以2-磷酸抗坏血酸酯为底物检测碱性磷酸酯酶的微分脉冲伏安法

提出以2_磷酸抗坏血酸酯为碱性磷酸酯酶(ALP)底物微分脉冲伏安法测定ALP的方法 ;2_磷酸抗坏血酸酯在ALP的催化作用下发生水解反应生成抗坏血酸 ,抗坏血酸在玻碳电极上 +0.40V(vsAg/AgCl)被氧化而产生一个灵敏的氧化峰 ,氧化峰电流随着酶浓度的增大而增大 ,借助此氧化峰电流可以测定ALP ,进而可用于以ALP为标记酶的酶免疫分析 ;用微分脉冲伏安法对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究 ,建立了以2_磷酸抗坏血酸酯为底物的伏安酶联免疫分析新体系 ,测定游离ALP的线性范围是0.4～2.0×103 U/L,检测限为0.3U/L,对游离的IgG_ALP的测定最大稀释比为1∶200000。     

The design of a peptide linker group to enhance the SERS signal intensity of an atto680 dye‐nanoparticle system

The Surface enhanced resonance Raman spectroscopy (SERRS) spectra of three modified atto680 dyes were recorded using Au nanoparticles and an excitation laser operating at 670 nm. The dyes were modified with linker groups based on the small peptides, Cys, Cys–Gly and Cys–Gly–Gly. The Cys thiol group acted as the coupling point to the Au surface and the Gly  NH₂ group used to attach the dye. The maximum signal was recorded for the Cys–Gly linker. This gave a signal intensity for the 577 cm⁻¹ Raman peak of the atto680 dye that was more than 27 times greater than the unmodified dye. The Au nanoparticles used had a diameter of 49.8 ± 1.2 nm and were synthesised by the citrate reduction method. The Raman dye‐AuNP probes were also used in an immunoassay to detect mouse IgG in the femto mole range. Copyright © 2010 John Wiley & Sons, Ltd.     

Multiplexed Electrochemical Cancer Diagnostics With Automated Microfluidics

Microfluidic platforms can lead to miniaturisation, increased throughput and reduced reagent consumption, particularly when the processes are automated. Here, a programmable microcontroller is used for automation of a microfluidic platform configured to electrochemically determine the levels of 8 proteins simultaneously in complex liquid samples. The platform system is composed of a programmable Arduino microcontroller that controls inexpensive valve actuators, pump, magnetic stirrer and electronic display. The programmable microcontroller results in repeatable timing for each step in a complex assay protocol, such as sandwich immunoassays. Application of the platform is demonstrated using a multiplexed electrochemical immunoassay based on capture at the electrode surface of magnetic particles labelled with horseradish peroxidase and detection antibody. The multiplexed assay protocol is completed in less than 30 mins and results in detection of eight proteins associated with prostate cancer. The approach presented can be used to automate and simplify high‐throughput screening campaigns, such as detection of multiple biomarkers in patient samples.     

An estimated quantitative lateral flow immunoassay for determination of artesunate using monoclonal antibody

There have been reports of fake artesunate (ART), which has led to deaths from untreated malaria in South East Asia. To rapidly screen for fake and adulterated ART products in the drug market, a lateral flow immunoassay (LFIA) based on a colloidal gold–monoclonal antibody probe for detection of ART within samples was developed. With this method, the calibration curve for ART was determined by the intensity ratio of the test and control bands at various ART concentrations. The linearity range was 12.5–200 μg/ml of ART. Samples were tested by the developed LFIA and can be calculated for ART contents. The levels of ART in the samples were also confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good conformance. The proposed LFIA was demonstrated to be a simple and rapid analytical method for detecting ART in the pharmaceutical formulation.     

新城疫病毒流式微球免疫检测新方法

本研究基于新城疫病毒多克隆抗体的制备及优化,以聚苯乙烯微球作为蛋白质载体建立免疫检测体系,建立了快速检测新城疫病毒的流式细胞术新方法。以藻红蛋白荧光染料对新城疫多克隆抗体荧光标记,取1μL荧光微球与100μL新配制的单克隆抗体溶液反应,依次加入一定量待测抗原和藻红蛋白标记的多克隆抗体,漩涡振荡10s,室温振荡反应充分,形成双抗体夹心复合物,用流式细胞仪进行检测。采用ELISA法对免疫试剂进行了匹配性筛选实验,优选了试剂用量,并与传统ELISA方法进行了对比分析。实验结果表明,单克隆抗体350mg/L、多克隆抗体300mg/L时可获得最佳分析效果,与传统的ELISA方法呈现出良好的相关性,不与鸡传染性支气管炎病毒、鸡痘病毒、鸡马立克氏病病毒等发生交叉反应。     

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.     

基于可控相转变温度热敏高分子的人IgG酶联荧光免疫分析

以温度敏感高分子聚N－异丙基丙烯酰胺－丙烯酰胺[P(NIP-AA)]作为载体,建立了酶联荧光免疫分析人IgG的新方法。AA摩尔含量为8％的高分子P(NIP-AA)其临界溶解温度为37 °C。竞争型免疫测定中,被固定的IgG和标准溶液（或样品）在33 °C均相条件下竞争性地与辣根过氧化物酶标记抗体反应,升高温度分离出高分子免疫复合物,沉淀重新溶解后通过偶联过氧化氢与对羟基苯乙酸的荧光反应进行定量,线性范围为100-1000 ng/mL,检测限为2.0 ng/mL。方法灵敏、快速操作简便,且提高了免疫反应效率。此外,灵敏度与用传统微孔板做载体相似,但测定时间更快（从100-120分钟减少到30分钟）。该法用于测定人血清中IgG的含量,结果满意。     

Time-resolved luminescent lateral flow assay technology

We here report a detection technology that integrates highly sensitive time-resolved luminescence technique into lateral flow assay platform to achieve excellent detection performance with low cost. We have developed very bright, surface-functionalized and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. The CRP assay can achieve a detection sensitivity of <0.2 ng mL⁻¹ in serum with a linear response from 0.2 to 200 ng mL⁻¹ CRP. We have also developed a low cost time-resolved luminescence reader for the lateral flow immunoassay (LFIA) devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The reader can detect less than 2.5 ng phosphorescent particles captured on a nitrocellulose membrane strip with more than three orders of magnitude linear detection dynamic range. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.     

Determination of estradiol in human serum using magnetic particles-based chemiluminescence immunoassay

A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E₂) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E₂, without cross-reaction for the major steroids, including estrone (E₁), estriol (E₃), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E₂ in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E₂-6-HRP conjugate and anti-E₂ polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51 pg mL⁻¹ with a larger working range of 15-1000 pg mL⁻¹. The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E₂ in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E₂ in human serum.     

Flow cytometric immunoassay for sulfonamides in raw milk

Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automated FCI, a previously developed biotinylated multi-sulfonamide mutant antibody (M.3.4) was applied in combination with fluorescent beads, directly coated with a sulfathiazole derivative, and streptavidin–phycoerythrin (SAPE) for the detection. With this FCI, at least 11 different sulfonamides could be detected (more than 50% inhibition at the 100 ng mL⁻¹ level) and, after an incubation of 1 h, measurements were rapid (10 s per sample). For the application with raw milk, a 96-well microplate-based filtration step was included into the protocol to remove disturbing milk fat particles. Because of differences in sensitivity towards different sulfonamides, the FCI was considered and validated as a qualitative screening assay. For sulfadoxine, the most applied sulfonamide in Dutch dairy cattle, the detection capability (CCβ) was <50 μg L⁻¹ and this level seems feasible for five other sulfonamides. For sulfadiazine, the CCβ was <200 μg L⁻¹ and this level seems feasible for four other sulfonamides. A major advantage of the applied xMAP-technology, with its 100 different color-coded bead sets, is the possibility to develop multiplex immunoassays for the simultaneous detection of several antibiotics.