title: Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.
abstract: In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples.
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: Y S
authors.initials: YS
authors.lastname: Li
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: X Y
authors.initials: XY
authors.lastname: Meng
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China. Electronic address: zhouyu69@sina.com.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Zhang
authors.affiliation: Grain and Oil Food Processing Key Laboratory of Jilin Province, Jilin Business and Technology College, Changchun 130062, PR China.
authors.forename: X M
authors.initials: XM
authors.lastname: Meng
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: L
authors.initials: L
authors.lastname: Yang
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: P
authors.initials: P
authors.lastname: Hu
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: S Y
authors.initials: SY
authors.lastname: Lu
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: H L
authors.initials: HL
authors.lastname: Ren
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: Z S
authors.initials: ZS
authors.lastname: Liu
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China.
authors.forename: X R
authors.initials: XR
authors.lastname: Wang
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized; D001560:Benzidines; D015374:Biosensing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D002364:Caseins / Q000032:analysis* / Q000276:immunology; D002417:Cattle; D006046:Gold; D006735:Horseradish Peroxidase; D007118:Immunoassay / Q000379:methods* / Q000706:statistics & numerical data; D060328:Magnetic Phenomena; D053768:Metal Nanoparticles / Q000648:ultrastructure; D008892:Milk / Q000737:chemistry*
chemical_list: D055910:Antibodies, Immobilized; D001560:Benzidines; D002364:Caseins; C021758:3,3',5,5'-tetramethylbenzidine; D006046:Gold; D006735:Horseradish Peroxidase
keywords: Bovine milk; Immunoassay; Probe; β-casein