Centre for Biotechnology, Anna University, Guindy, Chennai, Tamil Nadu, 600025, India.
Mar Biotechnol (NY). 2018 Jun;20(3):375-384. doi: 10.1007/s10126-018-9815-7. Epub 2018 Apr 14.
In ELISA, a popular analytical diagnostic tool, the stable non-covalent immobilization (coating) of hydrophilic proteins/peptides on to hydrophobic polystyrene surface has remained a major common challenge. Recombinant bacterial lipid modification of proteins in Escherichia coli system has been shown in this study to solve this problem owing to the hydrophobic anchorage provided by three fatty acyl groups in N-acyl-S-diacylglyceryl Cys at the N-terminus. Exploiting this first post-translational protein engineering, the most abundantly expressed white spot syndrome viral protein ICP11 was lipid-modified and tested as a new target in a new ELISA method useful to shrimp farming. The lipid served as a potent adjuvant to enhance the titer (16 times) of higher affinity antibodies where amino terminal lipoamino acid N-acyl-S-diacylglyceryl cysteine of bacterial lipoproteins induce inflammatory responses through TLR and stimulate humoral immune responses without additional adjuvant and also aided in the immobilization of even a few nanograms of ICP11. Competition between the immobilized and the free antigen from the sample provided a sensitive measure of antigen in the infected shrimp tissues. The detection limit for ICP11 protein using competitive ELISA was 250 pg and the linear range of the assay was 15-240 ng.
在 ELISA 中，一种流行的分析诊断工具，稳定的非共价固定（涂层）亲水蛋白/肽到疏水性聚苯乙烯表面仍然是一个主要的共同挑战。本研究表明，在大肠杆菌系统中，重组细菌脂质修饰蛋白可以解决这个问题，因为在 N 端的 N-酰基-S-二酰基甘油半胱氨酸中有三个脂肪酸酰基提供了疏水锚定。利用这种首次翻译后蛋白质工程，最丰富表达的白斑综合征病毒蛋白 ICP11 被脂质修饰，并作为一种新的 ELISA 方法的新靶标进行测试，该方法对虾养殖有用。脂质作为一种有效的佐剂，可提高高亲和力抗体的效价（16 倍），其中细菌脂蛋白的氨基末端脂氨酰基 N-酰基-S-二酰基甘油半胱氨酸通过 TLR 诱导炎症反应，并刺激体液免疫反应，而无需额外的佐剂，同时还有助于固定甚至只有几个纳克的 ICP11。固定化抗原与样品中游离抗原之间的竞争提供了一种敏感的方法来检测感染虾组织中的抗原。使用竞争 ELISA 检测 ICP11 蛋白的检测限为 250 pg，测定的线性范围为 15-240 ng。
