Objective To establish a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) assay for the quantitative detection of suilysin (SLY) and provide evidence for the evaluation of SLY producing ability and pathogenicity of Streptococcus suis.
Methods Antisera was prepared by using 4 kinds of cholesterol-dependent cytolysin (CDC) in BALB/c mice and New Zealand white rabbits. The reactivity of antisera with four recombinant CDCs, including suilysin (rSLY), pneumolysin (rPLY), listeriolysin (rLLO) and perfringolysin (rPFO), was evaluated by indirect ELISA. The concentrations of human-sourced monoclonal antibody (mAb) 1-10F (patent no. CN 113957057B), as trapping antibody, and antiserum, as testing antibody, were optimized by orthogonal test for the establishment of a double-antibody sandwich ELISA assay for SLY detection. The specificity of this assay was assessed by testing the cell-free mediums of S. suis, S. Pneumoniae, Listeria monocytogenes, Clostridium perfringens, hemolytic Escherichia coli and Staphylococcus aureus.
Results The results of indirect ELISA showed that rabbit anti-rSLYP353L serum could react with rPLY, mouse anti-rPLY serum could react with rSLY and rLLO, and the 4-valent serum had strong reaction with rPLY, rSLY and rLLO, while rPFO had weak reaction with the three types of antisera. The optimized conditions of double antibody sandwich ELISA assay were 100 ng 1-10F IgG with mouse anti-rPLY serum dilution as 1∶1 000. In this condition, the measurement of rSLY showed a linear correlation at concentrations of 25–800 ng/mL (R2=0.986). Moreover, the specificity test of this assay showed strong positive result for cell-free medium of S. suis, weak positive result for S. pneumoniae and negative result for others.
Conclusion The established double antibody sandwich ELISA assay can detect SLY quantitatively, which can be used as a new option for the diagnosis of highly pathogenic S. suis infection.
Home
DownLoad: