The Development Of An Immunoassay For The Rapid Detection Of κ-casein In Bovine Milk Based On Nano-magnetic Beads

Protein is the most valuable nutrients in milk and the caseins account for80%among totalproteins. There are four major caseins in milk, namely αs1-, αs2-, β-, and κ-casein.The κ-casein isspecific as antigen,so the quantity of κ-casein could be used to measure the quality of bovinemilk.In this study,the BALB/c mice were immunized with κ-casein using foot-pad immunizationand intraperitoneal injection. Popliteal lymph node cells or splenocyte from the immunized micewere fused with SP2/0myeloma cells in the presence of PEG. Three hybridoma strains (1C4、3G3、3E6) which secreted the antibody specific for Kappa casein were obtained．The sub-class ofthe antibodies were IgG1.The ascites were purified by Protein G affinity layer absorptioncolumn.The antigenic epitope of3G3and3E6were different and it was close between1C4and3E6.The titer of purified ascites (1C4) was1.28×106and the affinity constant was2.89×108mol/L. A monoclonal antibody against kappa Casein with good affinity has been achieved。Aimmunoassay based on nano-magnetic beads and a mode of McAb-antigen-labeled PcAb wereestablished,which provided foundations for the rapid detection of casein in bovine milk samples．The standard curve of the ELISA based on nano-magnetic beads was obtained. Theregression equation of κ-casein was y=2.814x+1.24with correlation coefficient R2=0.984，thelinear detection range was1-32μg mL-1, the LOD was0.42ng mL-1.And the regression equationof κ-casein which used another method was y=8.684x–14.11and the correlation coefficientwas R2=0.991, the linear detection range was0.25-8μg mL-1, the LOD was0.043μg mL-1.Comparing two kinds of detection methods, the nano-magnetic beads could accelerate the speedof reaction.Using the methods for detecting real samples,the results showed that both of themcould be used for the actual detection,and the immunoassay based on nano-magnetic beads wasbetter.