Immunoassay Technology To Identify Velvet Antler Varieties

Purpose:(1)The differences of water soluble proteins in four kinds of Sika deer,red deer,reindeer and New Zealand red deer were compared by electrophoresis,which provided the theoretical basis for the study on the immunological identification of pilose antler.(2)Using protein separation and purification technology to extract the target protein from sika deer antler.(3)Using sika deer antler target protein to prepare the polyclonal antibody.(4)Using the method of colloidal gold immunochromatographic assay to prepare an immune reagent box for quickly detecting the authenticity of sika deer antler.Materials and methods:(1)Materials: The experimental materials about velvet were purchased from Xifeng County Tieling city in Liaoning Province,the deer and red deer pieces by various specifications were supported by the limited company of the source of ginseng and deer pieces of Liaoning in Xifeng County,reindeer and New Zealand red deer were collected on the ginseng market in Xifeng country.Liaoning province and drug inspection institute director Wang Weining pharmacist identified its base source.(2)Methods:(1)the extraction of water soluble protein from pilose antler :Using tissue lysate method,first putting the antler pieces into the ball mill,freezing and crushing into fine powder,over 6 sieve,and then ultrasounding 3 minutes(25Hz)after adding the tissue lysate(1:30),Low temperature standing for 2.5 hours,shaking for 1 minutes every 20 minutes,high-speed low temperature centrifugation,last taking the supernatant reserve.(2)One way SDS-PAGE gel electrophoresis method: The blood ejection tablets and tablets on sika deer antler in different specifications;Velvet antler blood and blood tablet;Deer antler blood and blood tablet;New Zealand red deer blood were respectively carried out in the same system according to SDS-PAGE gel electrophoresis analysis which was provided by 2015 Pharmacopoeia.As a result to find the specific proteins between different kinds of pilose antler which could provide the theoretical basis for further study on immunity.(3)Molecular sieve chromatography: According to the molecular weight of each protein in Sika deer antler samples,using the principle of molecular sieve,differences in the size of the protein molecular weight,so as to achieve the purpose of separation and purification of protein.(4)Ion exchange chromatography: Through the effect of the pH of the solution on the net charge of the protein,the purpose of separating and purifying the protein was achieved.(5)Hydrophobic interaction chromatography: The purpose of separation and purification of proteins was achieved by using the hydrophobic degree and nature of hydrophobic groups on the surface of protein molecules.(6)Kit Method: In the process of electrophoresis,the protein could be kept intact,and the target protein was separated and purified according to the difference of the molecular weight of the protein.(7)Preparation of polyclonal antibody: Selecting the immunized animals and taking the specific protein as antigen,so as to obtain high quality antibodies for immunoassay.(8)Colloidal gold immunochromatographic assay: The specific antibody was immobilized in a certain area of the nitrocellulose membrane the sample by capillary action along the nitrocellulose membrane moved forward to the fixed antibody region the corresponding antigen in the sample could be specifically combined with the antibody,and a certain color could be observed by the naked eye so as for realizing the diagnostic specificity of immune.Results:(1)By comparing the water soluble protein electrophoresis patterns of four sika deer,sika deer,New Zealand red deer and reindeer the difference of protein was found which could as identification based on different varieties of antler velvet.(2)The soluble protein of sika deer antler 50 KD with molecular weight of 34 KD and PAGE was detected by the method of chromatography and the method of micro recovery kit.(3)Polyclonal antibodies were used to prepare antibodies with specific proteins as antigens.(4)The use of colloidal gold immunochromatographic assay kit could accurately and quickly identify sika deer antler.Conclusion:(1)The water soluble proteins of four species of sika deer,sika deer,red deer and reindeer were compared by electrophoresis method and the specific protein of sika deer antler was found.(2)Two kinds of water-soluble proteins with molecular weight of 34 KD and 50 KD were isolated and purified by chromatography method and kit extraction method.(3)Rapid identification of sika deer antler by immune analysis technology could provide a valuable reference for proteomics research of traditional Chinese Medicine.