Rasri Natchaya, Tabtimmai Lueacha, Kraiya Charoenkwan, Yamabhai Montarop, Sinthuvanich Chomdao, Rattanasrisomporn Jatuporn, Choowongkomon Kiattawee
Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Department of Biotechnology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand.
ACS Omega. 2023 Jul 19;8(30):27688-27696. doi: 10.1021/acsomega.3c03581. eCollection 2023 Aug 1.
For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual's immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 μM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit.
几十年来，猫传染性疾病一直是猫最常见的健康问题之一，也是导致猫死亡的主要原因。这些疾病包括弓形虫病、猫白血病病毒（FeLV），尤其是猫免疫缺陷病毒（FIV）疾病。早期诊断对于提高成功治疗的几率至关重要。一般来说，IgG水平的测量被认为可指示个体针对特定病原体的免疫状态。猫IgG特异性抗体是检测试剂盒开发的关键组成部分。在本研究中，使用噬菌体展示技术筛选出与猫IgG结合的单链抗体片段（scFv）。针对纯化的猫IgG进行了三轮生物淘选。通过间接酶联免疫吸附测定（ELISA），选择了两个对猫IgG具有最佳结合能力的scFv克隆进行生化特性分析。此外，所选的scFv（N14）在细菌系统中表达并纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示纯化后的N14大小为29 kDa。采用夹心ELISA评估纯化后的scFv与猫IgG的结合能力。正如预期的那样，纯化后的N14具有结合猫IgG的能力。此外，对N14进行修饰以创建一个scFv-碱性磷酸酶（scFv-AP）融合平台。表面等离子体共振（SPR）结果显示，N14-AP与猫IgG结合，亲和结合值为0.3±0.496μM。此外，直接ELISA证明了N14-AP在细胞裂解液和纯化蛋白中均具有与猫IgG的结合能力。而且，N14-AP可基于电传感用于检测猫IgG，检测限为10.42 nM。总体而言，本研究成功筛选出一种与猫IgG结合的scFv，并开发了一个scFv-AP平台，该平台可进一步改造并应用于猫传染性疾病检测试剂盒。
