重组蝎毒抗神经兴奋肽的ELISA分析方法建立及稳定性的初步研究

目的建立重组蝎毒抗神经兴奋肽(ANEP)的ELISA分析方法并对ANEP的稳定性进行初步研究。方法以分别建立的间接ELISA和间接竞争ELISA分析方法进行ANEP含量测定,并通过方法学的优化,最终以建立的间接法ELISA方法测定了温度、pH值、反复冻融、超声对ANEP稳定性的影响。结果间接ELISA相对于间接竞争ELISA有着更好的线性关系和灵敏度,线性范围0.025~1.60μg/mL。检测限为10 ng/mL。稳定性实验表明ANEP在37,60℃的条件下放置24 h含量基本不变,pH 5~11的缓冲液中保持稳定,反复冻融、超声4 min后含量降低。结论所建立的间接ELISA方法能很好的用于ANEP的测定。ANEP的热稳定性较好,强酸性下不稳定,对反复冻融与超声等稳定性较差。

Establishment of enzyme-linked immunosorbent assay for anti-neuroex citation peptide (ANEP)and study on the stability of ANEP

Purpose To establish an effective ELISA method for the determination of anti-neuroexcitation peptide(ANEP) and to investigate the stability of ANEP.Methods ANEP was determined by using two kinds of ELISA.After comparison,indirect ELISA was chosen and optimized for the research on stability of ANEP.The effects of temperature,pH value,freeze-thaw,and sonification on the stability of ANEP were investigated.Results The indirect ELISA showed better linearity and sensitivity than indirect competitive ELISA.A standard curve was obtained and the regression equation was y=1.026 6 x+0.044 3.The assay result was linear over a concentration range of 0.025-1.60 μg/mL with a limit of detection(LOD)of 10 ng/mL.ANEP didn′t degrade during 24 h incubation period at 37℃ and 60℃ and was found to be stable at the pH range from 5 to 11.ANEP obviously degraded after repeated freeze-thawing and sonification for more than 4 min.Conclusion The established indirect ELISA meets the requirements in determination of ANEP.ANEP is thermo-stable and sensitive to low pH value,repeated freeze-thawing and long time sonification.