Development of a rapid polymerase chain reaction-ELISA assay using polystyrene beads for the detection of Toxoplasma gondii DNA

AIMS: To develop a rapid colourimetric assay for the detection of Toxoplasma gondii DNA using polystyrene beads as solid support. METHODS AND RESULTS: A nested-polymerase chain reaction (PCR)-ELISA assay for the detection of T. gondii DNA was standardized by optimizing the hybridization time and probe concentration. Its detection threshold was then determined and compared with Southern blotting hybridization. These were found to be equivalent, but the PCR-ELISA-beads test is easier to perform and the turnaround time is much shorter than with Southern blot. CONCLUSIONS: The PCR-ELISA-beads assay is a valuable tool for the detection of T. gondii DNA. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Our results demonstrate that this PCR-ELISA assay, using polystyrene beads, can be used as a routine diagnostic test for the detection of T. gondii in clinical laboratories.     

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads.

The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430 nM for thrombin was achieved. A lower detection limit for the protein (175 nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45 nM of thrombin demonstrating the best analytical performances.

With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.     

Transforming growth factor type beta in normal human urine

TGF beta has been identified in normal human urine specimens from five individuals studied for five consecutive days. The peptide was extracted from urine using Sepralyte C1 beads. Detectable levels of [125I]TGF beta competing activity as measured by radioreceptor assay was found in about half of the specimens studied. The protein isolated from urine using C1 Sepralyte beads was further purified using Biogel P-60 column chromatography. Fractions were tested for TGF beta and EGF competing activity using radioreceptor assays. TGF beta and EGF extracted from urine are clearly separated by column chromatography. Two distinct EGF peaks and a single TGF beta peak were observed. Fractions having [125I]TGF beta competing activity were pooled and further purified using reverse-phase HPLC. HPLC fractions having [125I]TGF beta competing activity were tested for bioactivity using a soft agar assay. The fractions were capable of stimulating soft agar growth of AKR-2B (clone 84A) cells and cross reacted with a TGF beta antibody in a radioimmunoassay. The presence of TGF beta in normal human urine was also demonstrated by immunoblotting. These results also suggest that C1 bead extraction of urine specimens can be used as a rapid first step in purification of TGF beta.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.     

不同严重急性呼吸综合征冠状病毒2抗体检测试剂盒的诊断应用评价

本文评估了6个品牌的严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS­CoV­2)特异性抗体检测试剂盒的性能,以指导临床合理选用。收集2020年1月30日至2020年5月11日期间上海市(复旦大学附属)公共卫生临床中心的住院患者样本资料共245例,其中包括SARS­CoV­2感染确诊患者122例,排除SARS­CoV­2感染的其他疾病患者123例。选用6个品牌的抗体检测试剂盒(3种为胶体金法,3种为化学发光法)检测所有患者的血清样本,同步采用聚合酶链反应(polymerase chain reaction,PCR)检测SARS­CoV­2核酸,统计临床灵敏度、特异性、阴性预测值和阳性预测值等指标,比较各检测方法间的差异。 结果显示,各检测试剂盒在临床特异性方面表现相近,但临床灵敏度差异明显,IgG的灵敏度高于IgM。化学发光试剂灵敏度为72.1%～85.2%,整体优于胶体金试剂的47.5%～84.4%。所有试剂检测结果与SARS­CoV­2核酸诊断结果相比均有统计学差异(P<0.05)。SARS­CoV­2抗体的特异性检出率随时间而上升,核酸确诊患者≥16 d抗体检出率最高可达96%。 结果表明,SARS­CoV­2抗体检测可作为核酸诊断的辅助手段,IgG和IgM 联合诊断可提高检测的灵敏度。但是不同试剂盒性能表现有差异,应根据不同临床需求和应用场景选择合适的试剂盒。     

Recognition of the cellular beta1-chain integrin by the bacterial AfaD invasin is implicated in the internalization of afa-expressing pathogenic Escherichia coli strains

The afa operons from Escherichia coli associated with extra-intestinal and intestinal infections have been characterized and the AfaD protein has been shown to be involved in the low internalization of laboratory strains expressing the afa-3 operon. The aim of this study was to determine the role of the AfaD invasin during the interaction of pathogenic E. coli with epithelial cells. We show that AfaD is implicated in the entry of a clinical isolate into both HeLa and undifferentiated Caco-2 cells. Once in the cytoplasm of these cells, the bacteria formed inclusions in which they were able to survive for at least 72 h. Internalization assays using polystyrene beads coated with His6-tagged purified AfaD (rAfaD) demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for afa-positive strains. Consistent with the previous observation that an antibody blockade involving anti-alpha5beta1 integrin abolishes bacterial internalization, we show here that the entry of rAfaD-coated beads was dependent on the production and accessibility of beta1 integrins on the cells. The AfaD proteins belong to a family of invasins that are at least 45% identical. Despite their differences, the recombinant rAfaD-III and rAfaD-VIII proteins both bound to beta1 integrins. Our results suggest that beta1 integrin is a common receptor for AfaD invasins and that additional AfaD-type-specific receptors exist.     

Preparation and characterization of calibration beads for sorting cells expressing a beta-lactamase gene reporter.

BACKGROUND: Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate. METHODS: To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer. RESULTS: We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate. CONCLUSION: We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.     

Enzymatic solid-phase assay for biotin and a biotin-benzodiazepine conjugate

A novel enzymatic ligand binding assay for biotin and its benzodiazepine conjugate is based on their binding to horseradish peroxidase-avidin conjugate (A-P) followed by the uptake of biotin-unsaturated A-P onto polystyrene beads coated with biotin-BSA. The detection limit is 1.3 x 10(-16) mol per tube (300 microL) with a 3.3 x 10(-12) M A-P solution and varies with the conjugate concentration employed. The coefficient of variation for 10 repetitive assays of 10(-15) mol of biotin is 6.22%.     

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

Effective separation of peptides using highly dense thermo-responsive polymer brush-grafted porous polystyrene beads

For the development of well-defined highly dense thermo-responsive polymer grafted surface as an improved stationary phase for thermo-responsive chromatography, poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads were prepared by surface-initiated atom transfer radical polymerization (ATRP). The PIPAAm grafted region of polystyrene beads was adjusted by the addition of isooctane as a poor solvent for polystyrene upon the reaction of ATRP initiator immobilization. Using a thermo-responsive HPLC column containing the prepared beads with PIPAAm brush grafted on the inside pores nearby the outer surfaces, angiotensin subtypes were effectively separated with aqueous mobile phase, because the densely grafted PIPAAm on nearby the outer surface effectively interacted with the peptides hydrophobically. Retention of basic peptide was achieved by the beads with basic mobile phase. These results indicated that the prepared beads with grafted PIPAAm nearby the outer surface became an effective chromatographic stationary phase for retaining basic peptides using wide pH range of mobile phase.     

丙型肝炎病毒抗体化学发光免疫检测方法的建立及其临床应用简

目的：建立丙型肝炎病毒（rmv）抗体化学发光免疫检测方法,并分析其临床应用价值。方法：应用基因工程重组的HCV抗原包被微孔板,以辣根过氧化物酶标记的羊抗人IgG为二抗,并结合鲁米诺化学发光底物系统,建立HCV抗体化学发光免疫检测方法;应用HCV抗体诊断试剂国家参考品分析所建立方法的特异性、灵敏度、稳定性和精密性,并-9北京万泰公司的ELISA试剂盒同时检测临床血清样本350份,比较检测结果。结果：检测结果符合国家参考品质量标准。批内变异系数5．1％。6．6％,批间变异系数9-5％;试剂盒置37℃考核3d,其稳定性良好;与万泰公司的ELISA检测结果对照,阳性符合率分别为99．0％,阴性符合率分别为100％,总符合率为99．4％;Kappa值为0．986,一致性强度最强。结论：建立了特异、敏感和稳定的HCV抗体化学发光免疫检测方法,适用于HCV感染的批量筛查,具有较大的临床应用价值。     

Binding of carbohydrates to solid supports Part 3: Reaction of sugar hydrazones with polystyrene substituted with aromatic aldehydes

The hydrazones of glucose and N-acetylglucosamine, as models for the residues at the reducing termini of glycans, were covalently and reversibly bound in good yield to hydroxybenzaldehydo ligands attached to a polymer support. The binding, by a sugar azine linkage, occurred within two hours at room temperature at neutral pH, and efficient recoveries of sugars from the beads were achieved by displacement with aqueous hydrazine hydrate, ethanolic benzaldehyde, or aqueous acetone. Enzyme modification of glycans was demonstrated by separation of the products of hydrolysis of lactose hydrazone with -galactosidase, using hydroxybenzaldehyde-derivatized polystyrene beads. Addition of a spacer arm to aminopolystyrene beads, for binding of reducing sugars as Amadori compounds to the aromatic amine function, was also investigated.     

Characterization of the sensitivity of side scatter in a flow-stream waveguide flow cytometer.

BACKGROUND: We previously reported a new optical configuration, in which both the side scatter and the fluorescence are collected using the index-guided, total internal reflection of a flow stream in air (the flow-stream waveguide). METHODS: Using a mixture of 0.202-microm and 0.093-microm diameter polystyrene beads, we have characterized the side scatter (SSC) sensitivity of a custom-built flow cytometer (miniFlo) which incorporates a flow-stream waveguide. RESULTS: The SSC-triggered SSC signal of 0.093-microm polystyrene beads in water was almost baseline resolved from the background. We also measured the SSC-triggered SSC signal of the same beads in water on our FACScan, which is a commercial unit with the conventional optical arrangement that uses a custom imaging objective to collect light from a sheath flow cuvette in perpendicular direction-the signal from 0.093-microm beads was not resolved from the background. CONCLUSIONS: The SSC sensitivity of miniFlo is one of the best reported in the literature. Cytometry 37:160-163, 1999. Published 1999 Wiley-Liss, Inc.     

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics

A significant challenge for all biosensor systems is to achieve high assay sensitivity and specificity while minimizing sample preparation requirements, operational complexity, and sample-to-answer time. We have achieved multiplexed, unamplified, femtomolar detection of both DNA and proteins in complex matrices (including whole blood, serum, plasma, and milk) in minutes using as few as two reagents by labeling conventional assay schemes with micrometer-scale magnetic beads, and applying fluidic force discrimination (FFD). In FFD assays, analytes captured onto a microarray surface are labeled with microbeads, and a controlled laminar flow is then used to apply microfluidic forces sufficient to preferentially remove only nonspecifically bound bead labels. The density of beads that remain bound is proportional to the analyte concentration and can be determined with either optical counting or magnetoelectronic detection of the magnetic labels. Combining FFD assays with chip-based magnetoelectronic detection enables a simple, potentially handheld, platform capable of both nucleic acid hybridization assays and immunoassays, including orthogonal detection and identification of bacterial and viral pathogens, and therefore suitable for a wide range of biosensing applications.     

An improved method for anti-glycolipid antibody and glycolipid determination by an enzyme-linked immunosorbent assay using polystyrene beads

Sensitive determination of anti-glycolipid antibody titer and glycolipid content by an enzyme-linked immunosorbent assay (ELISA) using polystyrene beads was achieved. Glycolipid-coated polystyrene beads were used as the immobilized antigen. As antigen glycolipids, gangliotetraosylceramide (GA1), gangliotriosylceramide (GA2) and neolactotetraosylceramide (paragloboside) were used. Concentrations of 1-500 ng glycolipid in liposomes/ml or 0.1-100 micrograms glycolipid/ml could be used for the glycolipid determination. Glycolipid determination by the competitive inhibition method was not influenced by the presence of other glycolipids. A great advantage of this method is that the glycolipid-coated beads can be used repeatedly by washing the used beads with 3M NaSCN solution. The method was applied to the detection of auto-antibody against GA1 in ascitic fluid from cancer patients.     

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties

Novel -COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding -COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s--seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 microg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.     

Nucleation of polar actin filament assembly by a positively charged surface

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.     

Comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays

Using both experimental assays and fluid-dynamic finite element simulation models, we directly compared the achievable performance limits of four distinct assay configurations for label-free detection of an analyte from a test sample on a biosensor surface. The assay configurations studied in this work included a biosensor incorporated into the bottom surface of a microplate well and a microfluidic channel. For each configuration, we compared assay performance for the scenario in which the entire bottom surface of the fluid-handling vessel is coated with capture ligands with assay performance for the scenario in which the capture ligands are applied in the form of localized spots. As a model system, we used detection of the protein biomarker tumor necrosis factor-alpha (TNF-α) using immobilized TNF-α capture antibody. Results show that the microfluidic assay format dramatically reduces the time required to establish a stable equilibrium. Spot-based assays are advantageous for microplate-based detection for reducing the time required for equilibrium sensor response. The results derived are generally applicable to any label-free biosensor technology and any ligand-analyte system with adjustable variables that include sensor mass density sensitivity, analyte-ligand adsorption/desorption rate constants, immobilized ligand density, flow channel geometry, flow rate, and spot size.