牛白细胞介素-8单克隆抗体制备及应用

doi:10.16431/j.cnki.1671-7236.2025.08.031

Abstract

Abstract: 【Objective】 Interleukin-8 (IL-8) was a key chemokine that plays a role in the immune response,and the increase of its level was closely linked to multiple infectious diseases and autoimmune conditions.It had been recognized as a detection target for active pulmonary tuberculosis,tuberculous meningitis,and bovine tuberculosis.This study aimed to develop monoclonal antibodies against bovine IL-8 and create a sandwich ELISA kit for the quantitative detection of IL-8 in bovine serum or plasma,offering tools for diagnosing and researching bovine tuberculosis (bTB) and other inflammation-associated diseases. 【Method】 The bovine IL-8 gene was cloned into pGEX-6P-1 and pcDNA3.1 vectors and expressed in Escherichia coli and 293F cells.Recombinant proteins were purified using GST affinity and nickel columns.Eukaryotic recombinant protein was used to immunize BALB/c mice,and monoclonal antibodies against bovine IL-8 were generated and screened via lymphocyte hybridoma technology.The optimal antibody pairs were identified through checkerboard analysis,and a sandwich ELISA detection method was established.IL-8 level was quantitatively assessed in plasma samples from shedding and non-shedding tuberculosis-infected cattle as well as healthy cattle to evaluate the method’s effectiveness in detecting tuberculosis in cattle. 【Result】 SDS-PAGE analysis showed that IL-8 produced in both prokaryotic and eukaryotic expression systems was expressed in a soluble form,with purified proteins exhibiting a purity exceeding 90%.Five monoclonal cell lines stably secreting IL-8 antibodies including 2B8,2F5,and so on, were obtained after three rounds of subcloning,all confirmed as IgG1 subtype.Checkerboard analysis revealed that 4G10 and 3D5-HRP were the optimal antibody pairs.The ELISA method established with 4G10 and 3D5-HRP was applied for quantitative detection of IL-8 level in plasma from cattle with varying infection statuses,demonstrating that IL-8 level in plasma of tuberculosis-infected cattle was extremely significantly higher than that in healthy cattle (P＜0.01). 【Conclusion】 The monoclonal antibodies against bovine IL-8 were successfully prepared and a sandwich ELISA detection method was established，which could realize the quantitative measurement of bovine IL-8,with potential applications in screening for tuberculosis in cattle and analyzing immune and infection status.

Key words: cattle; interleukin-8; monoclonal antibodies; ELISA; bovine tuberculosis

CLC Number:

S852.4^＋3

YANG Rui, JIN Jiaxin, ZHANG Ze, SHI Wenjian, ZHANG Guangzhi, DING Jiabo, XIN Ting. Development and Application of Monoclonal Antibodies Against Bovine Interleukin-8[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3830-3837.

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Development and Application of Monoclonal Antibodies Against Bovine Interleukin-8

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