Preparation of Raloxifene Antibody and Preliminary Establishment of Enzyme-Linked Immunosorbent Assay
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Abstract:
The preparation of raloxifene antibody and the establishment of enzyme-linked immuno sorbent assay (ELISA) were mainly studied in this paper. Succinic anhydride was used for the raloxifene derivative and the raloxifene haptens were formed. The immunogen and inclusion antigen were synthesized by coupling the raloxifene hapten with bovine serum albumin (BSA) and ovalbumin (OVA) using the carbodiimide method, and then determined by UV spectroscopy. Results showed that raloxifene was successfully coupled with the carrier protein. Raloxifene- bovine serum albumin (Ra-BSA) was used to immune New Zealand white rabbits, and the antibodies showed a titer of 1.28×105 and a IC50 of 15.4 μg/L. There was no cross reaction with other anti-estrogens, indicating that the antibody specificity was good. The raloxifene ELISA method was initially established by optimizing the reaction concentration of antigen antibody, and the optimal conditions were as follows: antigen concentration, 300 μg/L, antibody concentration, 1:1.0×105. The standard curve was good (R2=0.9853) in the range of 0.4~102.4 g/L with a lowest detection limit, 0.4 μg/L. This research provided a technical reference for further development of the rapid test kit for raloxifene.
