嗜热脂肪芽孢杆菌试管法检测牛乳中β-内酰胺类抗生素残留

采用嗜热脂肪芽孢杆菌试管法对牛乳中6种最为常见的β-内酰胺类抗生素残留进行检测,指出嗜热脂肪芽孢杆菌试管法为半定量检测法,对牛乳中β-内酰胺类抗生素残留具有较好的灵敏性.牛乳中青霉素G、氨苄青霉素、羟氨苄青霉素的检测限为6μg/kg,头孢氨苄的检测限为150μg/kg,苯唑青霉素的检测限为15μg/kg、头孢匹林的检测限为30μg/kg.在检测时间、操作难易程度上较BSDA法具有一定的优越性,可应用于牛乳中β-内酰胺类抗生素残留的检测.     

鲜乳中抗菌素的Mell assay检测法与一些常用测定方法的比较

本文分析和对比了鲜乳中抗菌素残留量的四种检测方法、其中,应用 well assay检测法、DelvotestP检测法、Charm test II检测法测定鲜乳中苄基青霉素、双氢链霉素、土霉素及磺胺二甲基嘧啶的含量,应用Penzym检测法测定鲜乳中苄基青霉素、氨苄青霉素和奈基青霉素的含量。结果表明,Well assay检测法可有效地测定上述四种抗菌素,从最低可检浓度为;苄基青霉素0.005IU/ml乳、双氢链霉素0.2μg/m1乳、土霉素0.1μg/ml乳、磺胺二甲基俄院0.5μg/ml乳。因此认为Well assay 是一种简便、灵敏、经济、适于推广的检测方法。     

青霉素结合蛋白的重组表达及其在β-内酰胺类抗生素检测中的应用

目的 获得重组表达纯化的青霉素结合蛋白(penicillin binding proteins, PBPs),研究其在β-内酰胺类抗生素检测中的应用。方法 以来源于肺炎链球菌Streptococcus pneumoniae R6的PBP2x蛋白为对照,以PBP1a蛋白作为目标受体,研究其与β-内酰胺类抗生素的亲和作用。结果 经纯化后的重组蛋白PBP2x和PBP1a检测头孢噻呋、青霉素G、氨苄青霉素、喷沙西林、阿莫西林的半抑制浓度(inhibitory concentration, IC50)都在7.02 ng/mL以下,明确了PBP1a和PBP2x具有β-内酰胺类抗生素结合能力。结论 该研究为开发新的基于PBPs蛋白检测β-内酰胺类抗生素的免疫学方法奠定了基础。     

牛奶中β-内酰胺类和三聚氰胺检测方法的建立

建立一种快速、简便,同时针对牛奶中β-内酰胺类抗生素和三聚氰胺残留的检测方法。应用竞争抑制免疫层析原理,研制β-内酰胺类和三聚氰胺胶体金试纸条。该试纸条检测限分别为青霉素G2μg/L、氨苄青霉素3μg/L、阿莫西林3μg/L、苯唑青霉素6μg/L、邻氯青霉素6μg/L、双氯青霉素6μg/L、萘夫西林20μg/L、头孢喹肟20μg/L、头孢哌酮40μg/L、头孢曲松50μg/L、头孢噻呋90μg/L、头孢洛宁10μg/L、三聚氰胺50μg/L,假阳性率低于5%、假阴性率为0,检测时间不超过15min。该方法操作简便、灵敏度高、特异性强,适用于乳品流通环节中β-内酰胺类抗生素和三聚氰胺残留的快速检测。     

乳品中β-内酰胺类抗生素快速检测试纸条研制

通过β-内酰胺类抗生素-载体蛋白偶联物的合成,单克隆抗体的制备与纯化,胶体金标记物的制备,β-内酰胺类抗生素单克隆抗体-胶体金标记物的制备并冻干到微孔试剂,样品吸收垫和反应膜的制备等研究过程,研制出乳制品中β-内酰胺类抗生素的快速检测试纸条。检测限分别为:青霉素G2μg/L、氨苄青霉素4μg/L、阿莫西林5μg/L、苯唑青霉素/邻青霉素/双青霉素均为6μg/L、头孢洛宁10μg/L、萘夫西林20μg/L、头孢喹肟20μg/L、头孢曲松25μg/L、头孢哌酮50μg/L、头孢噻呋90μg/L;本试纸条特异性好、假阳性率不高于3%、假阴性率为0,检测时间不超过15min,检测限量达到了我国和欧盟的要求,适用于乳品流通环节乳品中β-内酰胺类抗生素残留的检测。     

黄鲫胃蛋白酶酶解液体外抗氧化、抑菌作用研究

采用胃蛋白酶水解黄鲫,测定酶解液的蛋白提取率、可溶性肽含量、水解度及氨基酸组成,并对酶解液体外抗氧化和抑菌作用进行研究。结果表明：黄鲫胃蛋白酶酶解液的蛋白提取率、可溶性肽提取率、水解度分别为 (110.28 ± 4.81)%、(81.78 ± 0.04)% 和 (18.12 ± 0.39)%,黄鲫胃蛋白酶酶解液的人体必需氨基酸相对含量可达37.91%。黄鲫胃蛋白酶酶解液具有很强的抗氧化能力,清除羟自由基和DPPH 自由基的ED50 分别为4.55μg/mL 和4.46μg/mL,还原力随着黄鲫胃蛋白酶酶解液质量浓度的增加而增大,在3.4 μg/mL 和13.6μg/mL 低、中质量浓度时螯合金属离子能力与EDTA 接近。体外抑菌实验显示：黄鲫胃蛋白酶酶解液具有广谱抑菌性,对大肠杆菌的抑菌效力分别与 13.21～15.44mcg/mL 的氨苄青霉素和 325.00～340.00U/mL 的硫酸链霉素相当 (P ＞ 0.05)。     

壳聚糖-活性红4缔合体系的荧光猝灭与共振瑞利散射光谱分析及其在壳聚糖定量分析中的应用

在弱酸性Britton-Robinson缓冲体系中,壳聚糖对活性红4存在荧光猝灭作用,在激发波长（λEx）/发射波长（λEm）=285 nm/341 nm处,在0.050～2.00 μg/mL质量浓度范围内,其荧光猝灭程度与壳聚糖质量浓度呈良好的线性关系。线性方程为ΔF=68.78c＋2.648（c,μg/mL）,R2=0.999 2,检出限为0.039 μg/mL。在相同的介质环境中,壳聚糖-活性红4离子缔合体系于波长342 nm处产生强烈的共振瑞利散射特征峰,共振瑞利散射强度与壳聚糖质量浓度c呈良好线性关系,在0.050～6.00 μg/mL质量浓度范围内,线性方程为：ΔI=681.31c＋114.95（c,μg/mL）,R2=0.999 1,检测限为0.033 μg/mL。据此,建立以活性红4为探针定量分析壳聚糖的2 种新方法：荧光猝灭法和共振瑞利散射法。同时,将2 种方法就壳聚糖分子质量对测定结果准确性的影响进行研究。将2 种新方法应用于实际样品的定量检测,测定结果基本一致,且回收率结果令人满意。     

应用酶联免疫法快速检测乳品中β-内酰胺类抗生素残留

建立乳品中β-内酰胺类抗生素残留酶联免疫快速检测方法。应用氨苄青霉素抗体,通过人工制备了氨苄青霉素(Amp)和辣根过氧化物酶(HRP)的结合物Amp-HRP,建立直接ELisa竞争法检测β-内酰胺类抗生素,确定了各种β-内酰胺类抗生素的检出限,并对检测条件进行了优化。检测了已知阴性和阳性的生鲜牛乳样品,结果表明对于阴性和阳性牛奶样品的检测未出现假阳性或者假阴性结果。该方法检测结果准确可靠,可广泛应用于检测乳品中β-内酰胺类抗生素残留的检测。     

固相萃取-高效液相色谱-串联质谱法测定牛乳和猪肉中5 种青霉素类抗生素

建立一种用固相萃取-高效液相色谱-串联质谱法检测牛乳和猪肉中苄星青霉素G、氨苄西林、苯唑西林、氯唑西林、双氯西林5 种青霉素类抗生素方法。样品经乙腈提取,Bond Elut C18固相萃取柱净化,用高效液相色谱-串联质谱检测,外标法定量。结果表明,本方法5 种青霉素在2.0～200 μg/L范围内呈良好线性关系,线性相关系数大于0.999;牛乳样品添加回收实验,回收率为85.2%～122.7%,相对标准偏差为3.43%～16.8%（n=6）;猪肉样品添加回收实验在50 μg/kg和100 μg/kg添加水平,除氨苄西林外,回收率在94.3%～116.4%,相对标准偏差为1.62%～5.09%（n=6）。方法定量限为1.0～2.0 μg/kg。该方法具有简便快捷、准确度高、定量限低的优点,适用于牛乳和猪肉中青霉素类抗生素的测定。     

葡萄籽和皮萃取物对小鼠乳腺癌细胞迁移的影响

目的：研究葡萄籽和皮萃取物对乳腺癌细胞迁移的抑制作用,以及对细胞活力的影响。方法：以小鼠乳腺癌细胞4T1为研究对象,四甲基偶氮唑盐(MTT)实验检测细胞增殖活性;细胞迁移实验检测细胞迁移能力;结果：质量浓度为5μg/mL的葡萄籽和皮萃取物对乳腺癌细胞的迁移具有明显的抑制作用,该抑制作用与药物质量浓度呈现剂量依赖关系;萃取物对癌细胞的活力有明显的抑制作用,葡萄籽和皮萃取物的半数有效质量浓度(IC50)分别为65.427μg/mL和72.408μg/mL。     

Detection limits of beta-lactam antibiotics in ewe milk by penzym enzymatic test.

The Penzym is an enzymatic test widely used for the detection of beta-lactam antibiotic residuals in milk. It is a specific method with good sensitivity to this group of antibiotics and enables results to be obtained within a short time. In the present work, the detection limits of 10 beta-lactam antibiotics were determined in ewe milk, given the lack of previous studies of the Penzym test in ovine milk. For each antibiotic, eight concentrations were tested on 20 ewe milk samples proceeding from individual ewes (160 analyses per drug). The limits of the Penzym test were determined by means of logistic regression models, as follows: 5 microg/kg amoxicillin, 4 microg/kg ampicillin, 33 microg/kg cloxacillin, 3 microg/kg penicillin "G," 43 microg/kg cephadroxil. 10 microg/kg cephalosporin "C," 16 microg/kg cephalexin, 900 microg/kg cephoperazone, 120 microg/kg Ceftiofur, and 77 microg/kg cephuroxime. The percentages of positive results for those antibiotics at the maximum residue limit (MRL) concentration established by the European Union (EU) were: 100% (penicillin "G"), 93.3% (ampicillin), 93.3% (cloxacillin), 56.7% (Ceftiofur), and 56.7% (amoxicillin).     

Inactivation of penicillin G in milk using hydrogen peroxide

Milk antibiotic residues have been a public concern in recent years. The Grade A Pasteurized Milk Ordinance mandates that raw Grade A milk will test negative for beta-lactam antibiotic residues before processing. The purpose of this research was to investigate the ability of various levels of peroxide and heat to inactivate penicillin G in raw milk. Whole milk spiked to a mean of 436 +/- 15.1 (standard error of the mean) ppb of potassium penicillin G was treated with hydrogen peroxide at levels of 0.0, 0.09, 0.17, and 0.34%. Samples at each peroxide level (n = 6 per treatment) were treated as follows: 1) incubated at 54.4 degrees C for 3 h, 2) pasteurized at 62.8 degrees C for 30 min, 3) incubated and pasteurized as in treatments 1 and 2, or 4) received no further treatment. A beta-lactam competitive microbial receptor assay was used for quantification of penicillin G. Concentrations of penicillin in selected samples were determined by HPLC for a comparison of test methods. Treatments were evaluated relative to their ability to reduce milk penicillin G levels to below the safe level of 5 ppb. The 0.09% hydrogen peroxide level was ineffective for all treatments. Hydrogen peroxide at 0.17% lowered the mean penicillin G (+/- SEM) from 436 +/- 15.1 to 6 +/- 1.49 ppb using the incubated and pasteurized heat treatment. The 0.34% concentration of hydrogen peroxide was the most effective, inactivating penicillin G to a level well below the safe level of 5 ppb with the pasteurized heat treatment, with or without incubation.     

Excretion of penicillins and cephalexin in bovine milk following intramammary administration

The elimination into bovine milk of beta-lactam antibiotic residues (procaine penicillin G, cloxacillin, ampicillin, oxacillin, cephalexin) following intramammary administration of 10 preparations marketed in France was studied. The quantitative analysis of residues was carried out by a microbiological agar diffusion method using Bacillus stearothermophilus. Sensitivity ranged from 0.001 I.U./ml for procaine penicillin G and 0.001 micrograms/ml for ampicillin to 0.02 micrograms/ml for cephalexin. The mean periods of elimination on which withholding times are based were between four and seven milkings according to the drugs administered.     

Detection limits of antimicrobials in ewe milk by delvotest photometric measurements

The Delvotest method detection limits per manufacturer's instructions at a fixed reading time of 3 h for 24 antimicrobial agents were determined in ewe milk by photometric measurement. For each drug, eight concentrations were tested on 20 ewe milk samples from individual ewes. Detection limits, determined by means of logistic regression models, were (microg/kg): 3, amoxycillin; 2, ampicillin; 18, cloxacillin; 1, penicillin "G"; 34, cefadroxil; 430, cephalosporin "C"; 40, cephalexin; 20, cefoperazone; 33, Ceftiofur; 18, cefuroxime; 6100, streptomycin; 1200, gentamycin; 2600, neomycin; 830, erythromycin; 100, tylosin; 180, doxycycline; 320, oxytetracycline; 590, tetracycline; 88, sulfadiazine; 44, sulfamethoxazole; 140, sulfametoxypyridazine; 48, sulfaquinoxaline; 12,000, chloramphenicol; and 290, trimethoprim. Whereas the beta-lactam antibiotics, sulphonamides, and tylosin were detected by Delvotest method at levels equal to those of maximum residue limits, its sensitivity needs to be enhanced to detect aminoglycosides, tetracyclines, streptomycin, chloramphenicol, and trimethoprim residues in ewe milk or to develop an integrated residue detection system for ewe milk with different sensitive microorganisms for each group of antiinfectious agents.     

基于量子点二抗偶联物的荧光免疫分析法测定牛奶中的氨苄青霉素

目的建立基于量子点二抗偶联物的荧光免疫分析法测定牛奶中的氨苄青霉素。方法采用共价偶联的方法将Qdot 655红色荧光量子点(quantum dots, QDs)与二抗偶联,利用制备好的QDs-二抗偶联物代替传统酶标二抗应用到检测牛奶中氨苄青霉素残留检测的荧光免疫分析方法中,并将该方法与酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)和高效液相色谱法(high performance liquid chromatography, HPLC)进行比较。结果该方法 50%抑制浓度(IC50)为8.3μg/L;检测限为2.5μg/L。空白牛奶加标回收率为94.0%～106.2%,变异系数为2.1%～9.2%。在实际样品的检测中,该方法与ELISA方法和HPLC方法测定的结果相比无显著差异(P0.05)。结论该方法准确、灵敏,适用于牛奶中氨苄青霉素残留的检测。     

Evaluation of the Delvo-X-Press assay for detecting antibiotic residues in milk samples from individual cows.

Performance of the Delvo-X-Press beta-lactam antibiotic assay was examined using bulk-tank milk samples and milk samples from individual cows. Bulk-tank milk samples fortified with bovine lactoferrin at a concentration of 1 mg/ml or more consistently tested positive. False-positive results were also obtained from bulk-tank milk samples fortified with bovine plasma at concentrations of 20 and 40%. The assay yielded positive results for milk with antibiotic concentrations as low as 2 ppb. Individual milk samples were collected from 144 healthy lactating cows and from 34 cows with chronic Staphylococcus aureus mastitis. Specificity estimates for samples from healthy and mastitic cows were 0.88 (95% confidence interval [CI], 0.82, 0.93) and 0.94 (95% CI, 0.86, 1.00), respectively. Individual milk samples were collected from three cows with experimentally induced mastitis for 21 consecutive days. False-positive results occurred as late as 12 days postchallenge. A moderate but significant (P < 0.01) positive linear correlation (r = 0.61) was observed between test result and somatic cell count (SCC) values in milk samples with SCCs of >10(6)/ml.     

Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk

The demand for commercially available human breast milk has significantly increased in recent years. For various reasons, a significant amount of commercially available human breast milk is being adulterated with other types of milk. This fraudulent practice poses a threat to consumers' health due to potential adulterants such as cow milk, which may put the infant at risk due to intolerance or allergy. A direct sandwich anti-bovine IgG ELISA has been developed for the sensitive and specific detection of cow milk in adulterated human breast milk. This assay uses polyclonal anti-bovine IgG antibody as a capture antibody and monoclonal anti-bovine IgG-alkaline phosphatase antibody as a detection antibody. Once optimized, the assay was found to be highly sensitive, and specific to bovine IgG. The assay had no significant cross-reaction with human breast milk, indicating that it was highly specific. The anti-bovine IgG ELISA was able to detect the presence of cow milk in adulterated human breast milk with a detection limit of 0.001% cow milk. The developed assay was highly reproducible (coefficient of variation <10%). The developed direct sandwich anti-bovine IgG ELISA is simple, reliable, and reproducible, making it an ideal test for this purpose.     

Biosensor assay for determination of haptoglobin in bovine milk

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.     

Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration

The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows' milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows' milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 microg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows' milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.     

胶体金免疫层析法快速检测配方羊奶粉中的牛β-乳球蛋白

建立了一种可快速检测配方羊奶粉中牛β-乳球蛋白(β-lactoglobulin,β-lg)的胶体金免疫层析检测方法。通过杂交瘤技术制备β-lg单克隆抗体(monoclonal antibody,mAb),半抑制浓度(50% inhibiting concentration,IC₅₀)为5.87 μg/mL。将胶体金标记的β-lg mAb包被于金标垫,β-lg和山羊抗小鼠IgG标记于硝酸纤维素膜(nitrocellulose membrane,NC膜)分别作为检测线(T线)和质控线(C线),开发了可检测β-lg的免疫层析试纸条。该试纸条对β-lg的检测限(limit of detection,LOD)值为50 μg/mL,与其他基质成分均未产生有效交叉反应,对全脂山羊乳粉中掺杂脱脂牛奶粉(nonfat skim milk,NFSM)、脱盐乳清粉(desalted whey powder,DWP)和乳清蛋白粉(whey protein powder,WPP)的LOD值分别为5%、5%和0.1%。运用该方法对9个市售配方羊奶粉进行分析,检测结果与商品化酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)试剂盒一致。该方法前处理快速简单,5 min即可裸眼判定结果,可用于配方羊奶粉商品的现场快速检测。