College of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food Quality and Safety, 100193 Beijing, China.
Key Laboratory of Molecular Epidemiology of Shenzhen, Shenzhen Center for Disease Control and Prevention, 518000 Shenzhen, China.
ACS Appl Mater Interfaces. 2022 Apr 20;14(15):17128-17141. doi: 10.1021/acsami.2c02299. Epub 2022 Apr 6.
Hybridoma technology is widely used for monoclonal antibody (mAb) discovery, whereas the generation and identification of single hybridomas by the limiting dilution method (LDM) are tedious, inefficient, and time- and cost-consuming, especially for hapten molecules. Here, we describe a single transgenic hybridoma selection method (STHSM) that employs a transgenic Sp2/0 with an artificial and stable on-cell-surface anchor. The anchor was designed by combining the truncated variant transmembrane domain of EGFR with a biotin acceptor peptide AVI-tag, which was stably integrated into the genome of Sp2/0 via a piggyBac transposon. To ensure the subsequent precise selection of the hybridoma, the number of on-cell-surface anchors of the transfected Sp2/0 for fusion with immunized splenocytes was further normalized by flow cytometry at the single cell level. Then the single antigen-specific transgenic hybridomas were precisely identified and automatically selected using a CellenONE platform based on the fluorescence assay of the on-cell-surface anchor with the corresponding secreted antigen-specific mAb. The STHSM produced 579 single chloramphenicol (CAP)-specific transgenic hybridomas with a positive rate of 62.7% in 10 plates within 2 h by one-step selection, while only 12 single CAP-specific hybridomas with a positive rate of 6.3% in 40 plates required at least 32 days using the LDM with multiple subcloning steps. The best affinity of mAbs from the STHSM was more than 2-fold higher than that of those from the LDM, and this was mainly due to the preaffinity selection based on the on-cell-surface anchors and more interactions between the mAb and CAP. Then the mAbs from the STHSM and LDM were used to develop an immunoassay for CAP in spiked and natural biological samples. The method displayed satisfactory sensitivity, accuracy, and precision, demonstrating that the STHSM we developed is a versatile, practical, and efficient method for mAb discovery.
杂交瘤技术广泛用于单克隆抗体（mAb）的发现，而通过有限稀释法（LDM）生成和鉴定单个杂交瘤则繁琐、低效且耗时耗力，尤其是对于半抗原分子。在这里，我们描述了一种使用带有人工且稳定的细胞膜表面锚定物的转基因 Sp2/0 的单转基因杂交瘤选择方法（STHSM）。该锚定物由 EGFR 的截断变体跨膜结构域与生物素受体肽 AVI 标签组合而成，通过 PiggyBac 转座子稳定整合到 Sp2/0 的基因组中。为了确保随后对杂交瘤进行精确选择，通过流式细胞术在单细胞水平上进一步对融合免疫脾细胞的转染 Sp2/0 上的细胞膜表面锚定物数量进行归一化。然后，使用基于相应分泌型抗原特异性 mAb 对细胞膜表面锚定物的荧光测定的 CellenONE 平台，精确鉴定和自动选择单个抗原特异性转基因杂交瘤。STHSM 在 10 个平板中在 2 小时内以一步选择产生了 579 个单个氯霉素（CAP）特异性转基因杂交瘤，阳性率为 62.7%，而使用 LDM 进行多个亚克隆步骤则需要至少 32 天才能在 40 个平板中获得阳性率为 6.3%的 12 个单个 CAP 特异性杂交瘤。来自 STHSM 的 mAb 的最佳亲和力比来自 LDM 的高 2 倍以上，这主要是由于基于细胞膜表面锚定物的预亲和力选择以及 mAb 与 CAP 之间更多的相互作用所致。然后，使用来自 STHSM 和 LDM 的 mAb 开发了用于 CAP 检测的免疫分析方法，该方法用于检测添加和天然生物样品。该方法显示出令人满意的灵敏度、准确性和精密度，表明我们开发的 STHSM 是一种通用、实用且高效的 mAb 发现方法。
