Enhanced Competitive Chemiluminescent Enzyme Immunoassay for the Trace Detection of Insecticide Triazophos

Abstract: A direct competitive chemiluminescent enzyme immunoassay (CLEIA) for triazophos was developed, which was based on the anti‐THHe IgG monoclonal antibody and a heterogeneous enzyme tracer (THHu‐HRP). Several components of chemiluminescent enhanced solution (CES) were optimized. The results showed that 1 mM of p‐iodo‐phenol, 0.625 mM of luminol, and 4 mM of H₂O₂ had the best performance. Based on the study of CES, the influence of several factors (assay buffer, blocking substance, and solvent) on the immunoassay was investigated. The sensitivity for detection, IC₅₀ value was 0.87 ng/mL at a practical working concentration range between 0.04 ng/mL and 5 ng/mL and the limit of detection for triazophos was 0.063 ng/mL. The average recovery of triazophos added to lettuce, carrot, apple, water, and soil were 78.71%, 67.52%, 118.3%, 117.2%, and 122.0%, respectively. Finally, comparison between the methods of CLEIA and high‐performance liquid chromatography‐tandem mass spectrum (HPLC‐MS/MS) was performed. The results obtained from CLEIA were in agreement with those of HPLC‐MS/MS. Practical Application: The method developed in the paper could take positive or negative test of vegetable, fruit, and environmental samples for triazophos with high sensitivity and efficiency.     

间接竞争化学发光酶免疫法检测动物源食品中的 呋喃西林代谢物

目的建立动物源食品中呋喃西林代谢物间接竞争化学发光酶免疫检测方法。方法采用活化酯法将衍生后的呋喃西林代谢物半抗原与卵清蛋白(ovalbumin,OVA)偶联成为包被原;其次,对化学发光液体系、包被原和单克隆抗体的最优稀释倍数及其他反应条件进行优化;最后对该法的灵敏度、特异性、精密度及准确度进行评价。结果化学发光液A液为8 mmol/L对碘苯酚溶液和10 mmol/L鲁米诺溶液(1:1,V:V)混合,B液为每10 m L三(羟甲基)氨基甲烷(Tris)-盐酸缓冲液加5μL 30%H_2O_2溶液,A液与B液临用前体积比1:1混匀;最优反应条件是包被原和单抗稀释倍数均为800,封闭液为1%脱脂乳,竞争时间30 min,酶标二抗孵育60 min;该法的线性方程为Y=-0.4654X+0.3768(r~2=0.993),线性范围为0.123~2.398 ng/m L,IC50为0.544 ng/m L,批内和批间变异系数分别为1.9%~4.1%和2.8%~5.3%,空白鸡肉样品添加回收率为89.6%~98.0%。结论该检测方法简单快速,可用于实验室或现场动物源食品中呋喃西林代谢物的筛查。     

Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SElM, SElO, and SElN

Staphylococcus aureus remains a leading cause of food-poisoning with substantial impact on public health. Using a multiplex polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), we studied the presence of genes encoding staphylococcal enterotoxin-like (SEl) superantigens sem, sen, and seo, associated with the enterotoxin gene cluster (egc), in 429 clinical Staphylococcus aureus isolates. 294 (68.5%) isolates tested positive for at least one of the three SEl genes. In contrast to the fixed gene combination seg/sei also located on egc, a substantial number of isolates (n = 108) were found to bear only one or two of the genes encoding SElM, SElN, and SElO. Regarding the origin of the S. aureus isolates, a significant difference (P = 0.022) was found for the possession of seo (61.2% of blood isolates versus 42.9% of nasal strains). Also sem (not significantly) was found more common in blood isolates (52.1% versus 40.5%). The survey of the newly described SEl genes sem-seo supports the concept that most clinical S. aureus isolates harbor subsets of pyrogenic toxin superantigens. The potential contribution of seo and sem to the pathogenic potential of S. aureus has to be further evaluated.     

A Fluorometric Enzymatic Assay for Quantification of Steryl Glucosides in Biodiesel

Steryl glucosides (SG) are common contaminants in biodiesel that form precipitates, which form and cause problems due to fouling during transport and storage. Therefore, their quantification is necessary to assess the quality of this fuel. The methods currently available for SG analysis require expensive instrumentation, need a previous concentration step by solid‐phase extraction (SPE) or are of limited use for the quantitative assessment. We developed an enzymatic method for SG quantification in biodiesel samples based on the hydrolysis of the glucoside catalyzed by a broadly specific beta glucosidase and the subsequent determination of the glucose released by the reaction. The method is non‐expensive, sensitive and was adapted to 96‐well format fluorescence plate reader, making it useful for the parallel assay of multiple samples. The enzymatic assay presented here represent a valuable tool for both quality control and the development of improved biodiesel production and purification procedures.     

化学发光免疫分析法在新疆地产动物源性食品中硝基呋喃代谢物检测的应用

目的探讨化学发光免疫分析法(chemiluminescence immunoassay,CLEIA)检测新疆地产动物源性食品中呋喃唑酮(furazolidone,AOZ)、呋喃它酮(furaltadone,AMOZ)、呋喃西林(nitrofurazone,SEM)、呋喃妥因(nitrofurantoin, AHD)的效果。方法运用化学发光免疫分析法检测采集自新疆不同地区的150份动物源性食品的AOZ、AMOZ、SEM和AHD;采用液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)进行结果验证。结果 CLEIA检测发现150份样品中AOZ检出率最高,为2.00%; AMOZ的检出率次之1.33%; SEM、AHD均未检出。鸡肉样品的硝基呋喃类药物残留检出率最高,为5.56%;牛肉和蛋类检出率分别为2.44%和3.57%;羊肉、猪肉和蜂蜜中未检出。UPLC-MS/MS检测结果与CLEIA检测结果一致。结论 CLEIA可用于动物源性食品的硝基呋喃代谢物的初筛检测。     

2020年饲料原料霉菌毒素污染状况调查

目的 为更好的指导饲料生产企业对原料质量的把控、采购及配方设计,汇总分析了2020年饲料原料霉菌毒素污染状况。方法 采用胶体金免疫层析法或上转发光免疫分析法（up-conversion immunoassays, UPT）对安佑集团各分子公司在2020年度所收集（含退货）的28519份大宗饲料原料中的呕吐毒素、玉米赤霉烯酮和黄曲霉毒素B1含量进行快速检测。结果 对比安佑集团企业标准,2020年饲料原料霉菌毒素污染总超标率为0.92%,污染整体情况较轻,其中上半年污染较重,主要由玉米副产物和次粉霉菌毒素污染超标所致,下半年霉菌毒素污染程度整体较轻,但第4季度玉米的玉米赤霉烯酮和黄曲霉毒素B1污染情况加重,玉米的霉菌毒素整体达中度污染;从产地来源看,2020年山东、湖北产地的麸皮和次粉呕吐毒素中度污染,四川、陕西产地的次粉重度污染;四川产地的米糠黄曲霉毒素B1达中度污染,山东产地的玉米呕吐毒素和江苏产地的玉米黄曲霉毒素B1达重度污染;且饲料原料中的霉菌毒素并非单一存在,多数情况下是多种毒素共存。结论 与2019年饲料原料霉菌毒素污染调查数据相比,2020年原料的霉菌毒素污染程度较轻。     

Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.     

免疫分析技术在食品农残药残检测方面的应用

介绍了酶免疫分析技术的简单分类;并叙述了酶免疫技术、放射免疫技术、金免疫技术等免疫学分析技术在食品中农药残留、药物残留检测方面的应用。     

Design and construction of two new polymers featuring macrocyclic subunits based on a rigid clamp-like ligand

Reaction of a rigid conjugated clamp-like ligand N,N′-bis(pyridin-3-yl)-2,6-pyridinedicarboxamide (bppdca) with ZnSO₄·7H₂O or CoSO₄·7H₂O resulted in the formation of two new polymers, namely, {[Zn₂(bppdca)(SO₄)₂(DMF)₂]·(DMF)}_n (1) and {[Co(bppdca)(SO₄)(CH₃OH)(DMF)]·H₂O}_n (2). Both 1 and 2 feature one-dimensional double-chain structures with macrocyclic subunits, and the chains further self-assemble into a higher-dimensional framework via the hydrogen-bonding and π–π stacking interactions. Fluorescence studies show that the free ligand displays a strong fluorescence emission in solid state at room temperature, but in 1 and 2, the complexation of the ligand with metal ions and weak intermolecular interactions make the fluorescence emission partial quenching.     

Occurrence and characterization of carbon nanoparticles below the soot laden zone of a partially premixed flame

An experimental study has been performed to detect the occurrence of nanosized carbon particulates below the soot laden zone of a co-flowing partially premixed flame. Samples have been extracted from different points across the flame and passed through DI water. Absorption and fluorescence spectroscopies have been performed with the collected water suspensions. The occurrence of carbon nanoparticles is evident across the inner flame front. In addition, evidence of naphthalene has also been found inside the inner rich premixed flame. The concentration of naphthalene decreases while that of the carbon nanoparticles increases as the inner flame front is reached. The stability of the nanoparticles in the sample has been ensured by observing that the change in fluorescence quantum yield from the sample over a long duration is small. The band gap energy has been evaluated using the absorption data to characterize the likely structures of the particles in the collected suspension. Two kinds of particles having different zones of band gap energy are found in the flame. Dynamic light scattering measurements show that the particle size grows with the increase in height in the lower part of the flame. While, at 3 and 6 mm elevations the particles are observed to be below 2.5 nm in diameter, the particles at 10 mm elevation are found in the size range of 2.5-5.5 nm.