Development of diagnostic kit for detection of pathogenic Leptospira- specific protein by water soluble chitosan derivatives

Abstract

Responsive polymers have been studied intensively in various field of applications. The aim of this study was to use water soluble chitosan derivatives conjugated with antigen or antibody for rapid detecting Leptospira-specific protein. First, water soluble phosphorylated chitosan (PCTS) was synthesized by phosphorylation of chitosan with phosphorus pentoxide in methane sulfonic acid. Fourier transform infrared (FT-IR) spectrum of PCTS revealed peaks of phosphoric groups at 1260 cm-1 (P=O stretching) and 800 cm-1 (P-O-C). Degree of phosphate substitution was determined of around 0.08 by scanning electron microscope equipped with energy-dispersive X-ray spectrometer (SEM-EDX). LipL32 and monoclonal antibody (MAb) were selected as antigen and antibody respectively. Then, LipL32 and MAb were conjugated onto PCTS through peptide linkage by using EDC/NHS as coupling agent in DI aqueous solution at 0-4oC for 12 hrs. The conjugation of PCTS with LipL32 and MAb was confirmed by FTIR and SDS-PAGE gel electrophoresis. FTIR spectra of PCTS-LipL32 and PCTS-MAb revealed characteristic peaks of amide I at 1655 cm-1 and amide II at 1555 cm-1 indicating successful conjugation at amino groups of PCTS. Furthermore, SDS-PAGE gel electrophoresis shown the band retardation in wide range of molecular weight with decreasing in amino groups, indicating covalently conjugation. Isoelectric point of PCTS-LipL32 and PCTS-MAb were also shifted from 5.80 (PCTS) to 10.64 and 9.47, respectively. PCTS-MAb and PCTS-LipL32/PCTS-MAb complex exhibited specific binding in the presence of LipL32 free antigen by rapidly aggregation within 1 min. This can be concluded that PCTS-MAb and PCTS-LipL32/PCTS-MAb complex could be utilized as rapid diagnostic kit for detecting antigen of leptospires.