Test system for determination of human lactoferrin in milk of transgenic rabbit with integrated construction as1-Cn-hLf
Human lactoferrin (hLF) was isolated from woman’s milk by the methods of ion exchange and gel chromatography. The degree of hLF purification was estimated by electrophoresis in polyacrylamide gel in denatured condition. Rabbits and mouses were immunized by the purified hLF. The blood serum probes with high titer of antibodies were used to develop test system. Anti bodies from blood serum of immunized rabbits were purified by affine chromatography with the use of hLF immobilized on a carrier. With the aim to provide maximal sensitivity, accuracy and specificity of the test system, the sandwich variant of immune-enzymatic analysis was developed. The conditions were determined of planting primary (rabbit) anti bodies on the plate, concentration of secondary (mouse) anti bodies, and concentration of anti-specie conjugate with horse-radish peroxidase. To obtain a standard curve, hLF isolated from woman milk was used. As a negative control, the milk from un transgenic rabbits was used. With the use of test system developed, a series of milk probes from transgenic rabbits were analyzed.