Bacterial Lipid Modification of ICP11 and a New ELISA System Applicable for WSSV Infection Detection

Abstract

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.

Electrochemical detection of white spot syndrome virus with a silicone rubber disposable electrode composed of graphene quantum dots and gold nanoparticle-embedded polyaniline nanowires

“…Piezoelectric microcantilever sensors WSSV (DNA) 50 to 10 5 virions/ml 100 virions/ml [51] Lateral flow assay WSSV (DNA) 36-1784 viral copies/ng 356 viral copies/ng [52] Surface plasmon resonance WSSV (Antigen) 5 to 50 ng/ml 2.5 ng/ml [53] Loop-mediated isothermal amplification WSSV (DNA) 0.05 to 1 μg/reaction (LAMP products) 2 × 10 2 copies [54] Electrochemical WSSV (Antigen) 1.37 × 10 −3 to 1.37 × 10 7 copies/μL 1.36 × 10 −3 copies/μl [22] Enzyme-linked immunosorbent assay WSSV (Antigen) 15-240 ng/well 250 pg/well [55] Polymerase chain reaction WSSV (DNA) 9.0 × 10 1 -2.0 × 10 4 copies/μg 4 copies/sample [14] Impedance electrochemical detection WSSV (Antigen) 10 2 to 10 9 DNA copies/ml 48.4 DNA copies/ml This work a b The concentration of the recovered WSSV was calculated using a calibration curve (Fig. 3b) based on the obtained Rct values.…”

A review on molecular detection techniques of white spot syndrome virus: Perspectives of problems and solutions in shrimp farming

“…Murugan and Sankaran proposed a new ELISA approach for evaluating recombinant bacterial lipid change in proteins (Murugan and Sankaran, 2018). In that study, the most frequently generated white spot syndrome viral protein ICP11 was found as a new target in a novel ELISA method using a lipid-modified protein.…”

Microfluidic-Based Nucleic Acid Amplification Systems in Microbiology

“…Immunological diagnostics are based on specific biomarker antibodies and optical sensing to detect the corresponding bacteria. Some drawbacks still exist with these methods, such as low or lack of signal, high background noise, and inconsistent results between replicate samples or controls [3,4]. Molecular diagnostics has been further expanded to proteomic and genomic methods such as mass spectrometry (MS), polymerase chain reaction (PCR), isothermal amplification, and high throughput next generation sequencing (NGS) [5,6,7,8].…”