Detection of Triamcinolone Acetonide Indirect Competitive ELISA Based on Heterology Coating Strategy

An indirect competitive ELISA for triamcinolone acetonide(TCA)was developed to monitor TCA abuse. To obtain the TCA complete antigen,TCA hapten and keyhole limpet hemocyanin(KLH)were coupled by using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDC)and N-Hydroxysuccinimide(NHS). The homologous detection antigen TCA-bovine serum albumin(TCA-BSA)and heterologous detection antigen Dexamethasone-BSA(DEX-BSA)were synthesized in the same way. After immunization and cell fusion,one strain of hybridoma cell line that could steadily secrete anti-TCA monoclonal antibody was prepared and its antibody(ascites)was prepared and purified. By using the antibody and two different detection antigens,both homologous and heterologous strategy indirect competitive enzyme linked immunosorbent assay(ic-ELISA)standard curve for detection of TCA was built,recoveries of TCA added to liquid milk was detected by the heterologous strategy. The results showed that the IC₅₀ of homologous strategy ic-ELISA was 5.4 ng/mL,the IC₅₀ of heterologous strategy ic-ELISA was 0.53 ng/mL.The method did not cross reacted with other glucocorticoids except halcinonide,budesonide and amcinonide. The recovery rates of TCA added to animal tissues ranged from 75% to 90% using 20% methanol/water. The method can be applied in TCA fast screening in animal tissue.