时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌

建立了生物素-链霉亲和素时间分辨荧光免疫分析（BAS-TRFIA）高灵敏检测嗜水气单胞菌的新方法.以兔抗IgG包被微孔板,加入嗜水气单胞菌菌株B18和生物素化IgG,生成的夹心复合物用Eu³⁺-链霉亲和素作为示踪物,Eu³⁺可与离解增强液形成时间分辨荧光（TRF）,通过TRF值对病原菌定量.方法的检测限为1.0×10² cfu/mL,在1.0×10~1.0×10⁶ cfu/mL范围内线性良好,相关系数0.9716.用该法检测时,不同来源的嗜水气单胞菌以及其它气单胞菌均呈阴性.板内和板间的变异系数分别小于5.00%和9.00%.所有相关检测试剂37℃恒温放置6 d后,检测性能无明显改变.对60份人工感染的美洲鳗鲡组织样品,包括肝、肾、肠、鳃和肌肉等组织进行检测,阳性检测率为98.33%.结果表明,BAS-TRFIA法检测嗜水气单胞菌,灵敏度高、特异性好、操作简便,该方法为水产养殖病原菌检测提供了新的思路.     

基于铕螯合物纳米粒子标记的时间分辨荧光免疫法检测嗜水气单胞菌

以铕螯合物荧光纳米粒子标记嗜水气单胞菌菌株B15兔抗IgG,得到纳米粒子-IgG复合物,建立了新的嗜水气单胞菌双抗夹心时间分辨荧光免疫检测(TRFIA)法.采用正交试验优化了兔抗IgG的包被时间和包被浓度,以及纳米粒子-IgG的免疫反应时间和稀释度.偶合了铕螯合物的纳米粒子发光强度大,光稳定性高,因此灵敏度被改善.方法...     

时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌

从皮肤点状出血的日本鳗鲡中分离出产酸克雷伯氏菌菌株B12,经福尔马林灭活后,注射新西兰兔,制备免疫抗血清,G蛋白亲和层析纯化得到抗体（IgG）,以固定于微孔板的IgG作为捕获抗体,Eu³⁺螯合物标记的IgG作为检测抗体,建立了产酸克雷伯氏菌的夹心型时间分辨荧光免疫检测法（TRFIA）,并研究了抗体浓度、免疫时间及离解时间对检测的影响。本方法检出限为5.0×10³ cfu/mL;线性范围5.0×10³～1.0×10⁷ cfu/mL,相关系数达0.99以上。交叉反应表明其具有较高的特异性,板内和板间相对标准偏差分别为5.64%和2.27%。将本方法标准化后,检测了29份人工感染的日本鳗鲡样品,包括鳃、肾、肠、肝和肌肉组织,结果满意。     

pH控制相转变分离-荧光免疫分析嗜水气单胞菌外膜蛋白

以pH敏感高分子作为载体,建立了荧光免疫分析嗜水气单胞菌外膜蛋白(outer membrane protein,OMP)的新方法。pH敏感高分子通过碳二亚胺(EDCI)与抗OMP抗体偶联,在夹心型免疫测定中,OMP首先与固定在高分子上的抗体在37℃均相反应,然后进一步与异硫氰酸荧光素标记抗体均相反应,调整pH分离出高分子免疫复合物沉淀,重新溶解后根据荧光强度确定OMP浓度。OMP在0.4~30 mg/L范围内与体系相对荧光强度呈良好线性关系,检出限为31μg/L。方法灵敏、快速且操作简便,抗体通过EDCI活化的羧基与氨基反应固定到高分子上,固定化效率、固定抗体的免疫反应活性较之以前的固定方法均得到了提高。     

利用铕鳌和试剂建立超灵敏的胃蛋白酶原I时间分辨荧光免疫分析法

采用时间分辨荧光测量技术,以稀土离子为示踪材料,建立了PGI的时间分辨荧光免疫分析法(TR-FIA),该方法具有测量灵敏度高、操作简便、示踪物稳定、定量分析量程宽、无放射性污染和应用范围广等优点。研究用北京佳瑞生物技术公司提供的抗PGI单克隆抗体8003#和8016#,PEI参考标准。结果表明,方法的特异性PGII对PGI-TRFIA无交叉反应。精密度和回收率:本方法的批内和批间CV分别为1.9%和4.7%,精密度图显示可测范围为3.5~328 KU.L-1;回收率为102.65%。灵敏度和稳定性:以零剂量点发光值均值加2 s后的发光值在标准曲线上得到的相应值为0.2 KU.L-1。8条不同时间进行的PGI-TRFIA的效点均值ED20,ED50和ED80分别为(11.34±0.2)KU.L-1,(38.73±0.8)KU.L-1和(132.3±2.9)KU.L-1。     

铕标记抗原、激光时间分辨荧光免疫分析尿中白蛋白

放射免疫分析法(RIA)将免疫反应的特效性与放射性测量的灵敏性相结合,是当今最广泛采用的方法之一。但RIA药盒使用寿命受放射性同位素半衰期的限制以及操作放射性对人体和环境可能导致某些危害,促使人们力图改善以非放射性物质标记的其它免疫分析法的灵敏度。1979年Soini和Hemmila提出的时间分辨荧光免疫分析(TR—FIA),以稀土螯合物作标记物连接到抗体或抗原上,免疫反应完成后,用时间分辨技术测荧光,很容易将稀土螯合物的荧光与背景荧光压分开。由于TR—FIA能达到甚至超过RIA灵敏度,标记物没有辐照分解之患,测定动态范围广,方法简便快速,     

时间分辨荧光免疫分析法直接测定雌二醇

合成了雌二醇抗原,并获得了雌二醇单克隆抗体,通过酶联免疫吸附法测得效价为１．０×１０＾７。使用洗脱增强－时间分辨荧光免疫分析法测定了血清中的雌二醇,浓度范围为１．０×１０＾－２－１．０×１０＾３μｇ／Ｌ,最低检出限为５．６ｎｇ／Ｌ,批内ＲＳＤ％小于３．７％,批间ＲＳＤ％小于７．５％。将测定结果 放射免疫分析法作了比较。     

时间分辨荧光免疫分析法间接测定雌二醇

以氯磺酰基噻吩甲酰三氟丙酮（CTTA）为铕（Eu)的螯合剂,羊抗鼠（SAM）的IgG为二抗,用SAM－IgG-CTTA-Eu作标记二抗,建立了以竞争抑制为基础的时间分辨荧光免疫分析测定离雌二醇（E2）的新方法。同均相方法相比灵敏度有很大提高,测定雌二醇（E2）的线性范围为2．5－200pg/mL,检测限为2.5pg/mL。这一方法可望用于E2的临床检测。     

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1 Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was log Y = 3.65786 + 0.43863·log X (R = 0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4 ng mL⁻¹. By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.     

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Production in Recombinant Aeromonas hydrophila 4AK4 Harboring phbA,phbB and vgb Genes

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), can be synthesized by Aeromonas hydrophila strain 4AK4 using long chain fatty acids as the carbon source. The wild type A. hydrophila 4AK4 accumulated PHBHHx consisting of 12-15 mol% 3HHx regardless of growth conditions. When phbA, phbB and vgb genes encoding β-ketothiolase, acetoacetyl-CoA reductase and vitreoscilla hemoglobin, respectively, were introduced together into A. hydrophila 4AK4, the recombinant strain grew to over 20 g/L cell dry weight (CDW) after 48 h of shake flask cultivation in co-substrates of dodecanoate and gluconate (weight ratio 1:1), and the CDW contained 50% PHBHHx consisting of 9 mol% 3HHx. Under similar conditions, the wild type strain produced only 12 g/L CDW containing 32% PHBHHx with 15 mol% 3HHx. In comparison, recombinant harboring phbA and phbB produced 35% PHBHHx with 9 mol% 3HHx in 15 g/L CDW under the same conditions. The obvious differences in terms of the cell growth and PHBHHx production were attributed to the expression of vgb in A. hydrophila 4AK4, which was clearly observed in carbon monoxide difference spectra. The expression of vgb in the recombinant not only improved cell growth and PHBHHx accumulation, but also increased the plasmid stability during cell growth, especially under low dissolved oxygen tension in fermentors. PHBHHx production could be further increased to over 60% of the CDW by the over expression of phaC and phaJ from Aeromonas caviae encoding PHBHHx synthase and (R)-specific enoyl-CoA hydratase, respectively. Over expression of phaC, phaJ and phaP, alone or in various combinations, also increased the 3HHx content of PHBHHx from 14-34%. The above results showed that A. hydrophila was amenable to genetic manipulation, and that these modifications could be exploited to produce compounds with different properties for commercial and research applications.     

Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application

Silica-based functionalized terbium fluorescent nanoparticles were prepared, characterized and developed as a fluorescence probe for antibody labeling and time-resolved fluoroimmunoassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion containing a strongly fluorescent Tb³⁺ chelate, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)phenylpyridine] tetrakis(acetate)-Tb³⁺ (BPTA-Tb³⁺), Triton X-100, octanol, and cyclohexane by controlling copolymerization of tetraethyl orthosilicate (TEOS) and 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEPS) with ammonia water. The characterizations by transmission electron microscopy and fluorometric methods show that the nanoparticles are spherical and uniform in size, 45 ± 3 nm in diameter, strongly fluorescent with fluorescence quantum yield of 10% and a long fluorescence lifetime of 2.0 ms. The amino groups directly introduced to the nanoparticle’s surface by using AEPS in the preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticle-labeled anti-human α-fetoprotein antibody was prepared and used for time-resolved fluoroimmunoassay of α-fetoprotein (AFP) in human serum samples. The assay response is linear from 0.10 ng ml⁻¹ to about 100 ng ml⁻¹ with the detection limit of 0.10 ng ml⁻¹. The coefficient variations (CVs) of the method are less than 9.0%, and the recoveries are in the range of 84-98% for human serum sample measurements.     

Ultrasensitive detection of pepsinogen I and pepsinogen II by a time-resolved fluoroimmunoassay and its preliminary clinical applications

A fast and highly sensitive assay for pepsinogen I (PG I) and pepsinogen II (PG II) by using time-resolved fluoroimmunoassay (TRFIA) detection technique has been developed for the determination of serum PG I and PG II against gastrointestinal diseases. On the noncompetitive assay, one monoclonal antibody (McAb) coated on wells was directed against a specific antigenic site on the PG I or PG II. The McAb, called as labelling McAb, was prepared with the europium-chelate of N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N,N,N-tetraacetic acid and directed against a different antigenic site on the PG I or PG II molecule. After bound/free separation by washing, the fluorescence counts of bound Eu³⁺-McAb were measured. The levels of PG in sera from patients or healthy volunteers were determined by PG I and PG II TRFIA using the autoDELFIA₁₂₃₅ system. The measurement ranges of PG I-TRFIA were 3.5-328.0 μg L⁻¹ and those of PG II-TRFIA were 2.0-55.0 μg L⁻¹. The within-run and between-run CVs of the PG I-TRFIA were 1.9% and 4.7%, respectively, and those of PG II-TRFIA were 2.1% and 3.8%, respectively. The recovery rates of PG I-TRFIA and PG II-TRFIA were 102.7% and 104.6%, respectively. The detection limitations of PG I and PG II were 0.05 μg L⁻¹ and 0.02 μg L⁻¹, respectively. The dilution experiments showed the percentage of expected value of PG I-TRFIA was 93.2-102.3% and of PG II-TRFIA was 97.3-110.6%. The cross-reacting rate between PG I and PG II was negligible. The linear correlation of radioimmunoassay (RIA) and TRFIA measurements resulted in a correlation coefficient as 0.926 of PG I and as 0.959 of PG II. The europium-labelling McAbs were stable for at least one year at −20 °C, and the results of the TRFIA with same reagents were reproducible over one year as well. The means of 1600 healthy volunteers were 162.4 ± 52.1 μg L⁻¹ for serum PG I, 11.7 ± 6.8 μg L⁻¹ for serum PG II, and 13.8 ± 7.4 for the PG I/PG II ratio. The normal ranges of Serum PG I levels for healthy volunteers were 58.2-266.6 μg L⁻¹, and those of serum PG II levels were less than 25.3 μg L⁻¹. The availability of a highly sensitive, reliable, and convenient PG-TRFIA method for quantifying PG will allow investigations into the possible diagnostic value of this analysis in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastric ulcer and gastritis. The sensitivity and reproducibility of the assay were satisfactory for clinical applications.     

Determination of Diethylstilbestrol by Time-resolve Fluoroimmunoassay

Abstract

A time-resolved fluoroimmunoassay (TRFIA) was developed for the determination of diethylstilbestrol (DES). The method was based on a competitive immunoassay using europium-labeled anti-DES antibody and DES-bovine serum albumin (DES-BSA) as coated antigen. The TRFIA exhibited a typical response for DES at concentrations of 0.001–100 ng · mL^?1, the linear correlation coefficient is 0.9933, and the detection limit (LOD) is 0.595 pg · mL^?1. Some serum and water samples have been analyzed by using this method with satisfactory results. Compared with the routine fluorescence immunoassay (FIA), this method was more sensitive. The TRFIA may offer a valuable alternative method for the DES detection and could be applied to routine analysis.     

Antigonon leptopus: a potent biological source for extermination of fish bacterial pathogens Providencia and Aeromonas

This study pertains to the phytochemical components and the biological properties of the weed, Antigonon leptopus Hook. & Arn. (AUT/PUS/064). Phytochemical screening of methanolic leaf extract of A. leptopus revealed the presence of saponin, phenolic compounds, tannins, flavonoids, alkaloids, fixed oils and amino acids. Accordingly, 12 phytochemical components were analysed and characterised by GC–MS. Antibacterial activity was evaluated against fish and clinical pathogens. Fish pathogens, Providencia vermicola (MTCC 5578) and Aeromonas hydrophila (MTCC 646) were more sensitive to the methanolic leaf extract than clinical pathogens. A useful information was obtained from the phytochemistry of A. leptopus leaves, which would pave way to further applications to treat fish diseases and for utility in the pharmaceutical field.     

Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb(3+) fluorescent chelate with N,N,N('),N(')-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb(3+)) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb(3+) labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb(3+) labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ngml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement.