The Construction Of Trenbolone Antigenicity Following It's Chemical Structure Modification

Trenbolone (17-β-hydroxyestra-4,9,â…¡-trien-3-one-17-acetate, TR) is a synthetic steroid male hormone, also a growth promoter of livestock agents, and it is harmful to health. In addition, TR has become a possible source of exhilarant. The EU banned the import of food containing the TR, is also one of the 31 veterinary medicine banned by the Chinese customs. At present, the detection of TR in food depends on the mass spectrometry techniques, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and so on. With the development of monoclonal antibody technology, molecular biology and modern immunoassay technology, it is important to develop a rapid and sensitive detection method to monitor and control TR expansion, TR is a small molecule compound and has immunogenicity binding with macromolecular carriers (protein). The construction of TR was modified by succinic anhydride and then conjugated with BSA. The complex was injected into the rabbit to analyze the immunogenicity and establishing ELISA monitoring methods.Objective:TR is a small molecule hapten. In order to give their immunogenicity, TR was modified and conjugated with BSA. And specific antibody and detection methods to TR were produced.Method:(1) The infrared spectroscopy, mass spectrometry and nuclear magnetic resonance were used to identify the structure of TR.(2) Structural modification:TR and succinic anhydride were dissolved into pyridine solution for 3h at 80℃.Reaction solution was cooled to room temperature, adjusted the pH to 3.0. And the brown viscous material was obtained, namely TR-HS. TR-HS was purified by hot isopropanol and dried. The TR-HS molecular was identified by spectrometry and infrared spectroscopy nuclear magnetic resonance.(3) TR-HS and BSA Conjugation:mixed anhydride method and the carbodiimide method (EDC) were used to conjugate TR-HS and BSA. Mixed anhydride method:TR-HS and BSA reacted by 100:1. In details, 5.5 mg TR-HS was dissolved in 2 mL N, N-dimethylformamide (DMF) pre-cooled at 4℃, then 30μL tributylamine and 15μL isobutyl chloroformate were added at 4℃for 1h, also namely, I solution. II solution was constituted by 20.0 mg BSA dissolved in 2 mL pH 8.5 of 1 mol/L NaOH solution I was added to II, the TR-BSA conjugation was obtained at 4℃overnight. Carbodiimide method (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC) method:TR-HS and BSA reacted by 100:1, in details. TR-HS, NHS and EDC were dissolved in 1ml DMF by 1:1:1 and mixed for 1h at 4℃, this was the reaction I solution; 10 mg BSA was dissolved in 3mL distilled water and DMF precooled for 20min 4℃, the II solution was obtained. solution I was added toâ…¡, the TR-BSA conjugation was obtained at 4℃overnight.(4) Coating antigen:the coating antigen was obtained by OVA coupling with TR-HS.(5) MALDI-TOF and NMR were used to analyze the conjugation.(6)The antiserum to TR-BSA preparation:TR-BSA with complete Freund's adjuvant immunized the rabbits by routine methods, and the valence was identified by ELISA and double immunodiffusion.Results: (1) The IR peaks, NMR H and carbon spectrum of TR were constant with the theory.(2)The identification of TR-HSThe 2974 cm-1 was the-OH asymmetric stretching vibration on carboxyl and 1738 cm-1 was the C=O asymmetric stretching vibration on framework six-ring by KBr pressed infrared testing. The base peak, MS (EI):m/z= 270 by gas mass spectrometry was the strongest peak formed by peaks' fragmentation compounds.¹H NMR (400 MHz, CDC13, rt):δ= 0.96 (s,3H, CH3),1.32 (m,1H, CH₂),1.54 (m,3H, CH₂),1.61 (m,1H, CH,),1.93 (d, 1H,J= 12.4, CH2),2.27 (m,1H, CH₁),2.44-2.60 (m,5H, CH2),2.69-2.72 (m,4H, CH2),2.86 (m,2H, CH2),4.84 (t,1H, J= 7.6, OCH),5.81 (s,1H, CH),6.36 (d,1H, J= 9.6, CH),6.44 (d,1H, J= 10.0, CH). And the constructure was identified by 1H NMR,¹³C NMR, the two-dimensional NMR, IR and MS.(3) The molecular of BSA and TR-BSA produced by mixed anhydride method were 6,7146 and 7,4471, respectively. And the ratio of conjugation was 20.8.(4) UV/Vis spectrophotometer BSA, TR-HS and TR-BSA scan showed 278nm absorption peak decreased, while 343nm absorption significantly increased, which indicated that the conjugation by EDC and mixed anhydride method were successful. The conjugation ratios were 1:12 and 1:18, respectively by UV spectrophotometry adjusted by 20% ethanol.(5) The valence of antiserum was 1:320000 by ELISA, and 1:16 by double immunodiffusion.Conclusion:(1) TR-HS was successfully synthesized.(2) TR-BSA conjugation was successfully prepared, TR-BSA was immunized rabbits and high titers of anti-serum was obtained, which indicated that the TR-BSA had a nice immunogenicity, which made a foundation for the mAb preparation and establishment of TR rapid detection method from food.