Enzyme-Linked Immunoassay For The Determination Of Glycoprotein From The Edible's Nest

The goal of this research was to study on the preparation of antigen of glycoprotein from bird's nest and a sandwich enzyme-linked immunosorbent assay(ELISA) was developed and offered effective method for distinguishing bird's nest.Crude glycoprotein was extracted from bird's nest with 60℃water and precipitated by ethanol,Glycoprotein was prepared after further purification on DEAE-Sepharose fast flow ionic exchange chromatographic and Sephacryl S-200 gel filtration.It was identified to be homogeneous glycoprotein complex.The molecular weight of glycoprotein determined by SDS-PAGE electrophoresis was 60kD.The protein and carbohydrate were linked by O-linkage throughβ-elimination reaction.It indicated that ingredient obtained was glycoprotein.The rabbit antiserum of glycoprotein was prepared successfully by using the purified glycoprotein as immunogen and the titer was 64000,the antibody which was purified with Protein A-sepharose 4B was chosen for ELISA and acted as the first one,the secondary antibody was labeled with horseradish peroxidase(HRP).A sandwich ELISA was developed to detect sialic acid glycoprotein by the optimization of experimental conditions, the limit of detection was 0.031μg/mL with a linear range approxiamately from 0.0625-2μg/mL.Cross reaction between antibody and tremella extract,collagen,starch,chicken ovalbumin as well as agglutinin,BSA were done and the cross-reaction rate were very low. It showed that the sandwich ELISA was specific.Bird's nest was extracted with 60℃water bath for 3-4 hours and the extraction residue repeated once and the extract combined,filtered and centrifuged.Supernatant was diluted by PBS buffer,sample extract diluent was determined with the sandwich ELISA and samples dilution curve with the standard curve was parallel.It proved that this method can be accurately applied to measure bird's nest extract.The extract concentration was 558μg/mL by the established determination method of bird's nest sample.The results indicated that the bird's nest sample can be efficiently determined in the established method combining with cross reaction experiment.