Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection

Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was l-thyroxine (also known as T₄). In this technique, a sample containing thyroxine was first combined with an excess of anti-T₄ antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T₄. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T₄ in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20-45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.     

Capillary electrophoresis enzyme immunoassay for alpha-fetoprotein and thyroxine in human serum with electrochemical detection

A novel CE-based enzyme immunoassay (CE-EIA) method was developed in o-aminophenol (OAP)-H(2)O(2)-horseradish peroxidase (HRP) system and applied to benign liver disease and hyperthyroidism research in the clinical practical field. In the presented method, after the enzyme immunoreaction, the HRP-labeled antibody or HRP-labeled antigen catalyzed the enzyme substrate OAP and H(2)O(2). The product of the enzymatic catalysis reaction 2-aminophenoxazine-3-one (AP) was determined using electrochemical detection on a Pt electrode at the outlet of the reaction capillary. Factors influencing the performance, including running buffer concentration, separation, and detection voltage, were investigated to the optimum conditions. Noncompetitive and competitive models were utilized to detect alpha-fetoprotein (AFP) and thyroxine (T(4)) in human sera, respectively. The linear ranges and the detection limits (S/N = 3) were from 1.5 to 66.6 ng/mL and 0.48 ng/mL for AFP, and from 1.7 to 260.0 ng/mL and 1.0 ng/mL for T(4). The results of this method were linear proportional to those of spectrophotometric ELISA method, giving a good prospect for a new clinical diagnostic instrument.     

酪氨酸基于表面活性效应在碳糊电极上的伏安行为研究

研究了酪氨酸在表面活性剂存在下于碳糊电极上的电化学行为,发现表面活性剂能显著提高酪氨酸的氧化电流,在此基础上,建立了一种直接测定酪氨酸的电化学方法。优化了测定酪氨酸的试验参数,即介质的pH、扫描速度、富集电位和富集时间、表面活性剂的种类及其浓度等。峰电流与酪氨酸在1×10-7～3×10-5mol·L-1浓度范围内呈良好的线性关系,检出限为6×10-8mol·L-1。1×10-5mol·L-1酪氨酸平行测定8次的标准偏差为4.6%。用该方法测定了人体尿液中酪氨酸的含量,取得了满意的结果。     

Models for thyroxine: Aromatic iodine-assisted self-assemblies

Trying to understand the different recognition factors between thyroxine and its binding proteins, the chemistry of the ternary complexes between copper and o-iodobenzoylglycine (I-hip) [or o-iodobenzoylglycilglycine (B^IGG)] as simple thyroxine models were studied with 2,2′-bipyridyl as secondary ligand.     

Spectrophotometric Determination of Thyroxine Sodium with p-Benzoquinone

Abstract

A simple, sensitive and selective spectrophotometric method has been developed for the determination of thyroxine sodium. It is based on a reaction with excess of p-benzoquinone in ethanol (95%) whereby 1:1 (thyroxine sodium: quinone) coloured product is obtained, which has an apparent molar absorptivity of 2.911 × 10₃ 1mol_?1 and Beer's law is obeyed over the range of 40–200 μg.ml_?1. When applied to tablets labelled to contain 100 μg, the proposed method gave mean recoveries of 99,13 ± 0.36% for different amounts of added authentic drug.     

具有甲状腺素脱碘酶活性的抗体酶的制备及性质研究

首次以具有适当疏水性的甲状腺素衍生物O-methyl-T4为半抗原,用杂交瘤技术得到相应的抗体(6E8),又经过苯甲基磺酰氟和NaHSe的化学修饰引入了催化基团硒代半胱氨酸,制备出具有较高甲状腺素脱碘酶活力的抗体酶(Se-6E8).用SDS-PAGE电泳对Se-6E8的纯度进行了鉴定,同时研究了其解离常数、温度及pH等酶学性质.结果表明,Se-6E8活力为2010U/μmolprotein,纯度达到电泳纯,催化反应的最适温度为57℃,最适pH为82.     

Interaction of Thyroxine with 7 Hydroxycoumarin: A Fluorescence Quenching Study

The interaction between thyroxine hormone and 7 hydroxycoumarin (7HC) was investigated using fluorescence quenching method. The experimental results showed that thyroxine could quench the fluorescence of 7HC by forming the 7HC–thyroxine complex with static quenching. The apparent binding constants (K) between 7HC and thyroxine were determined to be 1.51 × 10⁴ (297 K) and 9.06 × 10³ (310 K). The binding sites (n) 0.98 ± 0.1. The thermodynamic parameters showed that the interaction between 7HC and thyroxine was driven mainly by hydrogen bonding interactions and van der Waals force. Calibration for thyroxine, based on quenching titration data, was linear in the concentration range 2.0 × 10⁻⁸ to 3.0 × 10⁻⁷ mol/l. The relative standard deviation was 2.58% for 2.0 × 10⁻⁷ mol/l thyroxine (n = 4) and the 3σ limit of detection was 3.42 × 10⁻⁸ mol/l in cationic surfactant CTAB medium.     

低剂量氯化钐对大鼠血清激素水平的影响

应用放射免疫法测定了经腹腔注射、灌胃入尾静脉注射等３种不同方式，隔日给预寺鼠低剂量氯化钐０．０５ｍｇ／ｋｇ，一个月后，测试血清中生长激素，甲状腺素Ｔ３、Ｔ３及胰岛素水平。结果显示各实验组动物血清中生长激素、胰岛素水平均明显增高，甲状腺素Ｔ４及腹腔注射增高，其余两组无明显变化。     

氢氧化钡水解HPLC法测定碘化酪蛋白中甲状腺素的含量

建立了碘化酪蛋白中甲状腺素 (T4)的氢氧化钡水解HPLC测定方法。实验发现 ,每毫升碘化酪蛋白溶液加入0.5～1.0g的八水合氢氧化钡 ,110℃水解8～12h ,控制用硫酸沉淀钡离子后的pH12～13,碘化酪蛋白释放的甲状腺素达到相对最高稳定得率。使用WatersSymmetryShieldTMRP18色谱柱 (5μm ,3.9mm×150mm) ,乙腈 (B ,0.1 %的甲酸 )和水 (A ,0.1%的甲酸 )为流动相 ,0.8mL·min -1的流速 ,B从10 %到50 %线性洗脱40min ,240nm的紫外吸收波长 ,能使碘化酪蛋白水解液中甲状腺素得到完全分离并定量测定。方法在考察的T4 质量浓度范围内 (0.5～25mg·L-1)线性良好 ,r>0.99 ,检出限可达0.2mg·g-1,实验条件下测定的商品碘化酪蛋白中T4含量为1.0 %