Peptide mimotopes of phomopsins: Identification, characterization and application in an immunoassay

Peptide mimotopes of plant-associated toxins offer the potential for improving analytical and diagnostic methodologies as well as providing candidates for potential protective vaccines against plant poisoning diseases. Monoclonal antibody (mAb) C₃C₁₁, which recognizes the antimicrotubule phomopsin mycotoxins, was used to isolate peptide mimics of phomopsin A from a random 15-mer phage display peptide library. A total of 46 clones were isolated that showed specific reactivity with the mAb. Amino acid sequence analysis revealed four different types of mimotope sequences, all of which contained a common motif V-A-L/V-C. Of the 46 clones isolated, 44 contained the motif V-A-L-C while 2 contained the V-A-V-C motif. All four types of phage clones inhibited the reactivity of the mAb with phomopsin A in a competition ELISA. The clone with the mimotope sequence CT VALCNMYFGAKLD demonstrated the strongest binding. It was further shown that synthetic peptides containing these mimotope amino acid sequences were able to inhibit the mAb-phomopsin A interaction, indicating that the peptide mimotopes were responsible for the specific binding, independent of the phage framework. The results also suggest that the mimotope peptides bind to mAb C₃C₁₁ at the same site as phomopsin A. The application of recombinant phage particles carrying phomopsin mimotopes in immunoassay was evaluated and the results demonstrated approximately 100-fold increase in sensitivity in comparison with a conventional immunoassay using a chemically linked phomopsin-horseradish peroxidase conjugate.     

一种新型多元均相时间分辨荧光免疫分析方法的建立

制备出具有光学特异性的3种不同颜色量子点纳米微球和铽螯合物，分别与CEA、AFP和HBsAg的两株抗体偶联，采用双抗体夹心法模式，充分利用两株抗体与抗原的相互作用，通过采集不同量子点发出的荧光信号，根据其信号的强弱判定标记在量子点纳米微球上的抗体结合的抗原的量，从而实现多元待分析物的定性定量分析．结果表明，量子点的荧光信号与CEA、AFP和HBsAg抗原浓度呈线性关系，其函数分别为：lgY-2．79173＋0．915841gX（R-0．99808）、lgY-3．69543＋0．456441gX（R=0．99848）和lgY-4．12898＋0．409451gX（R=0．99845），分析灵敏度分别为：0．44、0．24和0．02ng／mL．     

Determination of Aflatoxin M1 in Milk by a Magnetic Nanoparticle-Based Fluorescent Immunoassay

A sensitive and rapid magnetic nanoparticle-based fluorescent immunoassay for the determination of aflatoxin M1 in raw milk was developed. Aflatoxin M1 was converted to aflatoxin M1-o-carboxymethyl oxime. The aflatoxin M1-oxime was used for the preparation of aflatoxin M1-oxime-fluoresceinamine conjugate through the carbodiimide reaction. The aflatoxin M1-oxime-fluoresceinamine conjugate was characterized by ultraviolet–visible and infrared spectroscopy. Magnetic nanoparticles (Fe₃O₄) were synthesized and modified by 3-(aminopropyl)triethoxysilane. The size of initial (139?nm) and functionalized magnetic nanoparticles (147?nm) was determined by particle analysis. The optimal mass of immobilized antibody (25?µg) and optimal concentration of aflatoxin M1-oxime-fluoresceinamine conjugate (15?µg?mL^?1) for magnetic nanoparticle-based fluorescent immunoassay were determined. The developed immunoassay provided a linear aflatoxin M1 concentration range from 3.0 to 100?pg?mL^?1 in bovine milk. The detection limit was 2.9?pg?mL^?1. The results of aflatoxin M1 magnetic nanoparticle-based fluorescent immunoassay in heat-treated milk and phosphate-buffered saline at pH 6.6 were compared. The influence of the somatic cell count, pH, and fat concentration in bovine milk on the aflatoxin M1 immunoassay was investigated. The influence of the milk species on the immunoassay was also characterized. The high fat concentration ovine milk depressed the sensitivity of the aflatoxin M1 immunoassay.     

夹心法免疫层析试条的数学模型与仿真研究

基于夹心法免疫层析试条检测原理,结合对流扩散方程和流体动力学方程,建立了夹心法免疫层析试条动态反应过程的数学模型,并通过COMSOL软件对试条动态反应过程进行仿真.分别探究了目标待测物A浓度在0 ～ 20 mol/L,标记物P浓度在1×10-2～1×103 mol/L以及硝酸纤维素膜的孔隙率在0～1范围内变化时,检测线上夹心复合物浓度关于位置和时间的浓度变化情况,并分析了各物质初始浓度以及试条结构对于检测结果和检测时间的影响.结果表明,在一定浓度范围内,目标待测物A以及标记物P浓度的增加将提高试条的定量检测性能,而孔隙率通过影响混合液流速和混合液中各物质反应接触情况来影响检测结果.     

增强化学发光酶免疫法对猪肉中盐酸克伦特罗的检测

建立了猪肉中克伦特罗(CLB)的直接竞争化学发光酶免疫检测(dc-CLEIA)方法。通过优化离子强度、pH值确立了化学发光酶免疫法的标准曲线,优化后dc-CLEIA的检出限可达0.02μg/L。猪肉中的盐酸克伦特罗用高氯酸提取、SPE净化后绘制基质曲线,基质曲线与标准曲线吻合,说明基质影响基本消除。测定0.10、1.0、5.0μg/kg 3个水平的添加回收率,平均回收率为83%~98%,批内与批间的相对标准偏差均小于15%。进一步研究了dc-CLEIA与HPLC两种方法测定猪肉样品的相关性,结果显示两者测定结果相关性良好,r=0.964 7,说明所建立的直接化学发光酶免疫方法可用于实际样品的检测,结果准确可靠。     

Sample cleanup-free determination of mycophenolic acid and its glucuronide in serum and plasma using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Mycophenolic acid (MPA) is an immunosuppressant drug which powerfully inhibits lymphocyte proliferation. Since the early 1990s it has been used to prevent rejection in organ transplantation. The requirement of therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and its major metabolite mycophenolic acid glucuronide (MPAG).The authors developed a sample cleanup-free, rapid, and highly specific method for simultaneous measurement of MPA and MPAG in human plasma and serum using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry. MPA- and MPAG-determinations were performed during a 2.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05-100 (r > 0.999) mg L⁻¹ and 4-4000 mg L⁻¹ (r > 0.999), respectively. Limits of Detection were 0.014 mg L⁻¹ for MPA and 1.85 mg L⁻¹ for MPAG. Lower Limits of Quantification were 0.05 mg L⁻¹ for MPA and 2.30 mg L⁻¹ for MPAG. Interassay imprecision was <10% for both substances. Mean recovery was 103.6% (range 78.1-129.7%) for MPA and 111.1% (range 73.0-139.6%) for MPAG. Agreement was good for MPA and MPAG between the presented method and a validated HPLC-MS/MS method. The Passing-Bablok regression line for MPA and MPAG was HPLC-MS/MS = 1.14 UPLC-MS/MS—0.14 [mg L⁻¹], r = 0.96, and HPLC-MS/MS = 0.77 UPLC-MS/MS + 0.50 [mg L⁻¹], r = 0.97, respectively. This sample cleanup-free and robust LC-MS/MS assay facilitates the rapid, accurate and simultaneous determination of MPA and MPAG in human body fluids.     

Automated chemiluminescent dual-analyte immunoassay based on resolved immunosensing channels

A novel system of series-wound immunosensing channels (SWIC) was proposed for automated chemiluminescent (CL) dual-analyte immunoassay by immobilizing respectively different capture antibodies on the inner walls of series-wound glass channels. This system could use a single enzyme as label to perform multiplex immunoassay in one fluid way. Using α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model analytes, the mixture including AFP, horseradish peroxidase (HRP)-labeled anti-AFP antibody, CEA and HRP-labeled anti-CEA antibody was introduced into the SWIC for carrying out the on-line incubation. Upon injection of CL substrate the CL signals from the two immunosensing channels were conveniently resolved and near-simultaneously collected with the aid of optical shutter. AFP and CEA could be rapidly assayed in the ranges of 1.0-100 and 1.0-80 ng/ml with detection limits of 0.41 and 0.39 ng/ml, respectively. The assay results of clinical serum samples were in an acceptable agreement with the reference values. This designed flow-through immunosensing system based on SWIC provided an automated, reusable, simple, sensitive and low-cost approach for multianalyte immunoassay.     

Analysis of Enestroburin Residues in Wheat and Soil by Solid Phase Extraction and High-Performance Liquid Chromatography

A method was developed for the determination of enestroburin residues in wheat grain, wheat straw, and soil by solid-phase extraction (SPE) and HPLC-UV. The analytes were extracted with acetonitrile, cleaned up by PestiCarb/NH₂ cartridges and determined by HPLC with UV detector. This method is characterized by recovery >88.0%, precision (RSD) <7.8% and sensitivity of 0.005 mg/kg, in agreement with directives for method validation in residue analysis. The proposed method was successfully employed for the determination of enestroburin residue levels and its dissipation rates in a field trial in Beijing, China. Dissipation study shows that the half life of enestroburin in wheat straw was 5.35–5.81 days and in soil was 6.13–6.75 days. When enestroburin was applied according to the recommended dose and doubled dose, the final residue in wheat grain was both lower than 0.2 mg/kg. A harvest interval should be more than 7 d, and a dosage of 100–200 g (a.i.)/ha was suggested and considered as safe to human beings and animals.     

Monitoring of Mycophenolic Acid in the Plasma of Transplant Patients by Capillary Electrophoresis

Plasma samples from 60 transplant patients on mycophenolate mofetil therapy were analysed using validated capillary electrophoresis (CE) method to determine mycophenolic acid (MPA) and its main metabolite mycophenolic acid glucuronide. Enzyme multiplied immunoassay technique (EMIT) values were available for the same samples. The differences between the results from the two methods was clarified by using Bland–Altman analysis and further statistical analysis. More than 90% of the results showed a positive bias, with EMIT giving higher levels than CE.     

Coagulation of antibody-sensitized latexes in the presence of antigen

The paper describes a study of the kinetics and mechanism of the coagulation of two types of immunoassays using sensitized latexes. The positive response to the first test is based on the aggregation of the gamma globulin (IgG)-coated polystyrene latexes in the presence of IgM rheumatoid antigen. The second test is relative to the heteroaggregation of two types of sensitized latexes induced by the presence of human chorionic gonadotropin (HCG). In the latter test, two identical polystyrene latexes bearing carboxylic acid surface groups were sensitized by covalent coupling of monoclonal antibodies specific for the αHCG determinant on one type of latex and for the βHCG determinant on the other type. Using the Coulter Counter method, the aggregate size distribution c(n) was determined as a function of the number n of elementary constituents, thus enabling calculation of the number N(t) and weight S(t) average sizes of the aggregates. The temporal variations of the average sizes were compared with typical situtions of reaction-limited aggregation processes in order to characterize the mechanism of aggregation induced by antibody–antigen reactions.