动物性食品中盐酸克伦特罗ELISA检测方法的建立及应用

目的:建立动物性食品中盐酸克伦特罗残留快速检测技术,确保食品安全.方法:以盐酸克伦特罗重氮化后分别连接到牛血清蛋白(BSA)和卵清蛋白(OVA)上制得免疫原BSA-CL和包被抗原OVA-CL.通过免疫得到多抗血清,经硫酸铵沉淀,Sephrose 4B-proteinA对抗体进行纯化,在此基础上建立间接竞争ELISA方法.结果表明:包被抗原的最适质量浓度为10 μg/mL,抗体的最佳稀释度为1∶1000;该方法的IC50为2.18 ng/mL,最低检测限达0.05 ng/mL,与沙丁胺醇和妥洛特罗的交叉反应率分别为8.28％和7.75％,平均加标回收率为90.78％.结论:该法灵敏度高,样品前处理简便、快速,适用于动物源性食品中盐酸克伦特罗残留的大批量现场检测.     

金磁微粒介导多克隆抗体纯化的最优化条件研究

将牛血清白蛋白(BSA)包被在自制的金磁微粒表面,以金磁微粒与抗体比例、反应时间、反应温度为主要影响因素,依据均匀设计表,对盐酸克伦特罗(CL)多克隆抗体进行纯化,并对纯化前后抗体的效价以及抗体对CL的检测灵敏度进行了研究。结果表明,金磁微粒与抗体的比例以及两者的反应时间是影响抗体纯化的主要因素;纯化后多克隆抗体中抗BSA抗体明显下降,而抗CL抗体基本保持不变;对比纯化前后CL的检测曲线,纯化后的最低检测限(LOD)和半抑制浓度(IC50)分别下降了6.90%和24.37%。     

盐酸克伦特罗快速检测胶体金试纸的研制

为建立快速检测猪尿等样品中盐酸克伦特罗（CL）的胶体金免疫层析方法,用柠檬酸三钠还原氯金酸制备了胶体金,将其标记抗CL单克隆抗体,制备了金标抗体;以CL-人血清白蛋白（HSA）为包被抗原、羊抗鼠IgG为质控线二抗,制成胶体金试纸。优化了胶体金颗粒粒径、标记抗体用量和pH值等各项参数,最终确定胶体金粒径为15 nm、每毫升胶体金溶液中添加20 ?g抗体、胶体金溶液pH值为7.4、金标抗体稀释液为添加0.1 %牛血清白蛋白（BSA）的0.05 mol/L磷酸盐缓冲液（pH 7.4）、金标抗体喷涂量为50 ?L/cm2、CL-HSA和羊抗鼠IgG的包被浓度分别0.5 mg/mL和2 mg/mL。研制的CL胶体金试纸检测限为3 ng/mL,与莱克多巴胺、沙丁胺醇等六种?-兴奋剂类药物无交叉反应。对42份猪尿样品的检测结果与市售酶联免疫（ELISA）试剂盒的符合率为100 %。试纸无需仪器辅助,操作简便,可在5 min内完成,适用于对CL残留进行现场检测。     

Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean + 3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 µg kg^-1. Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 µg kg^-1. The detection capability (CC_β), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 µg kg^-1. Incurred prawn samples (n = 8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 µg kg^-1 were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 µg kg^-1 and a CC_β of below 0.4 µg kg^-1.     

ELISA检测盐酸克伦特罗残留的方法学评价

以盐酸克伦特罗(clenbuterolhydrochloride)重氮化后分别连接到牛血清蛋白(BSA)和卵清蛋白(OVA)上制得免疫原BSA-CL和包被抗原OVA-CL。通过免疫兔获得含有多克隆抗体的血清,经硫酸铵沉淀、纯化,得到兔源抗CL的抗体,在此基础上建立了间接酶联免疫检测方法。实验结果表明,抗CL抗体最适稀释度为1:1000,羊抗兔酶联抗体(HRP-IgG)的最适稀释度为1:1500。该检测方法的检测灵敏度为1.452μg/L,线性检测范围为7.26～90.75μg/L。     

动物源性食品及生物材料中克伦特罗测定方法的研究进展

克伦特罗是一种非法的饲料药物添加剂，含有其残留的食品可引起食物中毒。为给我国检测食品中盐酸克伦特罗提供借鉴，从样品提取、净化、检测方法及其测定的发展趋势等方面综述了国内外献中动物性食品及生物材料中的克伦特罗残留测定方法。     

Determination of clenbuterol in UHT milk in Turkey

Clenbuterol use is linked to its ability to induce weight gain and a greater proportion of muscle to fat. Clenbuterol residues can affect lung and heart function in persons who have eaten liver or meat of animals which were given the drug. Any residue of clenbuterol is regarded unacceptable in the EU. The purpose of the study was to determine the occurrence of clenbuterol in UHT milk samples in Turkey. The occurrence of hormone residues in Ultra Heat Treatment (UHT) milk samples was investigated by semi‐quantitative enzyme‐linked immunoassay technique. There was a high incidence rate of clenbuterol, with forty‐one (68.3%) milk samples being contaminated. 21.7% of the samples were over the permissible level for clenbuterol as accepted by the EU. Clenbuterol levels in the samples purchased in Central Anatolia Region appear to be serious public health problem at the moment.     

旋转生物传感器高灵敏检测盐酸克伦特罗方法研究

盐酸克伦特罗(clenbuterol hydrochloride, CL),又名双氯醇胺、氨哮素、克喘素,为白色结晶状粉末, 无味略苦,是一种人工合成的β2-肾上腺素受体激动剂。盐酸克伦特罗又称"瘦肉精",被非法用于肉用动物生产。彻底查禁CL 的非法滥用,必须要有高效、灵敏、特异的残留检测方法。本研究着重应用免疫学和生物化学方法,构建了基于量子点标记的ATP合酶分子马达免疫旋转生物传感器 (immuno-rotary biosensor, IRB),结合荧光技术,利用中国科学院生物物理研究所张仲伦老师研发的BPCL弱光检测器实现了对盐酸克伦特罗快速、高灵敏度的检测。     

磁性化学发光酶免疫法检测猪肉中的氯霉素

建立以磁性微球为固定相的化学发光酶免疫法,定量检测猪肉中的氯霉素含量。此方法为在微孔中加入羊抗鼠磁性微球、样品和抗体,反应一定时间,之后补加酶标抗原,继续反应一定时间后清洗磁珠,加入发光液反应10 min后分析测定。通过磁珠用量、酶标抗原量、抗体量和反应模式的优化,成功建立了一种高灵敏度、高准确度的检测猪肉中氯霉素的磁性化学发光酶免疫方法。该方法的检出限为0.013μg/kg,线性范围在0.060~2.421μg/kg之间,回收率在85.24%~93.57%之间,相对标准偏差小于11.5%。     

Aflatoxin and ochratoxin A content of spices in Hungary

In October and November 2004, 91 spice samples (70 ground red pepper, six black pepper, five white pepper, five spice mix and five chilli samples), the majority of which originated from commercial outlets, were analysed for aflatoxins B₁, B₂, G₁ and G₂ (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) content by high-performance liquid chromatography (HPLC) after immunoaffinity column clean-up. Eighteen of the 70 ground red pepper samples contained AFB1, seven of them in a concentration exceeding the 'maximum level' of 5 µg kg^-1 (range 6.1-15.7 µg kg^-1). Of the other spices assayed, the AFB1 contamination of one chilli sample exceeded 5 µg kg^-1 (8.1 µg kg^-1). Thirty-two of the 70 ground red pepper samples contained OTA, eight of them in a concentration exceeding the 10 µg kg^-1 'maximum level' (range 10.6-66.2 µg kg^-1). One chilli sample was contaminated with OTA at 2.1 µg kg^-1. The AFB1 and OTA contamination of ground red pepper exceeding the 'maximum level' (5 and 10 µg kg^-1, respectively) was obviously the consequence of mixing imported ground red pepper batches heavily contaminated with AFB1 and OTA with red pepper produced in Hungary. This case calls attention to the importance of consistently screening imported batches of ground red pepper for aflatoxin and ochratoxin A content and strictly prohibiting the use of batches containing mycotoxin concentrations exceeding the maximum permitted level.     

Analysis of heat-processed corn foods for fumonisins and bound fumonisins

Thirty retail samples of heat-processed corn foods, i.e. corn flakes, corn-based breakfast cereals, tortilla chips and corn chips, were analysed for fumonisins — fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and hydrolysed FB₁ (HFB₁) — as well as for protein- and total-bound FB₁. Bound (hidden) fumonisins cannot be detected by conventional analysis. Improved methods for the determination of bound FB₁ were developed. The protein-bound FB₁ was extracted with 1% sodium dodecylsulfate (SDS) solution. The SDS, which interfered with high-performance liquid chromatography (HPLC) analysis, was then separated from protein-bound FB₁ by complexing with methylene blue followed by solvent extraction and hydrolysis with 2 N KOH. To measure total-bound FB₁, the sample itself was hydrolysed with KOH. In both cases, clean-up was accomplished on an OASIS polymeric solid-phase extraction column and the bound fumonisins were determined by HPLC measurement of HFB₁. Fourteen of 15 samples of corn flakes and other corn-based breakfast cereals analysed contained detectable levels of FB₁ with a mean in positive samples of 67 ng g^-1 (13-237 ng g^-1). Two samples also had detectable levels of FB₂ (21-23 ng g^-1). Bound FB₁ was found in all samples; the mean protein-bound FB₁ measured was 58 ng g^-1 (22-176 ng g^-1) and the mean total-bound FB₁ measured was 106 ng g^-1 (28-418 ng g^-1), reported as FB₁ equivalents after correction for recoveries of HFB₁. There was an average of about 1.3 times more FB₁ in the bound form compared with extractable FB₁, and this was about twice as much as protein-bound FB_1. Seven of the 15 samples of alkali-processed corn-based foods, such as tortilla chips and corn chips, contained FB₁ and three contained HFB₁ with means in measurable positive samples of 78 (48-134) and 29 (13-47) ng g^-1, respectively. Five of these alkali-processed corn foods contained bound FB₁; the mean measurable protein-bound FB₁ was 42 ng g^-1 (39-46 ng g^-1) and the mean measurable total-bound FB₁ was 100 ng g^-1(54-209 ng g^-1). HFB₁ derived from bound FB₁ in selected samples was confirmed by HPLC with mass spectrometry (MS).     

Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.     

Content of mercury and cadmium in fish (Thunnus alalunga) and cephalopods (Eledone moschata) from the south-eastern Mediterranean Sea

Mercury and cadmium concentrations were measured in the flesh and liver (or hepatopancreas) of albacore (Thunnus alalunga) and horned octopus (Eledone moschata) to establish whether the concentrations exceeded the maximum levels fixed by the European Commission. In both species, mercury and cadmium mean concentrations were higher in liver (albacore: mercury = 2.41 μg g^-1 wet wt, cadmium = 9.22 μg g^-1 wet wt; horned octopus: mercury = 0.76 μg g^-1 wet wt, cadmium = 6.72 μg g^-1 wet wt) than in flesh (albacore: mercury = 1.56 μg g^-1 wet wt, cadmium = 0.05 μg g^-1 wet wt; horned octopus: mercury = 0.36 μg g^-1 wet wt, cadmium = 0.33 μg g^-1). Mercury concentrations exceeding the prescribed legal limit of 1 μg g^-1 wet wt were found in almost all albacore samples (flesh: 71.4%; liver: 85.7%). For horned octopus, concentrations above 0.5 μg g^-1 wet wt were observed solely in hepatopancreas, while in flesh, the concentrations were below this limit in all the samples examined. Of the flesh samples of albacore, 42.8% exceeded the proposed tolerance for cadmium for human consumption, whilst for horned octopus, the established limit was not exceeded in any sample.     

Migration of formaldehyde and acetaldehyde into mineral water in polyethylene terephthalate (PET) bottles

The levels of formaldehyde (FA) and acetaldehyde (AA) in polyethylene terephthalate (PET) bottles and in commercial mineral water are reported. All the water samples bottled in Japan contained detectable levels of FA (10.1-27.9 μg l^-1) and AA (44.3-107.8 μg l^-1). Of 11 European bottled water samples, eight did not contain either FA or AA, while the remaining three had detectable levels of FA (7.4-13.7 μg l^-1) and AA (35.9-46.9 μg l^-1). In three North American bottled water samples, two contained FA (13.6 and 19.5 μg l^-1) and AA (41.4 and 44.8 μg l^-1), and one did not. Regardless of the region of origin, all the sterilized water samples contained FA and AA, whilst in contrast, none of the unsterilized water without carbonate contained FA or AA. Of the carbonated water samples, three contained FA and AA, and one did not. When fortified with FA and AA, the commercial water sample without otherwise detectable FA and AA was able to reduce levels, although the commercial water sample containing FA and AA could not. The presence of bacteria in the commercial water samples was investigated using an ATP-based bioluminescent assay and heterotrophic plate count method. The commercial water without FA and AA contained heterotrophic bacteria, whilst the commercial water with FA and AA did not contain detectable bacteria. It is suggested that in this case both FA and AA migrated from PET materials, but were subsequently decomposed by the heterotrophic bacteria in the unsterilized water.     

气相色谱-质谱法测定动物性食品中三种β-激动剂的残留量

建立了用气相色谱-质谱同时测定动物性食品中克伦特罗、沙丁胺醇和莱克多巴胺残留量的检测方法。样品用乙酸胺缓冲液匀浆,加入β-葡萄糖苷酸酶水解,用异丙醇-乙酸乙酯(40:60)萃取,有机相浓缩,经LC-SCX固相萃取柱进行分离,用4%氨化甲醇洗脱,洗脱液氮气吹干后,经N、O-双三甲基硅烷三氟乙酰胺(BSTFA)衍生后于气质联用仪上进行测定。检测采用选择性离子监控模式(SIM),以特征离子克伦特罗(86,262,243)、沙丁胺醇(86,369,440)和莱克多巴胺(250,267,502)定性,以试样峰(m/z86,m/z369,m/z250)的峰面积外标法定量。本方法检出限为克伦特罗和沙丁胺醇0.5ug/kg,莱克多巴胺2ug/kg;3种组分的线性相关系数均大于0.99。该方法的回收率为75%-98%,相对标准偏差为5%-14%。     

Method of test and survey of caprolactam migration into foods packaged in nylon-6

An analytical method for the determination of the nylon-6 monomer caprolactam in foods is described. The foodstuff was extracted with ethanol : water (1:2) containing capryllactam as internal standard and the extract was defatted using hexane. The extract was analysed by liquid chromatography coupled with mass spectrometry. The test method was calibrated down to 0.7 mg kg^-1. The repeatability of the method was good, with a relative standard deviation of 9% at the 15 mg kg^-1 level. The method was demonstrated to be accurate in an independent external check sample exercise. The new method was applied to the analysis of 50 retail foodstuffs packaged in nylon-6. Caprolactam was detected and confirmed in nine of the 50 food samples, in the range 2.8-13 mg kg^-1. The presence of caprolactam was indicated in a further 15 samples, in the range 0.8-11 mg kg^-1, but these samples did not meet all of the five confirmation criteria applied. All migration levels (both confirmed and unconfirmed) were below the European specific migration limit for caprolactam, which is 15 mg kg^-1. The average migration for all 50 samples, setting non-detectables at half the limit of detection, was 2.6 mg kg^-1 with a standard deviation of 3.1 mg kg^-1 (n = 50). All samples found to contain detectable levels of caprolactam migration were for applications involving heating the food in the packaging. They were packs of, for example, sausage meat for which the food would have been heat processed in the nylon casing, or they were nylon pouches for heating foods by boiling, microwaving or roasting.     

Distribution and elimination of fumonisin analogues in weaned piglets after oral administration of Fusarium verticillioides fungal culture

The distribution and elimination of fumonisins after oral administration of 50 mg FB₁, 20 mg FB₂ and 5 mg FB₃ per animal day^-1 for 22 days was studied in weaned barrows. At the end of the trial, the lung, heart, liver, kidney, spleen, brain, serum, bile, muscle, fat, urine and faeces samples were collected and their content of fumonisins (FB₁, FB₂) determined by LC-MS. The highest FB₁ concentrations were found in the liver (99.4 ± 37.5 ng g^-1) and kidneys (30.6 ± 10.1 ng g^-1), whilst the highest average amount of FB₂ was in the liver (1.4 ± 2.3 ng g^-1) and fat (2.6 ng g^-1 ± 4.8) samples. Comparing the FB₁/FB₂ ratio in different organs (19/1), it was found that the ratio in the abdominal and subcutaneous fat samples (4/1) was markedly different from those in all other tissues, namely the relative proportion of FB₂ was higher in latter cases. Of the total quantity of FB₁, the 13% taken up during 5 days was excreted unchanged with the faeces and urine. On average, in the urine and faeces, FB₁ was detected in nine- and 14-fold quantities, as compared with FB₂.     

荧光分光光度法测定猪肉中的盐酸克伦特罗的含量

利用荧光胺与盐酸克伦特罗（瘦肉精）生成高度荧光衍生物,检测猪肉中盐酸克伦特罗的含量.实验表明,荧光胺与盐酸克伦特罗反应生成的强荧光物质的最大激发波长和最大发射波长分别为500 nm、490 nm,利用L934）正交试验确定最佳衍生条件为衍生温度37℃,衍生时间40 min,衍生剂荧光胺（1μg/mL）加入量0.3 mL.荧光信号与盐酸克伦特罗的质量浓度变化呈良好的线性关系,线性方程为y=82 492x＋957.96,相关系数为0.993 3,此法可用于检测猪肉中瘦肉精的含量.     

Effect of clenbuterol on productive performance, body composition and muscle biochemistry in the rabbit

Twenty New Zealand White rabbits (INRA 1077 strain) were given a complete and balanced diet including a clenbuterol additive (100 μg per day) between 70 and 98 days. They were compared with 20 control rabbits. The treatment improved the growth performance (29.90 vs 26.07 g/day), the feed conversion (5.45 vs 6.46 g feed per g gain) and the carcass yield (64.37 vs 61.11%), by decreasing the relative weights of the skin and the digestive tract. Moreover, all organs in which development is precocious, were found to be relatively lighter. The muscle/bone ratio of the carcass was improved (7.56 vs 6.38), resulting in a greater relative development of muscle tissue, without any change in bone tissue weight. Perirenal and interscapular fat percentages in the carcass were reduced (3.23 vs 3.83 and 0.68 vs 0.86, respectively). Clenbuterol, a repartitioning agent, had therefore modified the growth allometry of the organs and tissues. In the hindleg region (Biceps femoris, Tensor fasciae latae, Semimembranosus accessorius), the ultimate muscular pH was increased, (+0.31 pH units on average), while the cooking loss was reduced (24.23 vs 24.88%). In the m. longissimus lumborum, the increase of ultimate pH (+0.31 units of pH), under the effect of clenbuterol, was explained by a relative increase in the oxidative metabolic pathway represented by aldolase/ICDH ratio (246 vs 284) and by a decrease in glucidic content (total glycosyl residues) of muscle (16.6 vs 26.2 μmol/g). Due to its effects on muscular biology, clenbuterol is thus likely to cause a change in meat quality.     

壳聚糖涂覆磁性纳米颗粒对纤维素酶的固定化

设计具有高稳定性、选择性的酶-载体复合物是固定化酶领域的研究重点。本研究以环氧氯丙烷作为表面活性剂,通过沉淀法制备磁性纳米颗粒并涂覆壳聚糖,用以固定化纤维素酶。通过SEM扫描电镜、VSM磁强计、FTIR红外光谱对 Fe₃O₄-壳聚糖磁性纳米颗粒进行表征,并研究其固定化纤维酶的表征及酶学性质。结果表明,制备的磁性纳米颗粒晶形完整,纤维素酶有效固定在Fe₃O₄-壳聚糖载体表面;固定化纤维素酶比游离纤维素酶具有更好的酸碱稳定性和热稳定性。固定化纤维素酶在pH 2~9范围内均有较好的活性,并且置于60和70 ℃条件下4 h后,仍然能保持将近50％的活性,经10次循环利用后,固定化纤维素酶仍然保持在52.6%的活性,说明Fe₃O₄-壳聚糖可作为固定纤维素酶的有效载体,为固定化酶的进一步应用提供了参考。