氯吡脲完全抗原的合成与鉴定

Synthesis and Identification of Complete Antigen of CPPU

氯吡脲(Forchlorfenuron)是半抗原,只有将其与载体蛋白偶联形成完全抗原才能刺激机体产生抗体。以2-氯异烟酸为基础设计并合成了半抗原CPPU-H1和CPPU-H2,并采用活性酯法分别将其与载体蛋白偶联制备完全抗原;采用质谱和核磁共振对半抗原CPPU-H1和CPPU-H2的结构进行分析,并用紫外光谱法和ELISA法对完全抗原进行鉴定。结果表明,半抗原CPPU-H1和CPPU-H2成功合成,且合成后的CPPU-H1-BSA和CPPU-H2-OVA具有吸收峰叠加效应,表明CPPU完全抗原合成成功,其偶联比分别为16.8∶1和22.3∶1;间接ELISA测定4次免疫后小鼠血清效价最高可达1∶25 600。CPPU完全抗原的制备为抗CPPU抗体的制备及免疫学方法的建立奠定了基础。

Forchlorfenuron(CPPU) was hapten which must be coupled with the carrier protein to form complete antigen in order to stimulate animals to secrect anti-CPPU antibodies. The hapten CPPU-H1 and CPPU-H2 were designed and synthesized on the basis of 2-Chloro-4-pyridinecarboxylic acid, and the complete antigen were prepared by coupling with CPPU-H1 and CPPU-H2 with carrier proteins using active ester method, respectively. CPPU-H1 and CPPU-H2 were identified by mass spectrometry and nuclear magnetic resonance spectroscopy. CPPU-H1-BSA and CPPU-H2-OVA were identified by ultraviolet spectrum and ELISA. The structure of CPPU-H1 and CPPU-H2 were consistent with the predictive structure by mass spectrometry and nuclear magnetic resonance spectroscopy. CPPU artificial antigen(CPPU-H1-BSA and CPPU-H2-OVA) showed the superposition phenomenon by ultraviolet spectrum, and the coupling ratios of CPPU-H1-BSA and CPPU-H2-OVA were16.8∶1 and 22.3∶1, respectively. The titer was high up to 1∶25 600 after four times immunization of antibody against CPPU in mice serum by detected with indirect ELISA. The preparation of complete antigen of CPPU contributes to the anti-CPPU antibody production and immunoassay development.