检测牛乳κ-酪蛋白的间接竞争ELISA法与间接ELISA法比较

doi:10.16431/j.cnki.1671-7236.2018.04.035

Abstract

Abstract:

The study was aimed to develop a rapid and simple enzyme-linked immunosorbent assay (ELISA) method for the detection of milk κ-casein (κ-CN) content and provide technical services for solving the problem of adulterated milk protein.The indirect competition ELISA method and the indirect ELISA method were respectively established by taking the milk κ-CN as the envelope antigen and the enzyme labeled antibody (HRP-IgG) as the detection antibody,and the two detecting methods were analyzed and compared.The results showed that the κ-CN antigen concentration was 2.5 μg/mL,the linear range was 62.5 to 1 000.0 ng/mL,the coefficient of variation was less than 2%,the recovery rate was 98.46% to 101.68% when using the indirect competitive ELISA method; The κ-CN antigen concentration was 1.56 μg/mL,the linear range was 0.098 to 3.125 μg/mL,the coefficient of variation was less than 1%,the recovery rate was 99.10% to 101.06% for the indirect ELISA method.Comparing these two methods,the indirect competitive ELISA method took shorter time and the amount used of antibody was less.The indirect ELISA method did not need to cover the higher cost of protein,and the correlation coefficient was higher.The latter was difficult to form ELISA kit system because of its more components and samples need to be coated,so it was not suitable for field testing.The indirect competition method could be used for the large-scale preparation of ELISA kit system for rapid detection of protein content,because of its low dosage of antibodies,cost savings,fast and simple operation,was better for practical applications.

Key words: bovine κ-casein; indirect competitive ELISA; indirect ELISA

CLC Number:

TS252.7

SONG Hongxin, HAN Bo, LI Shan, XUE Haiyan. Comparison Between Indirect Competitive ELISA and Indirect ELISA in Detecting Bovine κ-casein[J]. , 2018, 45(4): 1114-1119.

References

[1] 宋芳.β-酪蛋白单克隆抗体的制备及两种ELISA检测方法的建立[D].长春:吉林大学,2013. SONG F.Preparation of monoclonal antibodies against β-casein and development of two-type ELISAs for β-casein[D].Changchun:Jilin University,2013.(in Chinese)
[2] MADUREIRA A R,PEREIRA C I,GOMES A M P,et al.Bovine whey proteins-overview on their main biological properties[J].Food Research International,2007,40(10):1197-1211.
[3] 张莹.基于纳米磁珠的牛乳中κ-酪蛋白快速检测方法的建立[D].长春:吉林大学,2014. ZHANG Y.The development of an immunoassay for the rapid detection of κ-casein in bovine milk based on nano-magnetic beads[D].Changchun:Jilin University,2014.(in Chinese)
[4] ZHANG Z,ARRIGHI V,Campbell L,et al.Properties of partially denatured whey protein products 2:Solution flow properties[J].Food Hydrocolloids, 2016,56:218-226.
[5] QI P X,ONWULATA C I.Physical properties,molecular structures,and protein quality of texturized whey protein isolate:Effect of extrusion moisture content[J].Journal of Agricultural & Food Chemistry,2011,94(5):2231-2244.
[6] TURCK D,SZAJEWSKA H,ShAMIR R.Cow's milk and goat's milk[J].World Review of Nutrition & Dietetics,2013,108(1):56-62.
[7] HORNE D S,Casein micelle structure:Models and muddles[J]. Current Opinion Colloid & Interface Science,2006,11(4):148-153.
[8] SINGH H.Interactions of milk proteins during the manufacture of milk powders[J].Dairy Science & Technology,2007,87(4):413-423.
[9] 杨楠,梁琪,甘伯中,等.热处理对牛乳酪蛋白胶束结构影响的研究进展[J].中国乳品工业,2012,40(1):46-49. YANG N,LIANG Q,GAN B Z,et al.Progress of effect on bovine casein micelles during heat treatment[J].China Dairy Industry,2012,40(1):46-49.(in Chinese)
[10] 汝成业.酶法降低牛乳αs1-酪蛋白致敏性[D].无锡:江南大学,2015. RU C Y.Enzymatic hydrolysis of milk αs1-casein for allergenicity reduction[D]. Wuxi:Jiangnan University,2015.(in Chinese)
[11] 张莹,周玉,李岩松,等.抗κ-酪蛋白单克隆抗体的制备及其特性鉴定[J].中国畜牧兽医,2014,41(3):24-27. ZHANG Y,ZHOU Y,LI Y S,et al.Preparation and characteristics identification of anti-kappa casein monoclonal antibody[J].China Animal Husbandry & Veterinary Medicine,2014,41(3):24-27.(in Chinese)
[12] BRAMBILLA A,FELIGNI M,ENNE G.αs1-casein in goat milk:Identification of genetic variants by capillary zone electrophoresis compared to isoelectric focusing[J]. Italian Journal of Animal Science,2011,2(1S):100-102.
[13] PESIC M B,BARAC M B,VRVIC M M,et al.The distributions of major whey proteins in acid wheys obtained from caprine/bovine and ovine/bovine milk mixtures[J].International Dairy Journal,2011,21(10):831-838.
[14] 陈惠,谢彩军,叶广辉,等.几种儿童牛奶酪蛋白含量的测定与比较[J].食品研究与开发,2014,35(20):101-103. CHEN H,XIE C J,YE G H,et al.Several children milk casein content determination and comparison[J]. Food Research and Developmen,2014,35(20):101-103.(in Chinese)
[15] HAM J S,JEONG S G,LEE S G,et al.Irradiation effect on α-and β-caseins of milk and Queso Blanco cheese determined by capillary electrophoresis[J].Radiation Physics & Chemistry,2009,78(2):158-163.
[16] LIU Z,MOATE P,COCKS B,et al.Comprehensive polar lipid identification and quantification in milk by liquid chromatography-mass spectrometry[J].Journal of Chromatography B,2015,s 978-979:95-102.
[17] VERCAMBRE M,CUVELIER H,GAYON Y A,et al.Development of a cow's milk detection sandwich ELISA kit using monoclonal antibodies[J].Nippon Shokuhin Kagaku Kogaku Kaishi,2014,61(1):1-8.
[18] 李媛媛,刘宾,曹凤波,等.测定三种乳蛋白抗原性间接竞争ELISA法的建立[J].食品工业,2015(3):290-295. LI Y Y,LIU B,CAO F B,et al.Indirect competieive ELISA for detection of the antigenic of three kinds of protein from cow's milk[J].Food Industry,2015(3):290-295.(in Chinese)
[19] 张咪,高璐,印伯星,等.基于αs-酪蛋白特异性的牛乳总蛋白质ELISA定量检测方法的建立[J].江苏农业学报,2015,31(1):87-92. ZHANG M,GAO L,YIN B X,et al.Establishment of a quantitative ELISA method for detection of total milk protein based on the specificity of αs-casein[J].Journal of Jiangsu Agricultural,2015,31(1):87-92.(in Chinese)
[20] STERN E,VACIC A,LI C,et al.A nanoelectronic enzyme-linked immunosorbent assay for detection of proteins in physiological solutions[J].Small,2010,6(2):232-238.
[21] 张涛, 庞广昌. 酶联免疫快速测定原料乳中κ-酪蛋白的含量[J].中国乳品工业.2006,27(11):179-181. ZHANG T,PANG G C.Enzyme-linked immune content of kappa-casein predominate in rapid determination of raw milk[J].China Dairy Industry,2006,27(11):179-181.(in Chinese)
[22] BITRI L,ROLLAND M P,BESANCON P.Immunological detection of bovine caseinomacropeptide in ovine and caprine dairy products[J].Milchwissenschaft,1993,48:367-370.

Comparison Between Indirect Competitive ELISA and Indirect ELISA in Detecting Bovine κ-casein

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