酶联免疫法快速筛查牛奶中的二噁英

二噁英是最受人们关注的持久性有机污染物之一。本研究建立了ELISA快速筛查牛奶基质中二噁英的检测方法。牛奶样品经真空冷冻干燥,加速溶剂萃取法萃取、用酸性硅胶柱、活性炭柱净化后,浓缩氮吹后复溶,进行ELISA检测。结果表明,方法回收率为72.0%~125.2%,相对偏差<30%。本法对牛奶中二噁英毒性当量(TEQ)测定值与高分辨气相色谱-高分辨质谱法(HRGC-HRMS)测定值吻合度高,检测结果误差<20%。本方法具有操作简便、快速、检测结果可靠等特点,适用于基层检测机构对牛奶中二噁英的快速筛查。     

Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).     

Development of a stable isotope dilution assay for an accurate quantification of protein-bound N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine using a (13)C-labeled internal standard

Syntheses of the labeled Amadori compound [(13)C(6)]-N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine ([(13)C(6)]-DFLys) and the labeled glycated tetrapeptide Ala-[(13)C(6)]-DFLys-Leu-Gly are presented. The compounds were used in the development of stable isotope dilution assays for the quantification of the degree of glycosylation of bovine serum albumin treated for 20 min at 95 degrees C in the presence of glucose. The experiments revealed that the use of the labeled standards in combination with LC/MS allowed the exact quantification of protein-bound DFLys with the high recovery rate of 95% (at a spike level of 150 nmol/mg of protein) and a low detection limit of 5 nmol/mg of protein. The data revealed, however, that DFLys is significantly degraded during the enzymic hydrolysis of the protein backbone generally needed in the quantification procedure and, furthermore, incomplete digestion of the protein was observed. Both sources of errors were clearly overcome by using in particular the labeled peptide as the internal standard.     

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.     

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers.     

Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M1 in milk

Using a highly specific antibody against aflatoxin M1, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation of M1 in milk. RIA was sensitive in the range of 5-50 ng per assay but was subject to interference by whole milk. Extraction and cleanup were therefore necessary for the detection of M1 in milk at 0.5 ng/mL. An ELISA procedure was developed by using an aflatoxin M1-carboxymethyl-horseradish peroxidase conjugate as the ligand. Competitive assays revealed that this system was relatively more sensitive for M1 than for B1, and had a much lower degree of cross-reactivity for aflatoxins B2, G1, G2, B2a, and aflatoxicol. As low as 0.25 ng M1/mL in artificially contaminated milk (raw, whole, skim) could be detected by ELISA in 3 h without extraction or cleanup. Because of its simplicity, sensitivity, and specificity, ELISA is the preferred method for monitoring aflatoxin M1 in milk.     

Measurement of radioactivity in contaminated crops (Part 1)

It is a prerequisite to settle down how to subtract the counts based on natural radio-potassium in crops, finding out an accurate and efficient method. Considerable effort has been made so far with the investigations which have been conducted In various parts of Japan in accordance with this purpose. The writers wish to present a tentative method for measuring the radioactivity, reporting on the results obtained in comparison of this method with here-to-fore methods.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Origin of acetaldehyde during milk fermentation using (13)C-labeled precursors

Acetaldehyde formation by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus during fermentation of cow's milk was investigated using (13)C-labeled glucose, L-threonine, and pyruvate with a recent static-and-trapped-headspace technique that does not require derivatization of acetaldehyde prior to gas chromatography-mass spectrometry. Over 90% and almost 100% of acetaldehyde originated from glucose during fermentation by L. delbrueckii subsp. bulgaricus and S. thermophilus, respectively, taking into account both singly and doubly labeled acetaldehyde. As both microorganisms showed threonine aldolase activity and formed labeled acetaldehyde from (13)C-labeled threonine during the fermentation of milk, this amino acid should also contribute to the acetaldehyde produced.     

Quantification of beta casein in milk and cheese using an optical immunosensor

beta-Casein was quantified in milk and cheese, using an optical immunosensor, based on surface plasmon resonance (SPR) measurement. The assay consists of a two-step sandwich strategy, with two anti-beta-casein antibodies directed against each extremity of the casein. This strategy permits only native beta-casein to be quantified and not its degradation products. The calibration curve was obtained with a reference milk powder of known beta-casein concentration. The analysis time per sample was less than 10 minutes. The antibody-coated surface could be used for more than 250 determinations. The detection limit was established at 85 ng x mL(-)(1) and the intra- and inter-assay variation coefficients were 2.6 and 6.2% respectively. The method was applied to raw milk to quantify intact beta-casein, with no pretreatment of the sample. A second application was realized with cheese, to follow the proteolysis of beta-casein during ripening.     

边鸡胰岛素样生长因子1基因（IGF-1）外显子3的多态性及其与繁殖性能的关系

根据GenBank中发表的鸡胰岛素样生长因子1基因(IGF-1)的序列针对外显子2和外显子3分别设计1对引物,采用PCR-SSCP技术检测边鸡(Gallus gallus)及2个对照群体(京海黄鸡(Gallus gallus)和尤溪麻鸡(Gallus gallus))的单核苷酸多态性,并与边鸡的繁殖性能进行相关性分析.结果表明,3个品种在IGF-1外显子2中未检测到多态,而在外显子3中都检测到3种基因型(AA,AB和BB).在边鸡和京海黄鸡中A等位基因为优势等位基因,而在尤溪麻鸡中B等位基因为优势等位基因.克隆测序表明,AA型与BB型相比有两处突变(93A→G和148G→A),其中148 bp处的突变导致氨基酸由谷氨酸变为赖氨酸,通过与Gen-Bank中的鸡IGF-1基因序列比对,发现两处突变分别位于IGF-1基因外显子3的62 bp和117 bp.最小二乘分析表明,AA与BB基因型个体300日龄的产蛋数存在显著差异(P<0.05).     

皖西白鹅肝脏中GHR，IGF-1和 IGF-1R基因的表达及与朗德鹅的比较

选择90日龄的皖西白鹅和朗德鹅各20只,取肝脏组织。采用半定量逆转录多聚酶链式反应（RT-PCR）的方法,以β-actin为内标,对肝脏组织中生长激素受体（GHR）、胰岛素样生长因子-1（IGF-1）、胰岛素样生长因子-1型受体（IGF-1R）的mRNA表达量分别进行比较分析,并分别与鹅的肝脏重及肝重率进行相关性分析。结果表明：皖西白鹅的肝脏重和肝重率都显著大于朗德鹅（p<0.05）;皖西白鹅肝脏中IGF-1的mRNA表达明显高于朗德鹅（p<0.01）;而GHR、IGF-1R mRNA在两品种间没有显著差异。体重与肝脏重显著正相关（r = 0.35,p<0.05）,肝重率与肝脏中IGF-1的mRNA的表达显著正相关（r = 0.67,p<0.01）,而与GHR、IGF-1R mRNA没有显著的相关性。结果提示：（1）90日龄的皖西白鹅肝脏生长优于朗德鹅;（2）两品种鹅肝脏生长的差异可能与肝脏中IGF-1 mRNA表达的差异有关。     

Determination of aflatoxin M1 in milk and milk powder using high-flow solid-phase extraction and liquid chromatography-tandem mass spectrometry

Animal feeds occasionally have some degree of contamination by Aspergillus spp. Even pasteurized milk at times contains the toxic liver carcinogen aflatoxin M1 (AFM1). Confirmation of its presence is now done with solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC)-fluorescence, using a small enough sample that SPE time is reasonable. In this study 200 mL of milk was extracted using a C18 disk at a flow rate of approximately 100 mL/min and AFM1 quantified by HPLC-tandem mass spectrometry with negative electrospray ionization. The effectiveness and cleanup efficacy of immunoaffinity columns (IAC) was compared with that of Mycosep multifunctional cleanup columns (MFC). Average recovery and detection limits of whole milk and low-fat milk cleaned up by IAC were significantly superior to those obtained with the MFC (78-87% and 0.59-0.66 ng/L, respectively). The new procedure improves extraction speed, sensitivity, and specificity.     

Liquid chromatographic determination and identification of morantel-related residues as precursors of 3-(3-methyl-2-thienyl) acrylic acid (CP-20,107) in bovine milk

A simple liquid chromatographic (LC) method was developed to determine and identify incurred morantel-related residues in bovine milk by converting them to 3-(3-methyl-2-thienyl) acrylic acid (CP-20, 107). Key techniques in this method involve short-term digestion of milk in HCl to release residues convertible to CP-20, 107, isolation and alkaline hydrolysis of these precursors to CP-20, 107, and recovery of the product for LC analysis. Photochemical conversion of CP-20, 107 to its cis-isomer and separation by LC identifies the residue. A homolog (pyrantel), which is used as an internal standard, is hydrolyzed to 3-(2-thienyl) acrylic acid. These acrylic acid isomers are readily resolved by LC. The method was evaluated over the 1-4 ppb (ng/mL) range for accuracy and precision to assess its utility for withdrawal studies. Bovine milk supplemented with morantel at 1, 2, and 4 ppb and assayed in replicate (n = 7-8) over 4 trials gave mean values and standard deviations of 1.0 +/- 0.11, 2.0 +/- 0.24, and 4.0 +/- 0.44 ppb, respectively. A milk specimen containing physiologically incurred residues of morantel assayed 2.1 +/- 0.19 ppb in replicate (n = 5).     

Naturally occurring estrogens in processed milk and in raw milk (from gestated cows)

The occurrence of the steroid hormones estrone (E1), 17alpha-estradiol (alphaE2), 17beta-estradiol (betaE2), and estriol (E3) in processed bovine milk with different fat contents and in raw milk from (non)gestated cows was investigated. Following liquid extraction, optional enzymatical deconjugation, C18 solid-phase extraction, and derivatization, estrogens were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free and deconjugated E1 (6.2-1266 ng/L) was the major estrogen followed by alphaE2 (7.2-322 ng/L) and betaE2 (5.6-51 ng/L), whereas E3 was detected regularly at the detection limit of 10 ng/L. The lowest and highest concentrations were determined in raw milk from nonpregnant and from cows in the third trimester of gestation, respectively. The estrogen concentration in processed milk coincides with that of raw milk between first and second trimesters, reflecting the contribution of lactating pregnant cows to the final consumable product. The daily intake of total investigated estrogens through milk is 372 ng, which is dramatically more than currently recognized.     

Detection of streptomycin residues in whole milk using an optical immunobiosensor.

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples ( approximately 3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).     

IGF-1和IGF-2在成年牦牛皮肤毛囊生长周期中的表达与定位

为探究胰岛素样生长因子1(IGF-1)和胰岛素样生长因子2(IGF-2)对成年牦牛皮肤毛囊生长周期转换的影响,本研究采集成年牦牛毛囊生长期、退行期和休止期的皮肤,通过实时荧光定量PCR(qRT-PCR)、蛋白免疫印迹(Western blot)和免疫组织化学(IHC)方法检测IGF-1和IGF-2在毛囊生长周期过程中皮肤的表达与定位。qRT-PCR结果显示,IGF-1和IGF-2 mRNA转录水平均在毛囊生长期皮肤中最高,在休止期皮肤中最低,且在各个时期之间差异显著(P<0.05)。Western blot结果表明,IGF-1和IGF-2蛋白表达量均在毛囊生长期皮肤中最高,且显著高于毛囊退行期和休止期皮肤(P<0.05)。IHC结果显示,IGF-1和IGF-2在毛囊生长期、退行期和休止期皮肤中表达位置基本相同,主要定位于皮肤表皮、毛囊外根鞘和毛母质细胞。综上所述,IGF-1和IGF-2都参与毛囊生长周期的调控,并具有协同作用的关系,且该过程可能通过影响上皮细胞的活性来促进毛囊的生长。本研究结果为进一步探讨IGF-1和IGF-2在牦牛毛囊周期循环中的调控机制提供了理论依据。     

Quantitation of (R)- and (S)-linalool in beer using solid phase microextraction (SPME) in combination with a stable isotope dilution assay (SIDA)

A stable isotope dilution assay (SIDA) was developed for the quantitation of both linalool enantiomers using synthesized [2H(2)]R/S-linalool as the internal standard. For enrichment of the target compound from beer, a solid phase microextraction method (SPME) was developed. In comparison to the more time-consuming extraction/distillation cleanup of the beer samples, the results obtained by SPME/SIDA were very similar, even under nonequilibration conditions. Analysis of five different types of beer showed significant differences in the linalool concentrations, which were clearly correlated with the intensity of the hoppy aroma note as evaluated by a sensory panel. In addition, significant differences in the R/S ratios were measured in the beers. The SPME/SIDA yielded exact data independently from headspace sampling parameters, such as exposure time or ionic strength of the solution.