Rare Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, South Korea.
Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, South Korea.
J Transl Med. 2018 Jun 28;16(1):177. doi: 10.1186/s12967-018-1546-z.
Tumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients' serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum.
To overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens.
In this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081).
ELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.
肿瘤相关（TA）自身抗体是免疫系统在识别异常 TA 抗原时产生的，是肿瘤早期检测的有前途的生物标志物。为了有效地检测自身抗体生物标志物，诊断试验中的抗体特异性表位应保持尽可能接近体内呈现的特异性构象。然而，当使用患者的血清作为 TA 自身抗体的来源时，由于患者来源的血清量有限，自身抗体特异性表位的表征并不容易。
为了克服这些限制，我们构建了一个源自肝细胞癌（HCC）模型 HBx 转基因小鼠的 B 细胞杂交瘤池，并将其衍生的自身抗体作为肿瘤标志物进行了表征。通过质谱法鉴定了它们的靶抗原，并检查了与 HCC 的相关性。基于在肿瘤小鼠模型中产生的 TA 自身抗体在人类癌症患者中诱导的假设，我们基于小鼠 TA 自身抗体的特征开发了酶联免疫吸附测定（ELISA），用于检测人血清中的自身抗体生物标志物。为了模拟天然抗原结构，从噬菌体展示环随机七肽文库中筛选针对自身抗体的特异性表位，并将与特异性表位融合的链霉亲和素抗原用作包被抗原。
在这项研究中，我们对源自 HBx 转基因小鼠的一种 HCC 相关自身抗体 XC24 进行了表征。其靶抗原被鉴定为剪接因子 3b 亚基 1（SF3B1），并在 HCC 组织中证实了 SF3B1 的高表达。选择了针对 XC24 的特异性肽表位，其中 XC24p11 环肽（-CDATPPRLC-）被用作抗 SF3B1 自身抗体 ELISA 的表位。使用该表位，我们可以有效地将来自 HCC 患者（n=102）和健康受试者（n=85）的血清样本区分开来，其灵敏度为 73.53%，特异性为 91.76%（AUC=0.8731）。此外，同时检测抗 XC24p11 表位自身抗体和 AFP 可提高 HCC 诊断的效率，其灵敏度为 87.25%，特异性为 90.59%（AUC=0.9081）。
使用针对抗 SF3B1 自身抗体的 XC24p11 肽表位的 ELISA 可以用作增强 HCC 诊断效率的新型检测方法。
