基于电化学聚合固定抗体的电化学发光免疫分析法检测人免疫球蛋白

基于电化学聚合在金电极表面固定兔抗人免疫球蛋白G抗体与人免疫球蛋白G及标记有Ru(bpy)2+3 的羊抗人免疫球蛋白G抗体之间发生特异性免疫反应,形成三明治结构,成功建立了用于测定人血清中免疫球蛋白G的电化学发光(ECL)免疫技术.利用此方法测定人免疫球蛋白G含量,浓度在50 μg/L～2 mg/L范围内与电化学发光强度呈良好的线性关系,线性回归方程为y(a. u.)=48.41+0.09x(μg/L) (n=7);检出限为20 μg/L (3σ).测得正常人血清中免疫球蛋白G平均含量为11.2 g/L ,结果令人满意.     

基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

Spectrophotometric Determination of Human Immunoglobulin G Based on Enlargement of Gold Nanoparticles

Based on the sandwich immunoreaction and enlargement of colloidal gold, a simple spectrophotometric determination of human immunoglobulin G (IgG) was developed. The sandwich immunoreaction among the goat‐anti‐human IgG, the human IgG and goat‐anti‐human IgG labeled with colloidal gold was completed on the surface of 96 well clear polystyrene high bind stripwell(tm) microplate. The immobilized colloidal gold was enlarged by the reaction of 0.01% HAuCl₄ with 0.4 mmol·L^?1 NH₂OH·HCl. A common UV‐Vis spectrophotometer was employed to measure the absorption of the colloidal gold enlarged. The absorption signal of colloidal gold is proportional to the logarithm of human IgG concentration ranging from 5.0×10^?9 to 2.0×10^?5 g·mL^?1, which is comparable with those obtained by other immunoassays. In the proposed method, the immobilized colloidal gold can be easily enlarged and a new absorption band is present at about 620 nm, which makes the signal be recorded with a common medical analyzer. This spectrophotometric determination can be made in a common laboratory without the well‐trained technician and the special equipment.     

Sandwich-type electrochemiluminescence immunosensor based on N-(aminobutyl)-N-ethylisoluminol labeling and gold nanoparticle amplification

A novel electrochemiluminescence (ECL) sandwich-type immunosensor for human immunoglobulin G (hIgG) on a gold nanoparticle modified electrode was developed by using N-(aminobutyl)-N-ethylisoluminol (ABEI) labeling. The primary antibody, goat-anti-human IgG was first immobilized on a gold nanoparticle modified electrode, then the antigen (human IgG) and the ABEI-labeled second antibody was conjugated successively to form a sandwich-type immunocomplex. ECL was carried out with a double-step potential in carbonate buffer solution (CBS) containing 1.5 mM H₂O₂. The ECL intensity increased linearly with the concentration of hIgG over the range 5.0-100 ng/mL. The limit of detection was 1.68 ng/mL (S/N = 3). The relative standard deviation was 3.79% at 60 ng/mL (n = 9). The present immunosensor is simple and sensitive. It has been successfully applied to the detection of hIgG in human serums.     

基于酪胺信号放大的新型免疫传感器

将酪胺应用于酶联免疫分析,建立了一种新的高灵敏伏安型免疫传感器。利用纳米金的静电吸咐和己二硫醇、巯基乙胺的自组装,将羊抗人IgG抗体固定到金电极表面上,以辣根过氧化物酶标记羊抗人IgG抗体为酶标抗体,以生物素化酪胺为酶底物,利用催化酪胺沉积反应,在传感界面沉积大量生物素,使原始信号得到几何级数的放大。结果表明,通过生物素化酪胺催化放大后,制得的免疫传感器对H2O2的催化能力增大近20倍,检测hIgG在1.5μg/L～22 mg/L范围内有良好的线性关系,检出限为0.1μg/L。用于实际试样的回收率的测定,结果良好。     

Ultrasensitive Immunoassay Based on Amplified Inhibition of the Electrochemical Stripping Signal of Silver Nanocomposite by Silica Nanoprobe

A silver nanocomposite was one‐step synthesized in chitosan solution and used to prepare an immunosensor with the aid of gold nanoparticles (Au NPs) assembly. The Ag NPs at the immunosensor exhibited sensitive electrochemical stripping signal in KCl solution. After a sandwich immunoreaction, the current response of the immunosensor decreased due to the formation of antibody‐antigen immunocomplex on its surface, which was greatly amplified by the captured silica nanoprobes and thus enabled an ultrasensitive electrochemical immunoassay method. This method showed excellent analytical performance for human IgG measurement including wide linear range, low detection limit, cheap cost, satisfactory reproducibility and stability.     

A novel immunoassay based on the dissociation of immunocomplex and fluorescence quenching by gold nanoparticles

This study reports a novel, simple and sensitive immunoassay using fluorescence quenching caused by gold nanoparticles coated with antibody. The method is based on a non-competitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by antibody labeled with gold nanoparticles. The sandwich-type immunocomplex was subsequently dissociated by the mixed solution of sodium hydroxide and trisodium citrate, the solution obtained, which contains gold nanoparticles coated with antibody, was used to quench fluorescence. The fluorescence intensity of fluorescein at 517 nm was inversely proportional to the logarithm of the concentration of human IgG in the dynamic range of 10-5000 ng mL⁻¹ with a detection limit of 4.7 ng mL⁻¹. The electrochemical experiments and the UV-vis measurements were applied to demonstrate whether the immunoglod was dissociated completely and whether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigen and DNA strands, and has broad potential applications in disease diagnosis.     

纳米金免疫凝集比率光度法检测IgG

利用柠檬酸钠还原法制备了13nm胶体金,透射电子显微镜表征其具有良好的单分散性,通过它与牛血清蛋白(BSA)作用的紫外-可见光谱表明其对蛋白具有良好的生物亲和性。以羊抗人免疫球蛋白G(IgG)修饰的金纳米粒子作探针,基于金纳米粒子免疫聚集导致其消光系数和分散度的变化建立了人IgG的比率光度分析方法。结果表明所制备的纳米金标记探针在人IgG浓度100ng/mL～100μg/mL范围内有良好的线性响应,加标法测定结果表明该法具有良好的回收率和精密度。以细胞色素C(CytoC)和辣根过氧化物酶(HRP)两种蛋白质作对照实验,发现所制备的金纳米探针对IgG具有高度的特异性。     

A sensitive immunosensor using colloidal gold as electrochemical label

A sensitive immunosensor using colloidal gold as electrochemical label is described. In this method, the capture protein was first immobilized on a carbon paste electrode surface through passive adsorption to bind quantitatively with corresponding antigen and colloidal gold labeled antibody to perform a sandwich assay. To detect the amount of the colloidal gold captured on the electrode surface, the colloid was first oxidized electrochemically to produce AuCl₄⁻ ions which were adsorbed strongly on the electrode surface. Adsorptive voltammetry was then employed for the determination of the adsorbed AuCl₄⁻ ions. A linear relationship between reduction wave peak current and the antigen concentration (human IgG) from 10 to 500 ng/ml is obtained with a detection limit of 4.0 ng/ml.     

Homogeneous immunoassay based on aggregation of antibody-functionalized gold nanoparticles coupled with light scattering detection

A universal platform of homogeneous noncompetitive immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. The assay is based on aggregation of antibody-functionalized gold nanoparticles directed by the immunoreaction coupled with light scattering detection with a common spectrofluorimeter. In phosphate buffer (pH 7.0) solution, the light scattering intensity of the gold nanoparticles functionalized with goat-anti-human IgG can be greatly enhanced by addition of the human IgG. Based on this phenomenon, a wide dynamic range of 0.05-10 microg ml(-1) for determination of human IgG can be obtained, and the detection limit can reach 10 ng ml(-1). The proposed immunoassay can be accomplished in a homogeneous solution with one-step operation within 10 min and has been successfully applied to the determination of human IgG in serum samples, in which the results are well consistent with those of the enzyme-linked immunosorbent assay (ELISA), indicating its high selectivity and practicality. Therefore, the gold nanoparticle-based light scattering method can be used as a model to establish the general methods for protein assay in the fields of molecular biology and clinical diagnostics.     

Enhancement of the sensitivity of surface plasmon resonance biosensor with colloidal gold labeling technique

In our experiment colloidal gold was used to enhance the signal obtained from the surface-plasmonresonance biosensor. Fab′ fragments of human IgG molecules were immobilized on the gold surface and reacted with the sheep-anti-human IgG(SAH-IgG) molecules in solution. It was found that when the colloidal gold labeling technique was introduced the sensitivity of surface plasmon resonance instruments increased by a factor of about 300.     

Gold nanoparticle-based immunoassay by using non-stripping chemiluminescence detection

A novel microplate-compatible chemiluminescence (CL) immunoassay has been developed for the determination of human immunoglobulin G (IgG) based on the luminol-AgNO(3)-gold nanoparticles CL system. Polystyrene microtiter plates were used for both immunoreactions and CL measurements. The primary antibody, goat-anti-human IgG, was first immobilized on polystyrene microwells. Then the antigen (human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex. The gold label could trigger the reaction between luminol and AgNO(3), accompanied by light emission. Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng mL(-1), with a detection limit of 12.8 ng mL(-1) ( approximately 80 pM) at a signal to noise ratio of three (S/N = 3). Compared with other reported CL immunoassay method based on gold labels, the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes, making the method more simple, time-saving and easily automated. The present CL method is promising for the determination of clinically important bioactive analytes.     

Organic-inorganic matrix for electrochemical immunoassay: Detection of human IgG based on ZnO/chitosan composite

A new strategy to construct amperometric immunosensor for human IgG assay based on ZnO/chitosan composite as sensing platform has been described. This material, which combined the advantages of inorganic species, ZnO and organic polymer, chitosan, can maintain biological activity well. A sequential sandwich immunoassay format was performed on the ZnO/chitosan composite supported by glass carbon electrode (GCE) using goat-anti-human IgG antibody (IgG Ab) and human IgG as a model system. Amperometry was used to determine the amount of horse-radish peroxidase (HRP) fixed on the sensor surface, which was related to the content of the desired human IgG. Assay conditions that were optimized included the amount of labeled antibody, the incubation time and temperature, the pH of the substrate solution, etc. Using hydroquinone as a mediator, amperometric detection at −150 mV (versus SCE) resulted in a detection range 2.5-500 ng mL⁻¹, with a detection limit of 1.2 ng mL⁻¹. The simple manipulations of the construction of ZnO/chitosan composite, as well as low-cost and broad linear range, are the main features of the proposed immunosensing method.     

A Simple,Selective and Sensitive Immunoassay for Determination of Human Chorionic Gonadotrophin Based on Chemiluminescence Resonance Energy Transfer

A simple, selective and sensitive immunoassay was developed for the determination of human chorionic gonadotrophin (HCG) based on the chemiluminescence resonance energy transfer (CRET). Two kinds of antibodies were used to form a sandwich‐type immunocomplex to ensure the selectivity of immunoassay. Based on the simple magnetic separation, amplification feature of gold nanoparticles and CRET phenomenon, this method could be used for highly sensitive determination of HCG without tedious washing steps. Under the optimized conditions, a linear range was obtained when the concentrations of HCG were changed from 0.15 to 5 mIU mL^?1 (R = 0.992). This immunoassay could be used for the determination of HCG in serum samples, which provided a promising potential in clinical diagnosis.     

Determination of anti-canine IgG using a continuous filtration/dissolution system based on the formation of a high-molecular size immunocomplex

A method for the determination of monoclonal antibody anti-canine-IgG based on a continuous filtration/dissolution system is presented as prototype for further developments. The basis of the system is the continuous formation of a high-molecular immunocomplex, which is temporally retained on a microfilter located prior to the detector. The immunochemical method consists of the development of a sandwich type heterogeneous non-competitive reaction to yield a high molecular immunocomplex, as a result of the affinity interaction between streptavidin and biotincanine IgG and the immunoreaction between canine IgG and mAb anti-canine IgG, which occurs in solution. Goat anti-mouse IgG labelled with peroxidase is used as tracer. The extension of the immunoreaction is monitored fluorimetrically via the condensation product between 4-hydroxyphenylacetic acid and hydrogen peroxide in the presence of the peroxidase retained on the filter. The method provides a dynamic range from 10(-4) to 500 mug l(-1) with an IC(50) of 0.554 mug 1(-1) (for a biotin-IgG dilution of 1:250, chi(2)=0.6085, r(2)=0.9991, n=14) and a precision, expressed as R.S.D.%, lower than 4.7%. After modifications, the method here proposed can be extended for monitoring analytes of interest in the agrochemical, food and environmental areas, as far as permitted by the availability to produce the corresponding monoclonal antibody.     

Electrochemical Metalloimmunoassay Based on Enlargement of Gold Nanoparticle Label Treated with Ag Enhancement System

A novel sensitive electrochemical immunoassay with colloidal gold as the antibody labeling tag and subse-quent signal amplification by silver enhancement is described. Colloidal gold was treated by a light-sensitive silver enhancement system which made silver deposit on the surface of colloidal gold(form Au/Ag core-shell structure), followed by the release of the metallic silver atoms anchored on the antibody by oxidative dissolu-tion of them in an acidic solution and the indirect determination of the dissolved Ag ions by anodic stripping voltamrnetry(ASV) at a carbon fiber microelectrode. The electrochemical signal is directly proportional to the amount of analyte(goat IgG) in the standard or a sample, The method was evaluated by means of a non-competitive heterogeneous immunoassay of immunoglobulin G(IgG) with a concentration as low as 0. 2 ng/mL. The high performance of the method is related to the sensitive ASV determination of silver( I ) at a car-bon fiber microelectrode and to the release of a large number of Ag^ ions from each silver shell anchored on the analyte (goat IgG).     

Investigation on the interaction between colloidal gold and human complement factor 4 at different pH by spectral methods

The interaction between colloidal gold and human complement factor 4 (human C4) at different pH was investigated by spectral methods, including absorption and resonance light-scattering spectrometry. According to the changes of color and absorption spectra of colloidal gold solution in presence of human C4, the interaction between colloidal gold and human C4 was quantitatively investigated using a semi-empirical "flocculation parameter". At the same time, the changes of resonance light-scattering spectra and transmission electron microscopy (TEM) images indicate that the aggregation of colloidal gold happens by electrostatic interaction in presence of human C4 in the pH range 5-6. However, the colloidal gold solution remains stable at pH >6 and pH <5 due to the repulsive electrostatic interaction between colloidal gold and human C4. The flocculation parameter is directly proportional to the concentration of human C4 in the range from 9.7 to 233.0 microgl(-1). In addition, the interactions between the colloidal gold and bovine serum albumin (BSA) as well as human serum albumin (HSA) were also investigated using the same methods. It was found that there was no aggregation of colloidal gold in presence of BSA and HSA in the pH range 5-6. However, when the pH of solution is 4, the aggregation of colloidal gold happens. Because BSA and HSA have different structure, the intensity of aggregation of colloidal gold in presence of BSA is greater than that in presence of HSA at pH 4.     

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles’ colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core–shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient’s sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.     

A novel electrochemical immunosensor based on colabeled silica nanoparticles for determination of total prostate specific antigen in human serum

A novel electrochemical immunosensor using functionalized silica nanoparticles (Si NPs) as protein tracer has been developed for the detection of prostate specific antigen (PSA) in human serum. The immunosensor was carried out based on a heterogeneous sandwich procedure. The PSA capture antibody was immobilized on the gold electrode via glutaraldehyde crosslink. After reaction with the antigen in human serum, Si NPs colabeled with detection antibody and alkaline phosphatase (ALP) was sandwiched to form the immunocomplex on the gold electrode. ALP carried by Si NPs convert nonelectroactive substrate into the reducing agent and the latter, in turn, reduce metal ions to form electroactive metallic product on the electrode. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for PSA. The parameters including the concentration of the ALP used to functionalize the Si NPs and the enzyme catalytic reaction time have been studied in detail and optimized. Under the optimum conditions of immunoreaction and electrochemical detection, the electrochemical immunosensor was able to realize a reliable determination of PSA in the range of 1–35 ng/mL with a detection limit of 0.76 ng/mL. For six human serum samples, the results performed with the electrochemical immunosensor were in good agreement with those obtained by chemiluminescent microparticle immunoassay (CMIA), indicating that the electrochemical immunosensor could satisfy the need of practical sample detection.     

Studies on the interaction of colloidal gold and serum albumins by spectral methods

The interactions of colloidal gold and serum albumins, including bovine serum albumin (BSA) and human serum albumin (HSA), were studied by fluorescence and absorption spectrometry. Fluorescence quenching spectrometry was applied to study the interactions between colloidal gold and serum albumins. At pH 7.4 phosphate-buffered saline (PBS), the intensity of fluorescence emission spectrum of serum albumins decreased in the presence of colloidal gold, which indicated that colloidal gold quenched the fluorescence of serum albumins. Experimental results indicated that the combination reactions of colloidal gold and serum albumins were static quenching processes. Based on the effect of colloidal gold on fluorescence intensity, the binding constants, the numbers of binding sites and the acting forces between colloidal gold and serum albumins were found.