Development, characterization, and technical applications of a fish lysozyme-specific monoclonal antibody (mAb M24-2).
Development, characterization, and technical applications of a fish lysozyme-specific monoclonal antibody (mAb M24-2).
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DOI：
10.1016/j.cimid.2009.10.002
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发表时间：
2010-12
期刊：
影响因子：
--
通讯作者：
Rice CD
中科院分区：
文献类型：
--
作者：
Marsh MB;Rice CD
溶菌酶是与所有脊椎动物的前线天然免疫相关的几种体液和细胞因子之一。从历史上看，在硬骨鱼中，循环溶菌酶的定量是通过在低pH值下使用全血清或血浆来测量抗热致死溶菌的酶活性来实现的。然而，标准溶菌酶活性所需的血清或血浆的量超过了从小鱼中容易获得的量，因此在有限的样本量下很难测量溶菌酶的终点。此外，尽管循环中的溶菌酶被认为是促炎吞噬细胞活性的指标，但这种蛋白的细胞来源在鱼类中很难检测到。虽然有几种抗溶菌酶的抗体可以在商业上用于高等脊椎动物，但它们都不能与鱼类中的溶菌酶发生反应。本研究从能识别鸡蛋溶菌酶的骨髓样细胞--乳鼠中制备了一种检测和定量溶菌酶的单抗，并对不同种类的硬骨鱼进行了交叉反应试验，在鱼细胞裂解物和血浆样品中用该单抗鉴定了≈14-15 kDa的单一蛋白，以及变性的鸡溶菌酶。使用标准的ELISA法将循环中的总溶菌酶蛋白与溶菌酶活性进行比较，发现与酶活性相关。用mAbM24-2在福尔马林固定和渗透的淋巴样细胞中检测到细胞内溶菌酶蛋白。此外，mAbM24-2将溶菌酶定位于髓系细胞。结果表明，M24-2单抗可在塑料包埋的淋巴组织中检测到溶菌酶，适合用于免疫组织化学。
Lysozyme is one of several humoral and cellular factors associated with front line, innate immunity in all vertebrates. Historically, circulating lysozyme has been quantified in teleosts by measuring enzymatic activity against heat-killed Mycococcus lysodieticus using whole serum or plasma at a low pH. However, the amount of serum or plasma required for standard lysozyme activity exceeds that which can be easily acquired from small fish, thus making lysozyme a difficult endpoint to measure in limited sample volumes. Moreover, while circulating lysozyme is considered to be an indicator of proinflammatory phagocyte activity, the cellular source of this protein is not easily detected in fish. While several antibodies against lysozyme are commercially available for use in higher vertebrates, neither reacts with lysozyme in fish. In this study, a monoclonal antibody (mAb) for detecting and quantifying lysozyme was developed from mummichog, Fundulus heteroclitus, myeloid cells that also recognizes hen egg lysozyme (HEL), then tested for cross-reactivity in different species of teleosts, A single protein of ≈ 14–15 kDa mass was identified by the mAb in fish cell lysates and plasma samples, as well as denatured HEL. Total circulating lysozyme protein was compared to lysozyme activity using standard ELISA procedures and was found to correlate with enzymatic activity. Using mAb M24-2, intracellular lysozyme protein was detected in formalin-fixed and permeabilized lymphoid cells adhered to glass cover slips. Moreover, mAb M24-2 localizes lysozyme to myeloid cells. Finally, it was demonstrated that mAb M24-2 is suitable for immunohistochemistry in that lysozyme could be detected in plastic-embedded lymphoid tissues.
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DOI：
10.1016/0959-8030(92)90060-b
发表时间：
1992-01-01
期刊：
Annual Review of Fish Diseases
影响因子：
--
作者：
Ainsworth, A. Jerald
通讯作者：
Ainsworth, A. Jerald
DOI：
10.1152/ajpendo.00349.2005
发表时间：
2006-05-01
影响因子：
5.1
作者：
Liu, HX;Zheng, F;Vlassara, H
通讯作者：
Vlassara, H
影响因子：
2.8
作者：
Mazon, AF;Kemenade, BMLV;Huising, MO
通讯作者：
Huising, MO
影响因子：
2.6
作者：
Liu, F;Wen, ZL
通讯作者：
Wen, ZL
影响因子：
56.9
作者：
KOKOSHIS, PL;WILLIAMS, DL;DILUZIO, NR
通讯作者：
DILUZIO, NR