Fundamental and clinical evaluation of an immunoradiometric assay procedure "Amerwell AFP" for estimation of serum alpha-fetoprotein

The efficacy of assay procedure for estimation of serum alpha-fetoprotein (AFP) was examined on "Amerwell AFP" immunoradiometric assay (IRMA) using monoclonal AFP antibody. The condition of incubation was most satisfactory for 2 hours at 37 degrees C. Coefficients of variance for intraassay and interassay were 5.1-10.0% and 8.4-9.9% respectively. Dilution test gave satisfactory results. The binding capacity of microplate-well tagged monoclonal AFP antibody with AFP antigen was satisfactory for assay reactions. This method showed a good correlation to the AFP RIA bead (Dinabot Co.) method. The normal range (reference value) was within the level of 5.0 IU/ml due to Hoffmann's method in the examination of 860 subjects. Estimation of AFP with "Amerwell AFP" IRMA kit was a feasible routine method of clinical application for tumor marker.     

CdSe quantum dots as labels for sensitive immunoassay of cancer biomarker proteins by electrogenerated chemiluminescence

A sensitive and specific immunoassay method for detecting α-fetoprotein (AFP) based on electrogenerated chemiluminescence (ECL) was described. ECL could perform detection for a series of different concentrations of AFP. CdSe quantum dots (QDs) were used as labels and were linked to AFP antibody (anti-AFP, the secondary antibody, Ab2*). Immunoassay was carried out on a modified electrode using a sandwich assay approach, where anti-AFP (Ab1) was covalently bound to the surface of an Au electrode to be allowed to capture AFP specifically. Afterwards, Ab2* was allowed to bind selectively to the captured AFP. The non-specific adsorption was negligible. In the presence of H(2)O(2), the ECL intensity increased with the increase of AFP, which indicated that an immunosensor for AFP was constructed. The detection of AFP based on measuring the ECL intensity of CdSe without the enzyme and mediator can promote the stability of the immunosensor. The linear range of the AFP assay was from 0.002 to 32 ng mL(-1). Furthermore, the immunosensor showed high sensitivity, good precision, stability, and reproducibility and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method. The strategy was successfully demonstrated as a simple, cost-effective, specific, and potential method to detect AFP in practical samples.     

基于新型聚钙羧酸修饰电极的甲胎蛋白电化学免疫传感器研究

通过电聚合制得新型聚钙羧酸修饰电极并用于构建检测甲胎蛋白（AFP）的高灵敏电化学免疫传感器. 采用扫描电镜（SEM）、电化学交流阻抗（EIS）观察、表征修饰电极和AFP单克隆抗体（Ab1）固定前后的差异. 固定Ab1的电极与一定浓度的AFP、辣根过氧化物酶联AFP单克隆抗体（HRP-Ab2）反应,形成夹心型免疫复合物. 辣根过氧化物酶（HRP）催化3,3',5,5'-四甲基联苯胺（TMB）底物产生电流信号,实现AFP浓度的测定. 本检测方法灵敏度高,重现性好.     

New adjuvant design using layered double hydroxide for production of polyclonal antibodies in radioimmunoassay techniques

Various adjuvants have been used to enhance the immune response against specific antigens. So the objective of this work describes the immune stimulating activity of layered double hydroxide (LDH) particles incorporate with mineral oil as a new formulation of adjuvant as compared to known Freund’s adjuvant for production of alpha-fetoprotein polyclonal antibody (anti-AFP) for estimation of alpha fetoprotein (AFP) in human serum by radioimmunoassay technique. In this concern, the study comprised two groups of white New Zealand rabbits, 2?2.5 kg body weight and each group comprised three rabbits. The first group vaccinated with AFP antigen emulsified with Freund’s adjuvant and the second group vaccinated with AFP antigen emulsified with LDH formulation. The obtained data show that the highest displacement using LDH adjuvant reached (74.2, 61.7 and 66.5 %) while the corresponding values with Freund’s adjuvant recorded (64.8, 60.3 and 54.6 %) which indicates that the use of LDH adjuvant as a cellular vehicle is a more suitable choice. Also, the preparation of AFP tracer using lactoperoxidase oxidation method and its purification using gel chromatography on PD-10 column were carried out. Different factors affecting the optimization of the assay process were studied. Validation testes of the assay were carried out. The reproducibility as measured by the intra- and inter- assay variations is satisfactory. The recovery and dilution testes indicated accurate calibration and appropriate matrix. The present technique agreed well with the currently used commercial kit (Siemens, IRMA kit). In conclusion, the liquid phase double antibody RIA technique proved to be sensitive, specific, precis and accurate for routine laboratory use.     

高灵敏甲胎蛋白夹心免疫传感器的制备

构建了新型甲胎蛋白(AFP)夹心免疫传感器.采用金纳米粒子-氧化石墨烯-普鲁士蓝纳米立方体(AuNP-GO-PBNCs)纳米复合材料标记甲胎蛋白(AFP)二抗,将制备的金-聚多巴胺-四氧化三铁(Au-PDA-Fe3O4)磁性纳米复合物固定在自制的磁性电极表面,通过吸附作用固定AFP一抗,用牛血清白蛋白(BSA)封闭电极上的非特异性吸附位点.在37℃下与AFP抗原溶液孵育50 min,最后将电极放入AuNP-GO-PBNCs纳米复合材料标记的二抗溶液中孵育,基于此建立了采用普鲁士蓝(PB)标记的的夹心免疫传感器检测AFP的方法.在最佳实验条件下,PB催化H2O2氧化的响应电流与AFP的浓度表现出两段线性关系,线性范围分别为0.005~1.000 ng/mL和1~20 ng/mL, 检出限(LOD, S/N=3)为1.0 pg/mL.本方法具有灵敏度高、选择性好的特点.     

Prussian blue–gold nanoparticles-ionic liquid functionalized reduced graphene oxide nanocomposite as label for ultrasensitive electrochemical immunoassay of alpha-fetoprotein

In this work, poly(diallyldimethylammonium chloride) (PDDA) protected Prussian blue/gold nanoparticles/ionic liquid functionalized reduced graphene oxide (IL-rGO-Au-PDDA-PB) nanocomposite was fabricated. The resulting nanocomposite exhibited high biocompatibility, conductivity and catalytic activity. To assess the performance of the nanocomposite, a sensitive sandwich-type immunosensor was constructed for detecting alpha-fetoprotein (AFP). Greatly enhanced sensitivity for this immunosensor was based on triple signal amplification strategies. Firstly, IL-rGO modified electrode was used as biosensor platform to capture a large amount of antibody due to its increased surface area, thus amplifying the detection response. Secondly, a large number of Au-PDDA-PB was conjugated on the surface of IL-rGO, which meant the enrichment of the signal and the more immobilization of label antibody. Finally, the catalytic reaction between H₂O₂ and the IL-rGO-Au-PDDA-PB nanocomposite further enhanced the signal response. The signals increased linearly with AFP concentrations in the range of 0.01–100 ng mL⁻¹. The detection limit for AFP was 4.6 pg mL⁻¹. The immunosensor showed high sensitivity, excellent selectivity and good stability. Moreover, the immunosensor was applied to the analysis of AFP in serum sample with satisfactory result.     

Lectin affinity electrophoresis of alpha-fetoprotein. Increased specificity and sensitivity as a marker of hepatocellular carcinoma.

alpha-Fetoprotein (AFP) is widely used as a marker of hepatocellular carcinoma (HCC) for assisting diagnosis and also for screening purposes, even though its sensitivity has been decreased slightly as a result of the earlier detection of HCC by ultrasonography. Using lectin-dependent fractionation of AFP, the diagnostic sensitivity as well as the specificity of AFP can be increased compared with measurement of total AFP. Furthermore, lectin-reactive forms of AFP, AFP-L3 and AFP-P4, have been shown to serve as preclinical markers of HCC. Accordingly, AFP is still the most reliable marker of HCC in screening and monitoring high-risk patients.     

New Insights on Potentiometric Immunosensor at Carbon Fiber Microelectrode for Alpha-Fetoprotein in Hepatocellular Carcinoma

Herein, we investigated the analytical features of potentiometric immunosensors for detection of alpha-fetoprotein (AFP) in hepatocellular carcinoma at different electrodes, such as carbon fiber microelectrode (CFME) and carbon-disk electrode (CDE), respectively. To construct such an immunosensor, anti-AFP capture antibodies were first conjugated covalently onto the activated electrodes through typical carbodiimide coupling. Thereafter, one-step immunoreaction protocol was successfully introduced to develop a new potentiometric immunoassay upon addition of AFP. Accompanying the antigen-antibody reaction, the surface charges of the modified electrodes were changed for the readout of electric potential. Results indicated that the linear range of CDE-based immunosensor was 0.1–100 ng mL⁻¹ AFP, whereas the assay sensitivity by using CFME could be further increased to 3.2 pg mL⁻¹ with the linear range from 0.01 to 500 ng mL⁻¹ AFP. Meanwhile, CFME-based immunosensor showed high sensitivity, good reproducibility and specificity, and could be utilized for the analysis of human serum specimens with consistent results relative to commercialized ELISA kit.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Metal sulfide quantum dots-aggregated PAMAM dendrimer for cadmium ion-selective electrode-based immunoassay of alpha-fetoprotein

An ion-selective electrode(ISE)-based immunoassay has been innovatively designed for the sensitive detection of liver cancer biomarker(alpha-fetoprotein,AFP),using metal sulfide quantum dot(QD)-based nano labels.Cd S QDs-aggregated PAMAM dendrimer(QD-DE)was first synthesized and functionalized with polyclonal rabbit anti-human AFP antibodies.Thereafter,a sandwich immunoreaction was implemented on monoclonal mouse anti-human AFP antibody-coated microplate by using antibody-functionalized QD-DE as the secondary antibody.Accompanying the immunocomplex,subsequent potentiometric detection of cadmium ion dissolved from the QD-DE under acidic condition was conducted on a portable cadmium ion-selective electrode(Cd-ISE).Results revealed that the electrode potential of the Cd-ISE increased with the increment of AFP concentration from 0.1 to 100 ng m L~(-1)at a detection limit(LOD)of 68 pg m L~(-1).The relative standard deviations(RSD)were below9.09%and 10.54%for the intra-and inter-assay,respectively.Additionally,six human serum specimens were determined on CdISE-based immunosensor by using commercial human AFP ELISA kit as the reference,and gave good relationship between two methods.Importantly,Cd-ISE-based immunoassay offers the promise for simple and cost-effective screening of disease-related biomarkers.     

纳米金与磁性纳米复合物构建电流型免疫传感器的研究

制备了易于磁性分离、硫堇(Thi)包覆的四氧化三铁(Fe3O4)纳米复合物。通过静电吸附作用,将萘酚(Nafion)、Thi包覆的Fe3O4复合纳米粒子层层修饰到玻碳电极表面,再利用Thi分子中的氨基吸附纳米金,最后固载甲胎蛋白抗体,从而制得灵敏度高、稳定性好的无试剂电流型甲胎蛋白免疫传感器。实验通过透射电子显微镜(TEM)对该复合纳米粒子进行表征,并用循环伏安法考察了电极的电化学特性。结果表明,Fe3O4/Thi复合纳米粒子修饰的电极在实验过程中呈现出良好的氧化还原活性,其检测范围为0.05～20μg/L,检出限为0.03μg/L。     

Simultaneous multianalysis for tumor markers by antibody fragments microarray system

It is often necessary to measure a spectrum of tumor markers in oncology. We have developed a simultaneous multiplexing immunoassay method to determine six tumor markers: -fetoprotein (AFP), carcinoma embryonic antigen (CEA), beta-human chorionic gonadotropin (β-HCG), carbohydrate antigen 125 (CA 125), carbohydrate antigen 19-9 (CA 19-9) and carbohydrate antigen 15-3 (CA 15-3). F(ab′)₂ fragments of six capture antibodies were prepared and printed as microarrays on silylated slides to perform sandwich immunoassays with the use of an avidin–biotin system for amplified fluorescence signals. Each antigen with different concentrations was detected to assemble its calibration curve, and combinations of different markers were determined to examine the specificity of simultaneous detection based on the F(ab′)₂ microarrays. Some clinical samples were analyzed to compare with results obtained with the use of immunoradiometric assay (IRMA) method. Wide range calibration curve and its R-value were obtained for each analyte. Calibration curves concentrations were 0–640 μg/l for CEA, AFP and β-HCG, and 0–1280 kU/l for CA 125, CA 19-9 and CA 15-3. The antibody fragments microarray system bears comparison with conventional immunoassays and may find feasible application in measurement of series markers in oncology and other areas of medicine.     

磁共振扩散加权成像联合血清AFP对小肝癌临床诊断及疗效评估的价值

将194例小肝癌患者纳入小肝癌组,185例肝细胞结节患者纳入结节组,所有患者均行MRI扩散加权成像(DWI-MRI),评估患者在DWI-MRI上的形态学表现及ADC值,检测患者血清AFP水平,比较不同检测方法诊断小肝癌的准确度、灵敏度、特异度。结果显示,应用DWI-MRI联合血清AFP诊断小肝癌的特异度、灵敏度和准确度均显著高于单独应用DWI-MRI,或血清AFP(P<0.05)。表明DWI-MRI联合血清AFP对小肝癌的诊断价值显著,可为病情评估提供切实可行的参考。     

FeS2−AuNPs Nanocomposite as Mimicking Enzyme for Constructing Signal‐off Sandwich‐type Electrochemical Immunosensor Based on Electroactive Nickel Hexacyanoferrate as Matrix

In this work, a novel sandwich‐type electrochemical immunosensor with electroactive nickel hexacyanoferrate nanoparticles (NiHCFNPs) as matrix was constructed for α‐fetoprotein (AFP) detection in a signal‐off manner by using FeS₂?AuNPs nanocomposite catalyzed insoluble precipitation to significantly inhibit the electrochemical signal. Initially, the NiHCFNPs with excellent electrochemical property was modified on the electrodeposited nano‐Au electrode to obtain a strong initial electrochemical signal. Subsequently, another nano‐Au layer was formed for immobilization of capture antibody (Ab₁). In the presence of target AFP, the prepared FeS₂?AuNPs‐Ab₂ bioconjugate could be specifically recognized and immobilized on electrode through the sandwich‐type immunoreaction. The FeS₂ with large specific surface areas were used as scaffolds to load abundant mimicking enzyme AuNPs. With the help of hydrogen peroxide (H₂O₂), FeS₂?AuNPs with peroxidase‐like activity accelerated the 4‐chloro‐1‐naphthol (4‐CN) oxidation with generation of insoluble precipitation on electrode, which would greatly hinder the electron transfer and thus caused the decrease of electrochemical signal for quantitative determination of AFP. This approach achieved a wide dynamic linear range from 0.0001 to 100 ng mL^?1 with an ultralow limit detection of 0.028 pg mL^?1. Especially, the proposed AFP immunosensor can be applied to detect human serum samples with satisfactory results, indicating a potential application in clinical monitoring of tumor biomarkers.     

Electrochemical immunoassay for α-fetoprotein through a phenylboronic acid monolayer on gold

A novel reusable electrochemical immunosensor for α-fetoprotein (AFP) based on phenylboronic acid monolayer on gold was proposed. The sensor was fabricated by immobilizing of 3-aminophenylboronic acid (APBA) conjugated thiol-mixed monolayer on gold through 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) as linkage. AFP and enzyme-conjugated antibody were further trapped to the modified electrode surface through sugar-boronic acid and immunoaffinity interactions, respectively. The attached enzyme-conjugated antibody on the electrode surface could catalyze the reduction of hydrogen peroxide in the presence of thionine, which can be used to detect AFP in human serum by a competitive mechanism. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays were used to probe the assembly and regeneration process of the immunosensor. The influences of the competitive ratio of antigen and antibody, pH value of the measuring solution, incubation temperature and time were explored for optimizing the analytical performance. The whole assay process including incubation, detection and regeneration of the electrode could be completed in 35 min. The detection of AFP in five serum samples provided from clinically diagnosed patients with liver cancer showed acceptable accuracy. The proposed immunosensor enabled fast, low-cost and would be valuable for clinical diagnosis.     

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H₂O₂ catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL⁻¹ with a detection limit of 0.25 ng mL⁻¹. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.     

酶聚合体电流信号放大多通道电化学免疫传感体系及蛋白肿瘤标志物检测

本文构建了一种基于酶聚合体信号放大的多通道电化学免疫传感体系,并用于肝癌肿瘤标记物甲胎蛋白（AFP）的定量检测. 该传感体系由固定了抗AFP鼠单克隆抗体的多通道丝网印刷电极组成,可捕获肿瘤标记物抗原AFP,进而与抗AFP兔多抗特异性结合形成夹心免疫复合物,然后利用辣根过氧化物酶聚合体偶联的羊抗兔二抗（IgG-polyHRP）与三明治夹心免疫复合物结合,实现电流信号放大. 该体系结合多通道丝网印刷电极及自主研发的多通道电化学检测仪,可同时满足多通道电流信号的检测. 在最优化条件下,该传感体系检测AFP浓度的动态范围为64 pg·mL^-1 ~ 250 ng·mL^-1,最低检测下限为56 pg·mL^-1,具有检测灵敏度高、特异性强、操作简便以及仪器便携等优点.     

Selective response of antigen‐antibody reactions on chiral surfaces modified with 1,2‐diphenylethylenediamine enantiomers

This work reported a comparative analysis of the amperometric responses of antigen‐antibody reactions on two stable chiral surfaces which were modified with 1,2‐diphenylethylenediamine enantiomers. Alpha‐fetoprotein antibody and antigen (anti‐AFP and AFP) were selected as model systems. First, (1R,2R)‐1,2‐diphenylethylenediamine or (1S,2S)‐1,2‐diphenylethylenediamine was modified on the gold surface of the electrode through amide linkage to construct chiral surfaces. Then, anti‐AFP was immobilized on the chiral electrode surface by electrostatic and hydrogen bonding interactions. The electrochemical characteristics of the modified electrodes were studied via cyclic voltammetry. The selective current responses of antigen‐antibody reactions on chiral electrode surfaces for different incubation time and varying AFP concentrations were monitored. The antigen‐antibody reactions were greatly influenced by the chirality of 1,2‐diphenylethylenediamine enantiomers, and the amperometric responses obtained from the (1S,2S)‐1,2‐diphenylethylenediamine modified electrode was obviously stronger than that from the (1R,2R)‐1,2‐diphenylethylenediamine modified electrode. Such work may not only offer valuable reference to the research of chiral drugs, but also help to comprehend the high selectivity of chiral molecular species in biosystems. Copyright © 2011 John Wiley & Sons, Ltd.     

The sandwich-type electrochemiluminescence immunosensor for α-fetoprotein based on enrichment by Fe3O4-Au magnetic nano probes and signal amplification by CdS-Au composite nanoparticles labeled anti-AFP

A novel and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor was fabricated on a glassy carbon electrode (GCE) for ultra trace levels of α-fetoprotein (AFP) based on sandwich immunoreaction strategy by enrichment using magnetic capture probes and quantum dots coated with Au shell (CdS-Au) as the signal tag. The capture probe was prepared by immobilizing the primary antibody of AFP (Ab1) on the core/shell Fe₃O₄-Au nanoparticles, which was first employed to capture AFP antigens to form Fe₃O₄-Au/Ab1/AFP complex from the serum after incubation. The product can be separated from the background solution through the magnetic separation. Then the CdS-Au labeled secondary antibody (Ab2) as signal tag (CdS-Au/Ab2) was conjugated successfully with Fe₃O₄-Au/Ab1/AFP complex to form a sandwich-type immunocomplex (Fe₃O₄-Au/Ab1/AFP/Ab2/CdS-Au), which can be further separated by an external magnetic field and produce ECL signals at a fixed voltage. The signal was proportional to a certain concentration range of AFP for quantification. Thus, an easy-to-use immunosensor with magnetic probes and a quantum dots signal tag was obtained. The immunosensor performed at a level of high sensitivity and a broad concentration range for AFP between 0.0005 and 5.0 ng mL⁻¹ with a detection limit of 0.2 pg mL⁻¹. The use of magnetic probes was combined with pre-concentration and separation for trace levels of tumor markers in the serum. Due to the amplification of the signal tag, the immunosensor is highly sensitive, which can offer great promise for rapid, simple, selective and cost-effective detection of effective biomonitoring for clinical application.