Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese

The aim of this work was to develop an assay capable of detecting adulteration of soft goat, sheep and buffalo milk cheese with bovine milk from cheaper sources. A previously developed indirect competitive ELISA had a lower sensitivity when applied to cheese, compared with milk. A sandwich ELISA was developed utilising the same monoclonal antibody in combination with a polyclonal goat anti-bovine IgG antibody. Once optimised, the ELISA was found to be highly specific. Detection limits in milk were 0.001% cows’ milk adulteration of sheep or buffalo milk, and 0.01% cows’ milk adulteration of goat milk. Detection limits in soft cheese were 0.001% in goat cheese and 0.01% in sheep or buffalo cheese. The assay was highly reproducible with both intra- and inter-assay coefficient of variation <10%. The ELISA performance makes it suitable for development as a kit for use in routine surveillance of milk and soft cheese.     

Detection of cow milk adulteration in yak milk by ELISA

In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10–8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk.     

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.     

Detection of cows' milk in ewes' milk and cheese by an indirect enzyme-linked immunosorbent assay (ELISA)

An indirect enzyme-linked immunosorbent assay was successfully developed for the detection of defined amounts of cows' milk (1-50%) in sheeps' milk and cheese. The assay used polyclonal antibodies raised in rabbits against bovine caseins (BC). The anti-BC antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing BC. The antibodies were biotinylated and rendered cows' milk specific by mixing them with lyophilized ovine and caprine caseins. ExtrAvidin-peroxidase was used to detect the biotinylated anti-BC antibodies bound to BC immobilized on 96-well plates. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying mixtures of sheeps' milk and cheese containing variable amounts of cows' milk.     

A competitive enzyme-linked immunosorbent assay for detection of bovine milk in ovine and caprine milk and cheese using a monoclonal antibody against bovine beta-casein.

A competitive ELISA (enzyme-linked immunosorbent assay) was performed to detect and quantify bovine milk in ovine and caprine milk and cheese using a monoclonal antibody (AH4 MAb) against bovine beta-casein. Ovine or caprine milk and cheese containing bovine milk were added simultaneously with the AH4 MAb to the wells of a microtiter plate that had been previously sensitized with commercial bovine beta-casein. The bovine caseins in milk or cheese samples compete with the bovine beta-casein bound to the plate for the AH4 MAb binding sites. Further immunorecognition of AH4 MAb bound to the bovine beta-casein immobilized onto the plate was attained with rabbit anti-mouse immunoglobulin conjugated to peroxidase. Subsequent enzymic conversion of the substrate showed clear differences in absorbance values during assay of mixtures of ovine and caprine milk and cheese containing various amounts of bovine milk. The competitive ELISA developed in this work allows the quantitative detection of bovine milk in ovine and caprine milk and cheese samples in the range of 0.5 to 25% of substitution.     

Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes.

A direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking greater than 500,000 cells/ml as being indicative of mastitis, and the assay was 0.94, yielding an assay sensitivity of 95.2% and a specificity of 97.3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100,000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.     

羊乳中掺入牛乳的间接ELISA定量检测

制备抗β- 酪蛋白多克隆血清,免疫吸收封闭与羊乳反应的抗体,获免疫吸收抗体用于乳样的间接ELISA检测中,建立羊乳中掺入牛乳成分的定量免疫学方法。实验表明,该间接ELISA 法用于原乳检测时,掺入牛乳的百分含量与A450nm 在4%～50% 呈线性关系,该方法的最低检出量为4%。所建立的ELISA 方法变异系数＜ 5%,回收率在94%～105% 之间,符合方法学要求,可用于牛乳掺假的定量检测。对灭菌乳的检测表明,热处理不改变β- 酪蛋白与抗体反应的特性,方法还可用于经热加工的乳品检测中。     

牛β-乳球蛋白胶体金免疫层析试纸条的研制及检测应用

研制了一种可快速检测羊乳及制品中牛源β-乳球蛋白(β-lactoglobulin,β-lg)的胶体金免疫层析试纸条。通过杂交瘤技术制备牛β-lg特异性单克隆抗体(monoclonal antibody,mAb),利用柠檬酸钠还原氯金酸,形成30 nm胶体金颗粒,并用于标记牛β-lgmAb。采用竞争法研制免疫层析试纸条,将胶体金标记的牛β-lgmAb包被于金标垫,牛β-lg和羊抗鼠IgG标记于硝酸纤维素膜分别作为检测线(T线)和质控线(C线),牛β-lg和二抗的最佳包被浓度均为1.0 mg/mL。制得的单克隆抗体纯度都在90%以上,效价均在10000以上且特异性较好。该试纸条对牛β-lg的检测限(limit of detection,LOD)值为3.13μg/mL,对牛源α-CN,牛源β-CN,牛源κ-CN,牛源α-LA,BSA均未产生交叉反应,对脱脂羊乳粉中掺杂脱脂牛奶粉的LOD值为5%,并用该方法对市售羊奶及配方羊奶粉进行分析,检测结果与商品化的ELISA试剂盒一致。该方法前处理快速简单,可在5 min内对牛β-lg进行检测,可用于羊乳制品的现场快速检测。     

Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism

A new polymerase chain reaction (PCR)-based method was developed to detect cows milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0.1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.     

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。     

Application of an indirect ELISA and a PCR technique for detection of cows’ milk in sheep's and goats’ milk cheeses

We have applied a polymerase chain reaction (PCR) procedure and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting the presence of cows’ milk in sheeps’ and goats’ milk cheeses. The PCR used a cattle-specific primer set targeting a 223 bp fragment from the mitochondrial 12S rRNA gene. This technique was applied to experimental cheese mixtures, industrial cheeses produced with known amounts of cow's milk, and several commercial cheeses, enabling the detection of low percentages of cows’ milk (1%). An indirect ELISA using a monoclonal antibody (AH4) against bovine β-casein was also assayed in this study for the specific detection of bovine milk in cheese. Results suggested that both ELISA and PCR may provide specific and reliable tools for detection of low percentages of undeclared cows’ milk in sheeps’ and/or goats’ milk cheeses and other dairy products.     

恩诺沙星抗体免疫检测及其分子识别机制研究

兽药恩诺沙星对人体具有较强的毒副作用,检测恩诺沙星残留对保障食品安全具有重要的作用。本研究将恩诺沙星与牛血清白蛋白(BSA)的偶联物注射免疫BALB/c小鼠制备恩诺沙星单克隆抗体,并建立基于单克隆抗体的酶联免疫法检测样品羊奶中恩诺沙星的方法。将筛选出的杂交瘤细胞注射小鼠提取腹水,并以此单克隆抗体建立直接竞争ELISA方法。其半数抑制率(IC50)为0.71±0.05 ng/m L,最低检测限(IC15)为0.04±0.02 ng/m L。检测羊奶样品含恩诺沙星在10~200 ng/m L时回收率为96.56~105.10%,且与HPLC法检测呈良好线性关系(R2=0.9998)。最后利用计算机生物信息学构建恩诺沙星抗体的可变区三维结构并与抗原进行分子对接。结果显示Arg98、Asp99、Gly101、Thr50、Ser51、Ala82、Tyr85等氨基酸残基在抗体抗原识别过程中起关键作用。这些信息对今后抗体结构修饰具有理论指导意义。     

Fast biosensor immunoassays for the detection of cows' milk in the milk of ewes and goats

Two monoclonal antibodies (MAb) raised against bovine kappa-casein were developed and applied in an automated optical biosensor (Biacore 3000) to create easy and fast direct and inhibition biosensor immunoassays (BIA) for the detection of cows' milk in the milk of ewes and goats. With both assay formats, low limits of detection (<01%) and fast run times (around 5 min) were obtained. For sample preparation, milk was diluted in buffer (direct assay) or in an antibody-containing buffer (inhibition assay) only. For quantitative analysis, calibrants of cows' milk in ewes' or goats' milk were used. Advantages of the direct BIA are: the single reagent format (biosensor chip immobilized antibodies only); the use of small amounts of antibodies (2 microg for >350 tests); and the wide measurement range (0.1 to 10% cows' milk). Despite these advantages, the inhibition BIA (using kappa-casein immobilized on the chip) was preferred because of the possible application of non-purified Mab, the higher responses, the higher sensitivity at relevant low percentages of cows' milk and its robustness (>800 cycles per chip).     

Indirect ELISA for detection of goats' milk in ewes' milk and cheese

An indirect ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of goats' milk (1–25%) in ewes' milk and cheese. The assay uses polyclonal antibodies raised in rabbits against goats' caseins (GC). The anti-GC antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized goats' caseins. The anti-GC antibodies were biotinylated and rendered goats' milk specific by mixing them with lyophilized bovine and ovine caseins. The assay was developed in a non-competitive ELISA format and it comprised coating plates with extracts from samples. ExtrAvidin-peroxidase was used to detect the biotinylated anti-GC antibodies bound to goats' caseins immobilized on 96-well plates. The colour developed by the subsequent enzymic conversion of the substrate resulted in discernible differences in optical densities when assaying mixtures of ewes' milk and cheese containing variable amounts of goats' milk.     

Detection of cows' milk in ewes' milk and cheese by a sandwich enzyme-linked immunosorbent assay (ELISA)

A sandwich enzyme-linked immunosorbent assay (ELISA) has been successfully developed for the detection of defined amounts of cows' milk in ewes' milk and cheese. Polyclonal antibodies were raised in goats against bovine caseins (BC). The resultant antibodies were recovered from the crude antiserum by ammonium sulphate precipitation and further purified by immunoatisorption of the cross-reacting antibodies onto columns containing immobilised ovine, caprine and bovine caseins, followed by elution of the bovine caseins specific antibodies (anti-BC) from the column containing the bovine caseins. The anti-BC bound to the wells of a microtitre plate were used to capture the BC from milk and cheese mixtures. Further immunorecognition of the captured proteins was attained with the same specific antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying mixtures of ewes' milk and cheese containing variable amounts of cows' milk.     

牛初乳中免疫球蛋白G双抗夹心ELISA检测方法的建立

将牛免疫球蛋白G（immunoglobulin G,IgG）作为免疫原免疫BALB/c小鼠,通过细胞融合、筛选获得分泌抗牛IgG单克隆抗体的细胞株。制备小鼠腹水抗体,进一步纯化获得抗牛IgG单克隆抗体。建立双抗夹心酶联免疫吸附法检测牛初乳中IgG质量浓度,该方法在7.8～1 000 ng/mL范围内有良好的线性关系,最低检出限为7.06 ng/mL,批内变异系数为4.52%,批间变异系数为4.94%,回收率为91.85%～102.45%。此法操作简便、准确度高、稳定性好,可用于实际牛初乳样品的快速检测。     

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.     

Development of a cows' milk identification test (COMIT) for field use

A cows' milk identification test (COMIT) has been successfully developed for the detection of defined amounts of cows' milk (3-100%) in ewes' milk. The assay uses polyclonal antibodies raised in goats against bovine whey proteins. The test, an agar-gel immunodiffusion technique, uses agar plates with a printed template for correct placement of the test components (antisera discs, positive and negative milk reference discs and sample discs). The presence of cows' milk in the sample was recorded as positive when the precipitin lines appearing between the sample disc and the antisera disc fuse completely with those from the positive cows' milk reference disc. This test is reliable, practical and easy to perform in the field and in dairy factories. Furthermore, the nature and stability of the components of the test make it suitable for commercial development into kits which should be highly practical in any kind of inspection programme concerned with milk species identification.     

Production of a horse-specific monoclonal antibody and detection of horse meat in raw meat mixtures by an indirect ELISA

A stable hybridoma cell line (DD3) has been produced secreting a monoclonal antibody specific for horse muscle proteins. The DD3 monoclonal antibody (mAb) did not show significant cross-reactivity when tested against beef, chicken, pig and soya proteins or bovine caseins, gelatine and bovine serum albumin. The DD3 mAb was used in an indirect ELISA format for the detection of defined amounts of horse meat (10–500 g kg-¹) in beef meat mixtures. Immunorecognition of monoclonal antibodies adsorbed to horse meat adsorbed onto the ELISA plate was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of the substrate gave clear optical density differences when assaying mixtures of minced beef containing different amounts of horse meat.     

Studies on thermostable antigens, production of species-specific antiadrenal sera and comparison of immunological techniques in meat speciation

Heat-stable antigens (BE forms: resistant to heat and ethanol precipitation) of adrenal and muscle tissues of cattle, buffalo, sheep, goat and pig were prepared for use in detection of adulteration in meats. The physico-chemical characteristics of these antigens revealed that the antigens of adrenals had only one component corresponding to 'Troponin T'. Muscle antigens also contained a major troponin T component but were associated with low molecular weight fractions. Rabbit antiadrenal BE sera were developed and made species specific by immunoabsorption. The species-specific antisera were employed for identification of origin of fresh and cooked meats and their mixtures, using an immunodiffusion test-agar gel precipitation test (AGPT), counterimmunoelectrophoresis (CIEP), enzyme-linked immunosorbent assay (ELISA) and the unlabelled antibody peroxidase antiperoxidase (PAP) technique. The results indicated that absorbed antisera could successfully differentiate the fresh, cooked meats and the meat mixtures from the species under study. AGPT and CIEP were useful in identification of 5-10% addition, using water extracts of fresh meats and BE forms of cooked meats, whereas ELISA and PAP could detect adulteration down to the level of 1% when water extracts were used. Among the tests employed in the study, the PAP technique proved to be most sensitive. The antisera were also proved useful in identifying the species in canned meat products, milk, serum, plasma, semen, urine, organs, skin and spoilt flesh, employing AGPT and CIEP.