酶联免疫吸附分析法测定水产品及水中孔雀石绿和无色孔雀石绿

本文报道一种同时测定水产品及水样中孔雀石绿(MG)和无色孔雀石绿(LMG)的间接竞争酶联免疫吸附分析法。对无色孔雀石绿分子进行修饰,使其与载体蛋白交联,得到免疫原和包被抗原,经过多次免疫动物制得抗无色孔雀石绿的多克隆抗体。在优化的实验条件下,IC50值(标准曲线中吸光度抑制至最大吸光度值的50%时所对应的待测物浓度)为0.9~2.6μg/L,检出限为0.02~0.10μg/L,无色孔雀石绿在水样及水产品中的回收率为76.2~95.0%,与孔雀石绿的交叉反应率为95.25%。真实样品测定中,两种食用鱼养殖水样及一个鱼样中未检出孔雀石绿和无色孔雀石绿,但在观赏鱼养殖水样及另一鱼样中检出孔雀石绿和无色孔雀石,浓度分别为1.84μg/L和1.38μg/L。     

荧光偏振免疫分析方法分析磺胺二甲基嘧啶

建立了检测磺胺二甲基嘧啶的荧光偏振免疫分析方法。合成了3种结构不同的荧光标记物,并用薄层色谱法提纯。研究了不同结构的荧光标记物对FPIA方法灵敏度的影响。该FPIA方法在缓冲液中的检出限为1.6μg/L,半数抑制量(IC50)为41μg/L,检测范围为5~458μg/L,可以达到食品中SMZ最低残留限量的要求。研究了FPIA的动力学过程及对其它16种磺胺类药物的交叉反应,结果显示,SMZ、磺胺甲基嘧啶和磺胺二甲基恶唑的交叉反应率分别为100%、8.7%和2.4%,其它磺胺类药物的交叉反应率均低于1%。     

抗酮洛芬多克隆抗体的制备及其间接竞争ELISA检测方法的建立

分别将酮洛芬与牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联制得免疫原和包被原,经过免疫新西兰白兔制备多克隆抗体,抗体经纯化后效价为1:128000。使用自制的抗体,建立了测定酮洛芬的间接竞争酶联免疫吸附(ic-ELISA)新方法。ic-ELISA的线性范围为0.010～10.0μg/L,IC50为0.235μg/L,最低检测限为0.0040μg/L,线性回归方程为y=-22.97ρ+104.5(R2=0.980),与布洛芬、双氯酚酸的交叉反应率均小于4%,方法可用于水体中酮洛芬的检测。     

I3-共振瑞利散射法测定孔雀石绿及相关效应分析

在弱酸性介质中,孔雀石绿与I3-形成离子缔合物,并进一步聚集成纳米粒子,在λ=468nm下,激发产生强烈的共振瑞利散射。在0.012～0.900μg/L范围内,共振光强度与孔雀石绿的含量成线性关系;线性方程为y=6273.8x 37.1,r=0.9997;本工作研究了反应的适宜条件,建立了共振瑞利法测定孔雀石绿的新方法,方法灵敏度高,检出限为3.6μg/L。在线性范围内,选择低、中、高3组浓度进行精密度实验,RSD分别为2.3%、1.7%、4.2%(n=11)。     

虾肉中呋喃它酮代谢物化学发光酶免疫分析方法的建立

采用对碘苯酚增强的HRP-鲁米诺-H2O2化学发光体系,建立了虾肉中呋喃它酮代谢物5-吗啉甲基-3-氨基-2-恶唑烷基酮(AMOZ)残留的间接竞争化学发光酶免疫分析(icCLEIA)检测方法。检测所用抗体是基因重组的抗AMOZ衍生物单链抗体。优化的icCLEIA最佳工作条件为:AMOZA-OVA包被浓度62.5μg/L,抗AMOZ衍生物单链抗体最佳稀释度为1∶10,竞争免疫反应时间45 min,HRP酶标记羊抗鼠抗体最佳稀释度为1∶10000,孵育时间50 min。本方法的IC50为1.38μg/L;灵敏度为0.09μg/L;线性范围为0.26～9.08μg/L(IC20～IC80);批内和批间相对标准偏差均小于15%;抗AMOZ衍生物单链抗体与其它硝基呋喃类抗生素及其代谢物均没有交叉反应,特异性良好。4个不同添加量的AMOZ加标样品的平均回收率分别为72.2%,73.4%,72.6%和78.6%。与HPLC-MS/MS法测定值进行比较发现,两种方法相关性良好(R2=0.9997)。本方法可用于水产品中AMOZ残留的快速检测。     

化学发光酶免疫法检测猪肉中氯丙嗪残留

采用棋盘滴定法确定包被抗原浓度和抗体稀释倍数,通过单因素实验优化了竞争反应时间、磷酸盐缓冲液浓度、甲醇含量、pH值等参数,建立了氯丙嗪的间接竞争化学发光酶免疫检测方法,并考察了方法的特异性、灵敏度和稳定性.结果表明,最佳反应条件为包被抗原浓度为0.05 μg/L;氯丙嗪抗体稀释32000倍;体系缓冲液为含10％甲醇、0.1 mol/L磷酸盐缓冲液(pH 7.0).本方法的IC50为0.12 μg/L;检出限为0.02 μg/L;线性范围0.02～24.78 μg/L;批内和批间相对标准偏差均小于10％.与其它结构类似物没有明显交叉反应.猪肉检测的平均回收率为87.4％～105.6％,与高效液相色谱方法的相关性良好.本方法具有较高的灵敏度和较好的稳定性.     

化学发光酶免疫分析测定鱼肉中呋喃它酮代谢物方法研究

建立了呋喃它酮代谢物5-吗啉甲基-3-氨基-2-6唑烷基酮(AMOZ)间接竞争化学发光酶免疫分析(icCLEIA)检测方法,通过单因素实验优化了包被原浓度、抗体稀释倍数、反应缓冲体系及浓度、竞争反应时间等参数,结果表明icCLEIA最佳反应条件为:包被抗原浓度为10 ng/mL,抗体稀释60000倍,最佳竞争时间为50 min,体系缓冲液0.01 mol/L PBS(pH 7.4).在优化的条件下,本方法的线性检测范围为0.026 ～3.52 μg/L,IC50为0.29 μg/L,检出限(LOD,IC10)为0.012 μg/L.对鱼肉样品的平均添加回收率在101.4％～115.5％之间.建立的icCLEIA方法可用于实际样品中AMOZ残留检测.     

酱油与白酒中邻苯二甲酸二甲酯的化学发光酶免疫分析方法研究

本研究以邻苯二甲酸二甲酯( DMP)为研究目标,以4-氨基邻苯二甲酸二甲酯为半抗原,通过重氮化法偶联载体蛋白并免疫动物,制备针对DMP的特异性兔多克隆抗体。通过棋盘滴定法和单因素实验确定最佳的实验参数,即包被原浓度为50μg/L,一抗浓度为92.5μg/L,二抗浓度为1μg/mL,药物稀释液为pH 6.0的纯水,竞争反应时间为40 min,基于此建立了间接竞争化学发光酶联免疫分析法检测DMP。本方法对DMP的检出限为0.29μg/L,线性检测范围为0.74~30.32μg/L,与13种结构和功能类似物的交叉反应率均<5%,通过倍比稀释降低白酒、酱油样品基质干扰,对样品的平均回收率在80.2%~116.0%之间,平均相对标准偏差<3.6%,结果与GC-MS法的测定结果相符。本方法适用于食品样品中DMP的快速检测。     

呋喃唑酮代谢物单克隆抗体制备及酶联免疫吸附分析方法

本研究针对呋喃唑酮代谢物(AOZ),设计合成了系列半抗原,进一步通过偶联牛血清白蛋白(BSA)免疫Balb/c小鼠、细胞融合、筛选和亚克隆等过程成功获得了源于新颖半抗原H3的具有高亲和力(亲和力常数6.68× 1010L/mol)和高特异性(与其它功能类似物交叉反应小于0.1％)抗AOZ单克隆抗体.同时,基于设计合成的系列同/异源半抗原/包被抗原,考察了不同结构包被原对ELISA方法灵敏度的影响.另外,采用最佳的特征结构异源包被原H5 -OVA,建立了以对硝基苯甲醛(p-NP)为衍生剂的AOZ间接竞争ELISA(icELISA)和直接竞争ELISA(deELISA)检测方法.结果表明:icELISA模式的AOZ检测IC50为0.503 μg/L,定量检测线性范围(IC20～IG80)为0.06～14.0 μg/L,检出限(IC10)达0.017 μg/L; dcELISA模式的AOZ检测IC50为1.19 μg/L,定量检测线性范围为0.14～23.6 μg/L,检出限为0.056 μg/L.两种方法对AOZ的检测灵敏度和定量线性范围均达到相关检测限量要求,可满足不同需求的实际样品检测.     

加压毛细管电色谱-激光诱导荧光法检测4种黄曲霉毒素

建立了三氟乙酸( TFA)柱前衍生,加压毛细管电色谱-激光诱导荧光( pCEC-LIF)快速测定黄曲霉毒素B1、B2、G1、G2方法。使用粒径1.8μm的C18毛细管色谱柱,以甲醇-水(45：55, V/V,含0.05%甲酸)为流动相,泵流速为0.05 mL/min,分离电压为15 kV,激发波长为375 nm,发射波长为450 nm,黄曲霉毒素B1, B2, G1, G2达到基线分离。各组分的检出限(S/N=3)分别为0.02,0.016,0.008和0.01μg/L,在0.1~10μg/L,0.1~10μg/L,0.1~3.0μg/L,0.1~3.0μg/L 范围内分别呈线性相关,相关系数 R2分别为0.9999,1.0000,0.9995,0.9997。将本方法应用于花生酱的分析,加标回收率在90.0%~112.0%之间,RSD在0.5%~1.9%之间。     

Simultaneous determination of malachite green, brilliant green and crystal violet in grass carp tissues by a broad-specificity indirect competitive enzyme-linked immunosorbent assay

An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for brilliant green (BG), a triphenylmethane dye with a potential illegal use in fish feeding, was synthesized and used to produce polyclonal antibody (PcAb) against BG. Unexpectedly, the obtained PcAb showed high cross-reactivity (CR) to malachite green (MG) and crystal violet (CV) in an indirect competitive enzyme-linked immunosorbent assay (icELISA). After screening against three heterologous coating antigens, the icELISA exhibited good sensitivity and uniform response to BG (IC(50) of 1.98 ng mL(-1) and CR of 100%), MG (IC(50) of 1.61 ng mL(-1) and CR of 105%) and CV (IC(50) of 1.34 ng mL(-1) and CR of 142%) when using (4-(carboxymethoxy)phenyl)bis(4-(dimethylamino)phenyl)methylium as the coating hapten. Therefore, a broad-specificity icELISA for simultaneous determination of BG, MG and CV was developed. The recoveries of single analyte and mixture of three analytes from spiked grass carp tissues were estimated ranging from 74.94% to 110.39%. A statistically significant correlation of results was obtained between the developed icELISA and previously established HPLC approaches with the food-relevant three triphenylmethane dyes concentration range 1.83-200 ng mL(-1) (R(2)=0.9224), indicating good accuracy of the icELISA and suitability for the broad-specific detection of the three triphenylmethane dyes in grass carp tissues.     

细交链孢菌酮酸酶联免疫吸附分析方法研究

采用水合肼和乙醛酸依次对细交链孢菌酮酸(Tenuazonic acid,TeA)进行衍生化,设计合成了含有氮杂共轭双键偶联手臂,可增强免疫效果的半抗原TeAHGA.通过偶联载体蛋白BSA后的免疫原TeAHGABSA免疫新西兰大白兔,成功制备了特异性识别TeA水合肼衍生物TeAH的多克隆抗体;优化确立了ELISA最佳反应条件(TeAH-OVA为异源包被原、包被浓度0.156 μg/L、药物稀释及反应缓冲液为PBS、一抗反应时间40 min、二抗反应时间20 min),建立了TeA间接竞争ELISA(icELISA)检测方法,其抑制中浓度(IC50)为1.61 μg/L,检出限(LOD)为0.08 μg/L,定量线性检测范围为0.19～12.89 μg/L (IC20～IC80).番茄、面粉样品平均添加回收率分别为67.2％～89.8％和74.8％～93.7％.     

酸性红73多克隆抗体的制备及其应用

在酸性红73分子的羟基上引入一个带有羧基的“间隔臂”,采用N-羟基琥珀亚胺活性酯法将酸性红73分别与牛血清白蛋白(BSA)、卵清蛋白(OA)偶联,合成免疫原和包被原,经免疫新西兰白兔获得多克隆抗体,所得抗体最大效价可达2.56×105,建立了酸性红73的间接竞争ELISA检测方法.本方法的半数抑制浓度(IC50)为181.2 μg/L,检出限(LOD)为7.9 μg/L.交叉反应实验表明,除苏丹红3号(1.13％)外,抗AR73抗体与其它竞争物均无交叉反应.在虾仁中的空白添加回收率为63.5％～90.7％,RSD＜6.8％.说明本方法可用于虾仁中酸性红73的残留检测.     

Monoclonal antibody-based enzyme-linked immunosorbent assay for detection of total malachite green and crystal violet residues in fishery products

A rapid easy-to-use trace level direct competitive enzyme-linked immunosorbent assay (dc-ELISA) detection of total residual malachite green (MG), crystal violet (CV) and their corresponding primary metabolites leucomalachite green (LMG) and leucocrystal violet (LCV) in fishery products in a single assay was developed. The monoclonal antibodies, anti-MG and anti-CV mAbs, were prepared using carboxyl-malachite green (CMG) and cationized bovine serum albumin (cBSA) conjugates as immunogen. The linear range for the quantitative detection of total MG, CV and their primary metabolites LMG and LCV was between 0.15 to 4.5?ng?mL^?1 with a half maximal inhibitory concentration (IC₅₀) at 0.56?±?0.04?ng?mL^?1 (n?=?5). The anti-MG mAbs exhibited 98% cross-reactivity to CV, less than 0.1% cross-reactivity with LMG and LCV, and no cross-reactivity with chloramphenicol, enrofloxacin, sulfadiazine, and tetracycline. Application of the dc-ELISA in fish tissue samples gave a limit of detection (LOD) of 0.37?ng?g^?1. The improved total detection lead to a recovery of 74.60?±?8.38% at 0.5?ng?g^?1 and 87.47?±?12.83% at 2.0?ng?g^?1 that was better than existing techniques. The dc-ELISA showed total MG in 7 out of 44 field fish samples that were confirmed with LC-MS/MS. The easy-to-use, inexpensive, and rapid dc-ELISA for the detection of total MG, CV and their corresponding primary metabolites holds promise for field applications.     

免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

研究以双特异性核酸适配体A3作为传感探针、纳米金（AuNPs）为指示剂、NaCl溶液为聚集诱导剂,构建了一种新型的免标记AuNPs比色生物传感器,可实现水产品中孔雀石绿（MG）和无色孔雀石绿（LMG）的同步、快速、可视化检测。该方法的检测原理是核酸适配体A3对MG和LMG有双特异性识别能力,可作为MG和LMG理想的识别受体。它可通过静电作用吸附到AuNPs表面,保护AuNPs并抑制高盐溶液诱导的聚集,AuNPs溶液颜色不变,即为红色;当加入靶标MG或LMG后,该核酸适配体能够与靶标特异性结合,并从AuNPs表面上解离,AuNPs失去保护作用而在高盐溶液诱导下发生聚集,溶液颜色由红变蓝。根据颜色变化,可通过肉眼定性或通过光谱仪定量分析MG和LMG的残留量。该方法首先将50 μL的核酸适配体A3（终浓度150 nmol/L）与150 μL的AuNPs（终浓度1.25 nmol/L）混合,室温孵育6 min。随后加入50 μL待测液,室温孵育30 min。最后加入50 μL NaCl（终浓度150 mmol/L）,4 min后观察溶液颜色变化,并分别测定MG和LMG在520 nm和650 nm下的吸光度值。结果表明,在最佳反应条件下,该方法能够特异性检测MG和LMG,而对磺胺嘧啶（SDZ）和硝基呋喃妥因（NFT）无交叉反应;当MG、LMG的浓度为0~17.5 μmol/L时,吸光度比值与靶标浓度呈现良好的线性关系,相关系数（R ² ）分别为0.9938和0.9715。MG和LMG的检出限分别为6.93 nmol/L和6.38 nmol/L,加标回收率分别为88.60%~93.30%和101.80%~107.00%,相对标准偏差（RSD）分别为2.27%~3.55%和2.62%~3.75%。该方法操作简单,快速和灵敏,可为水产品中MG和LMG的同步快速检测提供一种新方法。     

Development of a sensitive monoclonalantibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L. It has been widely used in South-East Asia and Africa as an effective drug against sensitive and multidrug-resistant Plasmodium falciparum malaria. A monoclonal antibody (mAb), designated as 3H2, was generated with artesunate–bovine serum albumin conjugate as the immunogen. mAb 3H2 was used to develop a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemisinin. The concentration of analyte producing 50% of inhibition (IC₅₀) and the working range of the icELISA were 1.3 and 0.2–5.8 ng/mL, respectively. The mAb 3H2 recognized the artemisinin analogs artesunate, dihydroartemisinin, and artemether with cross-reactivity of 650%, 57%, and 3%, respectively, but negligibly recognized deoxyartemisinin and the artemisinin precursors arteannuin B and artemisinic acid. The average recoveries of artemisinin fortified in A. annua samples at concentrations from 156 to 5,000 μg/g determined by icELISA ranged from 91% to 98%. The icELISA was applied for the determination of artemisinin in different wild A. annua samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis. The correlation coefficient (R ²) between the two assays was larger than 0.99, demonstrating a good agreement between the icELISA and HPLC results. This ELISA is suitable for quality assurance of A. annua L. materials. Figure  Artemisia annua plant and antimalarial drugs derived from artemisinin     

Indirect Competitive ELISA for the Determination of Total Chromium Content in Food,Feed and Environmental Samples

Background: This study aimed to prepare monoclonal antibodies (mAbs) with high immunoreactivity, sensitivity, and specificity for the chelate (Cr(III)-EDTA) of trivalent chromium ion (Cr(III)) and ethylenediamine tetraacetic acid (EDTA). Further, the study established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for detecting the total chromium content in food, feed, and environmental samples. Methods: Hapten Cr(III)-iEDTA was synthesized by chelating Cr(III) with isothiocyanatebenzyl-EDTA (iEDTA). Immunogen Cr(III)-iEDTA-BSA formed by chelating Cr(III)-iEDTA with bovine serum albumin (BSA), and coating antigen Cr(III)-iEDTA-OVA formed by chelating Cr(III)-iEDTA with ovalbumin (OVA) were prepared using the isothiocyanate method and identified by ultraviolet spectra (UV) and inductively coupled plasma optical emission spectrometry (ICP-OES). Balb/c mice were immunized with the Cr(III)-iEDTA-BSA, and the anti Cr(III)-EDTA mAb cell lines were screened by cell fusion. The Cr(III)-EDTA mAbs were prepared by induced ascites in vivo, and their immunological characteristics were assessed. Results: The immunogen Cr(III)-iEDTA-BSA was successfully synthesized, and the molecular binding ratio of Cr(III) to BSA was 15.48:1. Three hybridoma cell lines 2A3, 2A11, and 3D9 were screened, among which 2A3 was the best cell line. The 2A3 secreted antibody was stable after six passages, the affinity constant (Ka) was 2.69 × 10⁹ L/mol, its 50% inhibition concentration (IC50) of Cr(III)-EDTA was 8.64 μg/L, and it had no cross-reactivity (CR%) with other heavy metal ion chelates except for a slight CR with Fe(III)-EDTA (1.12%). An icELISA detection method for Cr(III)-EDTA was established, with a limit of detection (LOD) of 1.0 μg/L and a working range of 1.13 to 66.30 μg/L. The average spiked recovery intra-assay rates were 90% to 109.5%, while the average recovery inter-assay rates were 90.4% to 97.2%. The intra-and inter-assay coefficient of variations (CVs) were 11.5% to 12.6% and 11.1% to 12.7%, respectively. The preliminary application of the icELISA and the comparison with ICP-OES showed that the coincidence rate of the two methods was 100%, and the correlation coefficient was 0.987. Conclusions: The study successfully established an icELISA method that meets the requirements for detecting the Cr(III)-EDTA chelate content in food, feed, and environmental samples, based on Cr(III)-EDTA mAb, and carried out its preliminary practical application.     

Development of a group selective molecularly imprinted polymers based solid phase extraction of malachite green from fish water and fish feed samples

A group selective molecularly imprinted solid phase extraction (MISPE) for malachite green (MG) from fish water and fish feed samples was developed. Using MG as template molecule, methacrylic acid as functional monomer, ethylene glycoldimethacrylate as linking agent and bulk polymerization as synthetic method, the molecularly imprinted polymers (MIPs) were synthesized and characterized with rebinding experiment. The Scatchard polt's analysis revealed that the template-polymer system showed the two-site binding behavior with dissociation constants of 0.3194 μmol L⁻¹ and 15.70 μmol L⁻¹, respectively. MG and two structurally related compounds, leucomalachite green (LMG) and crystal violet (CV) were employed for selectivity test. The MIPs exhibited the highest selective rebinding to MG, but also displayed 83.0% and 87.5% of cross-reactivity with LMG and CV, demonstrating that MIPs could be used as group recognition sorbents in solid phase extraction. The extraction conditions of MISPE column for MG were optimized. Tap water samples spiked with MG at concentration of 0.5-10 ng mL⁻¹ were extracted by MISPE column and analyzed by high performance liquid chromatography. The recoveries of MISPE column for MG extraction were found to be 76.8-93.7% with the relative standard deviations of 2.12-10.09%, indicating the feasibility of the prepared MIPs for MG extraction. No detectable MG was observed in one fish farming water sample and two fish feed samples; while the MG concentrations in two pet fishpond water samples were found at 1.50 ng mL⁻¹ and 0.67 ng mL⁻¹, respectively.