| Lactoferrin is an important iron-binding protein,and it belongs to the glycoprotein family. Due to its multi-biological functions,such as antibacteria and sterilization,antiviral and immune regulation, it has been widely used in medicine and food industry. Especially bovine lactoferrin added in the infant formula milk is a hot issue in current researches.At present there are such detection methods as High Performance Liquid Chromatography(HPLC),SDS-PAGE as well as Enzyme Linked Immunosorbent Assay(ELISA).Compared to other detection methods,ELISA method has rapid,high sensitivity and lots of samples can be handled at the same time, so it has more advantages in the detection methods.Six strains of hybridoma cells were got by the hybridoma cells technique.They were named 3H3,3A5,2B11,2B12,2E2 and 3D7.These six McAbs were measured for the antibody titer,specificities and affinity.The titer degree of ascites fluid of these five McAbs was above 1:106.There were no cross reactivities with other proteins in milk,such as casein, lactalbumin and β-lactoglobulin.The results of affinity showed that it was the highest for 2B12.PcAb against lactoferrin was achieved 55 mL by injection of rabbit with bovine lactoferrin,and the antiserum was purified by the saturated ammonium sulfate method.The titer degree of PcAb was above 1: 107. There was low reactivities with casein,lactalbumin and β-lactoglobulin.The experiment was operated by the double-antibody sandwich procedure.With purified antiserum as a coated antibody,and the McAb 2B12 as the detection antibody. The confining liquid was 1%OVA.The concentration of coated antibody was 0.5 μg/mL. The dilution of McAb was 1:5000.The standard curve was y=-0.2526x+1.7881.The lowest detectable limit of ELISA was 4.69 ng/mL,the range of detection was 4.69-200 ng/mL.The immunoassay provided an analytical method for the detection of lactoferrin in infant formula milk power.The study laid a foundation for studying the detection reagent box of bovine lactoferrin. |
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