Hapten Design and Development of Polarization Fluoroimmunoassay for Nonylphenol

Polyclonal antibodies to nonylphenol (NP), raised against two different haptenic derivatives were characterized by a rapid, homogeneous polarization fluoroimmunoassay (PFIA). The first hapten, ω-(4-hydroxyphenyl)nonanoic acid (NP9), was designed to mimic the linear NP isomer and contains hydroxyphenyl and linear alkyl chain moieties of the NP molecule. The second hapten, 4-aminophenol (4AP), contains the hydroxyphenyl moiety of NP molecule alone and thus potentially mimics various phenolic compounds with different side chain structures. A number of fluorescent labeled antigens (tracers) with various structures of the spacer arm between the antigen and the fluorescent dye was synthesized and used to optimize the competitor structure for NP-specific PFIA. The most sensitive assay with limit of detection (LOD) and IC₅₀ values of 8 and 53 mg L^–1, respectively, was obtained when anti-NP9 antibody and NP9-labeled antigen were used. Anti-NP9 resulted in more specific assay, where the cross-reactivity toward the relative phenolic compounds did not exceed 5%. Anti-4AP displayed substantial recognition of several bis-substituted phenols, including 2-amino-4-chlorophenol and 2,4-dinitrophenol.     

Development of Polarization Fluoroimmunoassay for the Detection of s-Triazine Herbicides

Abstract

A rapid, non-isotopic polarization fluoroimmunoassay (PFIA) for the monitoring of the simazine (striazine herbicide) level in water was developed. Polyclonal antiserum was raised in rabbits by immunization with simazine – Keyhole Limpet Haemocyanin conjugate. Sensitivity of the PFIA with the use of heterologous tracer with the shortest bridge between antigen and fluorescein proved to be the highest. All analytical criteria for PFIA were satisfied. The detection limit of simazine (3 ng/ml in 50 μl of sample) was comparable to that for liquid or gas chromatography method. The detection limit of ELISA using the same antiserum and conjugate derivative of atrazine with horseradish peroxidase was 0.1 ng/mL of simazine. The cross-reactivity for PFIA with widely used s-triazine herbicides: atrazine, propazine, terbuthylazine was 100%, 32% and 20%, respectively. The cross-reactivity for PFIA with some metabolites of s-triazines and other herbicides was negligible.     

Preliminary Screening Method for Dioxin Contamination Using Polarization Fluoroimmunoassay for Chlorinated Phenoxyacid Pesticides

Polarization fluoroimmunoassays (PFIA) were developed for the chlorinated pesticides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In order to optimize the PFIA procedures, a number of fluorescein-labeled 2,4-D and 2,4,5-T derivatives were synthesized and the influence of their structures on PFIA characteristics was studied. Also, several antisera were tested in developing the PFIA for 2,4,5-T. The assays were adapted for use with the Abbott TDx Analyzer and could be run in automatic mode by the adaptation of existing software and protocols. Dynamic ranges for 2,4-D and 2,4,5-T were 0.2-200 ng mL^–1 and 30-10 000 ng mL^–1, respectively. Total time for the automated assay of 20 samples was about 22 min. PFIA provides a suitable means for screening of a large number of samples. The rapid determination of 2,4,5-T, which is one of the precursors of polychlorinated dibenzo-p-dioxins, one of the most toxic groups of pollutants, may potentially be used to provide preliminary evidence of dioxin contamination.     

Preparation of Anti-Lomefloxacin Antibody and Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Lomefloxacin Residue in Milk

Lomefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a complicated instrumental method to detect lomefloxacin residue in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen of bovine serum albumin-lomefloxacin showed high sensitivity toward lomefloxacin with an IC₅₀ value of 0.35 ppb in PBS buffer and was suitable to be used as a screening assay to detect lomefloxacin residue in food products. The ELISA (Enzyme-Linked ImmunoSorbant Assay) developed in this study was compared with a commercial ELISA kit and significant improvement was achieved in terms of sensitivity and specificity. The antibody prepared showed excellent specificity with certain cross-reactivity with only two drugs including norfloxacin (17.5%) and fleroxacin (8.8%) among commonly used (fluoro)quinolones. The assay measured drug residue in defatted milk spiked with lomefloxacin with an inter-assay coefficient of variation <22.2% and an intra-assay coefficient of variation <18.2%. The average recovery rates at 0.1, 0.3, 0.6, 1.0, and 2.0 ppb were in the range of 120–85% for inter-assay and in the range of 113–90% for intra-assay.     

An Enzyme-Linked Immunosorbent Assay (Elisa) for the Detection of Acetochlor

Competitive enzyme-linked immunosorbent assays (ELISAs) were developed in hapten-homologous and hapten-heterologous formats for the detection of the chloroacetanilide herbicide acetochlor. ELISA systems were devised using antibodies generated against acetochlor conjugated to carrier proteins through a thioether moiety replacing the chlorine atom in the parent structure, while haptens modified both on the chloroacetyl moiety and on the ethoxymethyl group of acetochlor have been used for coating antigens. The optimized ELISA systems allowed the detection of acetochlor 0.2-65 µg/L, and cross-reactivity studies revealed high specificity of the immunoassay: only four (propisochlor, butachlor, alachlor and metolachlor) among 18 structurally related acetanilide herbicides, fungicides and intermediates showed significant (> 1%) cross-reactivity, with even the highest value (propisochlor) being below 10%. Assay performance was not affected detrimentally by methanol up to 10% (v/v) and ethanol up to 5% (v/v). Assay performance was tested by measuring acetochlor concentration in water samples and compared favorably ( r 2 = 0.976) with those detected by gas chromatographic method coupled with mass spectrometry (GC-MS) using solid-phase microextraction (SPME) for sample preparation.     

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug–antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.     

Development and Use of Antibodies in Surface Plasmon Resonance-Based Immunosensors for Environmental Monitoring

The interaction between antibody and antigen is characterised by relatively high affinity and specificity, making this type of reaction a prime candidate for use as an analytical tool. The interaction may be combined with biosensors in the production of immunosensors for environmental monitoring. Polyclonal and monoclonal antibodies have had a significant impact in analytical detection systems over the past few decades with antibody fragments becoming important in recent years. Production of antibodies to small haptens requires the initial conjugation of hapten to a larger carrier molecule. Once hapten-carrier conjugates have been produced, polyclonal, monoclonal and various antibody fragments may be produced by differing protocols. A critical step in the production of antibody fragments is the development of efficient screening procedures to identify suitable antibody-producing clones and this has been reviewed in this article. Various antibody types may then be used in the generation of immunosensors for the monitoring of environmental pollutants. The selection of the appropriate sensor technology applicable for the determination of an antibody-antigen interaction is of prime importance for immunosensor development. One example of such an application is surface plasmon resonance-based biosensors, as they provide real-time analysis of interactions between the antibody and antigen of interest.     

鸟嘌呤与氨基酸残基体系的极化力场

发展了应用于鸟嘌呤G和氨基酸残基体系的浮动电荷力场, 该力场明确定义了孤对电子和键的电荷和位置, 通过电荷随着环境的浮动来体现极化效应; 通过氢键拟合函数k_HB描绘了氢键键能. 应用量子化学方法, 对G与氨基酸残基体系从氢键、 几何结构及电荷分布3个方面展开计算及分析, 并以其为基准, 确定参数发展了适用于G与氨基酸残基氢键体系的ABEEMσπ PFF. 采用3种不同力场模拟目标分子的结构和性质. 模拟结果表明, 发展的ABEEMσπ PFF与量子化学方法具有最好的一致性, 可用于模拟生物大分子体系.     

带活性末端的热敏高分子的制备及其在荧光免疫分析中的应用

合成了带有活性末端的寡聚N-异丙基丙烯酰胺(ONIPAAm),并考察了其温度敏感性质.以ONI-PAAm作为免疫分析的载体与鼠IgG偶联,以四磺基铁酞菁为标记物,以羊抗鼠IgG抗体为分析模型,建立了竞争型热敏相分离荧光免疫分析新系统.羊抗鼠IgG抗体在0～1500ng/mL范围内与体系相对荧光强度呈良好的线性关系,检测限为2ng/mL.     

玉米赤霉烯酮人工抗原的合成以及抗体的制备

制备了玉米赤霉烯酮人工抗原,并且制备抗血清,为进一步建立酶联免疫分析方法奠定基础.应用活泼酯法和混合酸酐法分别合成人工免疫原和人工检测原,进行动物免疫获得抗血清.结果表明,经紫外吸收、红外光谱、荧光光谱和酶联免疫方法验证人工抗原合成成功,玉米赤霉烯酮-牛血清白蛋白、玉米赤霉烯酮-卵清蛋白偶联比分别为12:1和8:1,抗...     

聚芳醚酮在溶液中的分子构象与分子极化

经从头计算,得到4,4'-二氟苯酮二面角为44°,MES₃二面角为51°;用UV光谱法研究了单分散齐聚物在H₂SO₄中的分子构象与极化,结果表明:H₂SO₄使碳基质子化是产生极化的主要因素之一,由此产生的新吸收峰在λ=400nm左右;极化作用产生的平面构象经重叠后在λ=500nm左右产生另一新的吸收峰.由质子化模型并考虑组态相互作用的计算结果与文献一致.证明了聚芳醚酮高聚物在H₂SO₄中,于400nm左右处产生的峰(UV)为羰基极化的结果,指明在H₂SO₄中测其分子量时应按羰基含量校正.     

Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens

In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC₅₀ of the ELISA for enrofloxacin from 1.3 μg L⁻¹ to as low as 0.07 μg L⁻¹. The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step.     

Polarization and fluence dependence of the polarized emission in nanosecond laser-induced breakdown spectroscopy

Several studies have appeared in the past two years reporting that the continuum emission produced by the laser ablation of solid materials is strongly polarized. In a paper that appears to conflict with these findings, Asgill et al. report that they did not observe a significant amount of polarization produced by nanosecond laser excitation of nitrogen gas and laser ablation of copper and steel ( M.E. Asgill, H.Y. Moon, N. Omenetto, D.W. Hahn, Investigation of polarization effects for nanosecond laser-induced breakdown spectroscopy, Spectrochim. Acta Part B (2010) xxx-xxx [7]). Here we show that the apparent discrepancy is resolved when laser fluence and polarization are taken into account. Using a 532 nm Nd:YAG laser to ablate Al samples in air, we find that the degree of polarization, P, of the continuum is greater for s- vs. p-polarized excitation and that P decreases with increasing fluence. We show that P would be < 10% under the conditions of Asgill et al., whereas P > 60% is obtained at low fluences with s-polarized excitation. We also confirm that at high fluence the polarization of the discrete emission is much smaller than that of the continuum.     

基于适配体和抗体的生物传感器在雌二醇检测中的应用

雌二醇(Estradiol,E2)是一种主要的环境雌激素,具有作用强、危害广等特征,可扰乱人体及动物体的正常内分泌功能,从而产生不利影响,因此E2的快速、准确检测对于食品安全和公共卫生至关重要.传统的检测方法主要是基于大型仪器分析,这些方法具有高灵敏度、准确性的同时也存在操作繁琐、成本较高、仅限于在实验室内进行等缺陷....     

离子极化力连接性拓扑指数的应用

本文提出离子极化力连接性拓扑指数，定义为：mX=∑(gixgi……）^-0.5,gi为原子的极化力值，其0、1阶指数公式分别为：0X=∑(gixgj)^-0.5,0X,1X与56种金属卤化物的晶格能（U）呈优级线性关系，回归方程为：U=-631.58 1061.65^oX 1719.59^1X r=0.9996，另外，0X,1X与碱金属卤化物的磁化率，M-X键长等亦显著相关。     

Enzyme-Linked DNA Displacement (ELIDIS) Assay for Ultrasensitive Electrochemical Detection of Antibodies**

Here we report the development of a method for the electrochemical ultrasensitive detection of antibodies that couples the programmability and versatility of DNA-based systems with the sensitivity provided by enzymatic amplification. The platform, termed Enzyme-Linked DNA Displacement (ELIDIS), is based on the use of antigen-DNA conjugates that, upon the bivalent binding of a specific target antibody, induce the release of an enzyme-DNA hybrid strand from a preformed duplex. Such enzyme-DNA hybrid strand can then be electrochemically detected with a disposable electrode with high sensitivity. We applied ELIDIS to demonstrate the sensitive (limit of detection in the picomolar range), specific and multiplexed detection of five different antibodies including three clinically relevant ones. ELIDIS is also rapid (it only requires two reaction steps), works well in complex media (serum) and is cost-effective. A direct comparison with a commercial ELISA kit for the detection of Cetuximab demonstrates the promising features of ELIDIS as a point-of-care platform for antibodies detection.     

电泳毛细管的改性及其在酶和单克隆抗体分离分析中的应用

用壁处理办法对高性能毛细管电泳的毛细管柱进行改性,在Bio-Rad HPE-100中用肽测得组分的平均柱效为200000/m,使用寿命在200次以上。对纤维素酶.尿酶等一系列的样品作了分离和测定,尿酶5次重复测定的相对标准偏差小于1％。在10min内分离并测定了由细胞瘤培养的实际样品中的抗乙肝抗体(HBsAG),反映迁移时间重复性的相对标准偏差为1.87％,并作出了相应校正曲线。     

Development of a Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Analysis of the Organophosphorous Pesticide Fenthion in Real Samples Based on Monoclonal Antibody

A new specific monoclonal antibody against the organophosphorous pesticide fenthion was produced based on our previous study. A sensitive and specific Enzyme-Linked Immunosorbent Assay (ELISA) for fenthion based on the new immunizing/coating hapten combination was developed. In this study, the H₁ which attempts to expose the aromatic ring group was conjugated with bovine serum albumin (BSA) for the immunogen. All of the haptens that have been synthesized were conjugated with ovalbumin (OVA) for the coating antigen. The efficient antibody/coating conjugated combinations were selected, and the optimized ELISA was developed. In the optimized system, the IC₅₀ value was 5.8 ng · mL^?1 with the detection limit (IC₂₀) of 0.028 ng · mL^?1. The cross reactivity with all of the tested pesticides was lower than the 0.5%. Also, the system can detect the fenthion addition to the real samples such as soil, water, rice, and Chinese cabbages.     

QDs-labeled microspheres for the adsorption of rabbit immunoglobulin G and fluoroimmunoassay

This paper presents a study on the adsorption of rabbit immunoglobulin G onto CdTe quantum dots (QDs)/polystyrene microspheres. The adsorption appears to be sensitive to pH conditions and ionic strength. Maximum adsorption for protein was obtained near the isoelectric point. Adsorption isotherm analysis demonstrated that the electrostatic interaction plays an important role in the adsorption of protein. The thickness of adsorbed layer calculated from the maximal adsorption amounts (q(m)) is 6.5 nm, which indicates that the rabbit IgG molecules exist between the side-on and end-on mode in the monolayer. The bio-functional rabbit IgG/fluorescent microspheres were further used for the detection of antibody in fluoroimmunoassays. This approach allowed detection of goat anti-rabbit IgG in the range of 1-100 ng/mL.     

Production and characterisation of polyclonal antibodies to a derivative of 3-amino-2-oxazolidinone, a metabolite of the nitrofuran furazolidone

Polyclonal antibodies were produced to detect 3-amino-2-oxazolidinone (AOZ), a stable metabolite of the nitrofuran antibiotic furazolidone, following derivatisation with o-nitrobenzaldehyde. A carboxyphenyl derivative of AOZ was prepared, purified and conjugated to immunogenic carrier protein. Six antisera were produced from the immunisation of seven rabbits using various immunogen doses and time-scales. IC₅₀ values, as determined by competitive enzyme-linked immunosorbent assay (ELISA) suggested that reducing immunogen dose from 0.3 to 0.05 mg, while lengthening rest periods between booster immunisations from 2 to 8 weeks, increased the sensitivity of the antibodies to 3-{[(2-nitrophenyl)methylene]amino}-2-oxazolidinone (NPAOZ) from 3.8 to 0.3 μg l⁻¹. An IC₅₀ of 0.065 μg l⁻¹ (AOZ in the form of NPAOZ) was achieved with antiserum R670 by altering ELISA conditions. This antibody was highly specific for NPAOZ and did not cross-react with various nitrofuran metabolites, their nitrophenyl derivatives or a range of veterinary drugs. Antibody R670 is suitable for incorporation into an immunoassay for AOZ with sufficient sensitivity to satisfy current criteria for monitoring of veterinary drug residues. This is the first publication of an antibody for detection of a nitrofuran metabolite.