Establishment Of Accurate And Quick Detection Methods For Human Lactroferrin In Transgenic Milk

As the development and the maturation of biotechnologies, gene-manipulating targets are gradually transferred to animals, from plants and microorganisms. Of diverse biotechnologies, transgenesis is considered as an effective approach to change the traits of animals. By manipulating genes, we can change animals at a given direction, or prepare new breeds. As the attractive economic prospective, transgenic animals and related products exert powerful vitality, and guarantee their marketization. However, taking biology safety into account, it is necessary to strictly control every stage of transgenic animal-producing. Hence, a set of sound, scientific and normative system of safety assessment is indispensable, which is dependent on reliable detection approaches. Generally speaking, detection of transgenesis can go from nucleotide-based or protein-based levels. Because protein is the final product of a gene and always the functional substance, detecting the proteins can reveal the integration and the translation of exogenous genes. In addition, protein detection is somewhat simple and convenient. Therefore, in this study, we qualitatively and quantitatively detected the hLF (human lactoferrin) in milk, by establishing multiple methods, including SDS-PAGE, Dot blot, Western blot, ELISA and gold immunochromatography assay. Main results are as follows:1. By SDS-PAGE, Dot blot and Western blot methods, we qualitatively detected hLF in milk with accordant results, suggesting that these three methods were capable to identify hLF in transgenic milk;2. Indirect chemiluminescent ELISA was established with a linear range of 6.4×10-7mg/mL～2×10-3mg/mL, and subsequently qualified the concentration of hLF in the 3 transgenic samples, which were 1.31mg/mL,9.12mg/mL and 8.11mg/mL, respectively;3. To meet the demand of quick detection, we used gold-conjugated hLF protein and gold-conjugated polyclonal antibody as indicator, and preliminarily developed 2 types of competitive gold immunochromatography strips with consistent results to the former detection methods;In this study, we employed multiple methods to qualitatively and quantitatively detect the hLF in milk. Comparing the results derived from a certain method, we found all the assays concluded the same results, demonstrating that they were appropriate for the detection of hLF. Among these methods, ELISA was sensitive with a detection limit of 6.4×10-7mg/mL.