A new europium beta-diketone chelate for ultrasensitive time-resolved fluorescence immunoassays

4,4'-Bis(1",1",1"-trifluoro-2",4"-butanedione-6"-yl)-chlorosulfo-o-terphenyl (BTBCT) was synthesized by modifying the structure of the reported BHHCT. In comparison with the original BHHCT, the detection sensitivity of BTBCT-Eu chelate in aqueous solution was improved approximately 8 times by time-resolved fluorescence measurement. To construct sensitive TRFIAs with the use of BTBCT-Eu chelate as the fluorescent label, streptavidin-BSA conjugate was prepared by the maleimide-thiol method and labeled by BTBCT. The streptavidin-BSA conjugate and its BTBCT-labeled complex were affinity-purified using 2-iminobiotin-agarose as binding reagent. With streptavidin-BSA-BTBCT-Eu complex as signal generation reagent, a highly sensitive indirect serum hTSH TR-IFMA was developed. The low limit of detection (LLD) of the TSH TR-IFMA was 0.011 mIU/L with 10 microl of sample volume, corresponding to approximately 337,900 molecules per test. To evaluate the utility of BTBCT-Eu label in direct TRFIAs, a competitive serum T4 TRFIA was developed with T4-BSA-BTBCT-Eu complex as competing tracer. The measurements obtained by the present TSH TR-IFMA or T4 TRFIA correlated well with those obtained by commercial Wallac TSH DELFIA Ultra or T4 DELFIA, respectively. Primary results show that BTBCT can be employed as a powerful labeling material for constructing ultrasensitive TRFIAs.     

Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels

Based on a novel cocoating strategy and dissociation enhancement lanthanide fluorescence immunoassay technique, a sensitive time-resolved fluoroimmunoassay (TRFIA) has been developed for simultaneous quantification of human serum thyroid-stimulating hormone (TSH) and thyroxin (T4) in a one-and-the-same assay procedure. The new cocoating strategy for preparing highly active surface anti-TSH and anti-T4 monoclonal antibodies (McAbs) was performed by a three-step protocol. Namely, anti-TSH McAb at high concentration (10 micro g/ml) and extensively biotinylated bovine serum albumin (BSA) at low concentration (0.5 micro g/ml) were coated on microwells by passive adsorption, then streptavidin was captured by the surface BSA-biotin, and finally biotinylated anti-T4 McAb was immobilized by the remnant binding sites of the bound streptavidin. In the present TSH/T4 TRFIA, both sandwich- and competitive-type configurations were involved, and Eu(3+) and Sm(3+) were used as labels for TSH and T4 detection, respectively. The method showed rapid kinetics; the equilibrium was reached within 30min at 37 degrees C due to the use of high concentrations of reaction reagents, rapid agitation, and small reaction volume. The lower limits of detection of the method were 0.028 mIU/L for TSH and 4.1 nmol/L for T4 with 20 micro L of sample volume. The assay ranges for TSH and T4 were 0.21-80.00 mIU/L and 20-300 nmol/L, respectively. The correlation between the TSH/T4 values obtained by the present TSH/T4 TRFIA and those obtained by commercial chemiluminescence immunoassay was satisfactory.     

A sensitive immunoassay for determination of hepatitis B surface antigen and antibody in human serum using capillary electrophoresis with chemiluminescence detection

A sensitive and homogeneous immunoassay (IA) based on capillary electrophoresis (CE) with enhanced chemiluminescence (CL) detection has been developed for the determination of hepatitis B surface antigen (HBsAg) and antibody (HBsAb) in human serum. The conditions for the CL reaction and electrophoresis were investigated in detail using horseradish peroxidase (HRP) labeled HBsAg (HBsAg*) as a marker because of its catalytic effects on the luminol-hydrogen peroxide reaction. The CL reaction was enhanced by para-iodophenol and the CL detector was designed uniquely without any dead volume or diluents effect. The present method has been used for assaying HBsAg and HBsAb in human serum using a competitive format and a non-competitive format, respectively. Under the optimal conditions, the linear ranges were from 1 to 400 pmol/L (R=0.9988) for HBsAg and 2 to 200 mIU/mL (R=0.9981) for HBsAb. The detection limits were 0.4 pmol/L and 1 mIU/mL for HBsAg and HBsAb, respectively. The relative standard deviations of peak area were 4.2% and the errors of it were from -0.03% to +0.05% for 80 pmol/L HBsAg* (n=7). In this study, the free HBsAg* and the bound HBsAg* (HBsAg*-HBsAb) were separated in the separation capillary within 6 min using a borate run buffer. To verify the experimental reliability, the result was comparable with that of enzyme linked immunosorbent assay (ELISA) and demonstrated the feasibility of the CE-CL immunoassay method for clinical diagnosis.     

Development of an enzyme-linked receptor assay (ERA) for hCG.

An enzyme-linked receptor assay (ERA) for hCG was developed using horseradish peroxidase. The 1,500 g pellets, interstitial cell fraction, and solubilized homogenate from the rat testis linked to tanned sheep red blood cells (SRBC) were used as the binding fractions for hCG. In these ERA systems, the limit of detection for hCG was 30 IU/l (3-12 mIU/tube), which was almost equal to that of radio receptor assay (RRA). The ERA using SRBC linked solubilized receptor fraction showed the most satisfactory result in accuracy, reproducibility and easiness.     

Effect of polymers on enhanced chemiluminescent assays for peroxidase and peroxidase labels

Hydroxypropyl methylcellulose, hydroxyethyl cellulose, and hydroxybutyl methylcellulose stabilized light emission in a boronic acid-enhanced chemiluminescent assay for horseradish peroxidase. The stabilization of light emission was concentration-dependent and more effective with substituted boronic acid enhancers (e.g. 4-iodophenylboronic acid) than with substituted phenol enhancers (e.g. 4-iodophenol). Hydroxybutyl methylcellulose improved the linearity of the dose–response curve in a peroxidase-based antioxidant assay and stabilized light emission post-consumption of the antioxidant (Trolox). This polymer had no effect on the signal from a peroxidase label immobilized on a membrane (dot blot) or on the inside surface of a microwell in an enzyme immunoassay for thyrotropin.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II)

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.     

Magnetic beads-based electrochemical immunosensor for monitoring allergenic food proteins

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222 nM and a detection limit of 5 nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels

A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on horseradish peroxidase-doped nanosilica particles (HRP-SiO(2)) with the conjugation of anti-IgG antibodies (anti-IgG-SiO(2)-HRP). With the sandwich-type immunoassay format, the linear range of the developed immunosensor by using anti-IgG-SiO(2)-HRP as tracer and hydrogen peroxide (H(2)O(2)) as enzyme substrate is 0.01-15 nmol/L IgG with a detection limit of 5.0 pmol/L, while the assay sensitivity by directly using HRP-labeled anti-IgG as secondary antibodies is 1.0-10 nmol/L with a detection limit of 0.1 nmol/L IgG. The reproducibility, stability and specificity of the proposed immunoassay method were acceptable. The IgG concentrations of the clinical serum specimens assayed by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) method.     

Enzyme-based ultrasensitive electrochemical biosensors

Signal amplification in conventional enzyme-based biosensors is not high enough to achieve the ultrasensitive detection of biomolecules. In recent years, signal amplification has been improved by combining enzymatic reactions with redox cycling or employing multienzyme labels per detection probe. Electrochemical-chemical redox cycling and electrochemical-chemical-chemical redox cycling allow ultrasensitive detection simply by including one or two more chemicals in a solution without the use of an additional enzyme and/or electrode. Multiple horseradish peroxidase labels on magnetic bead carriers provide high signal enhancement along with a multiplex detection possibility. In both cases, the detection procedures are the same as those in conventional enzyme-based electrochemical sensors.     

Imaging of chemiluminescent reactions in mesoscale silicon-glass microstructures

Chemiluminescent reactions in mesoscale analytical structures (chips) containing micrometer-sized interconnecting channels and chambers (pL-nL total volume) were imaged. The chips were fabricated by bonding Pyrex glass to etched pieces of silicon using a high-temperature diffusive bonding technique. In initial experiments light emission from an enhanced chemiluminescent horseradish peroxidase reaction and from a peroxyoxalate reaction contained in straight channels (300 μm wide × 20μ deep; volume 70.2 nL) and open chambers (812 μm wide, 400 μm deep, 5.2 mm long) linked by channels (100μm wide, 20 μm deep) to an exit and entry port were studied using a specially modified microplate holder and an Amerlite microplate luminometer. Light emission from more complex structures (two chambers interconnected by a branching channel 100 μm wide, 20 μm deep) filled with a solution containing alkaline phosphatase, Emerald, and CSPD^TM was imaged using a Photometrics Star 1 CCD camera. Detailed investigation of the detection and spatial resolution of the signal was performed on a Berthold Luminograph LB 980 using both the enhanced chemiluminescent horseradish peroxidase reaction and a peroxyoxalate reaction. We successfully resolved light emission from silicon structures with dimensions 100 μm wide and 20 μm deep. These simple silicon structures served as models for more complex designs that will be used for simultaneous multi-analyte assays in which an imaging system resolves and quantitates light emission from different locations on a silicon-glass analytical device.     

桥本氏病合并甲状腺乳头状癌患者血清甲状腺相关激素水平的变化及意义

目的:探究桥本氏病(HT)合并甲状腺乳头状癌(PTC)患者血清甲状腺相关激素水平的变化及意义。方法:对我院148例HT患者的临床资料进行回顾性分析,根据其是否合并PTC分为HT合并PTC组(n=68)和单纯HT组(n=80)。比较两组患者性别、年龄及血清促甲状腺激素(TSH)、甲状腺功能指标[游离三碘甲腺原氨酸(FT3)、游离甲状腺素(FT4)]、抗甲状腺抗体[甲状腺球蛋白抗体(TGAb)、甲状腺过氧物酶抗体(TPOAb)]水平等临床资料差异,分析血清TSH水平变化及意义。结果:HT合并PTC组患者男性比例、年龄、病程及血清TSH水平均大于单纯HT组,血清TGAb、TPOAb水平则均小于单纯HT组(P0.05);血清FT3、FT4水平比较差异无统计学意义(P0.05)。HT合并PTC患者组血清TSH4.2 m IU/L患者占比高于血清TSH正常组(P0.05)。血清TSH4.2 m IU/L患者中HT合并PTC患者的占比大于血清TSH水平正常的患者(P0.05)。HT合并PTC患者中,血清TSH水平4.2 m IU/L患者中央区淋巴结转移发生率高于血清TSH水平正常患者(P0.05);血清TSH4.2 m IU/L与血清TSH正常患者多灶癌发生率比较差异无统计学意义(P0.05)。结论:HT患者血清TSH水平升高可能促进其甲状腺组织癌变,HT合并PTC患者血清TSH水平升高可能促进其中央区淋巴结转移。     

Flow injection chemiluminescence determination of isoniazid using the lucigenin-periodate system.

A weak chemiluminescence (CL) signal was observed during the mixing of isoniazid with lucigenin in alkaline aqueous solution. The CL signal was enhanced more than 100 times in the presence of potassium periodate. This CL system was developed for the determination of isoniazid using a flow injection mode. The CL intensity is proportional to the concentration of isoniazid in the range 0.005-1.0 mg/L. The limit of detection is 0.0034 mg/L and the relative standard deviation is 2.0% for 0.2 mg/L isoniazid solution in 11 repeated measurements. The method was applied to the determination of isoniazid in pharmaceutical preparations and satisfactory results were obtained.     

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP‐C) in the presence of 3‐(10'‐phenothiazinyl) propane‐1‐sulfonate (SPTZ) and 4‐morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP‐catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N‐azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.     

老年2型糖尿病患者血清促甲状腺激素水平及其与冠脉病变程度的相关性分析

目的:探讨老年2型糖尿病(T2DM)患者血清促甲状腺激素(TSH)水平及其与患者冠脉病变程度的相关性。方法:回顾性分析2015年6月～2017年6月我院收治的88例老年T2DM患者(T2DM组)、50例健康体检者(对照组)的临床资料,根据血清TSH水平将T2DM组分为四个亚组,A组(0.45～1.49 m IU/L,n=18)、B组(1.50～2.49 m IU/L,n=23)、C组(2.50～3.49 m IU/L,n=22)、D组(≥4.5 m IU/L,n=25)。比较T2DM组与对照组TSH水平的差异,并根据计算机断层血管造影(CTA)结果计算Gensini评分,分析Gensini评分与血清TSH水平的相关性。结果:T2DM组血清TSH水平显著高于对照组,且随着血清TSH水平的升高,T2DM患者的年龄、TC、TG、LDL-C明显增加,而HDL-C、T3明显降低(P0.05)。C组病变支数显著多于A组,重度病变的比例明显升高,而D组病变支数、病变程度与A组、B组、C组比较差异均有统计学意义(P0.05)。血清TSH水平与Gensini评分呈显著正相关(r=0.577,P0.05)。结论:老年T2DM患者血清TSH水平显著升高,且与患者冠脉病变严重程度呈显著正相关,血清TSH水平有助于评估老年T2DM患者冠脉病变的严重程度。     

Cross-reactivity of a commercial chemiluminescence immunoassay for human chorionic gonadotropin with the free β-subunit

An enhanced chemiluminescence immunoassay for the determination of serum human chorionic gonadotropin (HCG) in specimens from oncology patients has been assessed with respect to its cross-reactivity with the free HCG ββ-subunit (HCG-β). The assay, standardized against the First International Reference Preparation 75/537, had a crossreactivity with the free β-subunit of 625% (molar basis). Therefore this assay achieves high sensitivity for the detection of either intact HCG or free HCG-β in serum of patients with seminomatous or nonseminomatous testicular cancers. Results of both assays, the in-house immunoradiometric assay (+ HCG-β) and the Amerlite HCG-60 assay, showed a close correlation (R =0.854?0.960) when serum samples from tumour patients were analyzed. Moreover, the content of free β-subunit determined in a specific HCG-β assay, could be quantitatively measured in the enhanced chemiluminescence immunoassay. Thus, this assay is suitable for oncology use, but also highlights the limitations of measuring HCG in serum samples.     

Non‐competitive immunoassay for luteinizing hormone in human serum using capillary electrophoresis with chemiluminescence detection

A non‐competitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of luteinizing hormone (LH) in human serum. The work involved the development of separation and CL conditions, allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)‐labelled monoclonal anti‐LH can catalyse the luminol–hydrogen peroxide reaction. The determined LH can react with excessive amount of HRP‐labelled anti‐LH. Within 14 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by means of a high voltage (15 kV). To improve sensitivity, a series of measures were adopted, including the choice of para‐iodophenol as a CL enhancer, unique design in detect window. Under the optimal conditions, the calibration curve for LH was established in the concentration range 1–200 mIU/mL and the detection limit was 0.08 mIU/mL. Compared with ELISA, this method decreased the detection limit by about 12 times, and it has been successfully employed in the determination of LH in human serum. Copyright © 2007 John Wiley & Sons, Ltd.     

Ultrasensitive assay of azithromycin in medicine and bio-fluids based on its enhanced luminol-H(2)O(2) chemiluminescence reaction using flow injection technique

A simple flow injection chemiluminescence method with synergistic enhancement has been investigated for the rapid and sensitive determination of azithromycin. The synergistic action was significant in the chemiluminescence system of luminol–hydrogen peroxide with azithromycin as an enhancer. The enhanced chemiluminescence intensity was linear with the concentration of azithromycin over the range from 0.1 pg mL⁻¹ to 1.0 ng mL⁻¹ (r²⁼0.9988) with a detection limit (3σ) of 0.04 pg mL⁻¹. At a flow rate of 2.0 mL min⁻¹, a complete analytical process could be performed within 0.5 min, including sampling and washing, with a relative standard deviation of less than 3.0%. The proposed method was applied successfully in the assay of azithromycin in pharmaceutical preparations, human urine and serum without any pre-treatment procedure.