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.
Marine Biotechnology – Springer Journals
Published: Apr 14, 2018
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