摘要/Abstract

摘要： 目的·建立针对血清中抗体的整体质量评估体系，为血清自身抗体标志物相关研究提供量化的质控标准。方法·以50名健康者的血清为研究对象，分别经-80 ℃、-20 ℃、4 ℃、37 ℃、95 ℃处理后，与噬菌体随机肽展示库Ph.D.-12进行孵育，采用免疫沉淀方式获得噬菌体-IgG复合物。经高通量测序、生物信息学分析，统计每一种处理条件下血清结合肽的序列及频率，计算香农熵，评估血清中自身抗体的整体质量。结果·随着处理温度的升高，血清的香农熵逐渐降低；在同一温度条件下，血清的香农熵随着时间增长而逐渐降低，其中-20 ℃与-80 ℃处理的样品相比，香农熵波动不大；95 ℃处理10 min后的样品香农熵达到最低值。结论·联合噬菌体随机肽展示技术和高通量测序技术，可以用于解析血清中抗体的肽结合谱，并以香农熵为指征建立血清抗体整体质量评估体系。
关键词: 血清, 质量控制, 抗体, 噬菌体展示, 高通量测序, 香农熵
Abstract:
Objective · To establish a quality control system for serum antibodies and provide a quantitative standard for the quality control of serum autoantibody-based biomarker study. Methods · Sera from 50 healthy people were taken to prepare the samples and aliquoted. The aliquoted sera were stored at -80 ℃, -20 ℃, 4 ℃, 37 ℃, and 95 ℃ for a period of time. Samples were collected at different time points. The samples were incubated with Ph.D.-12, a phage display random peptide library. The phage-IgG complex was enriched through immunoprecipitation with protein G-coated magnetic beads. By sequencing the coding sequences of the displayed peptides, the sequence and frequency of the serum antibody-enriched peptides were determined. Using the frequencies of each enriched peptide, Shannon entropies were calculated for each sample. Shannon entropy was applied as an indicator to evaluate the quality of the serum autoantibodies. Results · With the increase of the treating temperature, the Shannon entropies of the sera gradually decreased, while there was no significant difference between the Shannon entropies of -20 ℃ and -80 ℃ . The Shannon entropy reached the lowest value after being treated at 95 ℃ for 10 min. At the same temperature, the Shannon entropies of the sera were inversely proportional to the length of the treating periods. Conclusion · It is practically applicable to decipher the profile of the serum antibody binding peptides, through the combination of a phage display random peptide library and next-generation sequencing. Shannon entropy can be calculated using the frequencies of each enriched peptide, and applied as an indicator to judge the overall quality of the serum antibodies.
Key words: serum, quality control, antibody, phage display, next generation sequencing, Shannon entropy


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