Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA–BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL⁻¹, and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74–110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL⁻¹ for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R ² = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance

To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 μg kg⁻¹, respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L⁻¹ phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 μg kg⁻¹ in liver and 2 μg kg⁻¹ in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r ²) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 μg kg⁻¹).     

Enzyme-linked immunosorbent assay in analysis of deoxynivalenol: investigation of the impact of sample matrix on results accuracy

Enzyme-linked immunosorbent assay (ELISA) represents a bioanalytical strategy frequently used for rapid screening of mycotoxin deoxynivalenol (DON) in cereals and derived products. Due to a considerable affinity of some anti-DON antibodies to structurally similar DON metabolites, such as DON-3-glucoside (DON-3-Glc) and 3-acetyl-DON (3-ADON), a significant overestimation of DON concentrations may occur. A validation study of six commercial DON-dedicated ELISA kits, namely Ridascreen DON, Ridascreen FAST, DON, DON EIA, AgraQuant DON Assay, Veratox 5/5, and Veratox HS was carried out on wheat, barley, and malt matrices. Performance characteristics of all tested ELISAs were determined using aqueous solutions of DON, DON-3-Glc, and 3-ADON analytical standards, further with extracts of artificially spiked blank cereals, and finally with matrix-matched standards of all three compounds. In the final phase, the accuracy of data was assessed through a comparison of DON concentrations determined by particular ELISAs and reference ultra-high-performance liquid chromatography–tandem mass spectrometry method. For this purpose, both quality control materials and a comprehensive set of naturally and artificially contaminated samples of wheat, barley, and malt were analyzed. High cross-reactivities were proved for both DON-3-Glc and 3-ADON in the majority of examined assays, and moreover, a considerable contribution of some matrix components to overestimation of DON results was confirmed.     

Enzyme-linked immunosorbent assays for the insecticide fenitrothion. Influence of hapten conformation and sample matrix on assay performance

This study aimed at developing competitive enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus (OP) insecticide fenitrothion using a monoclonal antibody. The hapten used to obtain the antibody had an ideal structural feature that allowed minimal functional group sacrifice. By using the antibody and a coating antigen, a competitive indirect ELISA was developed, which showed an IC₅₀ of 14 ng mL⁻¹ with a detection limit of 3.0 ng mL⁻¹. A competitive direct ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 17 ng mL⁻¹ with a detection limit of 1.6 ng mL⁻¹. The antibodies in both assays showed negligible cross-reactivity with the metabolites of fenitrothion and other OP pesticides except with the insecticides parathion-methyl and parathion-ethyl. Recoveries of fenitrothion from fortified rice and lettuce samples were determined and the bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.     

Enzyme-linked immunosensor based on super paramagnetic nanobeads for easy and rapid detection of okadaic acid

Okadaic acid (OA), a lipophilic phycotoxin highly toxic to humans is produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, competitive indirect enzyme-linked electrochemical immunosensor based on super paramagnetic nanobeads has been developed for the detection of OA. Streptavidin-coated magnetic beads were used as support to immobilize the biotinylated OA. Preliminary, colorimetric tests were performed in order to optimize different experimental parameters. Electrochemical detection was carried out by differential pulse voltammetry (DPV). The limit of detection (LOD) (0.38 μg L⁻¹), the mid point value (IC₅₀) (3.15 μg L⁻¹) and the time needed (60 min) for analysis of a real sample validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed, showing an excellent percentage of recovery.     

Hapten synthesis and enzyme-linked immunosorbent assay for phosmet residues: assay optimization and investigation of matrix effects from different food samples

In this study, a panel of haptens was synthesized for immunoconjugate preparation, and several haptens for heterologous tracer conjugates were also prepared. A highly sensitive polyclonal antibody against the organophosphorus insecticide phosmet was obtained and competitive direct enzyme-linked immunosorbent assays (cd-ELISA) for this pesticide were developed. In the cd-ELISA, the limit of detection (IC₁₅) was 0.6 μg kg⁻¹ and the sensitivity (IC₅₀) was 20 μg kg⁻¹. The suitability of the ELISA for pesticide quantification in peach, apple, orange juice, and apple juice was also studied. Good accuracy and precision were obtained with mean recoveries between 78% and 102.3% and mean coefficients of variation below 13.63%. Validation of the ELISA was conducted by high-performance liquid chromatography. The correlation between the data obtained using the microwell assay and the high-performance liquid chromatography was good (R ² = 0.9849). The developed immunoassay methods were suitable for the rapid quantitative or qualitative determination of phosmet in food samples.     

Enzyme-linked immunosorbent assay and immunochromatographic strip for rapid detection of atrazine in water samples

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic (ICG) strip for the determination of the herbicide atrazine in water samples. The ELISA had a half-maximum inhibition concentration (IC₅₀) of 0.12 ng mL^?1 and a limit of detection (LOD, calculated as the IC₁₅ value) of 0.01 ng mL^?1. The average of recoveries for all spiked water samples was 96.5%. There was a good correlation between the data determined by this ELISA and those obtained by high performance liquid chromatography (HPLC) (r ² ?=?0.996). The visual LOD of the ICG strip assay was 2 ng mL^?1. The assay process only took 10 min, and no sample pretreatment was required. Its high specificity, sensitivity and fast detection made the strip well suited for on-site screening of atrazine in water samples. Both the ELISA and the ICG strip assay are useful for rapid analysis of a large number of water samples at low cost.

Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic assay for several (fluoro)quinolones in milk

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a visual colloidal gold-based immunochromatographic assay (CGIA) for simultaneous determination of ofloxacin, marbofloxacin, and fleroxacin residues in milk using polyclonal antibodies. The half-maximum inhibition concentrations (IC₅₀) of ofloxacin, marbofloxacin, fleroxacin, and limits of detection (LODs; calculated as IC₁₅ values) are between 0.20 and 0.53?ng mL^?1, and between 0.02 and 0.05?ng mL^?1, respectively. The average recoveries range from of 78% to 113%, and the coefficients of variation of intra- and inter-assays are between 2 and 11%, and 3 to 19%, respectively. The LODs for ofloxacin, marbofloxacin, fleroxacin in milk are between 3.5 and 8.9?ng mL^?1. The visual minimum detection limit of the optimized CGIA is 2?ng mL^?1 for milk samples. The detection process can be completed within 10?min. The strips can be stored at 4?°C for 8?weeks without significant loss of activity. The results of the analysis of spiked samples showed that the CGIA can be applied to preliminary, fast, and on-site screening of milk samples. The ELISA and CGIA allow for a rapid, sensitive, and low-cost determination of (fluoro)quinolones residues in milk samples.

Enzyme-linked immunosorbent assay for the determination of T-2 toxin in cereals and feedstuff

A reliable enzyme-linked immunosorbent assay method was developed for the assay of T-2 toxin in cereals and feedstuff. A hapten of the T-2 toxin was synthesized, and a polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Compared to the other ELISA methods, the assay is simple, rapid and affordable. The concentration of T-2 causing 15% inhibition is 0.01?±?0.001 ng mL^?1, which makes the method more sensitive than others. The cross-reactivity against other mycotoxins is low, except for the HT-2 toxin. Sample extraction was achieved within 3 min. The recoveries from samples including barley, wheat, corn, oat, rice and feedstuff were between 75% and 102%, and the detection limit for T-2 toxin was lower than 4 ng g^?1. The method was validated by high-performance liquid chromatography with tandem mass spectrometry detection.     

Enzyme-linked immunosorbent assay for the determination of 20(S)-protopanaxatriol

A highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) method has been developed for the determination of 20(S)-protopanaxatriol (PPT), one of the major aglycones of dammarane-type ginseng saponins. Polyclonal antibodies raised against ginsenoside F₁ (GF₁)-bovine serum albumin showed high reactivities to PPT and GF₁, whereas they exhibited minor or even no cross-reactivities to other ginsenosides and protopanaxadiol (0.19%). The working range of this method extends from 50 pg ml⁻¹ to 20 ng ml⁻¹ of PPT. The assay reported here has been validated against an HPLC technique using PPT-containing samples and was shown to correlate closely (γ=0.993). This ELISA could be a useful tool for the determination of PPT contained in biological fluids and plant materials.     

Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents

Lowering of the detection limit of solid-state lead-selective electrodes was achieved by using the tuned galvanostatic polarization method. A Nernstian response was obtained down to nanomolar concentrations (low detection limit 10(-9) mol dm(-3)Pb(2+)). Good repeatability of the calibration curves was achieved by using a well established measuring procedure. Relatively high cathodic current densities were applied to the solid-state membrane in order to shorten the measurement time. Successful determination of lead in a synthetic sample (pPb(2+)=7.97±0.08) was achieved by introducing an analytical protocol and favourably compared to inductively coupled plasma mass spectrometry (pPb=7.93). By applying this method, a significant improvement in the detection limit of solid-state lead-selective electrodes was attained.     

Enzyme-linked immunosorbent assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection

Five different clones of antibodies developed against the aflatoxin M₁ were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM₁ in milk, in a wide linear working range (4-250 ng/L). The IC₅₀ value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM₁), showing an excellent percentage of recovery, close to 100%.     

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.     

Theoretical considerations on the estimation of sample matrix effects in analytical chemistry

Methods are suggested for evaluating the magnitude of matrix effects on the results of analysis.     

Effects of sample matrix on detection limits in analytical inductively coupled plasma-Fourier transform spectrometry

The feasibility of making analytical atomic spectrometry measurements by inductively coupled plasma-Fourier transform spectrometry (ICP-FTS) is demonstrated. Analytical working curves and detection limits are presented for Al, Ni, Fe and Ca. The effects of sample matrix composition on detection limits for analytical ICP-FTS are investigated. It is shown that a multiplex disadvantage may occur in the case of a spectral bandpass encompassing stroog emission lines of a matrix element. A “worse case” example of this problem is presented. Possible approaches to overcoming the multiplex disadvantage in analytical ICP-FTS and some ideal criteria for ICP-FTS instrumentation are presented.     

Enzyme-linked immunosorbent assays based on peroxidase labels and enzyme-amplified lanthanide luminescence detection

The enzyme-amplified lanthanide luminescence (EALL) detection is developed and applied for the determination of peroxidase as marker in enzyme-linked immunosorbent assays (ELISA). The detection scheme is based on the peroxidase catalysed dimerization of 4-hydroxyphenylpropionic acid (pHPPA) and the subsequent formation of a ternary complex with Tb(III)EDTA. Quantum yields and fluorescence lifetimes of the luminescent species are presented to give an estimate of the potential of this procedure. Two different ELISA were performed with the EALL detection scheme. For the first, a model ELISA for the determination of goat anti-rabbit IgG, a limit of determination of 3 micrograms dm-3 (2 fmol) of the antibody could be achieved. As second model assay, a commercial ELISA kit was successfully validated for the new detection scheme. Photometric and EALL detection were in good agreement for the determination of human anti-gliadin IgA in serum.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of glycyrrhizic acid

Glycyrrhizic acid (GL) is a major active compound of licorice. The specific monoclonal antibody (MAb) (designated as 8F₈A₈H₄2H₇) against GL was produced with the immunogen GL–BSA conjugate. The dissociation constant (K _d) value of the MAb was approximately 9.96×10⁻¹⁰ M. The cross reactivity of the MAb with glycyrrhetic acid was approximately 2.6%. The conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA adapted with a modified procedure were established using the MAb. The IC₅₀ value and the detect range by the conventional icELISA were 1.1 ng mL⁻¹ and 0.2–5.1 ng mL⁻¹, respectively. The IC₅₀ value and the detect range by the simplified icELISA were 5.3 ng mL⁻¹ and 1.2–23.8 ng mL⁻¹, respectively. The two icELISA formats were used to analyze GL contents in the roots of wild licorice and different parts of cultivated licorice (Glycyrrhiza uralensis Fisch). The results obtained with the two icELISAs agreed well with those of the HPLC analysis. The correlation coefficient was more than 0.98 between HPLC and the two icELISAs. The two icELISAs were shown to be appropriate, simple, and effective for the quality control of raw licorice root materials.     

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for monensin.

Monensin, a member of the ionophoric polyether antibiotics, is used primarily as a coccidiostat. A protein conjugate of monensin was prepared and utilized to produce monoclonal antibodies in the BALB/c-P3X63Ag8U.1 fusion system. Only one hybridoma that produces monoclonal antibody against monensin was isolated from one in 329 wells. The monoclonal antibody was used to develop quantitative assays for monensin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 1 ng ml-1 and the relative standard deviations were 2.1-6.3% intra-assay and 5.9-12.9% inter-assay. All ELISA results for assay of chicken plasma and cattle milk were confirmed using a bioassay to be used as the official method. The ELISA and bioassay results showed close correlations for plasma (r2 = 0.98, n = 25) and milk (r2 = 0.95, n = 25). Using the anti-monensin monoclonal antibodies produced, a rapid test kit based on the immunochromatographic method was developed. Detection limits of monensin for cattle milk, cattle plasma and chicken plasma were about 40, 40 and 160 ppb. respectively.