Automated chemiluminescent dual-analyte immunoassay based on resolved immunosensing channels

A novel system of series-wound immunosensing channels (SWIC) was proposed for automated chemiluminescent (CL) dual-analyte immunoassay by immobilizing respectively different capture antibodies on the inner walls of series-wound glass channels. This system could use a single enzyme as label to perform multiplex immunoassay in one fluid way. Using α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model analytes, the mixture including AFP, horseradish peroxidase (HRP)-labeled anti-AFP antibody, CEA and HRP-labeled anti-CEA antibody was introduced into the SWIC for carrying out the on-line incubation. Upon injection of CL substrate the CL signals from the two immunosensing channels were conveniently resolved and near-simultaneously collected with the aid of optical shutter. AFP and CEA could be rapidly assayed in the ranges of 1.0-100 and 1.0-80 ng/ml with detection limits of 0.41 and 0.39 ng/ml, respectively. The assay results of clinical serum samples were in an acceptable agreement with the reference values. This designed flow-through immunosensing system based on SWIC provided an automated, reusable, simple, sensitive and low-cost approach for multianalyte immunoassay.     

Synthesis of a new biacridine and its use as the chemiluminescent probe for immunoassay of carcinoembryonic antigen

A new biacridine compound, 10,10′-dimethyl-3,3′-disulfo-9,9′-biacridine (DMDSBA) was synthesized, and its chemiluminescent characteristics were investigated in detail. DMDSBA was used to label anti-CEA antibody. The labeling ratio was estimated to be 1.15-1.32, and the average labeling ratio was 1.25. The results show that there are no obvious changes in the immunoreactivity of the labeled anti-CEA antibody and the quantum efficiency of DMDSBA after attaching to the anti-CEA antibody. In addition, the conditions of labeling reaction, sandwich immunoassay and chemiluminescent reaction have been studied. A new sandwich chemiluminescent immunoassay method was firstly established, and used to determine CEA in human serum, the calibration range is 1.0-100 ng ml⁻¹ and the minimal detectable concentration of CEA is 0.53 ng ml⁻¹, the relative standard deviation is 6.5% for 20 ng ml⁻¹ CEA. This method was well-matched with radio immunoassay.     

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen

A micro-magnetic chemiluminescence (CL) enzyme immunoassay with high sensitivity, selectivity, and reproducibility was developed for the determination of the tumor marker, carcinoembryonic antigen (CEA) in human serum. A sandwich scheme assay has been utilized with fluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CL signal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was directly proportional to the amount of analyte present in a sample solution. The influences of the reaction time of antigen with antibody, the reaction time of substrate with label, the dilution ratio of ALP-labeled anti-CEA antibody, the concentration of FITC-labeled anti-CEA antibody, and other relevant variables upon the CL signal were examined and optimized. The CL responses depended linearly on the CEA concentration over the range from 2 to 162 ng mL⁻¹ in a logarithmic plot. Assay sensitivity as low as 0.69 ng mL⁻¹ was achieved. A coefficient of variance of less than 13% was obtained for intra- and inter-assay precision. This method has been successfully applied to the analysis of CEA in human serum. According to the procedure based on spiked standards, the recoveries obtained were 80–110%. Comparison experiments were carried out with the commercially available CEA chemiluminescence immunoassay. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level).     

Biofunctionalized dendritic polyaniline nanofibers for sensitive electrochemical immunoassay of biomarkers

Multi-armed dendritic polyaniline nanofibers (MPANFs) were first synthesized and functionalized with horseradish peroxidase (HRP) and carcinoembryonic antibody (anti-CEA) for highly efficient electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte here) in this work. Transmission electron microscope (TEM) and scanning electron microscope (SEM) techniques were employed to characterize the synthesized MPANFs. By using anti-CEA-conjugated core-shell gold-Fe(3)O(4) nanocomposites (GoldMag) as immunosensing probes and biofunctionalized MPANFs as molecular tags, a new sandwich-type homogeneous immunoassay strategy was developed for the determination of CEA by coupling with a home-made flow-through magneto-controlled microfluidic device. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of four orders of magnitude from 1.0 pg mL(-1) to 50 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) CEA at 3σ. Intra- and inter-assay coefficients of variation were below 10%. The assayed results for clinical serum specimens with the electrochemical immunoassay were received in good accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.     

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab₂)/thionine/nanoporous silver (HRP-Ab₂/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL⁻¹ with low detection limit of 0.35 pg mL⁻¹ and low limit of quantitation (LOQ) of 0.85 pg mL⁻¹. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules     

Simultaneous detection of two lung cancer biomarkers using dual-color fluorescence quantum dots

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, a novel protocol for performing a simultaneous dual-protein immunoassay, i.e. two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), based on dual-color QDs, is described. First, two capture antibodies (both with biotin tags), two antigens and two detection antibodies were mixed together and the sandwich complexes were thus formed in the homogeneous solution, and then streptavidin coated polystyrene beads were directly added into the resultant system. Bead aggregation can be made self-limiting by controlling the shaker speed during the immunoassay. A distinct transition occurs between limited and complete aggregation as a function of the shaker speed during the immunoassay. Second, dual-color QDs with emission maxima at 525 and 655 nm were added after washing and reacted with the corresponding detection antibodies. Third, the bead-QD conjugates were dissociated in the dissociation buffer and then free QDs were directly used for the fluorescence detection of CEA and NSE. The results show that CEA and NSE could be sensitively determined with a common 96-well fluorescence plate reader and with equal detection limits down to the 1.0 ng mL(-1) level. Within the calibrated amount, the protocol had excellent precision within 0.53% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual markers in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for CEA and NSE in 25 human serum samples.     

磁性纳米金共价固定癌胚抗原单克隆抗体的电流型免疫传感器

合成了Fe3O4/Au磁性复合纳米粒子, 在粒子表面通过自组装硫脲分子使表面氨基化, 再用戊二醛共价交联固定癌胚抗原抗体(anti-CEA). 在外加磁场的作用下, 将anti-CEA复合磁性粒子吸附在固体石蜡碳糊电极表面, 制成了新型电流型免疫传感器. 免疫电极在含有癌胚抗原CEA和辣根过氧化物酶标记的癌胚抗原(HRP-CEA)的混合溶液中温育, CEA和HRP-CEA与固定在电极表面的anti-CEA发生竞争反应, 导致HRP对H2O2的催化降解作用的改变, 从而可间接测定CEA. 由于标记的HRP可催化降解H2O2, 导致媒介体间苯二酚浓度改变, 使测定的灵敏度大大提高. 响应电流与CEA质量浓度的对数在2~160 ng/mL的范围内呈线性关系, 检出限为0.57 ng/mL(3σ法). 该免疫传感器具有制作简单、价廉及表面易于更新等特点.     

Nanoplatinum-enclosed gold nanocores as catalytically promoted nanolabels for sensitive electrochemical immunoassay

Here we designed a new electrochemical immunoassay protocol for determination of carcinoembryonic antigen (CEA) using nanoplatinum-enclosed gold nanocores (Pt@Au) as catalytically promoted nanolabels on the carbon nanospheres and graphene-modified immunosensor. The Pt@Au nanolabels were synthesized and functionalized with monoclonal anti-CEA antibodies and glucose oxidase (GOx). Using the functional Pt@Au nanolabels as molecular tags, the assay was implemented relative to glucose–hydroquinone system with a sandwich-type immunoassay. Initially, the added glucose was oxidized to gluconolactone and H₂O₂ by the labeled GOx, and then the generated H₂O₂ was reduced with the help of platinum nanoparticles, leading to the production of oxygen. The self-produced oxygen could promote the re-oxidation of the glucose, thus resulting in the dual amplification of the electrochemical signal. Several nanolabels, such as multiarmed star-like platinum nanowires, hollow platinum nanospheres and Pt@Au nanostructures, were investigated for CEA detection and improved analytical features were obtained with the Pt@Au nanostructures. Under optimal conditions, the Pt@Au-based immunoassay displayed a wide working range from 0.001 to 120 ng mL⁻¹ with a low detection limit of 0.5 pg mL⁻¹ CEA at 3s_B. Intra- and inter-assay coefficients of variation were <10.9%. The system was evaluated with 10 clinical serum samples, receiving good accordance with results from enzyme-linked immunosorbent assay method.     

A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL⁻¹ for CEA, with a detection limit of 0.3 ng mL⁻¹. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.     

A rapid and highly sensitive portable chemiluminescent immunosensor of carcinoembryonic antigen based on immunomagnetic separation in human serum

To detect a biomarker for lung cancer, carcinoembryonic antigen (CEA), a highly sensitive, selective, rapid and portable immunosensor based on immunomagnetic separation and chemiluminescence immunoassay was introduced. A sandwich scheme assay has been utilized with horseradish peroxidase (HRP) labeled anti-CEA antibody and immunomagnetic beads (IMBs). The presence of target protein CEA caused the formation of the sandwich structures (IMBs-CEA-HRP labeled antibody). IMBs were applied to capture CEA and immobilize CEA through the external magnetic field. The HRP at the surface of the antibody catalytically oxidized the luminescence substrate to generate optical signals which were detected by a portable home-made luminometer and which were directly proportional to the concentration of CEA in the samples. The signals were dependent on CEA concentrations in a linear range from 0 to 50 ng mL⁻¹. The limit of detection (LOD) of this method was as low as 5.0 pg mL⁻¹ (S/N = 3). The novel immunosensor was highly sensitive with an assay time of <35 min. The intra- and inter-assay coefficients of variation were <10%. The anti-CEA antibody can be bound to the bead efficiently with a conjugation rate of 73%. IMBs could be stored in 4 °C protecting from light for 2 months without obvious reduction of biological activity. Human reference sera mixed with various concentrations of CEA were tested with the proposed method and commercial enzyme-linked immunosorbent assay (ELISA) kit, and a good linear relationship was obtained. This proposed technique demonstrated an excellent performance for quantifying CEA and was expected to be used for clinical testing.     

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.

Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids

A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol-gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO₂/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO₂/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO₂/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12 ng mL⁻¹ in pH 5.5 HAc-NaAc containing 5 mM H₂O₂. At a signal-to-noise ratio of 3, the detection limit (LOD) is 5 pg mL⁻¹ CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay.     

Iridium Oxide Film-Enhanced Impedance Immunosensor for Rapid Detection of Carcinoembyronic Antigen

A simple, rapid and sensitive impedance immunosensor based on iridium oxide （IrOx） thin film for the detection of carcinoembyronic antigen （CEA） in human sera has been proposed. Gold electrode was electrochemically modified with IrOx thin film and simultaneously functionalized with protein A （PA） to bind anti-CEA antibodies in an orientated way. It has been found that the antibody loading amount was dependent on the PA concentration and the deposition time of IrOx matrix. Under the optimized experimental conditions, the electron transfer resistances obtained were linearly related to the CEA concentration ranging from 36.2 to 460.0 ng/mL, with a detection limit of 28.0 ng/mL. Analytical results of clinical samples from cancer patients show that the proposed immunoassay is reasonably comparable with the chemiluminescence immunoassay （CLIA）, indicating the feasibility of using the proposed method for CEA immunoassay in clinical laboratory.     

A novel piezoelectric immunosensor for detection of carcinoembryonic antigen

A simple, rapid, and highly sensitive immunosensor for the direct determination of carcinoembryonic antigen (CEA) in human serum using a piezoelectric crystal has been developed and optimized. In order to improve sensitivity of the immunosensor, a protein A-based orientation-controlled immobilization method for antibodies was adopted together with an immunoreactive accelerant of polyethyleneglycol (PEG) used to amplify the signal response of frequency. Human normal serum was utilized as a reference background. The linear range for CEA concentration obtained by the end-point method was 66.7-466.7 ng/mL. Clinical samples from cancer patients were analyzed by the proposed piezoelectric immunoassay, and the analytical results were reasonably comparable with those obtained by the chemiluminescence immunoassay (CLIA). The proposed immunosensor provides a new promising method for the highly sensitive immunoassay of CEA in clinical laboratory.     

Novel electrochemical sensor for the selective recognition of chlorogenic acid

In this article, we demonstrate the fabrication and simultaneous fluorescent detection of two biomarkers related to lung cancer. Polystyrene microspheres (PSM) were introduced as biomolecular immobilizing carriers and a 96-well filter plate was used as the separation platform. The whole experiment could be effectively carried out in a homogeneous system, as exemplified by the detection of carcinoembryonic antigen (CEA) and neuron specific enolase (NSE). First, two capture antibodies for CEA and NSE were immobilized on the PSM surface. Next, they reacted successively with two antigens and two modified detection antibodies. Finally, these two biomarkers could be recognized by streptavidin-conjugated quantum dots (QD) and goat-anti-FITC conjugated QD with a detection limit of 0.625 ng mL(-1), which was lower than the clinical cut-off level. The protocol showed good precision within 6.36% and good recovery in the range of 90.86-105.02%. Compared with several other assay formats reported previously, our new technique is competitive or even better. Furthermore, the immunosensor was successfully illustrated in 20 serum samples. Overall, this new immunoassay offers a promising alternative for the detection of biomarkers related to cancer diseases, taking advantage of simplicity, specificity, sensitivity and cost-efficiency.     

An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles

A quick and reproducible electrochemical-based immunosensor technique, using magnetic core/shell particles that are coated with self-assembled multilayer of nanogold, has been developed. Magnetic particles that are structured from Au/Fe₃O₄ core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The carcinoembryonic antibody (anti-CEA) was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode (SPCE) by an external magnetic field. This is an assembly of a novel immuno biosensor for carcinoembryonic antigen (CEA). The sensitivity and response features of this immunoassay are significantly affected by the surface area and the biological compatibility of the multilayered nanogold. The linear range for the detection of CEA was from 0.005 to 50 ng mL⁻¹ and the limit of detection (LOD) was 0.001 ng mL⁻¹. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA detection.     

Magnetic core-shell Fe3O4@Ag nanoparticles coated carbon paste interface for studies of carcinoembryonic antigen in clinical immunoassay

This study demonstrates a novel approach toward development of advanced immunosensors based on chemically functionalized core-shell Fe3O4@Ag magnetic nanoparticles, and the preparation, characterization, and measurement of relevant properties of the immunosensor useful for the detection of carcinoembryonic antigen (CEA) in clinical immunoassay. The immunosensor based on the combination of a magnetic nanocore and an Ag metallic shell shows good adsorption properties for the attachment of the CEA antibody selective to CEA. The core-shell nanostructure presents good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of CEA, and allows detection of CEA at a concentration as low as 0.5 ng.mL(-1). Importantly, the proposed methodology could be extended to the detection of other antigens or biocompounds.     

A highly sensitive label-free resonance light scattering assay of carcinoembryonic antigen based on immune complexes

As a kind of glycoprotein, carcinoembryonic antigen (CEA) is the important tumor marker for clinical diagnosis of the presence or recurrence of cancer. In this work, a novel label-free resonance light scattering (RLS) spectral CEA assay was developed based on the combination of highly selective immunoreaction and ultrasensitive RLS technique. In Tris–HCl buffer solution (pH 7.5), the specific immunoreaction between CEA antigen and mouse anti-CEA formed immune complexes which had a maximum RLS spectral peak at 389.0 nm, with the existence of physiological saline and polyethylene glycol 20,000 (PEG 20,000). Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI_RLS) was proportional to the concentration of CEA in the range from 0.1 to 60 ng mL⁻¹, with a detection limit (LOD, 3σ) of 0.03 ng mL⁻¹. The characteristics of RLS, the CEA immunocomplex, the immune response, the ratio of CEA antigen and mouse anti-CEA, and the optimum conditions of the immunoreaction have been investigated. The CEA concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) kit. And this method has many satisfying merits including label-free, sensitivity and high selectivity.     

Multianalyte immunoassay chip for detection of tumor markers by chemiluminescent and colorimetric methods

Most cancers developed an elevation of the level of at least two markers associated with their incidence. Simultaneous detection of multi-tumor markers associated with a particular type of cancer plays an important role in cancer diagnostic. Here, a multianalyte immunoassay chip for simple and sensitive detection of tumor markers with chemiluminescent and colorimetric methods was proposed, in which carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) that associated with colorectal cancer were detected as model. The immunoassay chip was fabricated by co-immobilization of CEA/CA19-9 antibody on a glass slide with γ-glycidoxypropyltrimethoxysilane as linkage. Through sandwiched immunoreactions, CEA, CA19-9, and their corresponding enzyme tracers, alkaline phosphatase-labeled anti-CEA and horseradish peroxidase-labeled anti-CA19-9, were introduced on the chip. Then, they were sequentially detected by chemiluminescent method in the range of 0.5–80 μg/L and 0.5–80 kU/L with the detection limits of 0.41 μg/L and 0.36 kU/L at 3σ for CEA and CA19-9, respectively. They could also be detected by colorimetric method in the range of 1–200 μg/L and 5–200 kU/L with the detection limits of 0.25 μg/L and 1.25 kU/L at 3σ for CEA and CA19-9, respectively. All these results demonstrated that the present work provided a promising analytical method for tumor markers’ analysis with the advantages of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for high-throughput detection.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.