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新型抗防御素5单克隆抗体克隆1A8和4F5在炎症性肠病结肠炎组织中的功能特性

Functional characterization of novel anti-DEFA5 monoclonal antibody clones 1A8 and 4F5 in inflammatory bowel disease colitis tissues.

作者信息

Thangaiyan Rabi, Sakwe Amos M, Hawkins Alexander T, Washington Mary K, Ballard Billy R, Izban Michael G, Chirwa Sanika S, Hildreth James E K, Shanker Anil, Blum David L, M'Koma Amosy E

机构信息

Department of Biochemistry, Cancer Biology, Neuroscience, and Pharmacology, School of Medicine, Meharry Medical College, 1005 D.B. Todd Jr. Blvd, Nashville, TN, USA.

Section of Colon and Rectal Surgery, Division of General Surgery, School of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.

出版信息

Inflamm Res. 2025 Jan 30;74(1):30. doi: 10.1007/s00011-024-01970-w.

DOI:10.1007/s00011-024-01970-w
PMID:39883179
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11782311/
Abstract

BACKGROUND

The aberrant expression of α defensin 5 (DEFA5) protein in colonic inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn's colitis (CC). It can serve as a biomarker for differentiating CC from Ulcerative colitis (UC), particularly in Indeterminate colitis (IC) cases into UC and CC. We evaluated the specificity of commercially available anti-DEFA5 antibodies, emphasizing the need to further validate their appropriateness for a given application and highlighting the necessity for novel antibodies.

METHODS

We established two mice monoclonal DEFA5 antibody clones, 1A8 and 4F5, by immunizing mice with purified recombinant protein. We validated the specificity, sensitivity, and cross-reactivity of these antibodies in recognizing both endogenous and recombinant DEFA5 protein, especially for use in Immunohistochemistry (IHC), Western blot (WB), Immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA).

RESULTS

Clones 1A8 and 4F5 effectively recognized the endogenous DEFA5 in active human colon tissue from patients with diverticulitis (DV), UC, CC, and IC disease samples, as well as in transiently transfected HEK293T cells expressing DEFA5 with minimal non-confounding cross reactivity.

CONCLUSIONS

The 1A8 and 4F5 clones are useful for a wide variety of immunoassays, including WB, IHC, IP/WB, and ELISA. Their specificity enhances their potential as valuable tools for research applications in IBD colitis.

摘要

背景

α防御素5（DEFA5）蛋白在结肠炎症性肠病（IBD）中的异常表达是克罗恩结肠炎（CC）独特发病机制的基础。它可作为区分CC与溃疡性结肠炎（UC）的生物标志物，特别是在将不确定性结肠炎（IC）病例区分为UC和CC时。我们评估了市售抗DEFA5抗体的特异性，强调需要进一步验证其在特定应用中的适用性，并突出了新型抗体的必要性。

方法

我们用纯化的重组蛋白免疫小鼠，建立了两个小鼠单克隆DEFA5抗体克隆，1A8和4F5。我们验证了这些抗体在识别内源性和重组DEFA5蛋白方面的特异性、敏感性和交叉反应性，特别是用于免疫组织化学（IHC）、蛋白质印迹（WB）、免疫沉淀（IP）和酶联免疫吸附测定（ELISA）。

结果

克隆1A8和4F5有效地识别了来自憩室炎（DV）、UC、CC和IC疾病样本患者的活性人结肠组织中的内源性DEFA5，以及在瞬时转染表达DEFA5的HEK293T细胞中，且交叉反应性极小，不会造成混淆。

结论

1A8和4F5克隆可用于多种免疫测定，包括WB、IHC、IP/WB和ELISA。它们的特异性增强了其作为IBD结肠炎研究应用中有价值工具的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/72966d73fd22/11_2024_1970_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/6abd1bdd3825/11_2024_1970_Fig1_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/13d0a8e5df81/11_2024_1970_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/72966d73fd22/11_2024_1970_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/6abd1bdd3825/11_2024_1970_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/968a46a8c7cd/11_2024_1970_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/4a709a8f1c73/11_2024_1970_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/a8cd0f62cf46/11_2024_1970_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/0541b0aa92b9/11_2024_1970_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/25465b7df9fe/11_2024_1970_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/13d0a8e5df81/11_2024_1970_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a028/11782311/72966d73fd22/11_2024_1970_Fig8_HTML.jpg

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The Multifactorial Etiopathogeneses Interplay of Inflammatory Bowel Disease: An Overview.炎症性肠病的多因素病因发病机制相互作用：概述
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Linking bacterial enterotoxins and alpha defensin 5 expansion in the Crohn's colitis: A new insight into the etiopathogenetic and differentiation triggers driving colonic inflammatory bowel disease.
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Redo IPAA After a Failed Pouch In Patients With Crohn's Disease: Is It Worth Trying?克罗恩病患者经肛门内镜微创手术（pouch）失败后再次行经肛门内镜微创手术（IPAA）：是否值得一试？
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