Immunoassay Of Carcinoembryonic Antigen Research Articles

Surface plasmon resonance-enhanced photoelectrochemical immunoassay with Cu-doped porous Bi2WO6 nanosheets

The development of rapid screening sensing platforms to improve pre-screening mechanisms in community healthcare is necessary to meet the significant need for portable testing in biomarker diagnostics. Here, we designed a portable smartphone-based photoelectrochemical (PEC) immunoassay for carcinoembryonic antigen (CEA) detection using Cu-doped ultrathin porous Bi2WO6 (CuBWO) nanosheets as the photoactive material. The CuBWO nanosheets exhibit a fast photocurrent response and excellent electrical transmission rate under UV light due to their surface plasmon resonance effect (SPR). The method uses glucose oxidase-labeled secondary antibody as a signal indicator for sandwich-type immune conjugation. In the presence of the target CEA, the electrons and holes generated at the surface of the photo-excited ultrathin porous CuBWO were rapidly consumed by the production of H2O2 from glucose oxidase oxidizing glucose, resulting in a weakened photocurrent signal. The photocurrent intensity increased logarithmically and linearly with increasing CEA concentration (0.02–50 ng mL−1), with a detection limit of 15.0 pg mL−1 (S/N = 3). The system provides a broader idea for inferring the electron-hole transport mechanism in ultrathin porous nanosheet layer materials and developing efficient PEC sensors.

Accurate method for value assignment of carcinoembryonic antigen reference materials.

In this study, we explored the commutability of reference materials (RMs) for carcinoembryonic antigen (CEA), selected the appropriate diluent matrix of the first International Reference Preparation (IRP) 73/601 of the World Health Organization (WHO 73/601) for CEA, and improved the comparability of CEA measurement results among different assay systems. Forty serum samples were divided into five aliquots. WHO 73/601 was diluted into nine concentrations using five diluents with different components, and the candidate RMs for CEA at five concentrations (C1-C5) were prepared by the Beijing Clinical Laboratory Center (BCCL). The samples were analyzed via five automated CEA immunoassays. Carcinoembryonic antigen candidate RMs were commutable among all immunoassays based on the CLSI approach and among 7 of 10 assay combinations based on the IFCC approach. WHO 73/601 diluted in phosphate-buffered saline (PBS) was commutable among all assays based on the CLSI approach and among 5 of 10 pairwise comparisons based on the IFCC approach with correction of bias at diluted concentrations, except for the lowest concentration, which had the smallest variation among systems. The median percentage biases among assays were decreased after calibration. The BCCL candidate RMs (C2-C5) for CEA were commutable among all immunoassays. WHO 73/601 RMs diluted in a PBS buffer matrix were selected as common calibrators for five immunoassays, which reduced bias, thereby effectively improving the harmonization of CEA detection; therefore, they could be used to assign values to CEA candidate RMs developed by BCCL. Our findings promote the harmonization of CEA detection in immunoassays.

Electrochemiluminescence Distance and Reactivity of Coreactants Determine the Sensitivity of Bead‐Based Immunoassays

AbstractHerein we report the study of electrochemiluminescence (ECL) generation by tris(2,2′‐bipyridyl)ruthenium (Ru(bpy)32+) and five tertiary amine coreactants. The ECL distance and lifetime of coreactant radical cations were measured by ECL self‐interference spectroscopy. And the reactivity of coreactants was quantitatively evaluated in terms of integrated ECL intensity. By statistical analysis of ECL images of single Ru(bpy)32+‐labeled microbeads, we propose that ECL distance and reactivity of coreactant codetermine the emission intensity and thus the sensitivity of immunoassay. 2,2‐bis(hydroxymethyl)‐2,2′,2′′‐nitrilotriethanol (BIS‐TRIS) can well balance ECL distance‐reactivity trade‐off and enhance the sensitivity by 236 % compared with tri‐n‐propylamine (TPrA) in the bead‐based immunoassay of carcinoembryonic antigen. The study brings an insightful understanding of ECL generation in bead‐based immunoassay and a way of maximizing the analytical sensitivity from the aspect of coreactant.

Nanozyme-Activated Synergistic Amplification for Ultrasensitive Photoelectrochemical Immunoassay.

At present, enzyme-mediated signal amplification strategies have been widely applied in photoelectrochemical (PEC) biosensing systems, while the introduction of natural enzymes onto the surface of photoelectrodes inevitably obstructs the electron transfer due to their insulating properties as proteins, leading to severe damage to photocurrent. In this work, the PdPt bimetallic nanozymes with the efficient peroxidase-like activity were used as alternatives to natural enzymes and amplified PEC biosensing signals via their efficient enzymatic reaction and remarkable enhancement in photocurrent. As a result, photoactive CdS nanorods modified with PdPt bimetallic nanozymes showed a boosted PEC performance compared with the pristine CdS nanorods due to the localized surface plasmon resonance effect and Schottky junction. On the basis of the as-prepared CdS/PdPt photoelectrode, a sensitive split-type glucose oxidase-mediated PEC immunoassay for carcinoembryonic antigen (CEA) detection was successfully constructed. Along with the sandwich immunocomplexing, the subsequently produced hydrogen peroxide (H2O2) can oxidize 4-chloro-1-naphthol into insoluble precipitates to inhibit photocurrent and simultaneously trigger the bio-etching of CdS to further restrain photocurrent signals due to the excellent peroxidase-mimicking activity of PdPt nanozymes. Owing to the synergistic signal amplification fulfilled by PdPt nanozymes, an ultrasensitive immunoassay of CEA was realized with a wider linear range from 1 to 5000 pg/mL and a low detection limit of 0.21 pg/mL, opening a new avenue for building ultrasensitive PEC biosensors with nanozymes.

Saw-Toothed Microstructure-Based Flexible Pressure Sensor as the Signal Readout for Point-of-Care Immunoassay.

A saw-toothed microstructure-based flexible pressure sensor was designed as the signal readout for point-of-care (POC) immunoassay of carcinoembryonic antigen (CEA). In this portable POC bioassay, the sandwich-type immunoreaction was first carried out to capture the target on a microplate, which simultaneously introduced the platinum nanoparticles (PtNPs). Upon adding hydrogen peroxide (H2O2), the pressure as the bridge between the molecular recognition event and detectable signal was increasing rapidly, resulting from the decomposition of H2O2 accelerated by PtNPs. Meanwhile, a flexible pressure sensor was fabricated with high sensitivity; the ability of the pressure response was dramatically improved by adopting the saw-toothed microstructure. By coupling with the pressure sensor, the pressure change could be monitored in real time to achieve the portable detection of CEA. Under the optimum conditions, the proposed pressure-based bioassay presented good sensing performance within 0.1-40 ng/mL at a detection limit of 87 pg/mL. The reproducibility, precision, and accuracy provided by the method were also studied and satisfied.

Paper Electrode-Based Flexible Pressure Sensor for Point-of-Care Immunoassay with Digital Multimeter.

A novel paper electrode-based flexible pressure sensor modified with multiwalled carbon nanotubes was designed for point-of-care (POC) immunoassay of carcinoembryonic antigen (CEA) with digital multimeter readout. The portable POC testing device consisted of flexible pressure sensor equipped with a paper electrode and connected through syringe tubing to a single-break microplate. The immunoreaction was initially carried out on the microplate with a sandwich-type assay format using platinum nanozyme-labeled secondary antibody for the gas generation. Upon addition of hydrogen peroxide (H2O2), platinum nanozyme (catalase-like mimic) reduced it into hydrogen oxide and oxygen (O2). The overflowing oxygen gas increased the pressure of the multiwalled carbon nanotube-functionalized paper electrode in a homemade pressure-tight system, and the increased pressure could be readily monitored using the paper electrode-based flexible pressure-tight sensor with a digital multimeter readout. The detectable signal mainly derived from the resistance change of pressure sensor because of its deformation with the assistance of the as-generated gas, and the shift in the resistance could be allowed to detect the gas pressure even as low as 80 Pa. Under optimum conditions, pressure sensor-based immunoassay exhibited good resistance responses toward target CEA within a linear range of 0.5-60 ng/mL at a detection limit of 167 pg/mL. Moreover, our strategy provided acceptable reproducibility, precision, high specificity, and good accordance with the commercial CEA ELISA kit for detecting human serum specimens.

A voltammetric immunoassay for the carcinoembryonic antigen using a self-assembled magnetic nanocomposite.

The authors describe a voltammetric immunoassay for the carcinoembryonic antigen (CEA). It is based on the use of a self-assembled magnetic nanocomposite as multifunctional signal amplification platform. The core of the nanocomposite consists of Fe3O4 microspheres, and the shell of zirconium hexacyanoferrate loaded with gold nanoparticles (AuNPs@ZrHCF@Fe3O4). The material was synthesized by an electrostatic self-assembly process which is caused by the strong interaction between cyano groups and AuNPs. The surface of the Fe3O4 microspheres was functionalized with amino groups to facilitate the immobilization of ZrHCF which acts as an electron mediator. The nanocomposite was placed on a glassy carbon electrode which then displays noteworthy electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2). The AuNPs serve as a support for the immobilization of antibodies by the interaction between AuNPs and amino groups on antibodies to construct a covalent Au-N bond. This facilitates electron transfer on the electrode surface using H2O2 as the electrochemical probe. Square wave voltammetry (measured typically at +0.2V vs. SCE) was carried out to record the electrochemical behavior. Under the optimal conditions, a response is linear in the 0.5pg·mL-1 to 50ng·mL-1 CEA concentration range, and the detection limit is as low as 0.15pg·mL-1 (S/N= 3). The method is selective, highly stable and acceptably reproducible. Graphical abstract A self-assembly magnetic nanocomposite for voltammetric immunoassay of CEA. GCE glassy carbon electrode; Au NPs gold nanoparticles; ZrHCF zirconium hexacyanoferrate; CEA carcinoembryonic antigen; Anti-CEA CEA antibody; BSA bovine serum albumin; SWV square wave voltammetry. A high sensitive voltammetric immunoassay method has been used for detecting CEA, It is based on a self-assembled magnetic nanocomposite (Au NPs@ZrHCF@Fe3O4) as multifunctional signal amplification platform.

Robust enzyme-free electrochemical immunoassay of CEA enhanced by porous PdCu nanoparticles

The interferences of dissolved oxygen towards the electrochemical immunosensors occurred constantly, which will restrict the practical clinical application of electrochemical immunoassay. Herein, a new enzyme-free strategy has been proposed for ultrasensitive electrochemical immunoassay of carcinoembryonic antigen (CEA), which can completely avoid the interference of dissolved oxygen. Porous PdCu nanoparticles can catalyze aniline to produce polyaniline by the inherited peroxidase-like activity for generating electrochemical detection signal. More interesting, with the assistance of electropolymerization strategy, the electrochemical measurement signal could enhance sharply. Accordingly, we established a sandwich-type electrochemical immunosensor based on porous PdCu nanoparticles produced polyaniline with electropolymerization strategy for enzyme-free and dissolved oxygen interference-free detection of CEA, affording a satisfied performance with a broad linear detection range (0.1pg/mL∼10ng/mL) and low detection limit (0.08pg/mL). This method might be considered as a robust approach for clinical applications.

Advanced nanomaterials for use in electrochemical and optical immunoassays of carcinoembryonic antigen. A review

This review (with 196 refs.) covers the state of the art in electrochemical and optical immunoassays for the carcinoembryonic antigen (CEA). In essence, it has sections on (a) frequently applied principles and types of CEA immunoassays; (b) aspects of sensor fabrication including immunological and immobilization procedures and the proper choice of nanomaterials; (c) electrochemical immunoassays, with subsections on assays based on the use of nanoparticles and other nanomaterials (such as conducting polymers and graphenes); (d) optical immunoassays based on the use of nanoparticles such as quantum dots, gold nanoparticles, upconversion nanoparticles, graphenes and their derivatives; (d) lateral flow and lab-on-a-chip (microfluidic) immunoassays; and (e) on multiplexed electrochemical and optical immunoassays with and without labels. Examples for applications to real samples are given. A final section discusses current limitations and trends in terms of sensing schemes and nanomaterials.

SERS tags-based novel monodispersed hollow gold nanospheres for highly sensitive immunoassay of CEA

Here, a highly specific, sensitive, and reliable immunoassay has been developed based on surface-enhanced Raman scattering for quantitative detection of carcinoembryonic antigen (CEA). A novel hollow gold nanospheres (HGNs) was firstly fabricated by seed-mediated methods instead of the one-step methods with high homogeneity and then, the monodispersed HGNs were embedded with 4-mercaptobenzonic acid to form surface-enhanced Raman scattering (SERS) tags and magnetic microspheres were synthesized as enrichment substrates. Based on this sandwich immunoassay strategy, CEA monoclonal antibody -conjugated HGNs-based SERS tags and CEA polyclonal antibodies -conjugated magnetic microspheres were used to detect CEA up to 100 ng/mL with a detection limit of 10 pg/mL, which were two orders of magnitudes lower than that of GNSs-based SERS tags. The monodispersed HGNs were desirable SERS tags for quantitative sandwich immunoassay assay, showing strong potential for the clinical diagnosis of cancer biomarkers.

An immune sandwich assay of carcinoembryonic antigen based on the joint use of upconversion phosphors and magnetic beads.

We herein report a sensitive and selective immunosensor for carcinoembryonic antigen (CEA) based on the joint use of upconversion phosphors (UCPs) and magnetic beads (MBs). UCPs as the signal probe were designed with a core-shell structure which provided a 40-fold enhancement of the luminescence intensity. Poly(acrylic acid) (PAA)-modified UCPs were covalently conjugated with the anti-CEA antibody (coating), and streptavidin functionalized magnetic beads were combined with another biotin-tagged anti-CEA antibody. With the assistance of a magnet, the as-formed immune sandwich in the presence of CEA can be readily separated from the assay matrix. The immunosensor showed a linear dynamic range for CEA within 0.05-20 ng mL(-1) in a buffered aqueous solution, and 0.1-20 ng mL(-1) in a human serum sample. The sensor was highly specific to CEA. Our results have suggested the potential application of the UCP-MB based immunoassay for CEA in clinical analysis.

Ultrasensitive electrochemical immunoassay for CEA through host–guest interaction of β-cyclodextrin functionalized graphene and Cu@Ag core–shell nanoparticles with adamantine-modified antibody

A novel non-enzymatic immunoassay was designed for ultrasensitive electrochemical detection of carcino-embryonic antigen (CEA) using β-cyclodextrin functionalized Cu@Ag (Cu@Ag-CD) core–shell nanoparticles as labels and β-cyclodextrin functionalized graphene nanosheet (CD-GN) as sensor platform. CD-GN has excellent conductivity which promoted the electric transmission between base solution and electrode surface and enhanced sensitivity of immunosensor. In addition, owing to supramolecular recognition of CD-GN for the guest molecule, quite a few synthesized adamantine-modified primary antibodies (ADA-Ab1) were immobilized on the CD-GN by supramolecular host–guest interaction between CD and ADA. Cu@Ag-CD as a signal tag could be captured by ADA-modified secondary antibody (ADA-Ab2) through a host–guest interaction, leading to a large loading of Cu@Ag nanoparticles with high electrical conductivity and catalytic activity. The fabricated immunosensor exhibits excellent analytical performance for the measurement of CEA with wide range linear (0.0001–20ng/mL), low detection limit (20fg/mL), good sensitivity, reproducibility and stability, which provide an enormous application prospect in clinical diagnostics.

A novel dual-template molecularly imprinted electrochemiluminescence immunosensor array using Ru(bpy)32+-Silica@Poly-L-lysine-Au composite nanoparticles as labels for near-simultaneous detection of tumor markers

A novel electrochemiluminescence (ECL) immunosensor array was fabricated on a screen-printed electrode (SPE) to perform multiplexed immunoassay of tumor markers (TMs), such as carcinoembryonic antigen (CEA) and carbohydrate antigen-199 (CA199). The SPE substrate consisted of a common Ag/AgCl reference electrode, a common carbon counter electrode and two gold nanoparticles modified carbon working electrodes. Firstly, dopamine (DA) as the functional monomer along with the corresponding TMs (CEA or CA199) as template molecules was electro-polymerized on the surface of different working electrodes. Then, the dual-template molecularly imprinted polydopamine (MIP-PDA) film was formed as the capture probe to recognize CEA and CA199. Secondly, a novel label was fabricated by conjugating the antibodies of TMs with Ru(bpy)32+-Silica@Poly-L-lysine-Au (Ru-Si@PLL-Au) nanocomposites, in which gold colloids (Au NPs) were doped on the surface of Ru-Silica (Ru(bpy)32+-doped Silica) using poly-L-lysine (PLL) as a bridging agent. PLL was also employed as a co-reactant of the luminophore. Based on a sandwich-type immunoassay, the MIP-PDA film, the corresponding antigen and labels were conjugated to produce the immunocomplex. The signals from the ECL immunosensor array were near-simultaneously detected by a photomultiplier tube (PMT) using a homemade single-pore-two-throw switch, which could avoid crosstalk between adjacent working electrodes. The obtained concentrations were 0.05-100pgmL−1 for CEA and 0.03-80 U L−1 for CA199, with detection limits of 0.02pgmL−1 and 0.01 U L−1, respectively. This novel ECL strategy provides a simple, economical, fast and sensitive approach for multiplexed immunoassay of CEA and CA199, and has significant potential for protein detection in a clinical laboratory setting.

Preparation of Protein-like Silver–Cysteine Hybrid Nanowires and Application in Ultrasensitive Immunoassay of Cancer Biomarker

Novel protein-like silver-cysteine hybrid nanowires (p-SCNWs) have been synthesized by a green, simple, nontemplate, seedless, and one-step aqueous-phase approach. AgNO3 and l-cysteine were dissolved in distilled water, forming Ag-cysteine precipitates and HNO3. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0 by addition of concentrated sodium hydroxide solution, leading to quick dissolution of the Ag-cysteine precipitates and sudden appearance of white precipitates of p-SCNWs. The p-SCNWs are monodispersed nanowires with diameter of 100 nm and length of tens of micrometers, and have abundant carboxyl (-COOH) and amine (-NH2) groups at their surfaces, large amounts of peptide-linkages and S-bonding silver ions (Ag(+)) inside, making them look and act like Ag-hybrid protein nanostructures. The abundant -COOH and -NH2 groups at the surfaces of p-SCNWs have been found to facilitate the reactions between the p-SCNWs and proteins including antibodies. Furthermore, the fact that the p-SCNWs contain large amounts of silver ions enables biofunctionalized p-SCNWs to be excellent signal amplifying chemiluminescence labels for ultrasensitive and highly selective detection of important antigens, such as cancer biomarkers. In this work, the immunoassay of carcinoembryonic antigen (CEA) in human serum was taken as an example to demonstrate the immunoassay applications of antibody-functionalized p-SCNWs. By the novel p-SCNW labels, CEA can be detected in the linear range from 5 to 400 fg/mL with a limit of detection (LOD) of 2.2 fg/mL (at signal-to-noise ratio of 3), which is much lower than that obtained by commercially available enzyme-linked immunosorbent assay (ELISA). Therefore, the synthesized p-SCNWs are envisioned to be an excellent carrier for proteins and related immunoassay strategy would have promising applications in ultrasensitive clinical screening of cancer biomarkers for early diagnostics of cancers.

Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen

A facile strategy by loading luminol and secondary antibody on gold nanoparticles (Au NPs) was described in the present work. The as-prepared luminol/antibody labeled Au NPs conjugates (LAAu NPs) were used as the chemiluminescent probe for the detection of carcinoembryonic antigen (CEA) in serum. The LAAu NPs were characterized by transmission electron microscopy (TEM), UV–vis spectrophotometry (UV–vis), and chemiluminescent method. Stable and efficient chemiluminescence (CL) was obtained when luminol molecules and secondary antibodies were coimmobilized on the Au NPs by using hydrogen peroxide (H 2O 2) as an oxidant, horseradish peroxidase (HRP) as a catalyst, and 4-(4′-iodo)phenylphenol (IPP) as an enhancer. The LAAu NPs were further evaluated via a sandwich-type CL immunoassay of CEA in serum. In this protocol, the CEA analyte was captured by the primary antibody immobilized on the surface of magnetic beads, and then was sandwiched by the secondary antibody loaded on luminol-labeled Au NPs. The chemiluminescent intensity was proportional to the concentration of CEA over the range of 5.0 × 10 −10 to 5.0 × 10 −8 g mL −1 and 5.0 × 10 −9 to 2.0 × 10 −8 g mL −1 by using HRP and Co 2+ as catalysts, respectively. The present chemiluminescent immunoassay based on the luminol/antibody labeled Au NPs conjugates has offered great promise for simple, highly biocompatible, and cost-effective analysis of biological samples.

Novel colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen

We developed a highly sensitive colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen (CEA). This method employed gold nanoparticles (AuNPs) as carriers of anti-CEA antibody labeled with biotin, which served as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP) binding. Using this strategy, about 12 HRP molecules were coated onto each AuNP. Through "sandwich-type" immunoreaction, the AuNP-anti-CEA-HRP complex was brought into the proximity of magnetic microparticle. As a result, HRP molecules confined at the surface of the "sandwich" immunocomplexes catalyzed the enzyme substrate and generated an optical signal. The spectrophotometric measurement confirmed effective signal amplification. The signals were linearly dependent on CEA concentrations from 0.05 to 50ngmL(-1) in a logarithmic plot, with a detection limit of 48pgmL(-1). Intra- and inter-assay coefficients of variation were <8.5%. The CEA concentrations of serum specimens assayed by the developed immunoassay showed consistent results in comparison with those obtained by a conventional enzyme-linked immunosorbent assay. The developed method thus proved its potential use in clinical immunoassay of CEA.

Detection of Carcinoembryonic Antigens Using a Surface Plasmon Resonance Biosensor.

Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection with high sensitivity, though its ability to detect CEA in human serum was highly dependent on the analytical conditions employed. We investigated the influences of various analytical conditions including immobilization methods for anti-CEA antibody and composition of sensor surface on the selective and sensitive detection of CEA. The results show that anti-CEA antibody immobilized via Protein A or Protein G caused a large increase in the resonance signal upon injection of human serum due to the interactions with IgGs in serum, while direct covalent immobilization of anti-CEA antibody could substantially reduce it. An optimized protocol based on further kinetic analysis and the use of 2nd and 3rd antibodies for the sandwich assay allowed detecting spiked CEA in human serum as low as 25 ng/mL. Furthermore, a self-assembled monolayer of mixed ethylene-glycol terminated alkanethiols on gold was found to have a comparable ability in detecting CEA as CM5 with thick dextran matrix and C1 with short flat layer on gold.

Electrochemical immunosensor and biochemical analysis for carcinoembryonic antigen in clinical diagnosis

A new electrochemical immunosensor and immunoassay method has been developed for the detection of carcinoembryonic antigen (CEA) by immobilization of biomolecules (CEA) onto nanogold/(3-mercaptopropyl) trimethoxysilane/nano-Fe3O4-modified carbon paste electrodes. A competitive immunoassay format was employed to detect CEA antigen with horseradish peroxidase-labeled CEA antibody (HRP-anti-CEA) as tracer, and hydrogen peroxide (H2O2) as enzyme substrate. After the immunosensor was incubated with a mixture of constant HRP-anti-CEA and CEA sample for 35 min at 25 ± 0.5 °C, the amperometric responses of the binding HRP relative to o-phenylenediamine-H2O2 system decreased with the increment of CEA concentration in the sample solution. The decreased percentage of the electrocatalytic current was proportional to CEA concentration ranging from 1.0 to 55 ng·mL−1 with a detection limit of 0.13 ng·mL−1 (S/N = 3). Intra- and interassay uncertainties were <5.5 and 7.5% for analyte concentrations, respectively. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross-talk uncertainty between other proteins was <9.5%. The immunosensor exhibited acceptable reproducibility and stability, and is shown to be applicable to clinical immunoassay of CEA. Importantly, the immunosensor is suitable for use in the mass production of miniaturized lab-on-a-chip devices and paves new opportunities for protein diagnostics and biosecurity.

Utility of Slot‐Blot‐ELISA as a New, Fast, and Sensitive Immunoassay for Detection of Carcinoembryonic Antigen in the Urine Samples of Patients with Various Gastrointestinal Malignancies

Carcinoembryonic antigen (CEA) is the most widely used clinical tumor marker. CEA immunoassay has found acceptance as a diagnostic adjunct in clinical diagnosis of gastrointestinal tumors (GIT). Several immunoassays have been established for detection of CEA in plasma, serum, tissue, feces, and urine of cancer patients using polyclonal or monoclonal antibodies raised against CEA. Some of these assays display both high sensitivity and specificity for the detection of CEA. However, these assays require special and highly expensive equipment and the procedures require long periods for their completion. In the present study, we established a Slot‐Blot Enzyme Linked Immunosorbent Assay (SB‐ELISA), based on anti‐CEA monoclonal antibody (CEA‐mAb), as a new, simple, fast, cheap, and non‐invasive immunodiagnostic technique for detection of CEA in the urine of GIT patients. Urine and serum samples were collected from 248 GIT patients (58 with pancreatic cancer, 20 with hepatoma, 23 with ampullary carcinoma, 15 with hilar cholangiocarcinoma, 28 with gastric cancer, 14 with esophageal cancer, and 90 with colorectal cancer). Moreover, urine and serum samples were collected from 50 healthy individuals to serve as negative controls. The traditional ELISA technique was used for determination of CEA in the sera of GIT patients using anti‐CEA monoclonal antibody. A comparison between the results of both techniques (ELISA and SB‐ELISA) was carried out. The traditional ELISA detected CEA in the sera of 154 out of 248 GIT patients with a sensitivity of 59.8%, 51.7% positive predictive value (PPV) and 75.37% negative predictive value (NPV). In addition, it identified 15 false positive cases out of 50 healthy individuals with a specificity of 70%. The urinary CEA was identified by a Western blotting technique and CEA‐mAb at a molecular mass of 180 Kda. The developed SB‐ELISA showed higher sensitivity, specificity, PPV, and NPV (70.1%, 78%, 62.4%, and 82.13%, respectively) for detection of CEA in the urine of GIT patients. The semi‐quantitative SB‐ELISA showed a higher overall efficiency of 72.8% versus 63.4% in the case of the quantitative ELISA, for detection of CEA. In conclusion, SB‐ELISA is more efficient for detection of CEA in gastrointestinal tumors. It is a simple, rapid, non‐invasive, and sensitive assay. Moreover, all steps of the SB‐ELISA are performed at room temperature, without the use of expensive equipment; this may enhance the application of this assay in field studies and mass screening programs.

A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen

A new simple immunoassay method for carcinoembryonic antigen (CEA) detection using a disposable immunosensor coupled with a flow injection system was developed. The immunosensor was prepared by coating CEA/colloid Au/chitosan membrane at a screen-printed carbon electrode (SPCE). Using a competitive immunoassay format, the immunosensor inserted in the flow system with an injection of sample and horseradish peroxidase (HRP)-labeled CEA antibody was used to trap the labeled antibody at room temperature for 35 min. The current response obtained from the labeled HRP to thionine-H(2)O(2) system decreased proportionally to the CEA concentration in the range of 0.50-25 ng/ml with a correlation coefficient of 0.9981 and a detection limit of 0.22 ng/ml (S/N=3). The immunoassay system could automatically control the incubation, washing and current measurement steps with good stability and acceptable accuracy. Thus, the proposed method proved its potential use in clinical immunoassay of CEA.