Aptameric system for the highly selective and ultrasensitive detection of protein in human serum based on non-stripping gold nanoparticles

A novel approach is proposed in this study for the development of an aptameric assay system for protein based on non-stripping gold nanoparticles (NPs)-triggered chemiluminescence (CL) upon target binding. The strategy chiefly depends on the formation of a sandwich-type immunocomplex among the capture antibody immobilized on the polystyrene microwells, target protein and aptamer-functionalized gold NPs. Introduction of target protein into the assay system leads to the attachment of gold NPs onto the surface of the microwells and thus the assembled gold NPs could trigger the reaction between luminol and AgNO(3) with a CL emission. Further signal amplification was achieved by a simple gold metal catalytic deposition onto the gold NPs. Such an amplified CL transduction allowed for the detection of model target IgE down to the 50 fM, which is better than most existing aptameric methods for IgE detection. This new protocol also provided a good capability in discriminating IgE from nontarget proteins such as IgG, IgA, IgM and interferon. The practical application of the proposed gold NPs-based immunoassay was successfully carried out for the determination of IgE in 35 human serum samples. Overall, the proposed assay system exhibits excellent analytical characteristics (e.g., a detection limit on the attomolar scale and a linear dynamic range of 4 orders of magnitude), and it is also straightforward to adapt this strategy to detect a spectrum of other proteins by using different aptamers. This new CL strategy might create a novel technology for developing simple biosensors in the sensitive and selective detection of target protein in a variety of clinical, environmental and biodefense applications.     

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。     

Gold nanoparticles catalyzed chemiluminescence immunoassay for detection of herbicide 2,4-dichlorophenoxyacetic acid

We present a novel immunoassay format utilizing the catalytic properties of gold nanoparticles in the luminol-silver nitrate-gold nanoparticle based chemiluminescence (CL) system for the detection of widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Highly sensitive anti-2,4-D antibody was produced and conjugated with gold nanoparticles of various sizes. In the present assay format, employing a competitive inhibition approach, a well-characterized hapten-protein conjugate (2,4-D-BSA) was used to coat the microtiter plates. The analyte (2,4-D) was pre-incubated with anti-2,4-D antibody labeled with gold nanoparticles and added to each well of the microtiter plate. The gold label triggered the reaction between luminol and silver nitrate generating a luminescence signal at 425 nm. Under the optimized conditions, the CL based immunoassay showed the detection limit of 2,4-D in standard water samples around 3 ng mL(-1). The CL based immunoassay format, based on gold nanoparticles as a catalyst, could be used as a fast screening methodology (<30 min) for pesticide detection.     

一种基于纳米金介导生物沉积铂催化氢还原的电化学免疫分析新方法

本文发展了一种基于纳米金介导生物沉积铂并以铂催化氢还原伏安法进行检测的高灵敏电化学免疫分析新方法。该方法采用夹心免疫分析模式,实现了人免疫球蛋白（HIgG）的测定。首先在聚苯乙烯微孔板中固定羊抗HIgG捕获抗体,HIgG捕获后,碱性磷酸酶标记的HIgG抗体修饰的纳米金探针通过与HIgG的形成的夹心复合物而结合在微孔板上。结合的碱性磷酸酶催化抗坏血酸磷酸酯底物水解产生抗坏血酸,后者在纳米金上介导下还原铂离子沉积于纳米金表面。沉积的金属铂用王水溶解并电富集于玻碳电极上。通过测定铂催化氢还原产生的阴极电流,可实现HIgG的高灵敏分析。催化氢还原电流与HIgG浓度对数在0.1~100ng/ml之间呈线性相关性,检测限达22pg/ml。由于铂催化氢还原的高灵敏度及纳米金介导的生物沉积放大反应,该法具有较高的分析灵敏度,且免疫分析微孔板模式使得该法可同时用于大量样品的分析。     

Spectral and fluorescent kinetics features of Nd3+ ion in Nb2O5, Ta2O5 and La2O3 mixed lithium zirconium silicate glasses

A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L⁻¹ HCl–0.10 mol L⁻¹ NaCl solution containing 0.10 mmol L⁻¹ Br₂. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL⁻¹ to 40 ng mL⁻¹ with a detection limit of 5.2 × 10⁻¹⁰ g mL⁻¹. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.     

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).     

Copper-enhanced gold nanoparticle tags for electrochemical stripping detection of human IgG

We demonstrate herein a novel electrochemical protocol for quantification of human IgG based on the precipitation of copper on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved copper. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on the wells of microtiter plates. The human IgG analyte was first captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. After dissolved with HNO₃, the released copper ions were then quantified by ASV. The detection limit is 0.5 ng/mL by 3σ-rule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were also examined by the method. To our knowledge, the copper enhancing procedure is the first time to be developed for immunoassay. The new strategy of using copper-enhanced gold nanoparticle tags for electrochemical stripping detection holds great promise for immunoassay and DNA detection.     

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.     

化学发光底物分辨双组分免疫分析法测定胶质瘤标志物NSE和CA15-3

本文建立了一种基于辣根过氧化物酶(HRP)和碱性磷酸酯酶(ALP)化学发光底物分辨的双组分免疫分析新技术,用以检测人胶质瘤血清标志物神经元特异性烯醇化酶(NSE)和糖链抗原15-3(CA15-3).实验详细考察了捕获抗体、检测抗体、HRP和ALP酶标记物的用量,结果发现NSE和CA15-3的线性范围分别为0.5～20ng/mL(R20.99)和0.5～20U/mL(R20.99),最低检出限分别为0.2ng/mL和0.2U/mL;高、中和低三个浓度的血清加样回收率良好;天内和天间相对标准偏差均小于10%;且一份血清,两组分同时检测无交叉干扰.综合而言:本法能够一次实验,高灵敏、高特异地同时检测两种疾病标志物,具有血清用量少、检测时间短、操作简单、结果可靠的优点,有望为胶质瘤的临床早期诊断提供坚实的支持.     

Homogeneous fluorescence-based immunoassay via inner filter effect of gold nanoparticles on fluorescence of CdTe quantum dots

Homogeneous immunoassays are becoming more and more attractive for modern medical diagnosis because they are superior to heterogeneous immunoassays in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. This assay relies upon the inner filter effect (IFE) of gold nanoparticles (AuNPs) on CdTe QDs fluorescence. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the IFE of AuNPs on CdTe QDs fluorescence is greatly enhanced, resulting in a decrease of fluorescence intensity in the system. Based on this phenomenon, a wide dynamic range of 1-100 pg mL(-1) for determination of IgG can be obtained. The proposed method shows a detection limit of 0.3 pg mL(-1) for human IgG, which is much lower than the corresponding absorbance-based approach and compares favorably with other reported fluorescent methods. This immunoassay method is simple, rapid, cheap, and sensitive. The proposed method has been successfully applied to measuring IgG in serum samples, and the obtained results agreed well with those of the enzyme-linked immunosorbent assay (ELISA).     

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.     

Gold(III) enhanced chemiluminescence immunoassay for detection of antibody against ApxIV of Actinobacillus pleuropneumoniae

We report, for the first time, a chemiluminescence immunoassay (CLIA) method based on AuCl(4)(-)-enhanced luminol chemiluminescence (CL) reaction for the highly sensitive detection of ApxIV antibody of Actinobacillus pleuropneumoniae (APP). The AuCl(4)(-), which was the dissolution product of the gold nanoparticle-rabbit anti-pig IgG conjugate, served as an analyte in the CL reaction for the indirect measurement of antibody against ApxIV. The optimal condition of gold dissolution was composed of a 5.0 x 10(-2) M HCl, 1.5 x 10(-2) M NaCl, and 2.5 x 10(-4) M Br(2) solution. Under the optimal conditions, a good correlation between the relative CL photon counting and the dilution coefficient of serum was obtained in the dilution range of 1:160-1:40 000. Based on the analysis of clinical samples, the results indicated that CLIA had remarkable advantages in terms of reliability and practical use compared with indirect hemagglutination (IHA) and enzyme-linked immunosorbent assays (ELISA). The proposed method provided a new tool for the indirect determination of antibody against ApxIV in pig serum samples and showed great potential for numerous applications in immunoassays.     

Determination of human IgG by solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing Rhodamine 6G luminescent molecules

Luminescent silicon dioxide nanoparticles (R-SiO2) with size of 50 nm containing Rhodamine 6G (R) were synthesized by sol-gel method. In the presence of Pb(Ac)2 as a heavy atom perturber, the particle can emit intense and stable room temperature phosphorescence signal of R, respectively, on polyamide membrane, with the lambda(ex)(max)/lambda(em)(max) = 470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO2 and human IgG can be carried on polyamide membrane quantitatively, and the phosphorescence intensity was enhanced after the immunoreactions. Thus, a new method of solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for the determination of human IgG was established basing on antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624-20.0 pg spot(-1) of human IgG (corresponding concentration, 0.156-50.0 ng mL(-1); sample volume, 0.40 microL spot(-1)). The regression equations of working curves are delta I(p) = 88.16. + 16.79 m(IgG) (pg spot(-1)) (485/646 nm, r = 0.9997). Detection limits calculated by 3Sb/k are 0.017 pg spot(-1). For samples containing 0.156 and 50.0 ng mL(-1) of IgG, we measured repeatedly for 11 times, RSDs are 3.9 and 2.8%, respectively. This method is sensitive, accurate and of high precision.     

Antibody-biotemplated HgS nanoparticles: extremely sensitive labels for atomic fluorescence spectrometric immunoassay

In this work, antibody goat anti-human IgG as a scaffold was employed for the synthesis and biofunctionalization of HgS nanoparticles (NPs) via a facile one-pot process. After a complete sandwich-type immunoreaction among primary antibody, human IgG and secondary antibody labeled with HgS NPs, a large number of mercury ions released from captured HgS NPs dissolution were quantitatively detected by chemical vapor generation atomic fluorescence spectrometry (CVG-AFS). Taking advantage of the signal amplification property of HgS NPs and the high sensitivity of CVG-AFS, the assay detected human IgG with a limit of detection (S/N = 3) of 0.6 ng mL(-1) (4.0 fmol mL(-1) or 0.4 fmol) and the response was linear over a dynamic range from 1.0 to 5.0 × 10(4) ng mL(-1) with a correlation coefficient of 0.996. A relative standard deviation (RSD) of 1.0 × 10(2) ng mL(-1) human IgG was 1.5% for within-batch (intra-assay) and 4.5% for between-batch (inter-assay). Other proteins, such as goat anti-rabbit IgG, goat anti-human IgG, rabbit anti-human IgG, carcinoembryonic (CEA), α-fetoprotein (AFP), human serum albumin (HSA) and bovine serum albumin (BSA) did not significantly interfere with the assay for human IgG. The analytical result of HgS NPs with AFS-based immunoassay technology for the quantification of human IgG in human serum from patients is in good agreement with the result obtained by conventional immunoturbidimetric method. The consequence shows that the novel immunosensor possessed satisfactory precision, extremely high sensitivity, high selectivity and could be applied for the quantification analysis of real samples.     

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.     

以pH敏感相分离高分子为载体的酶联荧光免疫分析法测定人IgG

p H敏感高分子因其独特的 p H敏感性质而在药物的控制释放 [1,2 ] 、分子分离 [3 ] 以及生物传感器 [4 ]等方面得到应用 .应用于免疫分析时 ,要求 p H敏感高分子在溶液 p H 7.4左右波动时做出响应 ,过多偏离生理 p H会对免疫反应生成的抗原 -抗体复合物造成不同程度的破坏 .目前 ,p H敏感高分子并未在免疫分析中广泛应用 ,这主要是由于高分子的相转变 p H大多在 4或 1 0左右[5,6] .我们 [7] 曾合成了3 7℃下相转变 p H在 5 .6左右的 p H敏感高分子 ,并将其作为免疫反应载体 ,建立了乙肝表面抗原的分析系统 ,虽然其相转变 p H比以往的高…     

基于金纳米微粒的化学发光金属免疫分析

建立了基于金纳米微粒溶解的化学发光反应体系,并探讨了金纳米微粒溶解及化学发光测定的最佳条件。首次将金纳米微粒引入生物素和IgG的化学发光金属免疫分析,比较了不同粒径金纳米微粒、不同检测系统对IgG测定的影响。在(一抗-IgG-二抗修饰金纳米微粒)检测系统中,基于10 nm和30 nm金纳米微粒测定IgG的线性范围分别为1~75 ng和0.5~25 ng,检出限分别为0.5 ng和0.1 ng。在(一抗-IgG-生物素化抗体-链霉亲和素修饰金纳米微粒)检测系统中,5 nm和10 nm金纳米微粒测定IgG的线性范围分别为10~250 ng和1~250 ng;检出限分别为5 ng和1 ng。     

Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification

A novel trace tag for chemiluminescent (CL) immunoassay was designed by using DNAzyme to functionalize antibody-labeled Au nanoparticles (AuNPs). The trace tag showed an excellent ability to catalyze the oxidation of luminol by hydrogen peroxide, leading to strong CL emission. By coupling the trace tag with a passive mixing accelerated immunoreaction system, a highly sensitive rapid flow-through CL immunoassay method was proposed. Using carcinoembryonic antigen (CEA) as a model analyte, the capture antibody for CEA was immobilized on paramagnetic microspheres, and DNAzyme-anti-CEA antibody functionalized AuNPs were prepared as trace tag. A three-dimensional helical glass tube kept at 37 °C in a water bath was used for passively mixing immunoreagents in a two-step sandwich immunoassay, with which each immunoreaction step could be finished within 150 s. With the help of a magnet, the immunocomplex could conveniently be separated from reactants. Compared with the horseradish peroxidase-based tag, the newly designed trace tag showed obvious signal amplification due to its strong catalytic ability and high loading ratio of DNAzyme on each AuNP. The proposed method showed a linear calibration range from 0.005 to 0.5 ng mL(-1) for CEA detection with a detection limit of 4.1 pg mL(-1) at a signal-to-noise ratio of 3 and acceptable detection reproducibility. The assay results of clinical serum samples were in acceptable agreement with the reference values. The designed immunoassay system with ultrahigh sensitivity provided a programmable and low-cost approach for high-throughput clinical application.     

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).     

基于pH敏感相分离技术的新型荧光免疫测定体系

In this paper, it was discovered that a novel pH-sensitive copolymer of N-isopropylacrylamide (NIP) and N-(3-dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pHtr) of P(NIP-DMAPM) polymer was found to be 7.4 at 37℃. The polymer was precipitated out of water above a critical pH=7.4 and re-dissolved below pH----7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP-DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pHtr of polymer to precipitate the polymer-immune complex,and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the FITC-labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100-1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pHtr of P(NIP-DMAPM), the damage to antigen-antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme-linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH-sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.