Poly(Ethyleneimine)‐Immobilized‐Cloth Enzyme Immunoassay for the Detection of Salmonella Lipopolysaccharide

Poly(ethyleneimine) (PEI) was immobilized on non‐woven polyester cloth and examined for application on a simple, rapid and economical “cloth enzyme immunoassay (CEIA)” which was developed originally as polymyxin‐CEIA for the detection of Salmonella lipopolysaccharide (LPS). PEI‐cloth regardless of the PEI molecular weight, but with the amine group contents of 0.1 ～ 0.35 meq/g immobilized either in a physisorption‐like or chemisorption‐like manner, adsorbed LPS rapidly, preferentially and effectively. The captured LPS was then able to be detected qualitatively and quantitatively as an antigen by enzyme immunoassay. PEI‐CEIA had a detection limit for Salmonella LPS of 10 ng/ml, which was equivalent to 1.6 × 10⁵ cell/ml and was ten times more sensitive than polymyxin‐CEIA. It was possible to detect Salmonella LPS in the presence of a 100‐fold excess of E. coli LPS. PEI‐CEIA was found to be more sensitive and much easier to carry out than polymyxin‐CEIA but had the same advantages as polymyxin‐CEIA.     

A Fluorescent Enzyme Immunoassay for Salmonella Detection

Abstract

A double antibody sandwich immunoassay (EIA) was developed for the detection of Salmonella. The assay utilizes a β-galactosidasemurine myeloma monoclonal antibody (M467) conjugate prepared with the heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and uses 4-methyl umbelliferyl β-D-galactoside as a fluorogenic substrate for the enzyme. The EIA is sensitive and rapid, and compared favorably with the conventional cultural technique in the analysis of 60 feed samples naturally contaminated with Salmonella. Proteins or natural contaminants from the sample did not interfere in the assay.     

A Cloth-based Enzyme Immunoassay for Detection of Peanut Proteins in Foods

A dot blot enzyme immunoassay was developed for detection of peanut proteins in foods based on the use of polyester cloth as the solid phase. Samples were spotted on polyester cloth pre-coated with anti-peanut antibodies (chicken IgY), and bound peanut proteins were detected by sequential reactions with peroxidase-conjugated anti-peanut antibodies and chromogenic substrate. This assay detected peanut proteins in a variety of foods, in some instances giving positive results with samples containing less than 1 ppm peanut protein.     

An Improved Preparation of Antibody-Coated Polystyrene Beads for Sandwich Enzyme Immunoassay

An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay of human thyroid-stimulating hormone (TSH) was described. Rabbit anti-TSH IgG was purified by eluting at pH 2.5 from a TSH-Sepharose column, diluted 3 or 9 fold with normal rabbit IgG and used for coating polystyrene beads by physical adsorption. In a sandwich enzyme inmunoassay of TSH using rabbit (anti-TSH) Fab-β-D-galactosidase conjugate, β-D-galactosidase activities specifically bound to thus prepared polystyrene beads in the presence of TSH was 2.8–6.3 fold higher than those bound to polystyrene beads coated with anti-TSH IgG before purification. A similar effect was observed when guinea pig anti-pork insulin IgG, rabbit (anti-human IgE) IgG and goat (anti-human IgE) IgG were treated at pH 2.5. This improvement may be based on a conformational change of Fc in IgG molecule which was caused by the treatment at pH 2.5. Other sandwich immunoassays such as fluoro-and radio-immunoassays may also be improved in the same way. (KEY WORDS: Sandwich enzyme immunoassay, TSH, insulin)     

Enzyme Immunoassay: Binding of Salmonella Antigens to Activated Microtiter Plates

Abstract

A heat extract prepared from radiolabeled Salmonella cells was used to determine if covalent binding to activated surface of polystyrene plates would improve antigen retention thus contributing to increase sensitivity in an enzyme immunoassay for Salmonella antigen. The effect of treatment with ethylchloroformate on the retention of antigens passively absorbed to polyvinylchloride and polystyrene plates was also investigated. Chemically modified plates retained more radiolabeled antigens after washing than did untreated plates in which the antigens had been physically adsorbed. However, improvement of assay sensitivity depended on the type of plate used for covalent binding of antigen. N-succiniraidyl 3-(2-pyridyldlthio) propionate (SPDP), was found to be potentially useful for mediation of covalent binding of antigens to activated plates.     

Mono-methoxy-poly(3-hydroxybutyrate-co-4-hydroxybutyrate)-graft-hyper-branched polyethylenimine copolymers for siRNA delivery

A class of non-viral siRNA vectors consisting of biodegradable poly(hydroxyalkanoates) (PHA) grafted onto branched poly(ethyleneimine) (bPEI, 25 kDa) was synthesized and evaluated for siRNA delivery. The mPHA-g-bPEI copolymers were synthesized through Michael addition between acrylated mono-methoxy-poly(hydroxyalkanoates) (mPHA-acrylated) and bPEI with various block length poly(hydroxyalkanoates) from 1300 to 2900 Da. Our research showed that mPHA-g-bPEI copolymers could effectively bind siRNA, protect it from degradation by nucleases and efficiently release the complexed siRNA in the presence of low concentrations of polyanionic heparin. The particle size of mPHA-g-bPEI/siRNA complexes was <200 nm with ζ-potential between 33 and 43 mV. mPHA-g-bPEI copolymers displayed low cytotoxicity compared to unmodified bPEI and efficient cellular uptake of Cy3-siRNA in A549 cells by flow cytometry and confocal microscopy. siRNA delivery efficiency of the copolymers was assessed by siRNA against luciferase in cultured A549-Luc and MCF-7-Luc cells. Those mPHA-g-bPEI copolymers revealed a higher transfection efficiency and lower cytotoxicity than bPEI in two cell lines. Furthermore, a remarkable knockdown of luciferase expression of mPHA-g-bPEI (mAP2) complex (up to 85%) in vitro was found to be equivalent to that of commercially available transfection agent Lipofectamine™ 2000.     

微粒子酶联免疫分析法与电化学发光免疫分析法测定甲型肝炎病毒抗体一致性评价

为对微粒子酶联免疫分析法（MEIA）测定甲型肝炎病毒抗体方法学进行验证,用MEIA与电化学发光免疫分析法（ECLIA）同时检测476份血清标本的甲型肝炎病毒抗体,对其测量准确性、特异性及重复性进行评估分析。结果表明,MEIA与ECLIA比较,阳性符合率为98.7%,阴性符合率为97.0%,总符合率为98.3%。类风湿因子（RF）阳性、重度溶血、脂血、黄疸血清对检测无明显影响。MEIA与ECLIA测定甲型肝炎病毒抗体一致性较高。     

Hs-CRP酶免疫分析与免疫比浊法灵敏度比较

利用我国首获批准文号的hs-CRP酶免疫分析试剂盒与hs-CRP免疫比浊法检测了80名正常人,比较两种方法的灵敏度。结果显示：酶免疫分析的检测下限为0.01mg/L,免疫比浊法为0.5mg/L。酶免疫分析法测得50岁以下组CRP值在1mg/L以下者占60%,用免疫比浊法检测,本组全部CRP测定值都大于1mg/L;酶免疫分析法测得50岁以上组CRP值在1mg/L以下者占35%,用免疫比浊法测得该组CRP值全部都大于1mg/L。对比两种方法的x±s,酶免疫分析法在50岁以下组为1.05±1.24mg/L,免疫比浊法为2.52±1.85mg/L;酶免疫分析法在50岁以上组为1.95±1.29mg/L,免疫比浊法为3.00±1.25mg/L。免疫比浊法测定的正常人群CRP值偏高（P〈0.01）。本文结果表明,免疫比浊法对80名正常人的CRP测定值全部大于1mg/L,说明其灵敏度不能满足国际上心血管事件风险评估新标准规定的1mg/L以下为低风险区的检测需要。     

Application of Cloth-Based Enzyme Immunoassay for the Characterization of Monoclonal Antibodies to Salmonella Lipopolysaccharide Antigens

The specificity, detection limit, and stability of twelve anti-Salmonella monoclonal antibodies (Mabs) were evaluated by cloth-based enzyme immunoassay (CEIA) and polymyxin-cloth based enzyme immunoassay (p-CEIA). Using the p-CEIA, five Mabs were found to react with cholate extracted lipopolysaccharide (IPS) antigens of all 44 Salmonella strains representing 19 different serogroups examined, with the exception of the one strain of serogroup-O tested. These five Mabs did not react with cholate extracts of any of 16 Gram-positive or Gram-negative non-Salmonella bacteria tested. The detection limit of purified S. typhimurium LPS antigen in the p-CEIA was approximately 40 ng for four of the Mabs and approximately 20 ng for the other Mab. Four of the five Mabs were stable during storage at 22°C-23°C for 24 h. These four Mabs are potentially useful for the immunodetection of Salmonella in foods and other samples.     

乙肝表面抗原吖啶酯化学发光定量免疫分析方法的建立

目的建立一种定量检测乙肝表面抗原(HBsAg)的化学发光免疫分析方法并评估其性能。方法利用双抗体夹心法实验原理,用吖啶酯标记多抗,用生物素标记两株单抗,通过磁颗粒链霉亲和素-生物素系统分离固相和液相完成检测。结果本方法的检测限为0.05IU/mL,线性范围为0.05~150IU/mL,可溯源至国际标准品,批内变异小于5%,总变异小于8%,浓度高达106IU/mL的强阳样本未出现HOOK效应;5套阳转盘对雅培Architect、西门子centaur和强生VITROS发光试剂相对敏感系数分别是3、0、-3;国家参考盘检出达标;与雅培Architect比较,2000例临床样本检测灵敏度为99.77%,特异性为100%,一致性达99.95%。结论本研究建立的HBsAg定量检测试剂,各项指标均满足临床检测的要求,灵敏度高,特异性好,适合临床推广应用。     

ELISA法和RT-PCR法在麻疹病毒检测中的应用比较分析

目的 探讨酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和实时荧光定量PCR(quantitative real-time PCR,RT-PCR)对疑似麻疹病例的麻疹病毒的检测,为疾病的尽早确诊及治疗提供实验依据.方法 以上海市宝山区2014年4月至2015年12月的250例疑似麻疹病例为研究对象,采集其血清标本和咽拭子标本,分别应用ELISA法检测患者血清中的麻疹特异性的免疫球蛋白M(immunoglobulin M,IgM)抗体和RT-PCR法检测咽拭子中麻疹病毒的核酸.结果 250例疑似麻疹病例,排除风疹病毒阳性的34例病例,其中麻疹病毒(measles virus,MV) IgM抗体呈阳性的有80例,阳性率为37.04％;MV RT-PCR呈阳性的有128例,阳性率为59.26％.RT-PCR的检测阳性率显著高于IgM检测(x2=41.14,P＜0.001).结论 RT-PCR法可快速诊断麻疹病毒的病原学,且对出疹初期麻疹IgM呈阴性的病例可进～步确诊,是一种有效可靠的方法.     

Adhesive Reactive Nanoparticles of Poly(ethyleneimine)/Poly(maleic acid‐co‐propylene) Complexes: A Novel Concept for the Immobilization of Pollutant Removing Laccase

Nanoparticles (NPs) based on the complexation of anionic poly(maleic acid‐co‐propylene) (PMAP) and cationic poly(ethyleneimine) (PEI) are introduced as novel strategy to immobilize pollutant removing enzymes under mild, aqueous, and nontoxic conditions, in few processing steps. PEI/PMAP NP size 80–230 nm and are colloidally stable. Dried PEI/PMAP NP at model substrates remain bound after water contact if molar mixing ratio between cationic and anionic groups is close to unity. This study claims deswelling by drying, hydrophobic nature, and merging of PEI/PMAP NP as prime factors for adhesive stability. Furthermore PEI/PMAP NPs bear uncomplexed free maleic acid groups of PMAP, which react to maleic anhydride groups above 100 °C. These maleic anhydride groups can be used for both additional intraparticle crosslinking of PEI/PMAP NP and intermolecular linkage of enzymes under formation of imide or amide chemical bonds. Herein, laccase is either bound to precasted binary PEI/PMAP NP films or integrated into casted ternary laccase/PEI/PMAP NP films. Bound or integrated laccase shows significant enzymatic activity toward model pollutant guaiacol. Laccase bound by electrostatic attraction shows slightly higher enzymatic activity compared to chemical linkage at PEI/PMAP NP films. After five consecutive cycles of guaiacol conversion above laccase/PEI/PMAP NP film activity remains dropping to 30% of initial value.

     

Two amino acid-based superlow fouling polymers: Poly(lysine methacrylamide) and poly(ornithine methacrylamide)

We developed and investigated two new antifouling zwitterionic polymers, poly(lysine methacrylamide) (pLysAA) and poly(ornithine methacrylamide) (pOrnAA), both derived from natural amino acids – lysine and ornithine, respectively. The pLysAA and pOrnAA brushes were grafted on gold via the surface-initiated photoiniferter-mediated polymerization, with the polymer film thickness controlled by the UV-irradiation time. Nonspecific adsorption from human blood serum and plasma was investigated by surface plasmon resonance. Results show that the adsorption level decreased with the increasing film thickness. With the thin films of ∼14.5 nm, the minimal adsorption on pLysAA was 3.9 ng cm⁻² from serum and 5.4 ng cm⁻² from plasma, whereas the lowest adsorption on pOrnAA was 1.8 and 3.2 ng cm⁻², from serum and plasma, respectively. Such protein resistance is comparable to other widely reported antifouling surfaces such as poly(sulfobetaine methacrylate) and polyacrylamide, with a much thinner polymer film thickness. Both pLysAA and pOrnAA showed better protein resistance than the previously reported serine-based poly(serine methacrylate), whereas the pOrnAA is the best among three. The pLysAA- and pOrnAA-grafted surfaces also highly resisted the endothelial cell attachment and Escherichia coli K12 bacterial adhesion. Nanogels made of pLysAA and pOrnAA were found to be ultrastable in undiluted serum, with no aggregation observed after culturing for 24 h. Dextran labeled with fluorescein isothiocyanate (FITC–dextran) was encapsulated in nanogels as a model drug. The encapsulated FITC–dextran exhibited controlled release from the pOrnAA nanogels. The superlow fouling, biomimetic and multifunctional properties of pLysAA and pOrnAA make them promising materials for a wide range of applications, such as implant coating, drug delivery and biosensing.     

New Complex Crystals of Chiral Poly(L‐lactic acid) and Different Tactic Poly(methyl methacrylate)

Based on evidence from differential scanning calorimetry (DSC), wide‐angle X‐ray diffraction (WAXD), and Fourier‐transform infrared (FTIR) spectroscopy, a new stereocomplex crystal (DSC T_m = 175 °C, with WAXD 2θ = 10.0° and 12.5°) is proven for the first time between structurally dissimilar chiral poly(L ‐lactic acid) (PLLA) and syndiotactic poly(methyl methacrylate) (sPMMA). There is a strong complexing capacity only between low molecular weight PLLA and sPMMA, in miscible state, at specific weight fractions (70:30). The complexing capacity is more significant when the mixtures are melt‐crystallized at T_c = 110 °C or lower, and the intensity of this complex can be further enhanced if it is annealed between 100 and 160 °C, below its T_m = 175 °C. The new complex crystal can be formed only between PLLA and sPMMA, but not with isotactic or atactic PMMA.     

Preparation of Poly(3-hydroxybutyrate) Porous Films by Microemulsion Templates Method

INTRODUCTION　　 The nature poly(3 -hydroxybutyrate) (PHB) is a biodegradable polyester pro-duced by a numberof bacteria as a reserve of carbon and energy. Due to its excellentproperties,for example,biodegradability,biocompatibility,optical activity,piezo…     

乳酸—乙醇酸共聚物（8：2）微球在大鼠体内的降解

制备了乳酸—乙醇酸(8:2)共聚物的空白微球,经过筛、~(60)Co灭菌后,注射于大鼠后腿肌肉中。微球直径65-80μm。每隔一定时间处死大鼠,并从注射部位分离得到微球。微球用电镜观察并用GPC测定其分子量。结果表明,此聚合物微球在大鼠体内完全降解约需6个月且分子量的下降与扫描电镜照片显示的微球形态的变化相符合。     

An Electrokinetically-Controlled Immunoassay for Simultaneous Detection of Multiple Microbial Antigens

An electrokinetically-controlled heterogeneous immunoassay microchip for multiple analyte detection was developed in this study. Numerical simulation was employed to study the transport process in a microfluidic network (μFN). The operation parameters obtained from numerical simulation was then applied to immunoassay experiment. The effectiveness of the automatic electrokinetic control was demonstrated in a separate experiment using fluorescein dye. The immunoassay microchip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using soft lithography and replica molding. Multi-antigen immobilization was accomplished by adsorbing the antigen molecules onto a PDMS-coated glass slide and by using a μFN. Immobilized lysate antigen of Escherichia coli O157: H7 at different concentrations was assayed and the lower detect limit was 3 μg/mL. The assay also displayed very good specificity, when different microbial lysate antigens were immobilized, including Escherichia coli and Helicobacter pylori, and the primary and secondary antibodies were mixtures of different species. The time required for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic-operating, high-throughput immunoassay microchip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.     

An Enzyme Immunoassay for the Detection of Antisperm Antibodies

ABSTRACT: A simple and reliable enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies has been developed in our laboratory. The antigen for the solid phase was produced by sperm sonication while antihuman globulin conjugated to alkaline phosphatase was used as the developing reagent. The conditions and reagents of the assay were chosen to give a mild treatment of the antigen, simple manipulation during washing steps, and nontoxic and readily available reagents. The results were compared to a conventional microscopical method routinely used in our laboratory that detects agglutinating antibodies to human spermatozoa. In 96% of all cases antibodies detected by the microscopical method were also detected by ELISA. Moreover there were some cases where no antisperm antibodies could be demonstrated by microscopy, but gave a positive reaction with ELISA. These were usually cases of unexplained oligospermia, agglutinates in the ejaculate, and bad motility or low viability of the sperms. These results, and also titration experiments of positive samples demonstrate the higher sensitivity of the ELISA by comparison with microscopical methods.     

Enzyme characteristics of recombinant poly(ADP-ribose) polymerases-1 of rat and human origin mirror the correlation between cellular poly(ADP-ribosyl)ation capacity and species-specific life span

Poly(ADP-ribosyl)ation is a posttranslational modification, which is involved in many cellular functions, including DNA repair and maintenance of genomic stability, and has also been implicated in cellular and organismal ageing. We have previously reported that maximum poly(ADP-ribosyl)ation capacity in mononuclear blood cells is correlated with mammalian life span. Here we show that the difference between a long-lived and a short-lived species tested (i.e. man and rat) is directly mirrored by the enzymatic parameters of recombinant poly(ADP-ribose) polymerase-1 (PARP-1), i.e. substrate affinity and reaction velocity. In addition, we have characterized two human PARP-1 alleles and assign their activity difference to their respective initial velocity and not substrate affinity.     

Enzyme immunoassays for the detection of sulfamethazine,sulfadiazine, sulfamethoxypyridazine and trimethoprim in milk

Polyclonal antibodies against sulfamethazine, sulfadiazine, sulfamethoxypyridazine and trimethoprim were prepared by using bovine serum albumin conjugates of the respective substances as antigens for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested by using the respective drug coupled to horseradish peroxidase as the labelled antigen in a competitive assay. The antiserum against sulfamethazine showed relative cross‐reactivities of 100 and 56% with sulfamethazine and sulfamerazine, respectively. The antiserum against sulfadiazine showed relative cross‐reactivities of 100, 12, 11 and 10% with sulfadiazine, sulfasalazine, sulfamerazine and sulfathiazole, respectively. The assays for sulfamethoxypyridazine and trimethoprim showed no relative cross‐reactivity above 1%. The detection limits (in buffer solution) for sulfadiazine, sulfamethazine, sulfamethoxypyridazine and trimethoprim were at 1.1, 1.3, 2.8 and 6.0 ng/ml, respectively. The recovery of the sulfonamides from milk samples, artificially contaminated at levels of 5, 10, 50 and 100 ppb, was between 68 and 99%. Recovery of trimethoprim from artificially contaminated milk samples was about 90% at concentrations of 50 and 100 ppb.