抗逆性好、特异、高效价抗体替代物的筛选及其在新型无标记肿瘤标志物电化学阻抗免疫传感器中的应用研究

The detection of tumor biomarkers is usually based on the immuno-reaction between antigen and monoclonal antibody (mAb). However, the inherent defects of mAbs (cockamamie preparation process, high production costs and instability) make the bioassay costly and unstable, and therefore limiting its large-scale application. Prostate-specific antigen (PSA), as the biomarker of prostate cancer, mainly exists in the active forms of free-PSA (f-PSA), PSA-α1-antichymotrypsin (PSA-ACT) in men's serum. The sum of fPSA and PSA-ACT is known as the total PSA (tPSA) on behalf of the level of PSA in serum. Thereafter, the content of fPSA, tPSA and ratio of fPSA/tPSA are important indexes in the early diagnosis of prostate cancer. In this project, first, the f8/8 landscape libray will be biopanned with fPSA to obtain a sub-library, in which lots of phage clones could bind with fPSA. Subsequently, the selected sub-library will be biopanned further with PSA-ACT, and two categories of phage clones (phage capable of binding fPSA and phage capable of binding PSA-ACT) will be obtained through elution and phage capture test. Then, seveal phage clones from both categories will be picked up and performed specificity testing, accordingly, fPSA-specific phage monoclone and tPSA-specific phage monoclone (phage to recognize tPSA) will be screened separately, which can be used as ideal antibody substitutes, because they are inexpensive (simple amplification for scale up), multivalent binding, stable and resistant to degradation under various harsh conditions such as pH2-12, 6 M urea and wide proteases. Finally, both fPSA-specific and tPSA-specific electrochemically modified bioelectrodes will be fabricated separately by using their corresponding antibody substitutes. The incubation condition and the regeneration of the sensing interface will be optimized, aiming at developing label-less, stable impedimetric immunosensors, which are affordable, sensitive, selective bioassay of fPSA and tPSA. This proposed approach would not only illuminate new idea to develop electrochemical immuno-sensors, but also will provide a strong technical platform for physical examination of healthy people, and may play an important role in early diagnosis prostate cancer. Moreover, on basis of the strategy proposed in this project, antibody substitutes specific to other tumor biomarkers can also be developed, which may find potential applications in establishing sensitive, specific, and affordable technique for tumor detection.

肿瘤标志物检测基于抗原与单克隆抗体(单抗)的特异性免疫反应，但是单抗存在制备过程繁琐、成本高且不稳定等固有缺陷。血清中游离的前列腺特异性抗原(fPSA)与PSA-ACT复合物之和为总PSA（tPSA），fPSA、tPSA的含量及其比值是确定前列腺疾病性质和诊断前列腺癌的重要指标。本项目拟以fPSA为靶标与风景噬菌体文库淘选，获得结合fPSA的噬菌体子文库。以PSA-ACT为靶标对上述子文库淘选，经洗脱和噬菌体捕获分别获得fPSA和PSA-ACT结合性噬菌体，经特异性测试，获得抗逆性好、廉价、效价高、特异识别fPSA和tPSA噬菌体单克隆作为抗体替代物。将这两种抗体替代物修饰电极，优化孵育条件，探讨传感界面的再生，构筑无标记、抗逆性好的PSA电化学阻抗免疫传感器，旨在建立低成本、灵敏、特异的fPSA和tPSA检测方法。本研究提出了构建电化学免疫传感器的新思路，也将为鉴别前列腺疾病提供新方法。

肿瘤标志物检测主要基于抗原与单克隆抗体(单抗)的特异性免疫反应，但是单抗存在制备过程繁琐、成本高且不稳定等固有缺陷。血清中游离的前列腺特异性抗原(f-PSA)与PSA-ACT复合物之和为总PSA（t-PSA）。f-PSA、t-PSA的含量及f-PSA/t-PSA比值是确定前列腺疾病性质和诊断前列腺癌的重要指标。 噬菌体具有多效价、制备简单、廉价，耐受极端环境而保持其活性等优点，将其开发为抗体替代物具有重要意义。本项目首先以f-PSA为靶标与f8/8噬菌体文库淘选，获得了结合f-PSA的噬菌体子文库，扩增噬菌体，经特异性测试，获得高亲和性、特异识别f-PSA的八肽配体ESNSVSPS融合的噬菌体单克隆P1。然后以PSA-ACT为靶标对上述子文库淘选，获得系列噬菌体，经特异性测试，获得了对f-PSA和PSA-ACT（即t-PSA）均具有高亲和性、又能有很好结合性的，但与其他抗原结合性差的、展示八肽配体ATRSANGM的噬菌体单克隆P5。将获得的两株噬菌体单克隆P1和P5分别作为f-PSA、t-PSA的抗体替代物修饰电极，优化孵育条件，构筑无标记PSA电化学阻抗免疫传感器，检测f-PSA的线性范围为0.05 pg/mL – 200 ng/mL ，检测下限（LOD）为0.03 pg/mL f-PSA（S/N = 3）；检测t-PSA的线性范围为0.05 pg/mL – 200 ng/mL ，LOD为0.025 pg/mL t-PSA。构筑的双免疫传感器分别对f-PSA和t-PSA具有很好的选择性，经含0.05％吐温20的Gly-HCl（pH 2.2）缓冲液处理可使传感界面再生。该方法用于实际血清样品中f-PSA、t-PSA分析，结果令人满意，f-PSA/t-PSA比值也可算出。本研究提出了构建电化学免疫传感器的新思路，也将为男士查体和前列腺癌的的早期准确诊断提供新的检测平台。