Developed Of Sandwich-Antibody Enzyme Linked Assay For The Detection Of Casein In Food

In this paper, the sanwich immunosorbent assay method for the determination of Casein residues in foods had been developed. One polyclonal antibody and two monoclonal antibodies which were both specific for casein were produced. After purified, the titer of polyclonal antibody was729000, and the monoclonal antibodies2H7and2D6were729000and2187000.We investigated the method of coupling the antibody-horseradish peroxidase (HRP)-The antibody-HRP produced was specific for casein.A indirect enzyme-linked immunosorbent assay (idELISA) and a sandwich enzyme-linked immunosorbent assay for the rapid detection of casein in foods were developed. The sensitivity of the method was higher. In the idELISA, the limit of detection for determination of casein residues was21ppb and the limit of sandwich ELISA was7ppb.In this paper, the plate coefficient of variation within and between plates were both lower than18%. The cross-reactivity experiments showed that casein cross-react with other related proteins were less than the rate of0.01%.The sandwich-ELISA was more sensitive than idELISA.We investigated11kinds of food samples such as milk, coffee to detect casein.A simple, rapid and efficient extraction method was developed and65%-80%recoveries of4samples were obtained.In order to validate sandwich ELISA,casein were analyzed by Bradford method. The correlation between data which was obtained by using sandwich ELISA assay and the one attained by Bradford was good (the R2value was0.9989).The ELISA method and the rapid extraction method developed in this paper is feasible for preparing ELISA kits, which can determine Casein residues in animal derived foods rapidly.