倍硫磷半抗原、人工抗原的合成和鉴定

为研究建立有机磷农药倍硫磷的免疫分析法,合成了1种倍硫磷半抗原和2种人工抗原。采用改进的方法将三氟硫磷与3-甲基-4-甲硫基苯酚、β-丙氨酸反应制备了倍硫磷半抗原,产物经薄层层析、^1HNMR和质谱鉴定为预期产物。分别将半抗原以活泼酯法、混合酸酐法与BSA、OVA偶联制备了人工抗原BZB和BGO,通过紫外光谱鉴定证明偶联成功。用TNBS比色法测定了BZB、BGO与载体蛋白的克分子结合比,分别为29：1和12：1。以BZB为免疫原免疫Balb／c小鼠,以BGO为包被抗原对小鼠血清进行间接竞争ELISA分析表明,小鼠经免疫后产生了针对倍硫磷的特异性抗血清,从而进一步证明抗原合成成功。本方法为研究建立倍碲磷的免疫分析方法奠定了基础。     

莱克多巴胺和土霉素人工抗原的合成及鉴定

研究莱克多巴胺和土霉素免疫抗原的制备。实验中用碳二亚胺法将莱克多巴胺(ractopamine,RAC)与载体蛋白牛血清白蛋白(bovine serum albumin,BSA)偶联,制备完全抗原;采用混合酸酐法将土霉素(oxytetracycline,OTC)与载体蛋白BSA偶联,制备完全抗原,并对合成产物进行表征。紫外扫描对合成产物RAC-BSA、OTC-BSA进行鉴定结果显示偶联成功,经重氮化后利用混合酸酐法制备完全抗原的偶联比为5:1;经衍生后利用碳二亚胺法制备RAC完全抗原偶联比为7:1。利用制备的免疫原免疫兔子可以获得相应的抗体。     

膝沟藻毒素GTX 2,3完全抗原的合成与鉴定

膝沟藻毒素GTX 2,3是一类小分子物质,不具备免疫原性,欲制备其免疫血清及抗体,则必须与偶联剂及载体蛋白偶联形成完全抗原。运用乙二醛作为偶联剂,分别与牛血清白蛋白(BSA)、血蓝蛋白(KLH)合成免疫抗原GTX 2,3-glyoxal-BSA和检测抗原GTX 2,3-glyoxal-KLH,并对经超滤纯化浓缩后的偶联产物进行BCA蛋白浓度测定、全波段紫外扫描、变形SDS-PAGE凝胶电泳和抗原免疫原性的鉴定。结果表明,免疫抗原和检测抗原的蛋白质量浓度分别为8.52 mg/m L和5.72 mg/m L;经全波段紫外扫描和变形SDS-PAGE凝胶电泳鉴定,半抗原GTX 2,3-乙二醛与载体蛋白BSA、KLH偶联成功,偶联比约为1︰16和1︰20,并制备出纯度较高的免疫抗原;用制备的人工抗原GTX 2,3-glyoxal-BSA对Balb/C小鼠进行免疫,通过间接ELISA检测6免后小鼠血清效价达到1︰20 000。成功制备了具有较强免疫原性的膝沟藻毒素GTX 2,3人工抗原。     

磺胺喹恶啉人工抗原合成及鉴定

国内首次用重氮化法将磺胺喹恶啉(SQ)分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联,制备免疫抗原SQ-BSA和包被抗原SQ-OVA.紫外扫描法与SDS-PAGE凝胶电泳法鉴定表明偶联成功,偶联比分别为10.1∶1、9.8∶1.将SQ-BSA免疫BALB/C小鼠,获得了高效价、敏感性好的抗SQ血清,表明ELISA方法鉴定偶联成功,为进一步制备抗SQ单抗奠定了基础.     

杀菌剂抑霉唑人工抗原的合成和鉴定

以咪唑乙醇为原料,与6- 溴己酸乙酯发生取代反应后再经水解反应合成抑霉唑半抗原(6-[1-(2,4- 二氯苯基)-2-(1- 咪唑基)乙氧基]己酸)。产物经薄层层析和1H-NMR 鉴定后,采用活化酯法分别与牛血清蛋白(BSA)和卵清白蛋白(OVA)偶联得到免疫原(HIA-BSA)和包被原(HIA-OVA),紫外光谱分析法计算得其偶联比分别为13:1 和7:1,初步说明人工抗原合成成功。通过免疫动物获得效价为1:64000 的抗体,采用间接竞争ELISA 法测定抗体的IC50 值为1.76μg/mL,从而进一步证明人工抗原合成成功,所得的抗体可用于ELISA 检测试剂盒的研制。     

两种司帕沙星完全抗原的合成鉴定及免疫效果研究

目的:用两种方法合成司帕沙星人工抗原,并制备相应的鼠源多克隆抗体。方法:用混合酸酐法和DCC法将半抗原SPFX与BSA偶联制备免疫原SPFX-BSA,通过DCC法制备人工包被原SPFX-OVA,采用紫外光扫描和聚丙烯酰胺凝胶电泳进行鉴定与分析。免疫小鼠后,采用间接ELISA法测定多抗血清效价,阻断ELISA,鉴定其抑制价和特异性。结果:UV和SDS-PAGE鉴定结果初步表明SPFX人工抗原成功合成;动物试验表明,4只小鼠多抗血清效价均在3.2×104以上,其中1号小鼠IC50值为163.87 ng/mL,与其他竞争物无明显交叉反应,抗体特异性良好。结论:两种偶联方法均能成功合成人工抗原,制备出鼠源抗SPFX多克隆抗体,其中混合酸酐法能明显提高完全抗原的合成和免疫效果。     

河豚毒素人工抗原的制备及其免疫原性鉴定

目的:选择血蓝蛋白(KLH)和牛血清白蛋白(BSA)分别作为载体蛋白与河豚毒素(TTX)进行偶联制备人工抗原,并对合成结果进行鉴定。方法:通过甲醛法将TTX分别与大分子载体蛋白,即血蓝蛋白(KLH)和牛血清白蛋白(BSA)进行偶联。利用紫外扫描法、电泳法对偶联产物进行鉴定,并对6周龄Balb/c雌性小鼠免疫注射检测其免疫原性。结果:紫外扫描显示偶联抗原较载体蛋白有轻微的红移现象出现,且经过琼脂糖凝胶及SDS-PAGE 2种方法电泳检测后发现,载体蛋白较人工抗原之间迁移率均不相同;对免疫小鼠进行间接酶联免疫吸附试验(iELISA)及间接竞争酶联免疫吸附试验(icELISA)联用检测其多抗血清效价及特异性,经过共计6次免疫后的效价能达到1:40w,且能有效抑制TTX。结论:河豚毒素人工抗原制备成功且具备良好的免疫原性。     

格列本脲人工抗原的合成与鉴定

目的：合成格列本脲人工抗原。方法：采用对氨基苯甲酸法制备半抗原,将半抗原分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)通过碳二亚胺法偶联制备免疫原(Gli-BSA)和包被原(Gli-OVA),利用紫外扫描进行抗原的化学鉴定,通过免疫原免疫Balb/c小鼠,间接酶联免疫吸附法测定抗血清进行生物鉴定。结果：制备了Gli-BSA、Gli-OVA的人工抗原,经紫外光谱扫描,偶联比分别为4:1和17.7:1。免疫小鼠后获得抗血清的效价均达到32000以上,半抑制质量浓度为10μg/mL。结论：成功合成了格列本脲人工抗原,并获得了格列本脲抗体,为格列本脲的免疫学检测方法进一步研究提供了实验基础。     

三唑磷多克隆抗体的制备及鉴定

采用活泼酯法将三唑磷半抗原(TZPM-Hap)与牛血清蛋白(BSA)和卵血清蛋白(OVA)偶联,制备出免疫抗原(TZPM-A-BSA)和包被抗原(TZPM-A-OVA),经紫外扫描证明人工抗原合成成功.用免疫抗原TZPM-A-BSA免疫雌性Balb/c小鼠,获得了特异性的三唑磷多克隆抗体.以所获得的抗体建立间接竞争酶联...     

伏马菌素B2人工抗原的制备研究

为制备伏马菌素B2人工抗原,通过戊二醛法和碳二亚胺法将伏马菌素B2偶联到栽体蛋白上,人工抗原免疫BALB/c小鼠后,酶联免疫吸附法测定抗血清的效价,竞争酶联免疫吸附法测定人工抗原的免疫原性.通过氨基化和羧基化的苏丹红作为内参,紫外可见光谱扫描图谱鉴定伏马菌素B2成功偶联到载体蛋白上,免疫原性鉴定证明,碳二亚胺法制备的人工抗原可刺激BALB/c小鼠产生抗伏马菌素B2的抗体.     

气相色谱法检测饮料中二羰基化合物

建立同时在线检测丙酮醛和乙二醛的气相色谱方法。确定检测二羰基化合物的最佳条件为：以丁二酮为内标,以邻苯二胺为衍生化试剂,邻苯二胺的用量67 倍于二羰基化合物、衍生化时间10 min、萃取溶剂二氯甲烷、超声时间15 min、萃取2 次、柱箱初始温度40 ℃、程序升温5 ℃/min,色谱柱载气流量2.0 mL/min,分流比1∶1。丙酮醛和乙二醛的定量限（RSN≈10）分别为0.06 mg/L和0.08 mg/L,检出限（RSN≈ 3）分别为0.02 mg/L和0.03 mg/L,方法灵敏度高。     

Trapping reactions of reactive carbonyl species with tea polyphenols in simulated physiological conditions

The carbonyl stress that leads to the formation of advanced glycation end products (AGEs) in diabetes mellitus has drawn much attention recently. Reactive alpha-dicarbonyl compounds, such as glyoxal (GO) and methylglyoxal (MGO), have been shown to be a high potential glycation agent in vitro and in vivo. In this study, epicatechins in green tea and theaflavins in black tea were found to be able to reduce the concentration of MGO in physiological phosphate buffer conditions. Modified MGO derivatization for GC/flame ionization detector (FID) method in quantification was systematically conducted. In molar ratio of 3 (MGO/polyphenol), theaflavin-3,3'-digallate (TF3) in theaflavins and (-)-epigallocatechin (EGC) in epicatechins showed the highest MGO reduction at 66.65 and 45.74%, respectively, after 1 h of incubation. In kinetic study (molar ratio of MGO/polyphenol = 1:1), rapid MGO reduction occurred within 10 min. Identities of primary adducts between (-)-epigallocatechin gallate (EGCG) and MGO were determined. Newly generated stereoisomers at the C8 position of EGCG A-ring were isolated with a chiral column, and structurally confirmed by 2-D NMR analyses.     

杨梅提取物抑制二羰基化合物活性研究

目的 分离纯化杨梅果实提取物并考察不同组分对模型及饼干中甲基乙二醛(methylglyoxal, MGO)、乙二醛(glyoxal, GO)的抑制效果。方法 乙醇浸提制备杨梅果实粗提物(crude extract, CE),采用AB-8大孔吸附树脂,以不同体积分数的乙醇(20%、40%、60%)洗脱,得到三种杨梅提取物;采用NaNO₂- Al(NO₃)₃-NaOH比色法、pH示差法、液相色谱-质谱法测定提取物中花色苷、总黄酮含量和主要成分,并用气相色谱法考察其对模型和曲奇中MGO/GO的抑制活性。结果 40%乙醇组分F₁得率最高,活性最好,主要成分为矢车菊素-3-O-葡萄糖苷(cyanidin-3-O-glucoside, C-3-Glu)和杨梅素-3-O-鼠李糖苷(myricetin-3-O-rhamnoside, M-3-Rha);60%乙醇洗脱组分F₂成分主要为槲皮素-3-O-鼠李糖苷(quercetin-3-O-rhamnoside, Q-3-Rha);当曲奇中提取物添加量为0.4%时,F_1、F₂对MGO和GO均有抑制活性,其中F₁效果最佳,分别为61.5%和40.4%。结论 杨梅果实提取物具有良好清除MGO/GO的能力,为其作为添加剂应用于抑制食品加工中的MGO和GO提供了一定的理论基础。     

Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand

The 1,2-dicarbonyl compounds 3-deoxyglucosulose (3-DG), glyoxal (GO), and methylglyoxal (MGO) were measured as the corresponding quinoxalines after derivatization with orthophenylendiamine using RP-HPLC and UV-detection in commercially available honey samples. Whereas for most of the samples values for 3-DG, MGO, and GO were comparable to previously published data, for six samples of New Zealand Manuka (Leptospermum scoparium) honey very high amounts of MGO were found, ranging from 38 to 761 mg/kg, which is up to 100-fold higher compared to conventional honeys. MGO was unambigously identified as the corresponding quinoxaline via photodiodearry detection as well as by means of mass spectroscopy. Antibacterial activity of honey and solutions of 1,2-dicarbonyl towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were analyzed using an agar well diffusion assay. Minimum concentrations needed for inhibition of bacterial growth (minimum inhibitory concentration, MIC) of MGO were 1.1 mM for both types of bacteria. MIC for GO was 6.9 mM (E. coli) or 4.3 mM (S. aureus), respectively. 3-DG showed no inhibition in concentrations up to 60 mM. Whereas most of the honey samples investigated showed no inhibition in dilutions of 80% (v/v with water) or below, the samples of Manuka honey exhibited antibacterial activity when diluted to 15-30%, which corresponded to MGO concentrations of 1.1-1.8 mM. This clearly demonstrates that the pronounced antibacterial activity of New Zealand Manuka honey directly originates from MGO.     

用于酶热传感器的酶标甲胺磷农药的制备

采用碳二亚胺(EDC)法进行酶标甲胺磷的制备研究。考察甲胺磷和丁二酸酐比例、反应时间对甲胺磷衍生物合成的影响,最终确定二者的最适物质的量比为1:1.5,最适反应时间为15h;甲胺磷衍生物与葡萄糖氧化酶比例对酶标甲胺磷偶联率及酶活的影响实验结果显示,随着甲胺磷衍生物与葡萄糖氧化酶二者比例的增加,酶标农药的偶联率增加,但同时也造成了酶活的降低。考虑到酶标农药还应具备足够的酯酶抑制活性,甲胺磷衍生物与葡萄糖氧化酶二者物质的量比为10:1时制得的酶标农药为最优。     

Changes in glyoxal and methylglyoxal content in the fried dough twist during frying and storage

Glyoxal (GO) and methylglyoxal (MGO) are important precursors for advanced glycation end products (AGEs) in foods and in vivo, which are also known as reactive carbonyls. Accumulation of AGEs may result in health risk. The effect of frying time and temperature on GO and MGO formation during fried dough twist processing was investigated. The changes in GO and MGO content of fried dough twist were also examined under the different storage condition. Sample extraction, derivatization, and cleanup conditions were evaluated to provide a sample preparation method for GO and MGO analysis by RP-HPLC–DAD. Quantification limits of GO and MGO were 0.01 and 0.02 mg/L for fried dough twist, respectively. The average recovery rate was 89 % with detection relative standard deviation of lower than 5.7 %. During frying and prolonged storage, GO and MGO contents in fried dough twist ranged from 6.5 to 11.8 mg/kg and from 5.1 to 12.0 mg/kg, respectively. The results indicated that content of GO and MGO in fried dough twist depended on the frying time and temperature. For GO, no significant differences (p < 0.05) among the all types of samples were found during the whole process of storage, with the exception of storage temperature after 60-day storage. Changes in MGO occurred during the 60-day storage, indicating storage temperature, oxygen content, lighting, and storage duration, exert a significant effect on the development of the MGO.     

Preparation of 1-Amino-1-deoxyfructose Derivatives by Stepwise Increase of Temperature in Aqueous Medium and Their Flavor Formation Compared with Maillard Reaction Products

A novel method of Maillard reaction performed under stepwise increase of temperature was proposed for the preparation of Maillard reaction intermediate (MRI) derived from fructose (Fru) and phenylalanine (Phe). The optimal formation conditions of MRI in aqueous medium were determined as follows: pH 7.4 and heating for 100 min at 100 °C. The purified MRI was characterized to be 1-amino-1-deoxyfructose derivative (C₁₅H₂₁NO₇, 327 Da) by mass spectrometry and NMR. The methodological effectiveness of this new developed method was further verified. Although almost similar types of volatile compounds were identified in the three heated solutions of Fru-Phe mixture, Maillard reaction products (MRPs) and 1-amino-1-deoxyfructose derivatives, the concentration of total volatile compounds was 118.30 μg/L relative to the internal standard for Fru-Phe mixture and increased by 64.49 and 1024.64 μg/L for MRPs and 1-amino-1-deoxyfructose derivative, respectively. The results showed that more fresh flavors were controlled formed from 1-amino-1-deoxyfructose derivative compared with those from MRPs or Fru-Phe mixture during the subsequent heating treatment; thus, the 1-amino-1-deoxyfructose derivatives could be a great potential substitute of unstable MRPs or Fru-Phe mixture in preparation of flavoring.     

没食子酸酯类化合物对食品中1,2-二羰基产物的清除效果

采用气相色谱法考察食品抗氧化剂没食子酸酯（没食子酸丙酯（propyl gallate,PG）、没食子酸辛酯（octyl gallate,OG）、没食子酸十二酯（dodecyl gallate,DG））对高活性糖基化因子1,2-羰基化合物（乙二醛（glyoxal,GO）、甲基乙二醛（methylglyoxal,MGO））的清除效果,并考察PG/OG/DG浓度、体系pH值及反应时间对清除效果的影响。此外,通过高效液相色谱-串联质谱（high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS）技术探究清除机理。结果表明：PG、OG、DG能有效清除GO和MGO,清除效果依次为PG＞OG＞DG;当清除剂浓度为0.5 mmol/L时,PG对GO、MGO清除率均分别为70.5%、67.6%,OG、DG对GO和MGO的清除率也均达到50%以上;且随着清除剂浓度增大、体系pH值提高、反应时间延长,PG、OG、DG对GO和MGO清除能力增强;在精氨酸-葡萄糖体系、食品体系中验证了清除效果,并在曲奇饼干体系中证明其清除机理为：PG、OG均可捕获1 分子GO或MGO形成相应加合产物,DG可捕获1 分子GO形成加合产物。可见,没食子酸酯类化合物在食品加工过程中能有效清除外源性1,2-羰基化合物,提高食品安全性。     

超高效液相色谱法快速检测麦卢卡蜂蜜中丙酮醛

目的利用超高效液相色谱建立一种麦卢卡蜂蜜中丙酮醛的快速检测方法。方法采用乙酸钠缓冲溶液直接提取蜂蜜中的丙酮醛,提取液与2,4-二硝基苯肼溶液室温避光衍生20 h后,经C18色谱柱分离后采用二极管阵列检测器(PDA)检测,外标法定量。结果丙酮醛的检出限为10.0 mg/kg,定量限为25.0 mg/kg,在1.0~60 mg/L之间线性关系良好,相关系数为0.9982,在蜂蜜中添加25.0、500、1000三个水平的回收率在93.9%~97.8%之间,相对标准偏差在1.8%~5.4%之间。结论 该分析方能够满足麦卢卡蜂蜜中丙酮醛的快速测定要求。