Bacterial Lipid Modification of ICP11 and a New ELISA System Applicable for WSSV Infection Detection
- 17 Downloads
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.
KeywordsLipid modification Shrimp WSSV ICP11 ELISA
The authors thank Dr. S. Kumar for providing the polyclonal antiserum and Dr. Shrikrishnan Sankaran for affinity quantification and valuable comments. The authors dedicate this article to (late) Dr. V. Murugan, Centre for Biotechnology, Anna University, Chennai.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interests.
- Leal CAG, Carvalho-Castro GA, Cottorello AC, Leite RC, Figueiredo HCP (2013) Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD. Braz J Microbiol 44(3):901–904Google Scholar
- Mendoza-Cano F, Sanchez-Paz A (2013) Development and validation of a quantitative real-time polymerase chain assay for universal detection of the white spot syndrome virus in marine crustaceans. Virol J 10(186):1–11Google Scholar
- Nagalakshmi G, Jyothi S (2013) White spot syndrome virus detection in shrimp images using image segmentation techniques. Int J Adv Res Comput Sci Softw Eng 3:107–112Google Scholar
- Reddy AD, Jeyasekaran G, Shakila RJ (2013) Morphogenesis, pathogenesis, detection and transmission risks of white spot syndrome virus in shrimps. Fish Aquac J 66:1–10Google Scholar
- Takahashi Y, Itami T, Maeda M, Suzuki N, Kasornchandra J, Supamattaya K, Khongpradit R, Boonyaratpalin S, Kondo M, Kawai K, Kusuda R, Hirono I, Aoki T (1996) Polymerase chain reaction (PCR) amplification of bacilliform virus (RV-PJ) DNA in Penaeus japonicus Bate and systemic ectodermal and mesodermal baculovirus (SEMBV) DNA in Penaeus monodon Fabricius. J Fish Diseases 19:399–403CrossRefGoogle Scholar
- Xia X, Yu Y, Weidmann M, Pan Y, Yan S, Wang Y (2014) Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay. PLoS One 9(8):1–8Google Scholar