| DMT, a steroidal synthetic anabolic hormones, is widely used in clinical treatment, livestock production and sports. Having many adverse effects on human body, DMT is strictly banned from using in food animals producing and sports in many countries. However, it is still used illegally. In order to guarantees safety of meat and fair and health of sports, it is necessary to establish a rapid and sensitive detection technology.GC-MS and LC-MS is the most common technology for the detection of DMT examination at present. But they are time-consuming, expensive and require well pretreatment. In actual examination, methods of fast, simple, cheap and high-throughput is required. Immunoassay is suitable for rapid detection of a large number of samples because of its high sensitivity, specificity, simple, rapid and low test cost. The main results of our research were as follows:1. DMT was oximated by carboxymethyl hydroxylamine, and was confirmed by TLC and IR spectroscopy. DMT-oxime was then coupled with different proteins carriers (BSA, OVA and KLH) by activated ester method and used as immunogen and coatingen. The conjugations were identified by UV spectroscopy, the ratios were is BSA:20, OVA:16, KLH:352 (molecular weight calculated by 106).2. Polyclonal antibodies were prepared by immunizing rabbits with DMT-OVA or DMT-KLH. Titers of two serums reached 1:64,000(DMT-KLH) and 1:256,000 (DMT-OVA) respectively, which were measured by indirect non-competitive ELISA method using following parameters:coating concentration is 0.25μg/mL,37℃for 1 h; blocking reagent is 2% gly,37℃for 2 h; the reaction conditions for primary and secondary antibodies is at 37℃for2 h; 10000-fold dilution for secondary antibody; color developing at 37℃for 10 min; H2O2 concentration in coloured solution is 1μL/mL IC50 and the limit of detection (IC15), determined by the indirect competitive ELISA, were 0.24μg/L,0.01μg/L (DMT-KLH) and 5.29μg/L,0.12μg/L (DMT-OVA). The cross reaction rate with testosterone propionate(TP) and trenbolone (TB) was less than 0.68%. By contrasting nature of two polyclonal antibodies, DMT-KLH was chosen to immunize Ballb/C mice.3. Ballb/C male mice of 6～8 weeks were immunized with DMT-KLH. Indirect non-competitive ELISA method was established using positive serum and parameters is as follow:coating concentration is 1μg/mL,37℃for 1 h;blocking reagent is 2% gly, 37℃for 3 h; the reaction conditions for primary and secondary antibodies is at 37℃for2 h; 5 000-fold dilution for secondary antibody; color developing at 37℃for 15 min4..Cell fusion was induced by polyethylene glycol (PEG). After three times subcloning, monoclone antibody (McAb), hybridoma cell 1D6F10B4 was obtained, which secreted IgG1 antibody subtypes. IC50 was 7.57μg/L, and the limit of detection (IC15) reached 0.30μg/L. The cross reaction rate with testosterone propionate (TP) and trenbolone (TB) was less than 1%. Titer of purified antibody is 1:32000,concentration is 0.868 mg/mL, affinity constant is 5.53x109 L/moL。... |
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