猪IL-15单克隆抗体的制备及双抗体夹心ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2025.07.036

摘要/Abstract

摘要：

旨在制备猪白细胞介素-15(IL-15)的单克隆抗体(McAb)，建立IL-15-ELISA定量检测方法。首先构建猪IL-15原核表达载体pET-28a-IL-15，应用大肠杆菌原核表达系统表达该重组蛋白，随后用其免疫6周龄的雌性BALB/c小鼠，在加强免疫后第3天，分离小鼠脾细胞，将脾细胞与SP2/0细胞融合，筛选阳性杂交瘤细胞，并对其分泌的IL-15单克隆抗体进行鉴定；然后基于单克隆抗体建立IL-15夹心ELISA方法。结果显示：获得9株分泌抗IL-15 McAb的杂交瘤细胞(1E8、1F4、2D3、2C10、2F3、2D10、3F4、3D4、3D5)，分泌的抗体均为IgG2bκ型。以制备的单抗2D10和2C10分别为捕获抗体及酶标抗体建立的夹心ELISA最低可检测31.25 ng·mL^-1的IL-15蛋白，与其它细胞因子或蛋白(IL-1、IL-2、IL-4、IL-6、IL-10、IL-12、Gzms-B、IFN-γ、TNF-α)无交叉反应，批内和批间重复的变异系数分别为2.56%和3.39%。该夹心ELISA可定量检测血清、组织液和细胞培养物等样品中的IL-15。综上，成功制备了猪IL-15单克隆抗体，建立了定量检测IL-15的双抗体夹心ELISA方法，为IL-15相关研究提供了重要工具。

关键词: 猪IL-15, 单克隆抗体, 双抗体夹心ELISA, 定量检测

Abstract:

A monoclonal antibody (McAb) to porcine interleukin-15 (IL-15) was prepared and a quantitative IL-15-ELISA was established. Porcine IL-15 prokaryotic expression vector pET-28a-IL15 was constructed, and the recombinant protein was expressed with Escherichia coli (E. coli) prokaryotic expression system, and six-week-old female BALB/c mice were immunized with recombinant IL-15. On day 3 after the booster immunization, splenocytes were isolated and collected from one mouse to fuse with SP2/0 cells. Positive hybridoma cells were screened and monoclonal antibodies to IL-15 secreted by the positive hybridoma cells were identified. Moreover, a sandwich ELISA for detection of porcine IL-15 based on a monoclonal antibody. Nine strains of hybridoma cells (1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4 and 3D5) secreting anti-IL-15 McAb were obtained, and all of the secreted McAb were of the IgG2bκ type. A sandwich ELISA using the prepared monoclonal antibody 2D10 as the capture antibody and 2C10 as the enzyme-labeled antibody, respectively, was able to detect a minimum of 31.25 ng·mL^-1 of IL-15 protein, with no cross-reactivity with the other cytokines or protein (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, and TNF-α). The mean coefficient of variation for intra-batch and inter-batch reproducibility experiments was 2.56% and 3.39%, respectively. This sandwich ELISA quantifies IL-15 in serum, tissue fluid and cell culture. Porcine IL-15 McAb was successfully prepared, and a double antibody sandwich ELISA method was established, which provided an important tool for IL-15 related studies.

Key words: porcine IL-15, monoclonal antibody, double-antibody sandwich ELISA, quantitative determination

中图分类号:

R446.61

王飞燕, 刘超凡, 张亚楠, 周晓甜, 任静, 袁晨, 李潭清, 宋勤叶. 猪IL-15单克隆抗体的制备及双抗体夹心ELISA方法的建立[J]. 畜牧兽医学报, 2025, 56(7): 3442-3452.

WANG Feiyan, LIU Chaofan, ZHANG Ya'nan, ZHOU Xiaotian, REN Jing, YUAN Chen, LI Tanqing, SONG Qinye. Preparation of Monoclonal Antibody to Porcine IL-15 and Establishment of Double Antibody Sandwich ELISA Method[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3442-3452.

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样本编号 Sample number	IL-15浓度 IL-15 concentration	样本编号 Sample number	IL-15浓度 IL-15 concentration
1	11.9	9	14.8
2	11.3	10	13.6
3	3.2	11	9.1
4	1.4	12	8.0
5	47.8	13	56
6	43.3	14	52.3
7	17.2	15	9.5
8	11.0	16	22.9

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