Identification of two neutralizing human single-chain variable fragment antibodies targeting Staphylococcus aureus alpha-hemolysin

Document Type : Original Article


1 Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran

2 Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran

3 Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

4 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran


Objective(s): The inability of the host immune system to defeat Staphylococcus aureus is due to various secreted virulent factors such as leukocidins, superantigens, and hemolysins, which interrupt the function of immune components. Alpha-hemolysin is one of the most studied cytolysins due to its pronounced effect on developing staphylococcal infections. Alpha-hemolysin-neutralizing antibodies are among the best candidates for blocking the toxin activity and preventing S. aureus pathogenesis. 
Materials and Methods: A human single-chain variable fragment (scFv) phage display library was biopanned against alpha-hemolysin. The selected phage clones were assessed based on their binding ability to alpha-hemolysin. The binding specificity and affinity of two scFvs (designated SP192 and SP220) to alpha-hemolysin were determined by enzyme-linked immunosorbent assay. Furthermore, the neutralizing activity of SP192 and SP220 was examined by concurrent incubation of rabbit red blood cells (RBCs) with alpha-hemolysin and scFvs.
Results: SP192 and SP220 showed significant binding to alpha-hemolysin compared with the control proteins, including bovine serum albumin, human adiponectin, and toxic shock syndrome toxin-1. Besides, both scFvs showed high-affinity binding to alpha-hemolysin in the nanomolar range (Kaff: 0.9 and 0.7 nM-1, respectively), leading to marked inhibition of alpha-hemolysin-mediated lysis of rabbit RBCs (73% and 84% inhibition; respectively). 
Conclusion: SP192 and SP220 scFvs can potentially be used as alpha-hemolysin-neutralizing agents in conjunction with conventional antibiotics to combat S. aureus infections. 


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