Analytical approach for monitoring endocrine-disrupting compounds in urban waste water treatment plants

The presence of endocrine-disrupting compounds in influent and effluent water samples from four waste water treatment plants located in Italy was studied. The estrogen-like activity of the water samples was measured using a chemiluminescent recombinant yeast assay which is based on genetically engineered yeast cells that express the human estrogen receptor. This receptor, once activated, elicits the expression of the reporter gene lac-Z and, consequently, the production of β-galactosidase, which is then measured by chemiluminescence. To control and minimize sample matrix effects, an external control based on a modified yeast strain stably expressing β-galactosidase was developed and also used in the assay. Rapid and sensitive chemiluminescent enzyme immunoassays were also developed and validated for the quantification of 17β-estradiol, estrone, and estriol in waste water samples. Results from both methods were compared with a reference high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC ESI-MS-MS) method developed for the quantification of natural estrogens. The recombinant yeast assay revealed a significant estrogenic activity in the influent samples, ranging from 80 to 400 pmol/L 17β-estradiol equivalents (EEQ), which was reduced by 70–95 % in the effluent samples. The yeast assay also showed a systematic 20–30 % overestimation of estrogenic activity relative to the HPLC ESI-MS-MS method, suggesting the presence of other compounds in the samples with estrogenic activity. The chemiluminescent enzyme immunoassays showed the presence of estrogens in the influent samples (mean concentrations: 350–450 pmol/L for estrone, 5–100 pmol/L for 17β-estradiol, 25–300 pmol/L for estriol), with significantly lower concentrations detected in the respective effluent samples. The waste water treatment was able to reduce natural estrogen concentrations by 40–95 %, although a high variability was observed. The enzyme immunoassay data correlated well with data obtained by the HPLC ESI-MS-MS method. Although the recombinant yeast assay represents a useful tool for a first-level screening of estrogenic activity due to its simplicity and high analytical throughput, sample matrix effects observed in waste water of industrial origin were found to strongly affect the yeast cells response, even when properly corrected for using the external control, thereby limiting its use to urban waste water. Its integration with chemiluminescent enzyme immunoassays would improve its performance by reducing false negative results, thereby enabling its use in extensive studies monitoring for the presence of endocrine-disrupting compounds in urban treatment plant effluents.     

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.     

基于铜增强纳米金标记物结合磁分离的溶出伏安免疫分析法用于蛋白质的高灵敏测定

应用铜原位化学放大纳米金颗粒的信号增强特性, 并结合磁分离技术, 提出了一种高灵敏的溶出伏安免疫分析方法. 实验中以人IgG为模式蛋白质, 将抗体修饰的SiO₂@Fe₃O₄核壳型磁性纳米颗粒和纳米金标抗体悬浊液混合, 用以均相免疫识别人IgG, 借助外加磁场分离纯化, 在免疫复合物悬浊液中加入铜增强试剂进行沉积放大反应, 再将铜用稀硝酸溶解并进行溶出伏安分析检测. 结果表明, 与基于固相反应的金属免疫分析法相比, 所提出的基于均相反应和磁分离原理的方法具有操作简单、分析时间短等优点. 该方法显示出明显增强的人IgG检测性能, 其线性检测范围为01~1000 ng/mL, 检出限为73 pg/mL. 此外, 将其用于实际样品的回收率测定, 结果令人满意.     

Flow-injection immuno-bioassay for interleukin-6 in humans based on gold nanoparticles modified screen-printed graphite electrodes

A flow-injection electrochemical immunoassay system based on a disposable immunosensor for the determination of interleukin-6 (IL-6) was proposed. The immunosensor was prepared by entrapping horseradish peroxidase (HRP)-labeled IL-6 antibody into gold nanoparticles-modified composite membrane at a screen-printed graphite electrode. With a non-competitive immunoassay format, the immunosensor was inserted in the flow system with an injection of sample, and the injected sample containing IL-6 antigen was produced transparent immunoaffinity reaction with the immobilized HRP-labeled IL-6 antibody. The formed antigen–antibody complex inhibited partly the active center of HRP, and decreased the immobilized HRP to H₂O₂ reduction. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal conditions, the current change obtained from the labeled HRP relative to thionine–H₂O₂ system was proportional to the IL-6 concentration in the range of 5–100 ng L⁻¹ with a detection limit of 1.0 ng L⁻¹ (at 3δ). The flow-injection immunoassay system could automatically control the incubation, washing and measurement steps with acceptable reproducibility and good stability. Moreover, the proposed immunosensors were used to analyze IL-6 in human serum specimens. Analytical results of clinical samples show the developed immunoassay has a promising alternative approach for detecting IL-6 in the clinical diagnosis.     

Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry

Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ_1–5, Aβ_6–16, Aβ_17–28 and Aβ_29–40(42) of either Aβ_1–40 or Aβ_1–42. Among those peptides, Aβ_17–28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL⁻¹ with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL⁻¹ of Aβ_1–40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.     

Multiplexed electrochemical immunoassay using streptavidin/nanogold/carbon nanohorn as a signal tag to induce silver deposition

An ultrasensitive multiplexed immunoassay method was developed by using streptavidin/nanogold/carbon nanohorn (SA/Au/CNH) as a novel signal tag to induce silver enhancement for signal amplification. The Au/CNH was prepared by in situ growth of nanogold on carboxylated CNH and functionalized with streptavidin. The SA/Au/CNH showed well dispersibility in physiological buffer and could sever as a common tracing tag to recognize biotinylated signal antibody. The immunosensor array was prepared on disposable screen-printed electrodes. Through sandwich-type immunoreaction and biotin-streptavidin affinity reaction, the SA/Au/CNH tag was captured on the immunoconjugates to induce silver deposition and amplify the electrochemical stripping signals. Using α-fetoprotein and carcinoembryonic antigen as model analytes, the proposed method showed wide linear ranges with the detection limits down to 0.024 pg mL⁻¹ and 0.032 pg mL⁻¹, respectively, and eliminated completely signal cross-talk between adjacent immunosensors. It provided a convenient, high-efficient and ultrasensitive electrochemical detection route for biological analytes, showing great potential in clinical application.     

农产品中三唑磷农药残留化学发光酶免疫分析方法研究

运用棋盘法确定直接竞争CLEIA法抗体和酶标半抗原的最适工作浓度,以4-(咪唑-1-基)苯酚(4-IMP)作为增强剂,运用L25(56)正交设计实验优化增敏液配方,建立了农产品中三唑磷农药残留的化学发光酶免疫分析方法。优化的最适工作条件:抗体包被浓度为2.0μg/mL,酶标半抗原浓度为0.006μg/mL;增敏液最佳配方为:6.4%DMF,0.01 mmol/L pH 9.0的Tris-HCl缓冲液,0.6 mmol/L鲁米诺,2 mmol/L H2O2,1 mmol/L 4-IMP。优化后方法灵敏度为0.489 ng/mL,线性范围为0.16~20.00 ng/mL,相关系数为0.996 4。该方法能实现对苹果、节瓜、大米、柑橘、荔枝及甘蓝等样品中三唑磷残留的快速、灵敏检测,加标回收率为82.8%~118.4%,与GC-MS法的相关性(r2)为0.957。结果表明,该方法可用于农产品中三唑磷农药的快速筛查。     

Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles

A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections.     

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.     

用多克隆抗体构建的夹心免疫传感器检测心肌肌钙蛋白I

用表面等离子体子共振生物传感器构建对心肌肌钙蛋白I特异性的免疫传感器检测心肌肌钙蛋白I,并建立两种检测方法:直接法的最低检测限为2.5μg/L,基于传感膜上的夹心免疫法的灵敏度为0.5μg/L,检测范围为0.5～20μg/L,批内及批间精密度分别为3.5%～4.9%,6.1%～7.4%;用夹心法及国外试剂盒对40名健康献血者和20例急性心肌梗死患者血清心肌肌钙蛋白I水平进行检测,两者符合率为95%.