Preparation of anti-pefloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of pefloxacin residue in chicken liver

Pefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a labor-intensive instrumental method to detect the residue of pefloxacin in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen cationized bovine serum albumin-pefloxacin showed high sensitivity toward pefloxacin with an IC50 value of 6.7 ppb in buffer and was suitable for a screening assay to detect the residue of pefloxacin in food products. The antibody has been assessed using rapid enzyme immunoassays to exploit its specificity. The antibody prepared shows cross-reactivity with a few other (fluoro)quinolones including fleroxacin (116%), enrofloxacin (88%), and ofloxacin (10%). The assay measured drug residue in chicken liver spiked with pefloxacin with an interassay coefficient of variation of 13.6% or less and an intra-assay coefficient of variation of 10.9% or less. The average recovery rates at 0.5, 5, 10, 50, and 100 ppb were in the range of 86-106% for interassay and in the range of 87-103% for intra-assay, respectively.     

Production and characterization of a monoclonal anti-anti-idiotype antibody against fumonisin B(1).

A monoclonal anti-anti-idiotype antibody (mAb3) against fumonisin B(1) (FmB1) was produced from the hybridoma cell line 7C7F4, which was generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse that had been immunized with the Fab fragments of affinity-purified anti-idiotype antibodies. The mAb3 belongs to the immunoglobulin M, kappa light chain. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were established for antibody characterization and toxin analysis. In an idc-ELISA using FmB1-ovalbumin (OVA) as the coating antigen, the concentrations causing 50% inhibition of binding (IC(50)) of mAb3 to the solid-phase FmB1-OVA by free FmB1, FmB2, and FmB3 were found to be 75, 95, and 450 ng/mL, respectively. In the dc-ELISA, the concentration causing IC(50) of FmB1-horseradish peroxidase to the solid-phase mAb3 by free FmB1 was found to be 233 ng/mL. Analysis of 12 samples naturally contaminated with fumonisins with mAb3-based idc-ELISA and polyclonal-based dc-ELISA showed a good correlation between these two methods with a correlation coefficient of 0.76 at p < 0.02. The linear regression slope was found to be y[polyclonal ELISA] = 0.87x[mAb3 ELISA] - 52 ppb.     

High pressure liquid chromatographic detection and estimation of furazolidone and nitrofurazone in animal feeds.

The feed sample is extracted with acetone or dimethylformamide-acetone (1 + 1) and the filtered extracts are evaporated to dryness. The residue is dissolved in chloroform and transferred to a silica gel column. The nitrofurans are eluted with methanol-chloroform (50 + 50). A portion of the eluate is evaporated to dryness and the residue is redissolved in a small volume of methanol. Aliquots of the methanolic solution are injected into a liquid chromatograph with a muBondapak C18 column, using 30% acetonitrile as the eluting solvent and ultraviolet detection at 365 nm. Several samples spiked with 0.5--50 ppm furazolidone or nitrofurazone and 2 commercial samples were analyzed by the proposed method.     

Preparation of polyclonal antibodies to a derivative of 1-aminohydantoin (AHD) and development of an indirect competitive ELISA for the detection of nitrofurantoin residue in water

Nitrofurans are used widely to treat animal diseases and were identified as the major compounds in many worldwide drug residue violations. To develop a rapid and convenient detection method to measure the residue of nitrofurantoin, we designed an immunogen and prepared a polyclonal antibody to develop an immunoassay in this study. The antibodies obtained were characterized by an indirect cELISA method and showed excellent specificity and sensitivity with IC50 of 3.2 ppb and no cross-reaction with most related species and compounds. Considering that nitrofurans often are used illegally to feed animals through drinking water, we measured the residue of nitrofurantoin in water spiked by the drug. The recovery rates are in the ranges of 88-103% for interassay and 90-103% for intra-assay. The CVs are in the ranges of 3.1-11.4% for interassay and 2.7-6.2% for intra-assay. The detection limit was determined to be 0.2 ppb. The immunoassay developed in this study is suitable to be used as a screening method to detect residues of nitrofurantoin in drinking water for animals.     

溴氰菊酯残留酶联免疫分析方法的建立

在前期设计合成了溴氰菊酯半抗原（DM）和人工抗原（DM-BSA和DM-OVA）的基础上,进一步利用人工抗原（DM-BSA）免疫新西兰大白兔获得了溴氰菊酯多克隆抗体。研究表明,新西兰大白兔产生的抗体对溴氰菊酯产生了灵敏的特异性免疫反应,所得多克隆抗体的效价为25600。以DM-OVA为包被原建立溴氰菊酯间接竞争ELISA检测方法,确定了抗原抗体最适工作浓度均为1∶12800。在含10%甲醇、pH7.4、盐浓度0.1mol·L^-1的缓冲液条件下制作了溴氰菊酯的标准抑制曲线,抑制中浓度IC50为3.999μg·mL^-1,检出限IC10为0.023μg·mL^-1,检测范围为0.015-100μg·mL^-1。抗体对包括氯菊酯、氯氰菊酯、甲氰菊酯在内的8种菊酯类农药的交叉反应率较低,在苹果中的添加回收率为86.2%-105.8%。成功制备出溴氰菊酯特异性抗体,并建立了水果中溴氰菊酯残留间接竞争酶联免疫分析方法。     

Development of a chemiluminescent ELISA for determining chloramphenicol in chicken muscle

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (CL) detection for chloramphenicol (CAP) in chicken muscle was developed. CAP-specific polyclonal antibody was raised in rabbit with a CAP-succinate derivative conjugated with bovine serum albumin. Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 6 ng/L. The CL-ELISA was 10 times more sensitive compared to the colorimetric-ELISA. When CAP was spiked in chicken muscle at levels of 0.05-5 microg/kg, recoveries ranged from 97 to 118% with coefficients of variation of 6-22%. In an actual residue study, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The residue levels of CAP in treated chicken decreased with time and dropped rapidly after the first 6 h from around 50 to 10 microg/kg. After 3 days, CAP was not detected in chicken muscle. The developed method is therefore suitable for screening of CAP in chicken muscle samples.     

高效液相色谱法测定土壤中的二氯喹啉酸

用50 ml甲醇︰0.05 mol/L硼砂溶液(p H=10)(9︰1,v/v)作为提取剂对20 g(或适量)土壤样品中残留的二氯喹啉酸振荡提取2 h,离心过滤后分取滤液25 ml浓缩,甲醇定容至2 ml,再用滤膜过滤后待测定;高效液相色谱仪(HPLC)以1.0 ml/min的甲醇︰1%乙酸水溶液(55︰45,V/V)为流动相,柱温45℃,在238 nm的紫外光波长下进行二氯喹啉酸的外标法定量。该方法精密度为6.0%,检出限为0.012 mg/kg,不同类型土壤的加标回收率74.9%~98.8%,能满足有机分析要求。该方法既简捷易操作,又能满足土壤中二氯喹啉酸低残留量的测定。     

Development of an enzyme-linked immunosorbent assay based a monoclonal antibody for the detection of pyrethroids with phenoxybenzene multiresidue in river water

A sensitive and broad class selective direct competitive enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (McAb) has been described for the detection of pyrethroids with phenoxybenzene group. One monoclonal antibody, 2G(2)E(7), was obtained and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice. The assay with the most selectivity for the family pyrethroids with phenoxybenzene group was optimized. The IC(50) values of the optimized immunoassay were 1.8 μg L(-1) for deltamethrin, 1.5 μg L(-1) for cypermethrin, 2.0 μg L(-1) for fluvalinate and fenvalerate, 2.2 μg L(-1) for phenothrin, 2.4 μg L(-1) for flucythrinate, 3.0 μg L(-1) for fenpropathrin, and 5.0 μg L(-1) for permethrin. River water samples fortified with pyrethroids were analyzed with the ELISA to evaluate the accuracy of the assay. The recoveries of pyrethroids in spiked water samples ranged from 74 to 108%. The results indicate that the ELISA developed can accurately simultaneously determine pyrethroids with phenoxybenzene group in water samples.     

Preparation of anti-gatifloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for the detection of gatifloxacin residue in milk

A polyclonal anti-gatifloxacin antibody has been prepared, and an indirect competitive enzyme-linked immunosorbent assay (cELISA) was developed on the basis of the antibody prepared for the first time. The antibody shows high sensitivity with an IC50 value of 2.6 ppb and excellent specificity with only a minor cross-reaction with lomefloxacin (3.0%) among common (fluoro)quinolones evaluated in this study. The high specificity of the antibody was explained by the molecular structures of related drugs by comparison with published research. The cELISA test kit developed has a detection limit of 0.05 ppb and could be used as a screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to the detection of milk samples spiked by gatifloxacin. The recovery rates were in the range of 86-106%, whereas the intra- and interassay coefficients of variation were <14.3 and <19.6%, respectively.     

苦参碱在黄瓜和土壤中的检测方法及其残留动态研究

为了解苦参碱在黄瓜和土壤中的残留状况及消解动态,建立了苦参碱在黄瓜和土壤中的气相色谱分析方法,并在天津和安徽两地开展了为期两年的苦参碱在黄瓜和土壤中残留状况和消解动态规律田间试验研究。结果表明,采用无水乙醇超声提取黄瓜和土壤中的苦参碱,使用大孔吸附树脂净化,甲醇定容,气相色谱带氮磷检测器（NPD）进行测定,外标法定量,在0.25-1.0 mg·kg-1添加水平范围内,苦参碱在黄瓜和土壤中的平均回收率为78.32％-98.06％,变异系数为3.72%-7.44％;黄瓜和土壤中苦参碱的最小检出量均为1.36×10-12 g,最低检出浓度为0.004 mg·kg-1（黄瓜）、0.008 mg·kg-1（土壤）。田间试验结果表明,苦参碱在黄瓜和土壤中的残留消解动态符合方程Ct=C0e-kt;苦参碱在黄瓜和土壤中的降解半衰期分别为5.19-7.24 d和6.70-9.18 d。在黄瓜中施用0.3%苦参碱乳油,其制剂施药量为0.18-0.27 g·m-2,施药3-4次,两次施药间隔期为7 d,距收获期为1 d时,苦参碱在黄瓜中的残留量为0.125 6-1.207 1 mg·kg-1,土壤中的残留量为0.045 0-0.183 7 mg·kg-1。目前,国内外尚无苦参碱在黄瓜中最大残留限量标准,该试验研究成果为0.3%苦参碱乳油在黄瓜上的登记、安全使用规则     

Effect of sampling size on the determination of accurate pesticide residue levels in Japanese agricultural commodities

The uncertainty in pesticide residue levels (UPRL) associated with sampling size was estimated using individual acetamiprid and cypermethrin residue data from preharvested apple, broccoli, cabbage, grape, and sweet pepper samples. The relative standard deviation from the mean of each sampling size (n = 2(x), where x = 1-6) of randomly selected samples was defined as the UPRL for each sampling size. The estimated UPRLs, which were calculated on the basis of the regulatory sampling size recommended by the OECD Guidelines on Crop Field Trials (weights from 1 to 5 kg, and commodity unit numbers from 12 to 24), ranged from 2.1% for cypermethrin in sweet peppers to 14.6% for cypermethrin in cabbage samples. The percentages of commodity exceeding the maximum residue limits (MRLs) specified by the Japanese Food Sanitation Law may be predicted from the equation derived from this study, which was based on samples of various size ranges with mean residue levels below the MRL. The estimated UPRLs have confirmed that sufficient sampling weight and numbers are required for analysis and/or re-examination of subsamples to provide accurate values of pesticide residue levels for the enforcement of MRLs. The equation derived from the present study would aid the estimation of more accurate residue levels even from small sampling sizes.     

Codex alimentarius approach to pesticide residue standards

To protect consumers' health, most countries have maximum legal limits for pesticide residues in foods. Trade difficulties can arise when limits differ between countries. The Codex Alimentarius Commission was established in 1962 to implement the Joint FAO/WHO Food Standards Programme, the purpose of which is to protect consumer health and ensure fair practices in international food trade. The Codex Committee on Pesticide Residues (CCPR), an intergovernmental body which advises the Commission on matters related to pesticide residues, is responsible for establishing maximum residue limits (MRLs) for pesticides in foods and feeds that move in international trade. Codex MRLs are based on residue data obtained mainly from supervised trials that reflect approved pesticide use in accordance with "good agricultural practice." MRLs must be toxicologically acceptable in terms of estimated pesticide intake by consumers. CCPR Working Groups examine problems related to establishing and implementing MRLs, including sampling and methods of analysis. Despite time and effort expended, acceptance and application of Codex MRLs face many problems in international trade.     

Development of an enhanced chemiluminescence ELISA for the rapid detection of acrylamide in food products

In this work, a polyclonal antibody for acrylamide (AA) was obtained by immunization of rabbits with N-acryloxysuccinimide (NAS) and keyhole limpet hemocyanin (KLH) conjugate. A direct enzyme-linked immunosorbent assay (ELISA) based on this antibody was developed with enhanced chemiluminescent (ECL) detection of AA in food samples. Assay conditions, such as concentrations of antibody and enzyme conjugate and competition time, were optimized. The effects of ionic strength and pH value were investigated. The optimized ECL-ELISA system allowed AA determination in a linear working range of 26.3-221.1 ng mL(-1) with an IC(50) value of 60.6 ng mL(-1) and a limit of detection of 18.6 ng mL(-1). Good recoveries with spiked food samples were obtained with a recovery range from 74.4 to 98.1%, and these results correlated well with those obtained using an HPLC method. This indicates that ECL-ELISA is applicable to the specific detection and routine monitoring of AA in food samples.     

几种蔬菜对硝态氮、铵态氮的相对吸收能力

采用溶液培养方法探讨了莴笋、菠菜、小白菜和大青菜 4种蔬菜作物对硝、铵态氮的相对吸收能力以及这两种氮源对它们生长发育的影响。结果表明 ,单独供给NO3-N ,4种作物均生长发育良好 ;供给NO3--N +NH4+-N(NO3-∶NH4+=1∶1) ,生长量均有所下降 ,而单独供给NH4+-N时 ,生长量则大幅度下降。莴笋单独供给NO3--N时 ,其吸氮量显著高于供给NO3--N +NH4+-N的处理 ,大青菜、菠菜供给NO3--N +NH4+-N与单独供给NO3--N相比吸氮量大体相当 ;小白菜同时供应NO3--N +NH4+N时吸氮量最高 ,供给NO3--N时次之 ,供给NH4+-N时显著降低。供给NH4+-N时 4种作物吸氮量均比其它氮源显著降低。 4种作物对NO3--N与NH4+-N的吸收具有明显的偏向性。供给等氮量铵、硝态氮 (NO3--N +NH4+-N处理 )时 ,菠菜、小白菜吸收的NO3-N显著多于NH4+-N ,表现出喜硝性 ,莴笋则与此相反 ,表现出喜铵性 ;而大青菜对两种形态氮素的吸收量相差不多 ,表现出兼性吸收的特点。但上述偏向性具有阶段特点 ,即喜硝作物可能在某一阶段表现出喜铵性状     

Synthesis of new glycosides by transglycosylation of N-acetylhexosaminidase from Serratia marcescens YS-1

Serratia marcescens YS-1, a chitin-degrading microorganism, produced mainly N-acetylhexosaminidase. The purified enzyme had an optimal pH of approximately 8-9 and remained stable at 40 degrees C for 60 min at pH 6-8. The optimum temperature was around 50 degrees C, and enzyme activity was relatively stable below 50 degrees C. YS-1 N-acetylhexosaminidase hydrolyzed p-nitrophenyl beta-N-acetylgalactosamide by 28.1% relative to p-nitrophenyl beta-N-acetylglucosamide. The N-acetylchitooligosaccharides were hydrolyzed more rapidly, but the cellobiose and chitobiose of disaccharides that had the same beta-1,4 glycosidic bond as di-N-acetylchitobiose were not hydrolyzed. YS-1 N-acetylhexosaminidase efficiently transferred the N-acetylglucosamine residue from di-N-acetylchitobiose (substrate) to alcohols (acceptor). The ratio of transfer to methanol increased to 86% in a reaction with 32% methanol. N-Acetylglucosamine was transferred to the hydroxyl group at C1 of monoalcohols. A dialcohol was used as an acceptor when the carbon number was more than 4 and a hydroxyl group existed on each of the two outside carbons. Sugar alcohols with hydroxyl groups in all carbon positions were not proper acceptors.     

Optimum Nutrient Rate and Nutritional Constraints in Tuber Crops Growing in Ultisol of India with Special Emphasis on Tannia

Tuber crops are generally grown in marginal lands with low native soil fertility. In India, laterite soils (acidic Ultisols) are the major soils for tropical tuber crops and are poor in innate fertility. Among tropical tuber crops, some have adapted to poor soils, such as cassava, whereas others such as tannia (Xanthosoma sagittifolium L.) cannot establish well in these soils and may manifest nutritional disorders, which ultimately result in the complete devastation of the crop. Therefore, we investigated the effects from a preliminary rate trial (PRT) and nutrient-omission pot trial (NOPT) using maize as a test crop and a NOPT with tannia to determine the optimum nutrient rate and limiting nutrients, as well as nutritional problems affecting the growth and yield of tannia. Each experiment was laid out in a complete randomized design with three replications and was conducted for both garden and paddy soils. The PRT revealed that the optimum nutrient requirements for the soils were different, with garden soils requiring nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), zinc (Zn), and molybdenum (Mo) at 200, 60, 160, 70, 60, 50, 4, 8, and 0.8 kg ha^?1, respectively, and paddy soil requiring twice these rates. The NOPT indicated that in addition to N, P, K, B, and Mo in both garden and paddy soils, Ca and Zn in paddy soils and S in garden soils were the constraining nutrients. The NOPT carried out with tannia indicated that the main nutritional problem was subsoil acidity-induced multinutrient deficiencies involving K, Ca, and Mg.     

肥料和稻草氮利用率的三年定位研究

对氮肥和稻草氮的利用率进行了3年6季同位素15N田间定位研究。结果表明,首季单季水稻对氮肥的利用率为37.02%,050cm土壤中15N的残留率为25.81%。经过连续3年6季的种植,作物肥料N的累计回收率分别为40.15(秸杆还田)41.63%(秸杆不还田),050cm土壤中15N的残留率仍达到23.62(秸杆不还田)28.33%(秸杆还田)。在不施氮肥条件下,小麦对稻草氮的吸收率为4.46%,第二季单季稻对稻草氮的吸收率为4.78%。5季作物累计吸收稻草氮11.76%,而土壤残留率为70.37%。     

Evaluation of a modified QuEChERS extraction of multiple classes of pesticides from a rice paddy soil by LC-APCI-MS/MS

A new method for the determination of clomazone, fipronil, tebuconazole, propiconazole, and azoxystrobin in samples of rice paddy soil is presented. The extraction of the pesticides from soil samples was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. Some extraction conditions such as salt addition, sample acidification, use of buffer, and cleanup step were evaluated. The optimized method dealt with a single extraction of the compounds under study with acidified acetonitrile, followed by the addition of MgSO(4) and NaCl prior to the final determination by liquid chromatography-atmospheric chemical pressure ionization-tandem mass spectrometry. Validation studies were carried out in soil samples. Recoveries of the spiked samples ranged between 70.3 and 120% with relative standard deviation lower than 18.2%. The limits of quantification were between 10 and 50 μg kg(-1). The method was applied to the analysis of real samples of soils where rice is cultivated.     

Quantitative determination of linoleic acid in infant formulas by gas chromatography

A method has been developed for the quantitative determination of linoleic acid in infant formulas by gas chromatography (GC). A known amount of triheptadecanoin was spiked into the sample. Total lipid was extracted from the product by an ethyl ether-petroleum ether-ethanol system in a Mojonnier flask. The sample was saponified by methanolic KOH after the solvents were evaporated. Methyl esters of the fatty acids were prepared by boron trifluoride (BF3) in methanol and analyzed by gas chromatography. A glass column packed with 10% SP-2340 (75% cyanopropyl silicone) was used to separate and identify the methyl linoleate and the methyl heptadecanoate. The quantity of methyl linoleate was calculated by comparing the integrated peak areas of these 2 fatty acid methyl esters. This method was satisfactory for both milk protein-based and soy protein-based matrixes. The results obtained by this method are comparable to those obtained by the AOAC spectrophotometric method 28.082-28.085.     

多菌灵在水稻及土壤中的消解动态和残留规律研究

采用田间试验方法,研究了多菌灵在稻田水、土壤和稻秆中的消解动态,测定了多菌灵在水稻和土壤中的最终残留量。样品采用甲醇和稀盐酸的混合溶液提取,经液-液分配净化,HPLC紫外分析测定。结果表明,田水、土壤、稻秆、谷壳、糙米中多菌灵添加浓度为0.05~1.0mg·kg-1时,平均回收率为83.16%~95.44%,变异系数在1.23%~5.32%之间,方法的最低检测浓度为：田水0.005mg·L-1,土壤0.005mg·kg-1,稻秆0.050mg·kg-1,谷壳0.050mg·kg-1,糙米0.025mg·kg-1。多菌灵在田水、土壤和稻秆中的消解动态均符合一级动力学方程,半衰期分别为2.53~3.41d、6.20~7.27d、3.27~3.91d,原始沉积量与施药量、施药次数密切相关。以231g·hm-2和346.5g·hm-2间隔7d施用多菌灵2次和3次,末次施药21d后多菌灵的最高残留量为：土壤未检出（≤0.005mg·kg-1）,稻秆0.524mg·kg-1,谷壳0.528mg·kg-1,糙米未检出（≤0.025mg·kg-1）。多菌灵在稻秆和谷壳中的残留量相对较高,以该稻秆和谷壳作为饲料有一定的风险;多菌灵在糙米中的残留量低于我国和食品法典委员会（CAC）及日本的最大残留限量（MRL）标准。