Electrochemical Enzyme-linked Immunosorbent Assay Research Articles

Aflatoxin M 1 determination and stability study in milk samples using a screen-printed 96-well electrochemical microplate

A highly sensitive method for the determination of aflatoxin M 1 (AFM 1) is described that involves the use of a disposable multichannel microplate coupled to intermittent pulse amperometry (IPA) for immunosensor development based on a direct competitive assay. Immunoassay parameters were evaluated and optimized to establish an electrochemical enzyme-linked immunosorbent assay (ELISA) procedure for AFM 1 in milk samples. The suitability of the immunosensors for the direct analysis of the toxin in milk was assessed. AFM 1 was measured with a working range of 5–250 pg mL −1 and a detection limit of 1 pg mL −1. Good recovery values (90–105%) were obtained. The method was used to study the recovery of this toxin in frozen (−30 °C) or lyophilized (4 °C) milk samples stored for up to 3 months. A significant, but variable, decrease (>50%) of the measured AFM 1 concentration with respect to the initial toxin contamination levels was found.

3,3′–diaminobenzidine (DAB)–H 2O 2–HRP voltammetric enzyme-linked immunoassay for the detection of carcionembryonic antigen

A new voltammetric enzyme-linked immunoassay system of 3,3′–diaminobenzidine (DAB)–H 2O 2–horseradish peroxidase (HRP) has been presented and used for the sensitive detection of carcinoembryonic antigen (CEA) in human serum. In this proposed procedure, DAB was firstly used as the electroactive substrate in the HRP catalyzed oxidation reaction in the present of H 2O 2. The generated product produced a sensitive second-order derivative linear sweep voltammetric peak at potential of − 0.62 V ( vs. SCE) in Britton-Robinson (BR) buffer solution. The free HRP could be measured in a linear range from 2.5 × 10 − 6 − 2.5 × 10 − 2 unit/ml and a detection limit of about 1.5 × 10 − 6 unit/ml. Under the optimal experiment conditions, CEA could be detected in the linear range from 0.50 to 80 ng/ml with a detection limit of 0.5 ng/ml. The proposed electrochemical enzyme-linked immunosorbent assay method is simple, inexpensive, reproducible and sensitive, which shows promising for detecting CEA in the clinical diagnosis.

Determination of Estradiol by Biotin‐Avidin‐Amplified Electrochemical Enzyme Immunoassay

A simple, sensitive biotin‐avidin‐amplified electrochemical enzyme‐linked immunosorbent assay (ELISA) for the determination of estradiol (E2) was proposed in this paper. The complex of biotinylated anti‐E2 antibody and horseradish peroxidase‐labeled avidin (HRP‐avidin) were regarded as a probe in this system. The activity of labeled enzyme was measured with electrochemical methods using o‐phenylenediamine as substrate. Coupled with the plate‐coated antigen, indirect ELISA format using E2‐ovalbumin, the electrochemical detection was performed for E2 with the detection limit of 21.0 pg/ml, and the linear range of determination of 50.0–500.0 pg/ml. The proposed method has been used for the determination of E2 in river water with satisfactory results. Compared with the traditionally spectrophotometric ELISA detection, this method shows greatly heightened sensitivity. The limit of detection improved by about two orders of magnitude, which is very suitable for the conditions with extremely low concentration of analyte or very small volumes of sample present.

Detection of prostate specific antigen with 3,4-diaminobenzoic acid (DBA)–H 2O 2–HRP voltammetric enzyme-linked immunoassay system

A new voltammetric enzyme-linked immunoassay system of 3,4-diaminobenzoic acid (DBA)–H 2O 2–horseradish peroxidase (HRP) for sensitive detection of prostate specific antigen (PSA) in human serum was present. In the proposed procedure, labelled HRP efficiently catalyzed the oxidation reaction of DBA by H 2O 2 and generated the electroactive product, 2,2′-biamino-4,4′-bicarboxyl azobiphenyl, which produced a sensitive second-order derivative linear sweep voltammetric peak at potential of −0.62 V (versus SCE) in Britton–Robinson (BR) buffer solution. The linear range for detection free HRP was from 4.0 × 10 −12 to 4.0 × 10 −9 g ml −1 and the detection limit was about 6.0 × 10 −13 g ml −1. The proposed new system could be used for detection of PSA ranging from 0.20 to 16.0 ng ml −1 with a detection limit of 0.10 ng ml −1, which was five times lower than that of the traditional o-phenylendiamine spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. The proposed electrochemical ELISA method is simple, inexpensive, reproducible and sensitive, which shows potential for detecting PSA in clinical diagnosis.

Detection of Aflatoxin B1 in Barley: Comparative Study of Immunosensor and HPLC

In the present work, an indirect competitive electrochemical enzyme linked immunosorbent assay (ELISA) has been used for determination of aflatoxin B1 (AFB1) in barley. The method involves the use of disposable screen‐printed carbon electrodes (SPCEs) and anti‐aflatoxin B1 monoclonal antibodies (MAb) for immunosensor development. The specificity of the assay was assessed by studying the cross‐reactivity of the MAb relative to AFB1. The results indicated that the MAb could readily distinguish AFB1 from other toxins, with the exception of AFG1. The stability of the coating reagents was evaluated using SPCEs coated with AFB1‐bovine serum albumin (BSA) conjugate. The results showed that the coated electrodes could be used for up to one month after their preparation and storage at 4°C. Prior to evaluating the performance of the electrochemical immunosensor for AFB1 with spiked samples, the effect of barley extract on assay performance was tested. Using this calibration method, the limit of detection (LOD) was found to be 90 pg mL−1. The linear range was 0.1–10 ng mL−1, and recoveries ranged from 100%–125%. The results obtained were confirmed by high performance liquid chromatography (HPLC) coupled with fluorescence detection. These results demonstrated the suitability of the proposed method for routine screening of AFB1 in barley.

Development of an Electrochemical Immunosensor for Ochratoxin A

A direct, competitive electrochemical enzyme‐linked immunosorbent assay (ELISA) has been developed for the quantitative determination of ochratoxin A (OTA) using polyclonal antibodies. The assay is carried out on carbon‐based screen printed electrodes (SPE). Optimisation of the ELISA competitive conditions allowed us to realise an assay with improved analytical behaviour compared to the classical spectrophotometric ELISA based assay. The performance was comparable to a published monoclonal based assay. The assay gave a detection limit of 180 pg mL−1 and sensitivity of 6.1 ± 0.1 ng mL−1. The immunosensor was challenged with wine to assess a matrix effect. Recoveries obtained were in the 70–118% range. The method appears to be suitable for OTA contamination screening in food samples.

Analysis of erythromycin and tylosin in bovine muscle using disposable screen printed electrodes.

A disposable electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed using a screen printed electrode (SPE) system as a differential pulse voltammetry (DPV) transducer with mouse anti-erythromycin (and anti-tylosin) monoclonal antibodies (MAb) serving as molecular recognition elements. The immunochemical system makes use of the competition assay principle, and employs an erythromycin (or tylosin)-BSA conjugate as coating molecule. After competition between free and coated analyte for the antibodies, the activity of the alkaline phosphatase labelled antiglobulins was measured electrochemically using 1-naphthylphosphate as substrate. Using standard solutions of erythromycin and tylosin, the detection limit of the assay was 0.2 ng mL(-1) determined to be for erythromycin and 2.0 ng mL(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.0 ng mL(-1) for erythromycin and 3.0 ng mL(-1) for tylosin. The suitability of the assay for quantification of erythromycin and tylosin in bovine muscle was also studied. Spiked and real samples were analysed using the immunosensor system developed here. The ELISA showed precision values (relative standard deviation, RSD%) ranging from 4 to 9% for erythromycin and from 8 to 15% for tylosin; the accuracy (relative error, RE%) ranged from -11 to 6% and from -4 to 12% for erythromycin and tylosin, respectively. Results obtained on real samples were confirmed by micro-liquid chromatography coupled on line with tandem mass spectrometry (micro-LC-MS-MS), using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest.

Electrochemical ELISA for the screening of DDT related compounds: analysis in waste waters

An electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 1,1,1-trichloro-2,2-bis( p-chlorophenyl)ethane ( p, p′-DTT), 1,1-dichloro-2,2-bis( p-chlorophenyl)ethylene ( p, p′-DDE), 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane ( p, p′-DDD) and o, p-DDT was developed. Optimization of the ELISA competition conditions, led to similar response for the p, p′-isomers. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3′,5,5′-tetramethylbenzidine as substrate. The use of purified antiserum for p, p′-DDT resulted in a sensitive assay with a detection limit of 40 pg ml −1 and R.S.D. ranging from 1 to 3% intra-day and 3 to 6% inter-day. No matrix effect for waste water samples of different origin has been evidenced. The ELISA was used to detect DDTs in 20 samples after extraction in diethylether. This method appears suitable for routine screening of DDTs without sample pre-treatment other than dilution in PBS or after organic solvent extraction if high sensitivity is required.

An electrochemical ELISA procedure for the screening of 17β-estradiol in urban waste waters

A sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) has been used for the detection of 17beta-estradiol in waste waters. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3',5,5'-tetramethylbenzidine as electrochemical substrate. The detection limit was estimated to be 5 pg mL(-1), interday and intraday precision (RSD), ranged from 1 to 3% and from 3 to 6%, respectively. Analysis of waste waters from three different treatment plants demonstrated no matrix effect both for samples diluted 1:1 in buffer and diethyl ether extracted. Data on 36 samples analysed by an LC-ESI-MS-MS procedure and by the electrochemical ELISA assay were compared. Results correlated well. The electrochemical enzyme immunoassay appears suitable as a screening tool for analysis of estradiol in waste waters.

Electrochemical ELISA for the detection of cucumber mosaic virus using o-pheneylenediamine as substrate

A sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the determination of cucumber mosaic virus (CMV) was proposed in this paper. The activity of labeled enzyme, horseradish peroxidase, was measured with electrochemical methods using o-phenylenediamine as substrate. The enzymatic reaction product is 2,3-diaminophenazine, which can be easily reduced on the dropping mercury electrode with improved sensitivity. Coupled with the plate trapped antigen indirect ELISA format using polyclonal rabbit antibody of CMV, the electrochemical detection was performed for CMV with the detection limit of 0.5 ng ml −1, which is ten times more sensitive than the colorimetric ELISA method. The conditions for enzymatic reaction and immunoassay were carefully optimized.

A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat.

A new sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed, using the mouse monoclonal antibodies anti-erythromycin and anti-tylosin. The competitive indirect assay was performed using an erythromycin (or tylosin)-BSA conjugate as a coating molecule; after competition between free and coated analytes for the antibodies, the activity of the horseradish peroxidase-labelled antiglobulins was measured electrochemically using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. The detection limit of the assay was 0.4 ng ml(-1) for erythromycin and 4.0 ng ml(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.4 ng ml(-1) for erythromycin and 13.0 ng ml(-1) for tylosin. The specificity of the assay was assessed by studying the cross-reactivity of various macrolides other than erythromycin and tylosin. The results indicate that the monoclonal antibodies anti-erythromycin and anti-tylosin can readily distinguish the target compound from other macrolides, with the exception of roxithromycin, a semisynthetic macrolide antibiotic derived from erythromycin. Fortified and real samples were analysed by the developed ELISA method and results confirmed by micro-LC-MS-MS using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest. The ELISA assay showed precision (RSD) values ranging from 6.3 to 11.4% for erythromycin and from 7.5 to 12.6% for tylosin; the accuracy (relative error, RE) ranged from -16.0 to -9.8% and from -9.5 to 8.0% for erythromycin and tylosin, respectively. All results obtained demonstrate that the electrochemical ELISA is a suitable method for a sensitive, simple, rapid and reliable screening of the two macrolides in animal tissues.

Development of an electrochemical ELISA for the screening of 17 beta-estradiol and application to bovine serum.

A sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 17 beta-estradiol (17 beta-E2) was developed. Optimisation of two ELISA competition assays, using monoclonal or polyclonal antibodies anti-17 beta-estradiol, coupled with the electrochemical detection was firstly performed. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3',5,5'-tetramethylbenzidine as substrate. The use of the polyclonal antibody resulted in a more sensitive assay and the detection limit of the assay was estimated to be 20 pg ml-1. The analytical performances of the method were compared to those obtained using a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA). Although sample extraction is not usually required by DELFIA, both extracted and non extracted samples were assayed. The comparison between the two screening techniques revealed similar results for the extracted samples and showed a comparable precision (RSD%), ranging from 6.2 to 13.4 and from 6.7 to 14.3 for DELFIA and ELISA, respectively. The results obtained by these screening assays were confirmed by liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry which is currently used to confirm illegal hormone administration for regulatory purposes. The electrochemical enzyme immunoassay appears suitable as a screening tool for routine analysis of bovine serum estradiol and can be extended to other anabolic hormones using appropriate antibodies.