One-step Immunoassay Research Articles

Vertical flow immunoassay (VFA) biosensor for a rapid one-step immunoassay

A highly rapid, one-step immunoassay of high sensitivity C-reactive protein (hsCRP) using a biosensor with a vertical flow immunoassay (VFA) was developed. The VFA biosensor was primarily composed of a sample pad, conjugate pad, FTH film and nitrocellulose (NC) membrane, which were all vertically stacked upon one another. Anti-hsCRP and secondary antibodies were consecutively immobilized on the NC membrane at the position below the holes. Gold nanoparticles (AuNPs) conjugated with another anti-hsCRP antibody were encapsulated in the conjugation pad. Various assay conditions, including the size of the hole and the sample volume, were optimized. Under optimized conditions, hsCRP concentrations from 0.01 to 10 μg mL(-1) were detected within 2 min. In comparison with a lateral flow assay (LFA) system, the VFA sensor showed a gradual increase of signal in a concentration-dependent manner without a hook effect in the tested range.

A Lateral Flow Immunoassay for the Rapid Detection of Ochratoxin A in Wine and Grape Must

A one-step lateral flow immunoassay was developed for semiquantitatively detecting ochratoxin A (OTA) in wines and grape musts. Matrix-matched calibration curves carried out in blank wines showed a detection limit of 1 μg L(-1) and IC(50) of 3.2 μg L(-1). Relative standard deviations for intra- and interday precision were in the 20-40% range. A simple treatment of samples, which only included dilution with sodium bicarbonate and polyethylene glycol (4% w/v) for red and white wines and the further addition of ethanol (12% v/v) for grape musts, was established. The developed assay allowed OTA detection in 5 min and proved to be accurate and sensitive enough to allow the correct attribution of samples as compliant or noncompliant according to EU legislation. Results agreeing with those of a reference chromatographic method were obtained on 38 wines and 16 musts. Although some lateral flow devices aimed at detecting OTA have been previously described, this is the first assay capable of measuring the toxin in wine and grape must, which represent a major source of OTA dietary intake. Analytical performances of the method are comparable to or better than previously reported assays showed. In addition, the assay, including sample treatments, is extremely simple and rapid and can be effectively regarded as a one-step assay usable virtually anywhere.

One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01–50 ng mL−1) and the same lower detection limit (0.01 ng mL−1). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data.

One-step and label-free detection of alpha-fetoprotein based on aggregation of gold nanorods

In this study, a novel one-step label-free immunoassay using gold nanorods (GNRs) for sensitive detection of alpha-fetoprotein (AFP), one of the major markers for hepatocellular tumors, was successfully developed. GNRs were fabricated through seed-mediated growth procedure, and their surface was activated by complete replacement with alkanethiols for the attachment of antibodies to obtain GNR-antibody conjugates with special space conformation. Dramatic aggregation of GNRs was induced by the interaction between target proteins and capture antibodies, and confirmed by transmission electron microscopy (TEM) and Ultraviolet–visible (UV–vis) analysis. Associated wavelength red-shifts and intensity decreases in the absorption peak of GNRs were observed with increasing concentrations of target AFP. Therein the absorption intensity as sensing response showed excellent linearity with the concentration of AFP in logarithmic scale. It was illustrated that this method can be used reliably to detect AFP protein in phosphate-buffered saline (PBS) buffer at concentrations of picomolar level with a wide quantification range from 0.25 to 4.0 nM. The complication of interfering proteins in the sample showed negligible effects on the detection results. Furthermore, the simple procedure, cost-effective and label-free features of this method offer advantages over common immunoassays. It is also expected that this new method would be applicable for detection of other tumor markers at low concentrations.

Fabrication and optical characterization of p-type single macro-porous silicon for detection of nano-sized functionalized superparamagnetic beads

Porous silicon (PSi) shows tremendous potential for applications including optical devices and biochemical biosensors due to the large surface area and convenience of surface functionalization of pores. Recently, variations of the Fabry–Pérot fringes shift with the refractive index of the material filling the pores of PSi have been used for real time monitoring of biorecognition processes, where the optical shift is determined by the magnitude of the optical thickness (2nL) by fast Fourier transform (FFT). The development of biosensing systems using functionalized superparamagnetic beads (SPBs) or ‘magnetic labels’ is expected to enable a fast, one-step immunoassay protocol. However, magnetic labels are difficult to infiltrate into small nanopores in silicon due to blocking effects such as surface tension, wettability, and stomatal morphology. Here, we investigated the optimal fabrication parameters of PSi with large pores into which magnetic labels easily infiltrate, and confirmed the penetration of SPBs by the changes in the optical thickness 2nL by reflectivity measurements. Finally, we show the potential for real-time point of care diagnostic system by utilizing our method.

Preparation of immuno-probes based on europium-chelate-adsorbed silica nanoparticles and magnetic nanoparticles and their application in detection of hepatitis B surface antigen

In this paper, a simple and reproducible method to synthesize intense fluorescence and photostable silica nanoparticles has been developed. Firstly, silica nanoparticles were prepared based on the Stober method, and hydrophobic europium chelates were adsorbed into their micro-holes by ultrasonication and phase-transfer solvent. Then they were modified by cationic polyelectrolyte polydiallyldimethylammonium chloride (PDAC) and anionic polyelectrolyte poly(acrylic acid) (PAA) layer by layer. Using them as detection probes and magnetic nanoparticles as capture probes, a novel immunoassay for determination of hepatitis B surface antigen (HBsAg) was proposed. Under the optimal experimental conditions, the immuno-probes performed with a good linear range over 10–200 ng mL−1 with a calibration curve equation of y = −549.24 + 652.44x (R = 0.9901). Based on the hydrophobicity and intense fluorescence europium chelates absorbed into silica nanoparticles, one-step fluorescence immunoassay has been realized and may have potential applications in clinical assays.

Development of a sensitive time-resolved fluoroimmunoassay for organophosphorus pesticides in environmental water samples

Due to the large-scale use of organophosphorus pesticides (OPs), their contamination in the environment is widespread throughout the world, especially in developing countries. With increasing public concern, there is an urgent requirement for simple, rapid, high-throughput and highly sensitive analytical methods for the screening of OP residues in the environment. In this work, a monoclonal antibody-based sensitive one-step direct competitive time-resolved fluorescence immunoassay (TRFIA) with broad specificity to a class of OPs was developed. After optimization of the assay conditions, this method can detect thirteen OPs with a limit of detection below 10 ng mL−1. The recoveries of OPs from spiked environmental water samples ranged from 74.8% to 121.3%, with the coefficient of variation (CV) ranging from 6.4% to 15.1%. The correlation coefficient between the TRFIA results and standard HPLC-MS/MS results was 0.964. The proposed TRFIA was capable of high-throughput analysis of a large number of samples prior to chromatographic analysis, with good sensitivity, simplicity and rapidity.

Rapid, Multiparameter Profiling of Cellular Secretion Using Silicon Photonic Microring Resonator Arrays

We have developed a silicon photonic biosensing chip capable of multiplexed protein measurements in a biomolecularly complex cell culture matrix. Using this multiplexed platform combined with fast one-step sandwich immunoassays, we perform a variety of T cell cytokine secretion studies with excellent time-to-result. Using 32-element arrays of silicon photonic microring resonators, the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), and tumor necrosis factor alpha (TNFα) were simultaneously quantified with high accuracy in serum-containing cell media. Utilizing this cytokine panel, secretion profiles were obtained for primary human Th0, Th1, and Th2 subsets differentiated from naïve CD4+ T cells, and we show the ability to discriminate between lineage commitments at early stages of culture differentiation. We also utilize this approach to probe the temporal secretion patterns of each T cell type using real-time binding analyses for direct cytokine quantitation down to ∼100 pM with just a 5 min-analysis.

Fast measurement of serum amyloid A in different specimens from swine by using a new one-step time-resolved fluorescent immunoassay

The objective of the present study was to develop a one-step, fast, competitive time-resolved fluorescent immunoassay to determine porcine serum amyloid A (SAA) by using species-specific reagents. The assay consisted of an all-in-one format involving only 55 min of incubation that was adapted and validated for use in 3 different specimens: serum, saliva, and meat juice. The method had overall within- and between-run coefficients of variation under 8% and 12%, respectively, and coefficients of determination higher than 0.93 for linearity under dilution analysis for all specimens. The limits of detection were 0.32 mg/l, 0.28 mg/l, and 1.74 mg/l for serum, saliva, and meat juice measurements, respectively. Upper and lower limits of quantification were determined for each sample type and resulted in wide assay ranges that allowed a precise SAA measurement in all the fluids investigated. Statistically significant differences (P = 0.0004 for serum and P < 0.0001 for the saliva and meat juice samples) in SAA levels were found when healthy (n = 20) and diseased (n = 20) pigs were compared. The obtained results indicate that this fast, sensitive, and robust assay for SAA measurement could be of use to determine health and welfare status in swine by employing alternative samples to serum.

GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein

A new flow-through electrochemical immunosensor was designed for sensitive detection of alpha-fetoprotein (AFP) in human serum by using nanogold-functionalized magnetic graphene nanosheets as immunosensing probes. Initially, amino functionalized magnetic beads were covalently immobilized on the surface of graphene oxide nanosheets (MGPs), then nanogold particles were adsorbed on the amino groups of the MGPs to construct GoldMag nanocomposites functionalized graphene nanosheets (GMGPs), and then horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP) were assembled onto the surface of nanogold particles (bio-GMGP). With the aid of an external magnet, the formed bio-GMGPs were attached onto the base electrode in the flow system. With a non-competitive immunoassay format, the injected sample containing AFP antigens was produced transparent immunoaffinity reaction with the immobilized HRP-anti-AFP on the bio-GMGPs. The formed immunocomplex inhibited partly the active center of HRP, and decreased the labeled HRP toward the reduction of H2O2. The performance and factors influencing the performance of the immunosensor were investigated in detail. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.01–200ngmL−1 with a low detection limit (LOD) of 1.0pgmL−1 AFP (at 3sB). Intra- and inter-assay coefficients of variation (CV) were below 10%. In addition, the methodology was validated with real serum samples, receiving a good correlation with the results obtained from commercially available electrochemiluminescence automated analyzer.

A Renewable C Reactive Protein Amperometric Immunosensor Based on Magnetic Multiwalled Carbon Nanotubes Probles Modified Electrode

A disposable, magnetism-controlled, and amperometric immunosensor was fabricated for the determination of C reactive protein (CRP). Firstly, Fe3O4 (core)/Au (shell) nanoparticle (GMP)–coated multiwalled carbon nanotubes (MWCNT–GMP) were prepared. Then, monoclonal antibody (anti-CRP) was immobilized on MWCNT–GMP to prepare MWCNT–GMP/anti-CRP composite magnetic probes. At last, the probes was adsorbed on the surface of N,N''-bis-(2-hydroxy-methylene)-o-phenylenediamine cobalt (CoRb) modified, screen-printed carbon electrodes through external magnetic field.The amount of CRP in the serum sample was determined by one-step immunoassay. When different concentrations of CRP were added into25μL of phosphate-buffered solution (pH7.0) containing10-4M H2O2, the percentage of DPV cathodic peak current decrease (CR%) exhibited a linear relationship with the concentration of CRP in the range of 0.3–100 μg L-1.The detection limit was 0.16 μg L-1(3б).The immunosensor was used to determine CRP in serum samples of patients with heart diseases, and the results were consistent with those of the traditional ELISA method. The proposed amperometric immunosensor was sensitive, rapid, magnetic field–controlled, and disposable; therefore, it could be used to determine even traces of CRP in the blood serums of patients with heart diseases.

Supercritical Angle Fluorescence Immunoassay Platform

An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle fluorescence (SAF) has been built into the tubes allowing for the extremely sensitive readout of solid phase immunoassays in real time and without washing steps. One-step sandwich immunoassays with interleukin 2 (IL-2) were carried out with capture antibodies immobilized in the tubes. At a turn around time of 12 min, the limit of detection for IL-2 was 0.27 pM (4.5 pg/mL) and the linear range covered 3 orders of magnitude. The developed technology is also adaptable to well plates and has great potential of replacing the work-intensive and time-consuming enzyme-linked immunosorbant assay (ELISA).

One-step electrochemical immunoassay of biomarker based on nanogold-functionalized graphene sensing platform

A rapid and sensitive one-step electrochemical immunoassay for the determination of α-fetoprotein (AFP, as a model biomarker) was developed by immobilizing anti-AFP antibody on a nanogold-functionalized graphene interface. Initially, thionine molecules as electron mediators were conjugated onto the surface of graphene nanosheets, then gold nanoparticles were sandwiched onto the graphene, and then horseradish peroxidase-anti-AFP (HRP-anti-AFP) conjugates were immobilized on the nanogold surface. The presence of two-dimensional graphene nanosheets with high conductivity not only increased the surface coverage of the electrode, but also enhanced the immobilized amount of biomolecules. With the AFP analyte, the formed antigen–antibody complex was coated on the electrode surface, which partially hindered the immobilized HRP toward the catalytic reduction of H2O2 in the solution. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1–200 ng mL−1 with a detection limit (LOD) of 0.05 ng mL−1 AFP at 3sB. The reproducibility, selectivity and stability of the immunosensor were investigated with the acceptable results. In addition, the one-step immunoassay was evaluated and validated by assaying 5 positive and 5 negative serum specimens, and the obtained results were compared with those by the referenced electrochemiluminescent, receiving a good correlation between the two methods.

Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis

Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 µm, 0.5 µm and 1 µm diameter were tested and 1 µm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.

Single particle technique for one-step homogeneous detection of cancer marker using gold nanoparticle probes

In this paper, we reported a single particle technique for the one-step homogeneous immunoassay of a cancer marker by resonance light scattering correlation spectroscopy (RLSCS). The setup of RLSCS was similar to fluorescence correlation spectroscopy (FCS), and its principle was based on measuring the resonance light scattering fluctuations in a small volumes (less than 1 fL) due to Brownian motion of single particles. In homogeneous immunoassay, we used a sandwich strategy and conjugated two different antibodies (Ab) with gold nanoparticles (GNPs) respectively. When two different GNPs labeled with antibodies are mixed in a sample containing antigen (Ag) targets, the binding of targets will cause GNPs to form dimers (or oligomers), which leads to the significant increase in the characteristic diffusion time of GNPs in the detection volume. The RLSCS method can sensitively detect the change in the characteristic diffusion time of GNPs before and after immune reactions. We used this technology in homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP). The conditions of the immune reaction were investigated systematically. In the optimal conditions, the linear range of this assay is from 1 pM to 1 nM and the detection limit is 1 pM for AFP. This new method was successfully applied for the direct determination of AFP levels in sera from healthy subjects and cancer patients. Our results were in good agreement with ELISA assays.

A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays

The limitation of current dissociative fluorescence enhancement techniques is that the lanthanide chelate structures used as molecular probes are not stable enough in one-step assays with high concentrations of complexones or metal ions in the reaction mixture since these substances interfere with lanthanide chelate conjugated to the detector molecule. Lanthanide chelates of diethylenetriaminepentaacetic acid (DTPA) are extremely stable, and we used EuDTPA derivatives conjugated to antibodies as tracers in one-step immunoassays containing high concentrations of complexones or metal ions. Enhancement solutions based on different β-diketones were developed and tested for their fluorescence-enhancing capability in immunoassays with EuDTPA-labelled antibodies. Characteristics tested were fluorescence intensity, analytical sensitivity, kinetics of complex formation and signal stability. Formation of fluorescent complexes is fast (5 min) in the presented enhancement solution with EuDTPA probes withstanding strong complexones (ethylenediaminetetra acetate (EDTA) up to 100 mM) or metal ions (up to 200 μM) in the reaction mixture, the signal is intensive, stable for 4 h and the analytical sensitivity with Eu is 40 fmol/L, Tb 130 fmol/L, Sm 2.1 pmol/L and Dy 8.5 pmol/L. With the improved fluorescence enhancement technique, EDTA and citrate plasma samples as well as samples containing relatively high concentrations of metal ions can be analysed using a one-step immunoassay format also at elevated temperatures. It facilitates four-plexing, is based on one chelate structure for detector molecule labelling and is suitable for immunoassays due to the wide dynamic range and the analytical sensitivity. Figure

A Non-enzyme Amperometric Immunosensor for Rapid Determination of Human Immunodeficiency Virus p24 Based on Magnetism Controlled Carbon Nanotubes Modified Printed Electrode

Abstract A magnetism-controlled, disposable amperometric immunosensor was fabricated for the determination of human immunodeficiency virus p24 antigen. First, Fe3O4 (core)/Au (shell) nanoparticle [gold magnetic particle (GMP)]-coated multiwalled carbon nanotubes (MWCNT-GMP) were prepared. Then, monoclonal antibody (anti-p24) was immobilized on MWCNT-GMP to prepare MWCNT-GMP/anti-p24 composite magnetic particles. At last, MWCNT-GMP/anti-p24 was adsorbed on the surface of N,N'-bis-(2-hydroxy-methylene)-o-phenylenediamine copper (CuRb)-modified, screen-printed carbon electrodes through external magnetic field. Cyclic voltammetry and differential pulse voltammetry (DPV) were used to study the electrochemical properties of the modified electrode. The amount of p24 in the serum sample of patients with AIDS was determined by one-step immunoassay. When different concentrations of p24 were added into 25 μL of phosphate-buffered solution (pH 7.0) containing 1.0 μM H2O2, the percentage of DPV cathodic peak current decrease (CR, %) exhibited a linear relationship with the concentration of p24 in the range of 0.6-160 μg L−1. The detection limit was 0.32 μg L−1 (3σ). The immunosensor was used to determine p24 in serum samples of patients with AIDS, and the results were consistent with those of the traditional ELISA method. The proposed amperometric immunosensor was sensitive, rapid, magnetic field-controlled, and disposable; therefore, it could be used to determine even traces of p24 in the blood serums of patients with AIDS.

A high-affinity recombinant antibody permits rapid and sensitive direct detection of myeloperoxidase

Over the past 10 years, a growing field of research supporting the value of myeloperoxidase (MPO) as a prognostic indicator in acute cardiac pathophysiologies has emerged. The availability of a rapid and disposable MPO detection platform would enable research clinicians to more readily assess MPO indications for guiding therapy and also facilitate clinicians at the patient interface to readily adopt MPO testing and potentially drive more informed prognoses. Here we describe the isolation of a high-affinity avian MPO-specific recombinant antibody panel using phage display. Rapid isolation of a suitable single-chain variable fragment (scFv) antibody was facilitated using a surface plasmon resonance (SPR)-based “off-rate ranking” screening process. The selected scFv was then successfully incorporated into a rapid, simple, and sensitive one-step lateral flow immunoassay (LFIA) for the detection of MPO. This “one-step” feature of the developed assay was made possible by the scFv’s strong affinity for MPO, obviating the need for sandwich signal enhancement steps. The assay’s rapid performance was also further enhanced by exploiting the intrinsic enzymatic properties of MPO in its final detection. Use of the optimized LFIA facilitated the sensitive detection of MPO in MPO-depleted serum within clinically relevant reference ranges.

A One-Step Immunoassay for Carbohydrate Antigen 19-9 by Biosensor Based on Imaging Ellipsometry

Cancer markers can offer much more information for developing or developed cancers. Simple and short-time assay of cancer biomarker is essential for clinic diagnosis and management. In this study, a one-step immunoassay for carbohydrate antigen 19-9 (CA19-9) by biosensor based on imaging ellipsometry (BIE) is described and its analysis performances are evaluated. Anti-CA19-9 monoclonal antibody as ligand covalently immobilized on carboxyl-modified silicon substrate and its immobilization concentration concurrently screened. Then, bovine serum albumin (BSA) is used to block for acquisition of sensing layer. The detection linear range is from 15.0 to 120.0 U/mL with a minimum limit of 10.0 U/mL (S/N=3). The intra-slide and inter-slide reproducibility, expressed as coefficient variation (CV%), are 9.9, 5.9, 5.4% and 9.6, 7.6, 5.3% for 31.9, 57.4, and 99.2 U/mL of CA19-9, respectively. Accuracy determined by spiked-recovery is among 95.1-106.7%. Specificity expressed as cross-reaction rate are all below 5.8% evaluated by three analogous biomarkers alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigen 242 (CA242). Application of BIE to 25 clinic samples demonstrated that the results are in high correlate with electrochemiluminescent immunoassay (ECLIA, R2=0.997). The proposed immunoassay has the potential for clinic application.

Nanogold-actuated biomimetic peroxidase for sensitized electrochemical immunoassay of carcinoembryonic antigen in human serum

A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP- anti-CEA) on the nanogold/nano-Fe 3O 4 particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H 2O 2. Nanogold-actuated nano-Fe 3O 4 mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1–220 ng mL −1 with a relative low detection limit (LOD) of 10 pg mL −1 CEA (at 3 σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA.