Alpha-fetoprotein In Human Serum Research Articles

Gold nanoparticle labeling with tyramide signal amplification for highly sensitive detection of alpha fetoprotein in human serum by ICP-MS

In this work, we developed an immunoassay based on tyramide signal amplification (TSA) and gold nanoparticles (Au NPs) labeling for highly sensitive detection of alpha fetoprotein (AFP) by inductively coupled plasma mass spectrometry (ICP-MS). AFP was captured by anti-AFP1 coating on the 96-well plate and labeled by anti-AFP2-horseradish peroxidase (HRP), in which the HRP can catalyze the deposition of biotinylated tyramine on the nearby protein. Then the streptavidin (SA)-Au NPs was labeled on the deposited biotinylated tyramine as the intensive signal probe for ICP-MS measurement. Under the optimal experimental conditions, the limit of detection of the developed method for AFP was 1.85pg/mL and the linear range was 0.005–2ng/mL. The relative standard deviation for seven replicate detections of 0.01ng/mL AFP was 5.2%. The proposed method was successfully applied to the detection of AFP in human serum with good recoveries. This strategy is highly sensitive and easy to operate, and can be extended to the sensitive detection of other biomolecules in human serum.

An ultra-sensitive label-free immunosensor toward alpha-fetoprotein detection based on three–dimensional ordered IrOx inverse opals

Three-dimensional ordered macroporous iridium oxides (3DOM IrOx 0≤x≤2), were synthesized through a simple self–assembly template method and developed as an electrode material for lable–free immunosensor of cancer biomarker alpha-fetoprotein (AFP). The 3DOM IrOx was well modified on flexible fluorine-doped tin oxide (FTO) electrode and such FTO electrodes based 3DOM IrOx immnuosensor enhanced the adsorption of electrolytes, the diffusion of reactants, and also could act as a rapid electron transfer channel benefited both from the advantage of 3DOM architecture and the nature of IrOx itself. These combined characteristics greatly increased the sensitivity, shortened the response time, reduced the interference, promoted the stability and moreover, demonstrated good accuracy to detect AFP in human serum. The optimum structure and sensing properties of immnuosensor were obtained based on 3DOM IrOx annealed at 500°C. It allows detecting AFP over a wide concentration range from 1 to 250ng/mL with a detection limit of 0.3ng/mL, and a higher slope of the calibration equation. The manufactured immunosensor was also used to determine AFP in human serum with satisfactory results. This kind of 3DOM IrOx modified and FTO-based immunosensor has great application potential for the AFP electrochemical detection.

Capillary electrochromatography immunoassay for alpha-fetoprotein based on poly(guanidinium ionic liquid) monolithic material

Alpha-fetoprotein (AFP) is widely used as a tumor marker for the serum diagnosis of primary hepatoma. Sensitive detection of AFP level plays an important role in the early diagnosis of disease and highly reliable prediction. In this study, a novel non-competitive immunoassay (IA) based on poly(guanidinium ionic liquid) monolithic material was developed for detecting ultra trace levels of AFP in capillary electrochromatography (CEC) mode. The AFP was mixed with an excess amount of fluorescently labeled antibody. After incubation, the immunocomplex was separated from the free labeled antibody and detected by CEC coupled with laser-induced fluorescence detector. Under the optimized conditions, the developed CEC-IA performed a low detection limit of 0.05 μg L−1 (S/N = 3) and a wide linearity ranging from 0.1 to 1000 μg L−1 for AFP, which can be largely attributed to the high separation and enrichment efficiency of poly(guanidinium ionic liquid) monolithic material for the targets. The application of this method was demonstrated by determining AFP in human serum.

Upconversion nanoparticle as elemental tag for the determination of alpha-fetoprotein in human serum by inductively coupled plasma mass spectrometry.

Upconversion nanoparticles (UCNPs) have received increasing attention due to their unique optical properties. Recognizing that UCNPs are lanthanide-doped nanoparticles, we incorporated UCNPs into an immunoassay with inductively coupled plasma mass spectrometry (ICP-MS) detection for the determination of specific proteins, e.g., alpha-fetoprotein (AFP). The sensitivity of the assay was enhanced because of the ICP-MS detection of UCNPs that contained large numbers of lanthanide elemental tags. Conjugates of UCNPs and antibodies were prepared and the morphology of the conjugates was characterized by transmission electron microscopy. After a sandwich immunoreaction, the AFP was determined by the ICP-MS analysis of UCNPs. Under the optimized conditions, a limit of detection (3σ) of 0.31 ng mL-1 based on 89Y signal and 0.22 ng mL-1 based on 174Yb signal was obtained for AFP, with a dynamic range of 0.5-35 ng mL-1 and a relative standard deviation of 4.8% (c = 5 ng mL-1, n = 9). The developed method was applied to the determination of AFP in human serum and the recovery for the spiked sample was in the range of 98.6-123%. The proposed method is simple, rapid, selective and sensitive, and has a good tolerance for the complex biological matrix, indicating great potential for the application of UCNP in biological research as an elemental tag.

A novel microchip electrophoresis-based chemiluminescence immunoassay for the detection of alpha-fetoprotein in human serum

A sensitive immunoassay method based on microchip electrophoresis chemiluminescence (MCE-CL) detection technology was developed for the detection of tumor marker alpha-fetoprotein (AFP). This method adopts the non-competitive immunoassay mode, and was conducted after AFP reacted with excessive horseradish peroxidase (HRP) labeled monoclonal antibody. The extreme pH value was adopted in the electrophoresis buffer solution. The use of brij 35 as an additive of electrophoresis buffer increased dramatically the resolution (Rs) and the reproducibility of the analysis. Under the optimized experimental conditions, effective separation of the immune complex Ag-Ab* and free Ab* was achieved within 60s. The peak height of the immune complex Ag-Ab* was taken as quantification of AFP. Good linearity was observed within AFP concentrations ranging from 10ng/mL to 150ng/mL, and the detection limit was found to be 7.2ng/mL (1.0×10−10M). The present method was successfully applied for the detection of AFP in human serum from both healthy and cancer patients, and the AFP levels in the both were found be in the range of 16.5–23.4ng/mL and 416.2–825.4ng/mL, respectively.

Fabrication of Gold Nanoparticle-Embedded Metal–Organic Framework for Highly Sensitive Surface-Enhanced Raman Scattering Detection

Surface-enhanced Raman scattering (SERS) signals strongly rely on the interactions and distance between analyte molecules and metallic nanostructures. In this work, the use of a gold nanoparticle (AuNP)-embedded metal-organic framework was introduced for the highly sensitive SERS detection. The AuNPs were in situ grown and encapsulated within the host matrix of MIL-101 by a solution impregnation strategy. The as-synthesized AuNPs/MIL-101 nanocomposites combined the localized surface plasmon resonance properties of the gold nanoparticles and the high adsorption capability of metal-organic framework, making them highly sensitive SERS substrates by effectively preconcentrating analytes in close proximity to the electromagnetic fields at the SERS-active metal surface. We discussed the fabrication, physical characterization, and SERS activity of our novel substrates by measuring the Raman signals of a variety of model analytes. The SERS substrate was found to be highly sensitive, robust, and amiable to several different target analytes. A SERS detection limit of 41.75 and 0.54 fmol for Rhodamine 6G and benzadine, respectively, was demonstrated. The substrate also showed high stability and reproducibility, as well as molecular sieving effect thanks to the protective shell of the metal-organic framework. Subsequently, the potential practical application of the novel SERS substrate was evaluated by quantitative analysis of organic pollutant p-phenylenediamine in environmental water and tumor marker alpha-fetoprotein in human serum. The method showed good linearity between 1.0 and 100.0 ng/mL for p-phenylenediamine and 1.0-130.0 ng/mL for alpha-fetoprotein with the correlation coefficients of 0.9950 and -0.9938, respectively. The recoveries ranged from 80.5% to 114.7% for p-phenylenediamine in environmental water and 79.3% to 107.3% for alpha-fetoprotein in human serum. These results foresee promising application of the novel metal-organic framework based composites as sensitive SERS-active substrates in both environmental and clinical samples.

Label-free immunosensor based on Pd nanoplates for amperometric immunoassay of alpha-fetoprotein

In this paper, Pd nanoplates were used as a kind of electrode materials for fabrication of an electrochemical immunosensor, which was applied for detection of cancer biomarker alpha-fetoprotein (AFP). Thanks to the unique structure and properties of Pd nanoplates, the antibody of AFP (Ab) was effectively immobilized onto the surface of the Pd nanoplates modified glassy carbon electrode (GCE). Moreover, the good electrochemical properties of Pd nanoplates greatly improved the electronic transmission rate and enhanced the electrochemical signal, which led to an increase of the detection sensitivity. Based on the specific antibody–antigen interaction, a label-free immunosensor based on Pd nanoplates was developed for sensing of AFP. The current method allows us to detect AFP over a wide concentration range from 0.01 to 75.0ng/mL with a detection limit of 4pg/mL. The proposed immunosensor has been used to determine AFP in human serum with satisfactory results.

Multiplexed detection of tumor markers with multicolor quantum dots based on fluorescence polarization immunoassay

A multicolor quantum dot (QD)-based nanosensor for multiplex detection of two tumor markers in a homogeneous format based on fluorescence polarization immunoassay was proposed. QDs520 and QDs620 were labeled alpha-fetoprotein(α-AFP) and carcinoembryonic antigen (CEA), respectively. After separated and purified by ultrafiltration, they were used in fluorescence polarization immunoassay for the simultaneous detection of human serum alpha-fetoprotein and carcinoembryonic antigen. Under the optimal conditions, the multi-analyte immunosensor had a wide linear range (from 0.5ngmL−1 to 500ngmL−1) for both two tumor markers and good correlation (0.996 for α-AFP and 0.993 for CEA). The detection limits (LOD) were 0.36ngmL−1 for CEA and 0.28ngmL−1 for α-AFP (S/N=3). The carcinoembryonic antigen and fetoprotein in clinical serum samples were simultaneously detected. The results from 28 serum samples had a good agreement with enzyme-linked immunosorbent assay (ELISA). The relative standard deviation and the recovery suggested that the precision and the accuracy of this analytical method were satisfactory. This strategy with high sensitivity, good specificity, easy procedures and short analysis time shows great promise for clinical diagnoses and basic discovery. The application of QDs with longer fluorescence lifetime and small fluorescence polarization can be used for the determination of high molecular-weight substances which cannot be analyzed using dye fluorescence polarization immunoassay.

New amperometric immunosensor with response enhanced by PAMAM-dendrimers linked via self assembled monolayers for determination of alpha-fetoprotein in human serum

A new amperometric immunosensor for alpha-fetoprotein (AFP), based on nanobiocomposite substrate and with response enhanced by polyamidoaminic (PAMAM) dendrimers was developed and characterized. The nanostructurated substrate obtained by electrochemical deposition of 100 nm-sized gold nanoparticles on glassy carbon electrodes (GCE) was functionalized by deposition of a SAM of 2-aminoethanethiol (AET), used as linker for the subsequent immobilization of polyamidoaminic dendrimers (PAMAM G.1.5). Two different modes were investigated for the reading of the assay: cyclic voltammetry (CV) or Double Step ChronoAmperometry (DSCA). Satisfying results in terms of response range and precision were reached with both methods. Immunosensors were tested and validated for AFP determination in human serum, showing a limit of detection of 3 ng/mL and a limit of quantitation of 15 ng/mL. The enhanced immunosensor has proved an attractive diagnostic tool able to match the needs of clinical monitoring purposes for AFP quantification in human serum at levels useful both for prognosis of pregnancy progression, and for the identification of the occurrence of neoplastic diseases.

Sandwich-type conductometric immunoassay of alpha-fetoprotein in human serum using carbon nanoparticles as labels

A simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) was designed using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of the immobilization of monoclonal anti-AFP primary antibodies on an interdigitated conductometric transducer, while the detection antibodies were prepared using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP-anti-AFP). With a sandwich-type immunoassay format, the conjugated CNP-HRP-anti-AFP on the transducer was increased with the increase of AFP in the sample, and the conductivity of the immunosensor was decreased in the H2O2–KI system. Under optimal conditions, the immunosensor exhibited a wide dynamic range of 0.1–500ng/mL with a detection limit of 50pg/mL AFP at 3σ. The reproducibility and recovery were <10% and 83.9–112.3%, respectively. Interestingly, 45 clinical serum specimens were assayed using the conductometric immunosensor, and the results were in accordance with those obtained from our Clinical Laboratory using Roche 2010 Electrochemiluminescent Automatic Analyzer.

Integrated Microfluidic Device for Serum Biomarker Quantitation Using Either Standard Addition or a Calibration Curve

Detection and accurate quantitation of biomarkers such as alpha-fetoprotein (AFP) can be a key aspect of early stage cancer diagnosis. Microfluidic devices provide attractive analysis capabilities, including low sample and reagent consumption, as well as short assay times. However, to date microfluidic analyzers have relied almost exclusively on calibration curves for sample quantitation, which can be problematic for complex mixtures such as human serum. We have fabricated integrated polymer microfluidic systems that can quantitatively determine fluorescently labeled AFP in human serum using either the method of standard addition or a calibration curve. Our microdevices couple an immunoaffinity purification step with rapid microchip electrophoresis separation in a laser-induced fluorescence detection system, all under automated voltage control in a miniaturized polymer microchip. In conjunction with laser-induced fluorescence detection, these systems can quantify AFP at approximately 1 ng/mL levels in approximately 10 microL of human serum in a few tens of minutes. Our polymer microdevices have been applied in determining AFP in spiked serum samples. These integrated microsystems offer excellent potential for rapid, simple, and accurate biomarker quantitation in a point-of-care setting.

Localized surface plasmon coupled fluorescence fiber-optic biosensor for alpha-fetoprotein detection in human serum

In this study, we demonstrated that the fiber-optic biosensor based on localized surface plasmon coupled fluorescence (LSPCF) is capable of detecting alpha-fetoprotein (AFP) in human serum. The sensitivity of LSPCF fiber-optic biosensor is not only enhanced but also the specific selectivity is improved since the fluorophores are excited by the localized surface plasmon with high efficiency. Experimentally, this fiber-optic biosensor is able to detect AFP concentration in phosphate buffered saline (PBS) solution from 0.1 ng/mL to 100 ng/mL whereas the linear relationship between the AFP concentrations and the fluorescence signals is shown. Furthermore, a linear response between the fluorescence signals and the concentrations of AFP in human serum from 2.33 ng/mL to 143.74 ng/mL is also obtained. As a result, the detection limit of the LSPCF fiber-optic biosensor on AFP detection is comparable with the conventional enzyme-linked immunosorbent assay (ELISA). Additionally, the LSPCF fiber-optic biosensor benefits on inexpensive, disposable and simpler optical geometry that can become a high efficient immunoassay comparable with the conventional ELISA and radioimmunoassay (RIA) clinically.

Non-competitive immunoassay for alpha-fetoprotein using micellar electrokinetic capillary chromatography and laser-induced fluorescence detection

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP–antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 m M tetraborate containing 100 m M SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.

Glycan composition of serum alpha-fetoprotein in patients with hepatocellular carcinoma and non-seminomatous germ cell tumour.

Although estimation of serum alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma (HCC) and non-seminomatous germ cell tumours (NSGCT), the clinical usefulness of this test is limited by a low specificity. However, there exist glycoforms of AFP which may be more specific for particular tumours. Previously, detailed analysis has been prevented by the low levels of AFP in human serum. We report here the application of fluorescence labelling, sequential exoglycosidase digestion, high-performance liquid chromatography and matrix-assisted laser desorption ionization in time-of-flight mass spectrometry, to determine the glycan structures of purified serum AFP from patients with HCC and NSGCT. Eleven major glycans were found, of which seven were N-linked, and four were O-linked, to the protein backbone. The structure of the N-linked glycans (all of bi-antennary complex-type with varying degrees of sialylation, fucosylation and galactosylation) were consistent with those previously reported. The O-linked glycans (three mucin O-GalNAc type glycans with variable degrees of sialylation, one O-HexNAc monosaccharide glycan) have not previously been reported. The finding of mucin O-GalNAc type glycans was supported by the prediction of potential O-GalNAc glycosylation sites on the protein backbone by analysis of the AFP structure by molecular modelling. With knowledge of these structures it may be possible to develop more specific assays for the detection of HCC and NSGCT. © 1999 Cancer Research Campaign © 1999 Cancer Research Campaign

The use of a CCD imaging luminometer in the quantitation of luminogenic immunoassays

AbstractWe describe the application of a CCD Imaging Luminometer (CCDIL) for the detection and quantitation of rapid, simultaneous, peroxidase‐linked chemiluminescent immunoassays of multiple samples of human serum alphafetoprotein (AFP). Results from some analogous immunoassays (total IgE, and thyroid stimulating hormone (TSH)) are included for comparison.Values for precision and antigen concentration obtained using the CCDIL and colorimetric versions of the immunoassays, on human serum samples, were in good agreement.The flexibility of the CCDIL is demonstrated; its ability to detect and quantitate antigen (particularly AFP) on a variety of solid phases is indicated.The work on the AFP immunoassays illustrates not only the flexibility of the CCDIL for sample presentation on a variety of solid phase systems, but also some relative merits of such systems.

Use of soluble and Staphylococcus aureus-immobilized second antibody compared in a radioimmunoassay for human alpha-fetoprotein.

Staphylococcus aureus cells coated with rabbit anti-goat immunoglobulin G were compared with soluble second antibody in a radioimmunoassay for human serum alphafetoprotein. Recovery of primary immune complexes with use of the bacterial immunoadsorbant (a) allowed second-antibody incubation time to be shortened from 3 h to 20 min at 37 degrees C, (b) decreased by 80% the amount of second antibody required per assay, thereby significantly reducing assay costs, and (c) provided a convenient and reproducible method for separation of free and bound antigen. The ease of preparation and the uniformity and stability of the S. aureus second-antibody reagent are additional advantages that may be useful in other radioimmunoassay procedures.

Comparison of double-antibody radioimmunoassay with Farr-technique radioimmunoassay and double-antibody enzyme immunoassay for alpha-fetoprotein.

We describe double-antibody procedures for determining alpha-fetoprotein in human serum. An equilibrium procedure can be done in 24 h with a sensitivity of at least 4 mug/liter and coefficient of variation of 5.5%. There are no interferences from normal human sera or sera with certain commonly seen chemical abnormalities. We also describe and discuss sequential procedures that range in sensitivity from 250 ng to 1mug/liter and require 24-48h incubation. The precise (mid-range)portion of the dose/response curve for sequential procedures can be shifted to higher or lower values by an adjustment of the time of preliminary incubation of antibody with unlabeled antigen. With a 37 degrees C incubation, a sequential procedure can be completed in 7 h. Sensitivity is 1 mug/liter, and coefficient of variation 8.0%. The relative merits of the above assay procedures are discussed. The double-antibody redioimmunoassay is twice as sensitive as the Farr procedure [J. Infect. Dis. 103, 239 (1958)], and it is free of the large and variable nonspecific precipitation that accompanies the precipitation of bound antigen with sodium sulfate solution. Double-antibody radioimmunoassay is superior to enzyme immunoassay in both sensitivity and precision.