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.     

Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones

This study shows the detection of (N-acyl) homoserine lactones (AHLs or HSL) with monoclonal antibodies via a surface plasmon resonance (SPR)-based immunosensor in comparison to conventional microtiter plate-based enzyme-linked immunosorbent assay (ELISA). An HSL derivative, named HSL2 (Table 1), was attached to bovine serum albumin (BSA) and the conjugate (HSL2-BSA-r2) was either covalently immobilised on the SPR sensor chip surface via free amino groups or via adsorption on the ELISA polystyrene plate surface. With a newly developed rat monoclonal antibody (mAb HSL1/2 2C10), AHLs were detected sensitively in a competitive format with SPR and ELISA. Well comparable experiments between SPR and ELISA could be obtained in buffers. Moreover, the SPR sensor surface with the immobilised conjugate HSL2-BSA-r2 could be regenerated at least 340 times (regeneration cycles) without loss of activity. The measurement time per cycle was approximately 15 min. The competitive detection format for SPR and ELISA allowed the detection in the μg L⁻¹ range.     

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71-27 ng ml⁻¹ with a limit of detection (LOD) of 0.32 ng ml⁻¹, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC₅₀) was 4.2 ng ml⁻¹. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67-27 ng ml⁻¹ with a LOD of 0.38 ng ml⁻¹ and an IC₅₀ of 3.7 ng ml⁻¹. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml⁻¹ were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.     

Immunoenzyme assay of nonylphenol: study of selectivity and detection of alkylphenolic non-ionic surfactants in water samples

Immunoenzyme assay (ELISA) is proposed and characterized for determination of alkylphenol ethoxylates, a primary class of manufactured non-ionic surfactants. The assay is based on the obtained polyclonal antibodies against nonylphenol (NP), the main stable intermediate of the decomposition of nonylphenol ethoxylates. A mixture of non-modified branched isomers of NP was applied as hapten coupled to protein carriers by Mannich reaction with the use of formaldehyde. The proposed ELISA format is based on immobilized NP-(soybean trypsin inhibitor) conjugate as a competitor of antigen molecules contained in the tested sample for binding with specific antibodies indirectly labeled via an anti-species immunoperoxidase conjugate. The developed ELISA allows to reveal NP with the limit of detection about 10 ng ml⁻¹ and NP-related compounds such as octylphenol, alkylphenoletoxylates, alkylphenolcarboxylates and their halogenated derivatives. The ELISA was applied for assaying polluted water samples, namely influents and effluents from different wastewater treatment plants (WWTP) and tap water. ELISA and chromatographic data demonstrate good correlation (r = 0.94), while ELISA gives higher values. Due to endocrine disrupting and other toxic activities of some metabolites of alkylphenolic non-ionic surfactants, the developed assay may be effectively used in ecological monitoring and sanitary control.     

Screen-printed immunosensor modified with carbon nanotubes in a continuous-flow system for the Botrytis cinerea determination in apple tissues

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. This paper describes the development of an immunosensor coupled to carbon-based screen-printed electrodes (SPCE) modified with multi-walled carbon nanotubes (CNTs), which show a rapid and sensitive determination of B. cinerea in apple tissues (Red-delicious) using a competitive immunoassay method.Both the infected plant tissue sample and the B. cinerea-specific monoclonal antibody are allowed to react immunologically with the B. cinerea purified antigens immobilized on a rotating disk. Then, the bound antibodies are quantified by a horseradish peroxidise (HRP) enzyme labeled second antibodies specific to mouse IgG, using 4-tertbutylcatechol (4-TBC) as enzymatic mediators. The HRP, in the presence of hydrogen peroxide, catalyses the oxidation of 4-TBC to 4-tertbutyl o-benzoquinone. The electrochemical reduction back to 4-TBC is detected on SPCE-CNT at −0.15 V. The response current is inversely proportional to the amount of the B. cinerea antigens present in the fruit sample. The time consumed per assay was 30 min and the calculated detection limits for electrochemical method and the ELISA procedure are 0.02 and 10 μg mL⁻¹, respectively. Moreover the intra- and inter-assay coefficients of variation were below 7%. This electrochemical immunosensor promises to be usefully suited to the detection and quantification of B. cinerea in apparently healthy plant prior to the development of the symptoms.     

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.     

High performance enzyme-linked immunosorbent assay for determination of miroestrol,a potent phytoestrogen from Pueraria candollei

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73–3000 ng mL⁻¹, which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80–104.37% and 98.31–106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R² = 0.9996) (Yusakul et al. [18]) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695 ± 0.037–12.108 ± 0.285 μg g⁻¹ dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.     

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.     

Lanthanide sensitized chemiluminescence determination of grepafloxacin in tablets and human urine

A flow-injection chemiluminescence (CL) method, based on the luminescent properties of the Ce(IV)-Na₂SO₃-lanthanide(III)-grepafloxacin system, was developed for the determination of grepafloxacin {1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid}. La(III), Tb(III), and Eu(III) ions were tested as possible chemiluminescence sensitizers. The best results were achieved when Tb(III) was used as lanthanide ion, so the technique was optimised working with this ion. Under the optimum experimental conditions, the linear range was 0.05-2.00 μg ml⁻¹ grepafloxacin, with a 0.01 μg ml⁻¹ detection limit and 2.0% relative standard deviation (n=10). The proposed procedure has been applied to the determination of grepafloxacin in tablets and spiked human urine.     

Quick analysis of baicalin in Scutellariae Radix by enzyme-linked immunosorbent assay using a monoclonal antibody

To establish an immunoassay for baicalin (BA), a hybridoma cell line (9D6) secreting a monoclonal antibody (MAb) against BA was prepared by cell fusion with splenocytes derived from a mouse immunized with BA-bovine serum albumin (BSA) conjugate and a myeloma cell line, SP2/0-Ag14. MAb 9D6 shows specific reactivity against BA and its aglycone, baicalein, but not against other natural products. We developed an enzyme-linked immunosorbent assay (ELISA) using MAb 9D6 in a competitive manner, ranging from 200 ng/mL to 2 μg/mL. After validating the developed ELISA on the basis of intra- and inter-assays and a recovery experiment, it was found that the ELISA was not only simple, but also sufficiently reliable and accurate for quality control of Scutellariae Radix. It allowed determination of BA in complex and mixed materials, such as Kampo medicines.     

Salmonella typhi determination using voltammetric amplification of nanoparticles: A highly sensitive strategy for metalloimmunoassay based on a copper-enhanced gold label

A highly sensitive electrochemical amplification immunoassay for Salmonella typhi (S. typhi) determination has been developed for the first time by using a copper-enhanced gold nanoparticle label coupled with anodic stripping voltammetry. Monoclonal antibodies for S. typhi were first immobilized on polystyrene microwells and then captured by S. typhi bacteria. After an immunoreaction occurred, a polyclonal, antibody-colloidal gold conjugate was added to bind to the S. typhi bacteria. Next, a copper-enhancer solution containing ascorbic acid and copper (II) sulfate was added into the polystyrene microwells. The ascorbic acid was employed to reduce the copper (II) ions to copper (0), which was subsequently deposited onto the gold nanoparticle tags. After the copper was dissolved in nitric acid, the released copper ions were detected by anodic stripping voltammetry. The amount of deposited copper was related to the amount of gold nanoparticle tag present, which was controlled by the amount S. typhi attached to the polyclonal antibody-colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration over concentration range of 1.30 × 10² cfu/mL to 2.6 × 10³ cfu/mL in a logarithmic plot, with a detection limit as low as 98.9 cfu/mL. The influences of the relevant experimental variables, such as the concentration of copper and the reaction time of S. typhi with antibody, were investigated. We also successfully applied this method to determine the presence of S. typhi in human serum. Our results are a step towards developing more sensitive and reliable nanoparticle immunoassays.     

FT-IR measurement of tagitinin C after solvent extraction from Tithonia diversifolia

Tagitinin C, an antiplasmodial compound, identified as one major compound of the subtropical medicinal plant, Tithonia diversifolia, was determined by FT-IR spectroscopy method. The crude ether extracts from aerial parts of the plant were evaporated to dryness and re-dissolved in tetrachloroethylene (C₂Cl₄) before analysis.The magnitude of the absorbance of the very specific CO stretching vibration (ν_CO) at 1664.8 cm⁻¹ was exploited in order to quantify tagitinin C. The determination coefficient (r²) of the calibration scale was 0.9994, the detection limit was lower than 3 μg ml⁻¹ and the quantification limit was lower than 10 μg ml⁻¹.Recovery values from 100.5 to 101.7% were found for spiked concentration levels from 19.91 to 89.95 μg ml⁻¹. The main characteristics of the curves obtained from the calibration standards and from the standard addition technique were not statistically different (Student t-test) suggesting that matrix effects were negligible.The results obtained for the determination of tagitinin C in the crude ether extract from aerial parts of T. diversifolia by LC and FT-IR spectroscopic method agreed well: 0.76±0.02 and 0.773±0.009, of tagitinin C in dried plant respectively.     

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.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples

The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ng mL⁻¹. The values of IC₅₀ for nine standard curves were in the range of 1.1-2.0 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.07-0.14 ng mL⁻¹. The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀ value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n = 9). The mAb-based ELISA proven to be a feasible quantitative/screening method for Sudan I analysis in food samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput and low expense.     

Immunochemical Detection of Microquantities of Bacterial Formate Dehydrogenase

Abstract

Two immunochemical methods were developed for detection of NAD⁺?dependent formate dehydrogenase (EC 1.2.1.2, FDH) isolated from the methylotrophic bacteria Pseudomonas sp. 101:1) the dot-blot analysis using rabbit polyclonal antibodies; and 2) the indirect competitive ELISA using poly- or monoclonal mouse antibodies. The first method was used for screening the bacterial gene bank, the sensitivity is 5 and 1 pg enzyme per sample using the anti-rabbit antibodies - horse radish peroxidase conjugate or the biotinylated anti-rabbit antibodies and avidin - peroxidase conjugate, respectively. The second method was applied for precise determination of FDH concentration in cell-free extracts of selected recombinant clones. Mouse polyclonal antibodies to bacterial FDH have exibited a rather high affinity binding also to FDH from the methylotrophic yeast Candida methylica. In the indirect competitive ELISA the sensitivity of bacterial FDH determination is 1 ng per sample.     

Flow-injection catalytic spectrophotometic determination of molybdenum(VI) in plants using bromate oxidative coupling of p-hydrazinobensenesulfonic acid with N-(1-naphthyl)ethylenediamine

A novel flow-injection spectrophotometry has been developed for the determination of molybdenum(VI) at nanograms per milliliter levels. The method is based on the catalytic effect of molybdenum(VI) on the bromate oxidative coupling of p-hydrazinobenzenesulfonic acid with N-(1-naphthyl)ethylenediamine to form an azo dye (λ_max = 530 nm). Chromotropic acid (4,5-dihydroxy-2,7-naphthalenedisulfonic acid) acted as an effective activator for the molybdenum(VI)-catalyzed reaction and increased the sensitivity of the method. The reaction was monitored by measuring the change in absorbance of the dye produced. The proposed method allowed the determination of molybdenum(VI) in the range 1.0-20 ng mL⁻¹ with sample throughput of 15 h⁻¹. The limit of detection was 0.5 ng mL⁻¹ and a relative standard deviation for 10 ng mL⁻¹ molybdenum(VI) (n = 10) was 2.5%. The interfering ions were eliminated by using the combination of a masking agent and on-line minicolumn packed with cation exchanger. The present method was successfully applied to the determination of molybdenum(VI) in plant foodstuffs.     

Palladium@gold bimetallic nanostructures as peroxidase mimic for development of sensitive fluoroimmunoassay

In this paper, gold nanoparticles coated with palladium dots (Pd@Au) bimetallic nanostructures have been reported to have a peroxidase like activity which is not found in their monometallic counterparts. Based on this finding, we have developed an immunoassay in which antibody-modified Pd@Au nanostructure catalyzes the dimerization of a fluorogenic substrate for peroxidase, 3-(4-dihydroxy phenyl) propionic acid (HPPA), to generate high fluorescence signal. Specific antibodies against bensulfuron-methyl were generated by using a well characterized bensulfuron–protein conjugate as an immunogen, and the assay was performed in a competitive immunoassay format where Pd@Au nanostructure was bound to secondary antibody to show the peroxidase like activity. The developed immunoassay exhibited an excellent sensitivity showing a dynamic response range from 0.001 to 100 ng mL⁻¹ for herbicide bensulfuron-methyl with a detection limit of 0.01 ng mL⁻¹ (n = 3). The newly synthesized bimetallic nanostructure shows the advantages of low cost, easy synthesis and tunable catalytic activity, making it a promising substitution of enzyme peroxidase in different applications.     

Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography

Arnica montana preparations have been used in Europe for centuries to treat skin disorders. Among the biologically active ingredients in the flower heads of the plant are sequiterpenes, flavonoids and phenolic acids. For the simultaneous determination of compounds belonging to the latter two groups a micellar electrokinetic capillary chromatography (MEKC) method was developed and validated. By using an electrolyte solution containing 50 mM borax, 25 mM sodium dodecyl sulfate and 30% of acetonitrile the separation of seven flavonoids and four caffeic acid derivatives was feasible in less than 20 min. The optimized system was validated for repeatability (σ_rel ≤ 4.4%), precision (inter-day σ_rel ≤ 8.13%, intra-day σ_rel ≤ 4.32%), accuracy (recovery rates from 96.8 to 102.4%), sensitivity (limit of detection (LOD) ≤ 4.5 μg mL⁻¹) and linearity (R² ≥ 0.9996), and then successfully applied to assay several plant samples. In all of them the most dominant flavonoid was found to be quercetin 3-O-glucuronic acid, whereas 3,5-dicaffeoylquinic acid was the major phenolic acid; the total content of flavonoids and phenolic acids varied in the samples from 0.60 to 1.70%, and 1.03 to 2.24%, respectively.     

Analytical performances of validated chemiluminescent enzyme immunoassays to detect N-methylcarbamate pesticides

In the present work, enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection for the determination of carbofuran, carbaryl and methiocarb were developed and the analytical parameters of these assays were compared with those of ELISAs with colorimetric detection. Both were conjugate-coated formats based on identical monoclonal antibodies and homologous protein conjugates. In comparison with colorimetric ELISA, the ability of the chemiluminescent reagents to detect lower concentrations of horseradish peroxidase allowed to decrease the optimal antibody and conjugate concentrations and to reach better analytical parameters. The experimental comparison of the analytical performance of the ELISAs was carried out by analysing extracts of apple-strawberry baby food and simply diluted fruit juices, both spiked at different concentration levels with the above mentioned pesticides. Recovery values for both ELISAs were around 100% and no matrix effects were observed when fruit juices were diluted 1:20 or more. Results obtained by ELISAs correlated well, both in terms of accuracy and precision, with those obtained by a liquid chromatography-electrospray mass spectrometry (LC/ESI/MS/MS) analysis, used as reference method to validate the immunoassays results. The limits of detection reached by using the chemiluminescent assay were 0.03, 0.007 and 0.004 ng ml⁻¹ for carbofuran, carbaryl and methiocarb, respectively.     

In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin

The low therapeutic index of digoxin necessitates careful monitoring of its serum levels. Most of digoxin immunoassays suffer from interferences with digoxin-like immunoreactive substances. Since aptamers have been shown to be highly specific for their targets, the aim of this study was to develop DNA aptamers for this widely used cardiac glycoside. Digoxin was coated onto the surface of streptavidin magnetic beads. DNA aptamers against digoxin were designed using Systematic Evolution of Ligands by Exponential enrichment method (SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR amplification. The PCR product was cloned and sequenced. Binding affinity was determined using digoxin–BSA conjugate, coated onto ELISA plate. Inhibitory effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium. Three aptamers (D1, D2 and D3) were identified. Binding studies of fluorescein-labeled truncated (without primer binding region) D1 and D2 and full length D1 anti-digoxin aptamers were performed and their corresponding dissociation constants values were 8.2 × 10⁻⁹, 44.0 × 10⁻⁹ and 17.8 × 10⁻⁹ M, respectively. This is comparable to what other workers have obtained for interaction of monoclonal antibodies raised against digoxin. There was little difference in binding affinity between full length and truncated anti-digoxin D1 aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between digoxin and ouabain in both tissue study and binding experiments. Our finding indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further studies might show its suitability for use in digoxin assays and as a therapeutic agent in life-threatening digoxin toxicity.