检测伐地那非的单克隆抗体制备方法

为了能够获得检测伐地那非的单克隆抗体,采用戊二醛作为连接剂,将伐地那非连接到载体蛋白上,使伐地那非药效必需基团暴露在人工抗原表面作为主要抗原决定簇。经条件优化,偶联比为6.8:1的抗原免疫小鼠后产生的抗体滴度最高。通过杂交瘤技术获得了4株能够同时识别伐地那非及其类似物Piperidenafil的单克隆抗体。     

同时检测伐地那非及其类似物的免疫学方法

伐地那非是用于治疗阳痿的磷酸二酯酶(phosphodiesterase type-5,PDE-5)抑制剂药物。违法添加到保健品和中成药的伐地那非及其结构类似物对公众健康构成了威胁。单克隆抗体4B9被鉴别为能够同时检测伐地那非及其类似物。以这株抗体建立的间接竞争ELISA方法,对伐地那非的检测限为5.0 ng/mL,线性范围为5.0~40 ng/mL(R2=0.952),IC50值为18.2 ng/mL。对20个中成药进行检测没有发现假阳性,伐地那非最低检出浓度为0.08 mg/g;添加回收率76%~116%,批内差异为9.7%~16.2%。     

液相色谱串联质谱分析酒类产品中他达那非等11种非法添加药物方法研究

建立了配制酒产品中伐地那非、那红地那非、红地那非、羟基豪莫西地那非、西地那非、豪莫西地那非、氨基他达那非、他达那非、硫代艾地那非、伪伐地那非、那莫西地那非等11种药物残留量的高效液相色谱法测定和液相质谱联用法定性确认方法。对色谱柱选择、流动相组成、梯度洗脱条件进行实验,最终采用C18分离柱(150×2.1 mm×5 μm)乙腈和0.1%甲酸作为流动相,确定质谱分析方法。并检测了枸杞酒、苦瓜酒等十余种配制酒中的他达那非等11种非法添加物,效果良好。     

液相色谱-质谱联用法检测保健品中非法掺入的硫代伐地那非

目的：建立了保健品中非法掺入的硫代伐地那非HPLC-MS专属检测方法。方法：保健品样品经甲醇超声提取得提取液,再通过液相色谱-串联四级杆质谱进行分析,根据相对分子量、液相色谱保留时间等,对保健品中非法掺入的硫代伐地那非进行定性以及定量分析。 结果表明：硫代伐地那非在浓度为10ug/mL~200ug/mL范围内,呈良好线性关系,R2=0.998,精密度和稳定性良好,加样平均回收率为99.9%,RSD为0.613%,方法准确可靠,专属性强,并测得某市售保健品中硫代伐地那非的含量为0.34%。结论：液相色谱-质谱联用法专属性强,灵敏度高,可作为检测保健品中非法掺入硫代伐地那非的有效方法。     

超高效液相色谱-串联质谱法同时测定保健品中三种违禁成分

建立固相萃取-超高效液相色谱-串联质谱/质谱法(SPE-UPLC-MS/MS)测定保健品中伐地那非、西地那非、他达那非的分析方法。试样经甲醇或盐酸溶液提取,HLB固相萃取柱净化后,以乙腈和0.1%甲酸水溶液作为流动相,C18色谱柱分离,采用电喷雾离子源,正离子扫描多反应监测(MRM)模式进行检测。伐地那非、西地那非、他达那非在10~200μg/L浓度范围内呈良好线性关系,相关系数(r2)均大于0.99。空白样品加标回收率均大于75%,相对标准偏差小于8%。胶囊样品中伐地那非、西地那非、他达那非的方法检出限分别为10μg/kg、10μg/kg、20μg/kg;定量限分别为40μg/kg、40μg/kg、80μg/kg。口服液中分别为3μg/L、3μg/L、6μg/L,定量限分别为10μg/L、10μg/L、20μg/L。研究了基质效应的影响,样品基质对伐地那非、西地那非、他达那非的响应表现为抑制作用,伐地那非受到抑制作用最明显。     

食品中-种新型PDE-5抑制剂的快速筛查和定量测定

摘要：目的对食品中发现的一种非法添加的新型PDE-5抑制剂进行结构解析并建立定量测定方法。方法采用高效液相色谱-二极管阵列检测器法（HPLC-DAD）发现了一种未知的新型伐地那非类似物,采用超高效液相色谱-四级杆-静电场轨道阱质谱（UPLC-Q-Exactive Orbitrap-MS）对其结构进行质谱解析,并依据解析的化学结构购买对照品,确定该物质为O-丙基伐地那非。采用超高效液相色谱-三重四极杆质谱(UPLC-MS/MS)建立典型食品中O-丙基伐地那非的定量测定方法。结果O-丙基伐地那非在0.5025~50.25ng/mL质量浓度范围内线性关系良好,相关系数（r）为0.9998,方法检出限为0.025mg/kg,方法的定量限为0.05mg/kg,在咖啡、压片糖果、保健酒基质中3个水平加样回收率分别为92.5%、86.3％、98.6%,RSD均小于4.6％。采用该方法对市场监管部门送检的93批样品进行测定,结果其中17批检出O-丙基伐地那非,其含量为28.3~359mg/kg。结论所建立的方法快速、灵敏、准确,可用于O-丙基伐地那非的筛查和定量。研究思路可给其他非法添加未知物的鉴定提供参考。     

电喷雾-离子迁移谱法快速筛查保健酒饮料中3种壮阳类药物

目的建立电喷雾-离子迁移谱(ESI-IMS)快速筛查保健酒饮料中违法添加西地那非、硫代艾地那非和伐地那非3种壮阳类违禁药物的方法。方法样品经乙腈超声提取,稀释定容后直接测定。以ESI作为电离源,空气作为迁移气体,在正离子模式进行检测,基质外标法定量。结果西地那非、伐地那非、硫代艾地那非分别在1.25~12.50、2.00~20.00、1.00~10.00 mg/L范围内线性关系良好,相关系数(r)均大于0.99,3种违禁药物的检测限分别为0.50、0.80、0.40 mg/L。平均回收率为93.33%~121.17%,相对标准偏差(RSD)为2.65%~10.00%,实际样品检测与气相色谱-质谱联用法确证结果一致。结论该方法快速简便,结果准确可靠,适用于保健酒饮料中非法添加西地那非、硫代艾地那非和伐地那非3种壮阳类违禁药物的快速筛查。     

超高效液相色谱-二极管阵列检测器法快速测定保健食品中违法添加的14 种性功能药物

建立超高效液相色谱-二极管阵列检测器检测方法同时、快速测定抗疲劳类及增强免疫力类保健食品中违法添加的14 种性功能类化学药物（那红地那非、红地那非、伐地那非、羟基豪莫西地那非、枸橼酸西地那非、豪莫西地那非、氨基他达拉非、他达拉非、硫代艾地那非、伪伐地那非、那莫西地那非、康力龙、丙酸睾酮、苯丙酸诺龙）。采用ZORBAX SB-C18色谱柱,流动相A：乙腈-甲醇（90﹕10,V/V）,B：0.7%三乙胺溶液（pH 2.8）,梯度洗脱,流速0.35 mL/min。采集230 nm波长处的色谱图进行初筛和定量。根据14 种药物的光谱特征建立光谱库,确证检出化合物。结果表明：14 种性功能药物在15.0 min内可有效分离,在8～80 μg/mL的线性范围内相关系数均大于0.999 5,平均回收率为90.1%～106.5%,相对标准偏差为0.7%～4.9%,检测限为2～3 ng。该方法简便、快速、灵敏度高且重复性好,可作为检测抗疲劳类及增强免疫力类保健食品中违法添加化学药物的快速测定方法。     

超高效液相色谱-串联质谱同时测定露酒中11种那非物质

建立了一种使用超高效液相色谱仪和三重四极杆质谱仪联用同时检测露酒中11种那非类物质残留量的测试方法。样品经处理后,用超高效液相色谱在7 min内完成分离,三重四极杆质谱仪进行定量分析。对11种那非的线性、精密度、检出限(LOD)、定量限(LOQ)进行了验证。西地那非、那红地那非、红地那非、伐地那非、羟基豪莫西地那非、豪莫西地那非、氨基他达那非、他达那非、硫代艾地那非、伪伐地那非、那莫西地在1 μg/L~20 μg/L内线性良好,相关系数均大于0.999;用浓度为4 μg/L的混合标准溶液进行了重现性实验,实验结果表明连续6次进样保留时间和峰面积相对标准偏差分别在0~0.17%和2%~6%之间,检测方法重现性良好;在露酒中添加标液,回收率80%~115%。     

三聚氰胺偶联抗原制备及其免疫原性分析

探讨以2-氯-4,6-二胺-1,3,5-三嗪(CAAT)作为起始物,与6-氨基己酸(ACS)连接转化得到半抗原MelACS,再将其与牛血清白蛋白(BSA)偶联形成具有连接手臂的抗原BSA-ACS-Mel,用于单克隆抗体(m Ab)制备。经过液相色谱、Western blot及ELISA等抗原特性分析,证明半抗原Mel-ACS的转化效率达到了85.9%,且针对Mel特异性单抗获得率达到了42.9%,抗原BSA-ACS-Mel免疫原性较好,完全可以适用于三聚氰胺分子的特异性检测需要。这对其余小分子抗原的合成也起到一定的指导作用。     

An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml^–1. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml^–1, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml^–1. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.     

酚酞单克隆抗体的制备

目的 建立免疫学快速检测减肥类保健食品中非法添加的酚酞, 制备酚酞单克隆抗体并进行评价。方法 利用碳二亚胺(carbodiimide, EDC)法合成免疫原和包被原, 用免疫原免疫Balb/C小鼠, 取小鼠脾脏与SP2/0鼠骨髓瘤细胞融合。采用竞争结合双阳性两步筛选法, 筛选出能分泌特异性抗体的杂交瘤细胞, 利用有限稀释亚克隆方法得到单株细胞; 采用体内诱生法制备腹水型单克隆抗体。利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化, 利用酶联免疫吸附法鉴定纯化后的抗体。结果 成功合成了酚酞-BSA免疫原和酚酞-OVA包被原, 筛选获得酚酞杂交瘤细胞株FT/BSA/2019, 单克隆抗体的效价1×105。结论 本研究初步建立了特异性高的酚酞单克隆抗体的制备方法。     

呋喃西林代谢物单克隆抗体的制备及免疫学特性鉴定

为建立呋喃西林代谢物氨基脲（semicarbazide,SEM）的免疫学检测方法,采用碳化二亚胺法成功合成人工免疫抗原SEM-牛血清白蛋白（bovine serum albumen,BSA）（SEM-BSA）,并用该抗原免疫BALB/c小鼠,采用聚乙二醇介导的细胞融合技术制备SEM的单克隆抗体（SEM mAb）,采用辛酸-硫酸铵沉淀法纯化单抗,并对其效价、敏感性、特异性和亲和力等免疫学特性进行鉴定。结果表明：成功制备SEM单克隆抗体SEM-3D9,抗体效价达到1∶5×106,敏感性半抑制质量浓度为0.42 μg/L,亚型为IgG1,亲和常数Ka=2.1×108 L/mol,与呋喃唑酮代谢物2-NP-3-氨基-2-恶唑酮、呋喃它酮代谢物2-NP-5-甲基吗啉-3-氨基-2-唑烷基酮类似物的交叉反应率（cross reaction,CR）均小于0.01%,与呋喃妥因代谢物2-NP-1-氨基-2-乙内酰的CR为0.016 8%,特异性良好。     

Determination of Amaranth in Beverage by Indirect Competitive Enzyme-linked Immunosorbent Assay (ELISA) Based on Anti-amaranth Monoclonal Antibody

Amaranth (E 123) is a member of azo dyes, and it is allowed to use in various foods. The acceptable maximum addition of amaranth is strictly fixed because of its potential risk to physical health. The objective of this study was to prepare a specific anti-amaranth monoclonal antibody and develop an indirect competitive ELISA for amaranth quantification analysis. The immunogen and the coating antigen were designed by introducing a carboxyl group into amaranth for the conjugation with carrier proteins. Based on the immunogen, the monoclonal antibody exhibits satisfactory performances and the proposed ELISA shows an IC₅₀ of 20.33 ng mL^?1. The limit of detection is as low as 3.35 ng mL^?1, and the linear standard curve of the method ranges from 3.0 to 243.0 ng mL^?1. Additionally, the antibody reflects minimal cross-reactivity (<1 %) with six related food dyes (erythrosine, ponceau 4R, allura red, tartrazine, sunset yellow FCF, and brilliant blue). The recoveries of amaranth spiked beverage samples are in the range of 85.8–100.7 % with low coefficient of variation values (<11.5 %). The data shows that the developed ELISA provides a simple, sensitive, specific, and accurate alternative for amaranth determination and monitoring. Furthermore, it is the first time that icELISA of amaranth is developed based on monoclonal antibodies.     

Immunity Theory-Based High-Specific Monoclonal Antibody Preparation and Application of Fumonisin B<Subscript>1</Subscript>

Antibody preparation is the most important step in immunoassays. According to immunity theory (IT) “Antibody tends to recognize the furthest distance of side groups in the coupling league,”; immunogen must be well designed for high-specific antibody production for high-specific antibody preparation. In this research, a novel fumonisin B₁ immunogen was synthesized in which protein was conjugated at C2 position to expose the distant two type–feature groups of side chains at C14 and C15 position. After immunization, a high-specific anti-FB₁ monoclonal antibody (mAb) was produced with negligible CR value to analogues, including the most structure-related FB₂ and FB₃. Based on this mAb, a specific ELISA method was developed and validated for FB₁ detection in rice with limit of detection at 0.0339 ng kg^?1. In real-life samples, 20% were detected positive in 10 samples, with FB₁ concentrations at 84 and 20 ng kg^?1, respectively.     

A novel approach to simultaneous screening and confirmation of regulated pharmaceutical compounds in dietary supplements by LC/MS/MS with an information-dependent acquisition method

The commercial success of synthetic phosphodiesterase-5 (PDE-5) inhibitors (viz. sildenafil, vardenafil and tadalafil) for erectile dysfunction (ED) has led to their widespread use as adulterants in dietary supplements (DSs). Reports on adulteration by ED drugs or their analogues in DSs suggest they may cause a serious threat to human health. The problem is becoming more complex as hidden and structurally modified analogues are continuously being reported. To analyse known drugs and their analogues, three commonly used PDE-5 inhibitors, naturally existing icariin and yohimbin, and their 19 analogues were analyzed in this study. They were identified using ion-spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM). This MRM procedure gave a limit of detection of less than 0.02 ng ml(-1) for the 24 compounds, selectivity of fragmentation using MRM for 2.5 - 8.5 min in a single run and peak height repeatability of coefficient of variation of 3.9 - 31.8%. An IDA method using the MRM scans to detect the presence of known analytes was set up and added to a built-in library for screening for PDE-5 inhibitors. These MRM experiments were used to trigger product ion scans using a hybrid quadrupole-linear ion trap instrument. The product ion scan was compared and confirmed by a library search of MS/MS spectra acquired from a reference standard. To search for new analogues of PDE-5 inhibitors, a precursor ion scan of an expected ion m/z 283, which was one of the mass fragments from the analogues of sildenafil or vardenafil, was performed and fragmentation of the precursor ion, by combining a precursor ion scan with automatic confirmation using EPI spectra, was acquired. Of the 37 DSs tested, two were eventually found to be adulterated with yohimbin and vardenafil, respectively. The approach proposed in this study would be valuable in characterizing chemical constituents of drug residues and their analogues with identical chemical substructures from complex natural and synthetic sources in DSs using an information-dependent acquisition-enhanced product ion (IDA-EPI) scan.     

A novel approach to simultaneous screening and confirmation of regulated pharmaceutical compounds in dietary supplements by LC/MS/MS with an information-dependent acquisition method

The commercial success of synthetic phosphodiesterase-5 (PDE-5) inhibitors (viz. sildenafil, vardenafil and tadalafil) for erectile dysfunction (ED) has led to their widespread use as adulterants in dietary supplements (DSs). Reports on adulteration by ED drugs or their analogues in DSs suggest they may cause a serious threat to human health. The problem is becoming more complex as hidden and structurally modified analogues are continuously being reported. To analyse known drugs and their analogues, three commonly used PDE-5 inhibitors, naturally existing icariin and yohimbin, and their 19 analogues were analyzed in this study. They were identified using ion-spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM). This MRM procedure gave a limit of detection of less than 0.02?ng?ml^?1 for the 24 compounds, selectivity of fragmentation using MRM for 2.5?–?8.5?min in a single run and peak height repeatability of coefficient of variation of 3.9?–?31.8%. An IDA method using the MRM scans to detect the presence of known analytes was set up and added to a built-in library for screening for PDE-5 inhibitors. These MRM experiments were used to trigger product ion scans using a hybrid quadrupole-linear ion trap instrument. The product ion scan was compared and confirmed by a library search of MS/MS spectra acquired from a reference standard. To search for new analogues of PDE-5 inhibitors, a precursor ion scan of an expected ion m/z 283, which was one of the mass fragments from the analogues of sildenafil or vardenafil, was performed and fragmentation of the precursor ion, by combining a precursor ion scan with automatic confirmation using EPI spectra, was acquired. Of the 37 DSs tested, two were eventually found to be adulterated with yohimbin and vardenafil, respectively. The approach proposed in this study would be valuable in characterizing chemical constituents of drug residues and their analogues with identical chemical substructures from complex natural and synthetic sources in DSs using an information-dependent acquisition-enhanced product ion (IDA–EPI) scan.     

Structure determination of new analogues of vardenafil and sildenafil in dietary supplements

New analogues of vardenafil and sildenafil illegally added to dietary supplements were detected by high-performance liquid chromatography (HPLC) analysis with a photodiode array detector (PDA). These compounds were isolated and their structures elucidated by mass spectrometry (MS), infrared (IR) spectroscopy, one- and two-dimensional nuclear magnetic resonance (NMR). One of the new analogues given the trivial name pseudovardenafil (compound 1) was structurally elucidated and shown to be 1-[[3-(1,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f][1,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-piperidine. It was a vardenafil analogue isolated from a dietary supplement capsule. Compared with vardenafil, the piperidine ring was substituted for the ethylpiperazine group. The second new analogue, trivially named hydroxyhongdenafil (compound 2), was separated from bulk powder used as a raw material for a dietary supplement. The piperazine and phenyl groups were connected through an acetyl group instead of a sulfonyl group, and hydroxyethylpiperazine was substituted for the methylpiperazine of sildenafil. It was structurally elucidated as 5-[2-ethoxy-5-[[4-(2-hydroxyethyl)-1-piperazinyl]acetyl]phenyl]-1,4-dihydro-1-methyl-3-propyl-7H-pyrazolo[4,3-d]pyrimidin-7-one.     

展青霉素免疫学检测方法研究进展

展青霉素(patulin, PAT)作为一种对人类和动物有致畸、致癌等多种危害的真菌毒素,极易污染水果、蔬菜和谷物及其制品等。鉴于PAT污染范围广、超标检出多等问题,针对该毒素的即时检测对保障食品安全至关重要。免疫分析是快速检测真菌毒素污染的有效手段之一。但是,尚未有灵敏性好、实用价值高的PAT单克隆抗体和基因工程抗体报道。本文综述了PAT免疫学检测方法研究现状,并通过梳理其免疫学检测发展历程,提出高效特异性PAT单克隆抗体制备的阻碍主要包括PAT存在较大的极性、非免疫原性、在动物体内易被降解的特性及抗体制备选用的类似物结构偏差过大等。因此,本文认为筛选或制备结构稳定且更接近PAT的类似化合物可能是特异性PAT抗体制备的关键。另一方面,随着新型材料和技术(如电化学传感器)的发展,应用性能优良的抗体替代物建立新型免疫学或非免疫学检测方法的前景非常广阔,本文为PAT快速免疫检测方法的建立和应用提供理论依据和参考。