Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural products

In this work, a micro-plate chemiluminescence enzyme immunoassay by antibody-coated for the determination of aflatoxin B1 (AFB1) in agricultural products has been established. Aflatoxin B1 antibody (AFB1-Ab) was adsorbed physically on polystyrene micro-plate hole as solid phase antibody, which took place immunity-reaction between antigen and antibody with AFB1 standard solution or samples by direct competition. Luminol-hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase (HRP) with p-iodophenol enhancement was used as signal detecting system. The effects of several factors, including composition and pH of coating solution, dilution ratio and amount of antibody and enzyme labeled antigen, time of antibody-coating, incubation and chemiluminescence reaction, and other relevant variables upon the immunoasaay were studied and optimized. The linear range of proposed method for AFB1 was 0.05-10.0 ng g⁻¹ with a correlative coefficient of −0.9997. The sensitivity of the proposed method was 0.01 ng g⁻¹. The RSDs of intra- and inter-assay were less than 12.2% and 10.0%, respectively. This method has been successfully applied to the evaluation of AFB1 in agricultural products with recoveries of 79.8%, 101.9% and 115.4% for low, middle and high concentration samples, respectively. It shows a good correlation with the commercial available ELISA kit for AFB1 with correlative coefficient of 0.9098 indicating that the established CLEIA method can be used to determine AFB1 in real samples.     

红细胞标记抗体的化学发光免疫分析

用红细胞代替辣根过氧化物酶作为双抗体夹心免疫分析中第二抗体的标记物, 建立了一种红细胞标记抗体的免疫化学发光测定乙型肝炎病毒表面抗原的新方法. 在免疫反应完成后, 结合了抗原-抗体免疫复合物的致敏红细胞在低渗溶液中溶血, 释放出血红蛋白. 基于血红蛋白对鲁米诺-H2O2体系化学发光具有催化作用的原理, 采用化学发光法测定血红蛋白含量. 测得的血红蛋白发光强度与待测抗原浓度呈线性关系. 采用这种方法可检测出0.5 ng/mL的乙型肝炎病毒表面抗原. 将该方法与酶联免疫吸附分析(ELISA)结合起来对乙型肝炎患者血清乙肝病毒表面抗原(HBsAg)进行检测, 两者符合率均为97%, 表明本法具有良好的灵敏度和特异性, 可用于临床标本测试.     

Sensitive detection of glycyrrhizin and evaluation of the affinity constants by a surface plasmon resonance-based immunosensor.

A sensitive and selective determination of glycyrrhizin (GC) based on surface plasmon resonance (SPR) was performed by using an anti-GC monoclonal antibody (GC-MAb) and GC-bovine serum albumin (GC-BSA) conjugate (antigen). GC-BSA was immobilized on an Au thin film of the SPR sensor chip by physical adsorption, and GC determinations were performed by an indirect competitive method. The addition of GC into the GC-MAb solution (5 microg/ml) was found to decrease the incident-angle shift sharply because of an inhibition effect of GC. The RSDs (n = 3) of each point were less than 4%. The lowest detection limit for GC by SPR was almost the same as that by ELISA, 60-75 ng/ml. An evaluation of the affinity constant between GC-MAb and GC using the data from ELISA and those from SPR measurements was performed. The values of the association constant (KA) from three different analyses of ELISA data and from SPR measurements are discussed in detail. As a whole, the affinity constant (KA) between GC-MAb and GC was on the order of 10(7) M(-1).     

Docking of B-cell epitope antigen to specific hepatitis B antibody

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.     

Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

Summary High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites. The adsorption properties of two different monoclonal anti-HSA antibodies immobilized on a chromatographic support were characterized by different approaches. The location of the epitope on the HSA molecule was determined by enzyme-linked immunoassay (ELISA) with albumin fragments. The chromatographic method was used to determine the column capacity and the adsorption rate constant of HSA on the immunoadsorbent. To compare the affinity of the antibodies for the antigen, an indirect ELISA method was used to determine the equilibrium constant of antigen-antibody association in solution Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th september, 1996.     

A Simple Method to Optimize Elution from Affinity Chromatography

Abstract

A rapid and convenient screening method to optimize elution from affinity chromatography is described. An immunoaffinity system employing antibody bound human thyroglobulin (hTg) served as a model. Anti - hTg was complexed to polystyrene microplates adsorbed hTg. Immune complex (IC) dissociation and elution of antigen by various agents was determined by enzyme linked immunosorbent assay (ELISA). The method permits simul taneous monitoring of residual antigenicity and of possible desorption of antigen from its solid support.     

Preparation of Anti—Cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

IntroductionIn fast and slow skeletal and cardiac muscles,troponin I,the inhibitory protein of the troponin-tropomyosin complex,exists in three isotype formsencoded by three separated genes.The amino acidsequences of the two skeletal and one cardiac Tn Iforms( s Tn I and c Tn I,respectively) exhibit40 %dissimilarity[1] .Moreover,human cardiac Tn I has31 additional residues on the N - terminal end,which do not exist in skeletal forms,thus it pro-vides a high potential for obtaining cardiac-…     

A flow-based enzyme-linked immunosorbent assay on a polydimethylsiloxane microchip for the rapid determination of immunoglobulin A

A flow-based enzyme-linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) microchip has been developed for the rapid determination of immunoglobulin A (IgA). The analytical principle of this integrated method is the same as the conventional sandwich-type ELISA. A primary antibody (anti-IgA) was adsorbed on the surface of a PDMS microchannel, and then an antigen (IgA) and a secondary antibody (anti-IgA HRP labeled) were reacted successively. The resulting antigen-antibody complex, fixed on the surface of the microchannel, was detected using Amplex^® Red and a fluorescent imaging system. The calibration curve of the IgA standard solution was linear in the range of 0-50 ng/mL at the flow rate of 10 μL/min. This flow rate corresponds to the reaction time of 4.8 s. Compared to the conventional assay on a 96-well microtiter plate, the present assay on the microchip dramatically shortened the reaction time necessary for the enzyme-substrate reaction from 30 min to 4.8 s, i.e., to 1/375. The amounts of the reagent and sample were also reduced to 1/100 compared to the 96-well microtiter plate.     

pH敏感相分离高分子的免疫分析应用研究

合成了溶解性受pH值影响的高分子化合物聚甲基丙烯酸甲酯-丙烯酸-顺丁烯二酸酐(MAM)及聚甲基丙烯酸一丙烯酰胺-N-羟基琥珀酰亚胺丙烯酸酯(MAN),分别与抗体进行了共价连接,将其用作免疫分析载体,利用其溶解性可调节特征,建立了新的免疫分析方法.以聚甲基丙烯酸-丙烯酰胺-N-羟基琥珀酰亚胺丙烯酸酯为载体,对血清中乙肝表面抗原进行了检测,阴阳性检出结果与常规ELISA方法相符.     

Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls

Detection of pollutants is of significant importance for environmental protection. However, conventional monitoring methods are often time-consuming, and require expensive equipments. Biosensors based on enzyme linked immunosorbent assay (ELISA) provide an alternative method to conventional ones. In this research, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a micro-flow immunosensor chip. The immunosensor chips were fabricated by micro-electromechanical system (MEMS) technology. The quantitative determination of coplanar polychlorinated biphenyls (Co-PCBs) was performed by using a micro-flow immunosensor chip. Polystyrene beads were used as the solid substrate for the immobilization of Co-PCB antibody. The antibody-immobilized beads were introduced into the flow channel. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP) conjugated antigen, and non-HRP conjugated antigen was allowed to react in the flow channel. After the antigen-antibody reaction, addition of phosphate buffer solution containing hydrogen peroxide and the fluorogenic substrate produced a fluorescent dye, which was monitored with the resulting change in the fluorescence intensity. By using our micro-flow immunosensor chip, it was possible to determine the sensing range of Co-PCB derivatives up to 0.1 ppt in 30 s. This immunosensor chip had a wide linear range for Co-PCB detection from 0.1 pg/ml to 1.0 μg/ml. The regression analysis provided the correlation coefficients of r = 0.982−0.964 with good reproducibility and precision. In a series of five measurements with immunosensor chips prepared with a new batch of antibody-immobilized polystyrene beads, a relative standard deviation of 21.3% was obtained. Our immunosensor chip design reported here has the potential to be implemented to several different detection methodologies for numerous analytes.     

SPR免疫竞争法研究小分子吗啡与其抗体间作用的动力学特征

表面等离子体共振技术(SPR)主要应用于生物大分子相互作用的研究,本文采用溶液竞争法,测定了小分子吗啡与其抗体作用的结合常数K₁,并计算了吗啡抗体与吗啡化牛血清白蛋白的结合常数K₂.证明了多抗对抗原的亲和力较单抗大,并表明大分子蛋白质的存在对抗体与待测物的结合有阻碍作用.     

An optical tweezers-based immunosensor for detection of femtomoles-per-liter concentrations of antigens

We used optical tweezers—optical trapping with focused laser beams—to pull microspheres coated with antigens off of an antibody-coated surface. Using this technique, we could quantify the force required to separate antigen to antibody bonds. At very low surface density of antigen, we were able to detect the single antigen to antibody binding. The force required to break the antigen-antibody bonds and pull the microsphere off the surface was shown to increase monotonically with increasing surface density of antigens. Using the force determination as a transducer, we were able to detect concentrations of free antigens in solution as small as 10⁻¹⁵ mol/L in a competitive binding assay.     

An immunosensor for syphilis

An immuno responsive membrane was prepared by immobilizing Wasserman antigen to acetylcellulose. The antigen-binding membrane was used in developing an immunosensor for determining specifically corresponding antibody in serum. The senor is characterized by electrochemical determination of the antigen-antibody complex formed at the membranesoluti on interface. Experimental data on the applicability of the sensor to serology tests for syphilis are given as well as an outline of a possible mechanism for evaluating the electrical potential difference across the antigen-binding membrane.     

化学发光酶联免疫分析测定血清中抗DNA抗体

建立起适于临床应用的抗ＤＮＡ抗体化学发光酶联免疫分析法。该法精密度良好，相对标准偏差为２．４％。比ＥＬＩＳＡ法更加简便、经济、省时，同时提高了灵敏度（８倍）和血清的阳性检出率。探讨了检测系统中对碘苯酚增强鲁米诺－过氧化氢－辣根过氧化物酶化学发光反应机理。     

Amplification immunoassay for the determination of Hepatitis B surface antigen

A sensitive sandwich immunoassay for the determination of Hepatitis B surface antigen (HBs) was developed, using a cascade system of Limulus amebocyte lysate as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to anti-HBs antibody. Anti-HBs antibody was adsorbed to polystyrene beads. First, HBs were reacted to solid phase anti-HBs antibody (a-HBs). After the reaction, the beads were rinsed, and were then reacted with a-HBs-LPS. Then, LPS activity specifically bound to the beads was measured. HBs could be measured in the range of 10(-10)-10(-12) g/mL.     

Solid-solute phase equilibria in aqueous solution. IV. Calculation of Phase Diagrams

The thermodynamic principles of conventional (T-x, P-T) phase diagrams and solubility (log ΣK-x) diagrams depicting solid-solute phase equilibria in aqueous solution are derived from a unifying point of view. It is shown that thermodynamic quantities necessary for the construction of conventional phase diagrams can be obtained from solubility measurements. The unary system calcite-aragonite and the binary system aragonite-strontianite, where solubility data are available over the whole compositional range, have been selected as examples. In the latter case, the constraint of constant composition of the solid phase leading to a metastable equilibrium with the respective solute species is an essential point in the thermodynamic derivation and was observed experimentally as well.     

A simplified radioimmunoassay for triiodothyronine (T3) using pre-incubated labelled antigen and antibody

We discribe the development of a simplified radioimmunoassay for triiodothyronine (T₃) using pre-incubated labelled T₃ and antibody. The assay is carried out by adding 50 l of standard or sample to 0.4 ml of pre-incubated reagent dispensed in assay tubes. The reaction is allowed to proceed for about four hours and the antigen-antibody complex precipitated by the addition of 1 cm³ of 22% polyethylene glycol solution. Due to the high dissociation constant of T₃-antibody complex at 37° C (2.83·10^–4 S^–1), the labelled antigen-antibody complex dissociates and thereby the unlabelled antigen binds with the antibody. With a four hour incubation the sensitivity of this assay is comparable to an assay done by the equilibrium method using the same antibody. Sixty serum samples were analyzed using this method and compared with the equilibrium assay (Y=0.94x+0.046 ng/cm³, r=0.98).     

活化高分子膜免疫电极检测乳腺癌抗原

报道了一种灵敏度高和使用简便的免疫电极,用于测定乳腺癌抗原（CA15－3）。CA15－3抗体固定在用羟胺活化的聚氯乙烯（PVC）－牛血清白蛋白（BSA）基体膜上。当抗体膜与CA15－3血清结合形成抗原体复合物膜后,将该复合物膜固定在自制Ag-AgCI基体电极顶端,利用毫伏计测定膜电位。该法可在15－240U／mL范围测定CA15－3,线性关系良好,回归方程：△E=-8、84 13、97Ig[CA15-3],相关系数0．9998。人血清中其他常见抗原对CA15－3测定基本无干扰。同时,探讨了免疫膜的电位响应机理。     

生物素-亲和素体系测定雌酮

雌酮与牛血清白蛋白共价结合,合成雌酮的完全抗原。利用此抗原免疫小鼠,通过细胞融合技术制备了雌酮的单克隆抗体,经纯化表征知,抗体是IgG1型,相对分子量为164000,与固定抗的亲和常数为8.2×10^8L/mol。以生物素化的羊抗鼠免疫球蛋白及辣根过氧化物酶标记的链亲和素为标记体系,通过竞争抑制的方式测定游离的雌酮,结果表明：雌酮在10～10000pg/mL内呈线性关系。     

Determination of aculeatisides based on immunoassay using a polyclonal antibody against aculeatiside A

An enzyme-linked immunosorbent assay (ELISA) was developed for determination of aculeatisides. Aculeatiside A was conjugated with bovine serum albumin (BSA) for immunization. The ratio of hapten in an antigen conjugate was determined by matrix-assisted laser desorption/ionization TOF mass spectrometry. Polyclonal antibody was developed in rabbits against an aculeatiside A-BSA conjugate. The antibody was specific for aculeatiside A and aculeatiside B. The range of the immunoassay extended from 100 ng ml(-1) to 5 pg ml(-1) of aculeatisides. Good correlation between ELISA and HPLC methods was obtained when crude extracts of plant samples were analyzed. The optimized ELISA was found to be applicable to the determination of total aculeatisides in various plant samples.