, has been developed. Magnetic particles that are structured from Au/Fe₃O₄ core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The carcinoembryonic antibody (anti-CEA) was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode (SPCE) by an external magnetic field. This is an assembly of a novel immuno biosensor for carcinoembryonic antigen (CEA). The sensitivity and response features of this immunoassay are significantly affected by the surface area and the biological compatibility of the multilayered nanogold. The linear range for the detection of CEA was from 0.005 to 50 ng mL⁻¹ and the limit of detection (LOD) was 0.001 ng mL⁻¹. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA detection.     

Flow-injection chemiluminescent determination of metoclopramide hydrochloride in pharmaceutical formulations and biological fluids using the [Ru(dipy)(3)(2+)]-permanganate system

A flow-injection (FI) methodology using (2,2′-dipyridyl) ruthenium(II) [Ru(dipy)₃²⁺] chemiluminescence (CL) was developed for the rapid and sensitive determination of metoclopramide hydrochloride. The method is based on the CL reaction of metoclopramide with Ru(dipy)₃²⁺ and KMnO₄ in a sulfuric acid medium. Under the optimum conditions, a calibration graph was obtained over the concentration range 0.005-3.5 μg ml⁻¹ with a limit of detection (S/N=2) of 1 ng ml⁻¹. The correlation coefficient was 0.99993 (n=8) with a relative standard deviation of 0.48% for 10 determinations of 1 μg ml⁻¹ of drug. The method was successfully applied to the determination of metoclopramide in pharmaceutical preparations and biological fluids after IP administration of 25 mg kg⁻¹ dose to rats. The elimination half-life was 2.5±0.4 h.     

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab₂)/thionine/nanoporous silver (HRP-Ab₂/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL⁻¹ with low detection limit of 0.35 pg mL⁻¹ and low limit of quantitation (LOQ) of 0.85 pg mL⁻¹. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules     

An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates

Based on direct hapten coated format a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for bisphenol A (BPA) was developed. Polystyrene surface was modified by 3-Aminopropyltriethoxysilane (APTES) to produce amino groups after H₂SO₄/HNO₃-pre-treatment. 4,4-bis (4-hydroxyphenyl) valeric acid (BVA) which is analogue of BPA, was successfully immobilized on the surface of microtiter plates by N,N′-dicyclohexylcarbodiimide (DCC) method. The essential steps of the assay were optimized, especially blocking procedure which is key step to prevent unspecific binding of antibody. The results indicated that compared with hapten-protein coated format (IC₅₀ = 176.67 ng ml⁻¹, LOD = 15.90 ng ml⁻¹), the direct hapten coated format (IC₅₀ = 23.50 ng ml⁻¹, LOD = 0.27 ng ml⁻¹) could improve assay sensitivity and the detection ranges were 2.30 ng∼157.60 ng ml⁻¹ with good signal reproducibility (P value > 0.05) after careful optimization of assay conditions. Tap water samples and seawater samples were spiked with a known amount of BPA and measured by ciELISA. The average recoveries were between 70 and 142%. As far as we are aware this is the most sensitive ELISA for BPA yet reported.     

Flow injection electrochemical enzyme immunoassay based on the use of an immunoelectrode strip integrate immunosorbent layer and a screen-printed carbon electrode

A flow injection amperometric immunoassay system based on the use of screen-printed carbon electrode for the detection of mouse IgG was developed. An immunoelectrode strip, on which an immunosorbent layer and screen-printed carbon electrode were integrated, and a proposed flow cell have been fabricated. The characterization of the flow immunoassay system and parameters affecting the performance of the immunoassay system were studied and optimized. Amperometric detection at 0.0 V (versus Ag/AgCl) resulted in a linear detection range of 30-700 ng ml⁻¹, with a detection limit of 3 ng ml⁻¹. The signal variation among electrode strips prepared from variant batch did not exceed 8.5% (n=7) by measuring 0.5 μg ml⁻¹ antigen standard solution.     

Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application

Silica-based functionalized terbium fluorescent nanoparticles were prepared, characterized and developed as a fluorescence probe for antibody labeling and time-resolved fluoroimmunoassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion containing a strongly fluorescent Tb³⁺ chelate, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)phenylpyridine] tetrakis(acetate)-Tb³⁺ (BPTA-Tb³⁺), Triton X-100, octanol, and cyclohexane by controlling copolymerization of tetraethyl orthosilicate (TEOS) and 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEPS) with ammonia water. The characterizations by transmission electron microscopy and fluorometric methods show that the nanoparticles are spherical and uniform in size, 45 ± 3 nm in diameter, strongly fluorescent with fluorescence quantum yield of 10% and a long fluorescence lifetime of 2.0 ms. The amino groups directly introduced to the nanoparticle’s surface by using AEPS in the preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticle-labeled anti-human α-fetoprotein antibody was prepared and used for time-resolved fluoroimmunoassay of α-fetoprotein (AFP) in human serum samples. The assay response is linear from 0.10 ng ml⁻¹ to about 100 ng ml⁻¹ with the detection limit of 0.10 ng ml⁻¹. The coefficient variations (CVs) of the method are less than 9.0%, and the recoveries are in the range of 84-98% for human serum sample measurements.     

Sensitive and real-time fiber-optic-based surface plasmon resonance sensors for myoglobin and cardiac troponin I

A sensor to detect markers of cardiac muscle cell death at less than 3 ng ml⁻¹ and in less than 10 min has been achieved. This fiber-optic-based surface plasmon resonance (SPR) sensor is being applied to detect myoglobin (MG) and cardiac troponin I (cTnI) in HEPES buffered saline solution. An in vivo sensor for the early detection of the onset of myocardial infarction (MI) will greatly enhance the patient care. MG and cTnI are two biological markers released from dying cardiac muscle cells during an MI, and their detection at biologically-relevant levels can be diagnostic of MI. Antibodies specific to an antigen of interest are attached to a carboxymethylated dextran layer on a gold SPR surface. With the method developed, the lower limit of detection (LOD) for MG is 2.9 ng ml⁻¹ at 25 °C. The biological level for MG reaches 15-30 ng ml⁻¹ in patient blood after myocardial damage. A Langmuir adsorption isotherm describes the binding well. For cTnI, a lower detection limit of 1.4 ng ml⁻¹ was achieved in preliminary tests. cTnI levels are in the range of 1-3 ng ml⁻¹ in patient blood after myocardial damage. The antibody reaction with the carboxymethylated dextran surface was optimized by modifying the reaction pH, the temperature, and the dextran chain length.     

Magnetic-particle-based,ultrasensitive chemiluminescence enzyme immunoassay for free prostate-specific antigen

We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1–30 ng mL⁻¹ with the detection limit of 0.1 ng mL⁻¹. The linear detection range could match the concentration range within the “diagnostic gray zone” of serum f-PSA levels (4–10 ng mL⁻¹). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL⁻¹). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum.     

Multiplex detection of tumor markers with photonic suspension array

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL⁻¹, 1.0-500 ng mL⁻¹, 1.0-500 U mL⁻¹ and 3.0-500 U mL⁻¹ with limits of detection of 0.68 ng mL⁻¹, 0.95 ng mL⁻¹, 0.99 U mL⁻¹ and 2.30 U mL⁻¹ at 3σ, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.     

A highly sensitive label-free resonance light scattering assay of carcinoembryonic antigen based on immune complexes

As a kind of glycoprotein, carcinoembryonic antigen (CEA) is the important tumor marker for clinical diagnosis of the presence or recurrence of cancer. In this work, a novel label-free resonance light scattering (RLS) spectral CEA assay was developed based on the combination of highly selective immunoreaction and ultrasensitive RLS technique. In Tris–HCl buffer solution (pH 7.5), the specific immunoreaction between CEA antigen and mouse anti-CEA formed immune complexes which had a maximum RLS spectral peak at 389.0 nm, with the existence of physiological saline and polyethylene glycol 20,000 (PEG 20,000). Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI_RLS) was proportional to the concentration of CEA in the range from 0.1 to 60 ng mL⁻¹, with a detection limit (LOD, 3σ) of 0.03 ng mL⁻¹. The characteristics of RLS, the CEA immunocomplex, the immune response, the ratio of CEA antigen and mouse anti-CEA, and the optimum conditions of the immunoreaction have been investigated. The CEA concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) kit. And this method has many satisfying merits including label-free, sensitivity and high selectivity.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

Sensitive sandwich immunoassay based on single particle mode inductively coupled plasma mass spectrometry detection

A sensitive sandwich type immunoassay has been proposed with the detection by inductively coupled plasma mass spectrometry (ICP-MS) in a single particle mode (time resolved analysis). The signal induced by the flash of ions (¹⁹⁷Au⁺) due to the ionization of single Au-nanoparticle (Au-NP) label in the plasma torch can be measured by the mass spectrometer. The frequency of the transient signals is proportional to the concentration of Au-NPs labels. Characteristics of the signals obtained from Au-NPs of 20, 45 and 80 nm in diameters were discussed. The analytical figures for the determination of Au-labeled IgG using ICP-MS in conventional integral mode and single particle mode were compared in detail. Rabbit-anti-human IgG was used as a model analyte in the sandwich immunoassay. A detection limit (3σ) of 0.1 ng mL⁻¹ was obtained for rabbit-anti-human IgG after immunoreactions, with a linear range of 0.3-10 ng mL⁻¹ and a RSD of 8.1% (2.0 ng mL⁻¹). Finally, the proposed method was successfully applied to spiked rabbit-anti-human IgG samples and rabbit-anti-human serum samples. The method resulted to be a highly sensitive ICP-MS based sandwich type immunoassay.     

Application of generalized artificial neural networks coupled with an orthogonal design to optimization of a system for the kinetic spectrophotometric determination of Hg(II)

A simple and sensitive kinetic method for the determination of traces of mercury (70-760 ng ml⁻¹) based on its inhibitory effect on the addition reaction between methyl green and sulfite ion is proposed. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of methyl green at 596 nm between 2 and 4 min using a fixed time method. Artificial neural networks with back propagation algorithm coupled with an orthogonal array design were applied to the modeling of the proposed kinetic system and optimization of experimental conditions. An orthogonal design was utilized to design the experimental protocol, in which pH, concentration of sulfite, temperature, and concentration of methyl green were varied simultaneously. Optimum experimental conditions in term of sensitivity were generated by using ANNs. The rate of decrease in absorbance is inversely proportional to the concentration of Hg(II) over entire concentration range tested (100-550 ng ml⁻¹) with a detection limit of 45 ng ml⁻¹ and a relative standard deviation at 200-400 ng ml⁻¹ Hg(II) of 3.2% (n=5). A simple preconcentration step improved the limit of detection and linear dynamic range of the method to about 8 and 12-760 ng ml⁻¹, respectively, by about 10 times enrichment of mercury between 12 and 75 ng ml⁻¹. The method was based on enrichment of Hg(II) from dilute samples on an anionic ion exchanger fixed on a plastic strip and was applied to the determination of Hg(II) in environmental samples with satisfactory results.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.     

Potassium permanganate-glyoxal chemiluminescence system for flow injection analysis of cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium in pharmaceutical preparations

A sensitive flow injection chemiluminescence (FL-CL) method for the determination of cephalosporin antibiotics, was developed. The method was based on that cephalosporin antibiotics could enhance the CL reaction of glyoxal and KMnO₄ in sulfuric acid. Method development included the optimization of reagent concentrations and flow-rate. Under the optimized conditions, three cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium, were determined. The detection limits of the method are 10 ng ml⁻¹ cefalexin, 2 ng ml⁻¹ cefadroxil, and 2 ng ml⁻¹ cefazolin sodium. The method was successfully applied to the determination of three cephalosporin antibiotics in pharmaceutical preparations.     

Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.