Monoclonal antibody to Shiga toxin 2 which blocks receptor binding and neutralizes cytotoxicity.
A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a kappa light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1. 1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producing E. coli infection.
Nakao
H
H
Department of Infectious Diseases Research, National Children's Medical Research Center, Tokyo 154-8509, Japan.
Kiyokawa
N
N
Fujimoto
J
J
Yamasaki
S
S
Takeda
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology* / Q000378:metabolism / Q000633:toxicity; D003603:Cytotoxins / Q000276:immunology*; D000939:Epitopes; D004926:Escherichia coli / Q000472:pathogenicity*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D022603:Shiga Toxins; D014281:Trihexosylceramides / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D003603:Cytotoxins; D000939:Epitopes; D022603:Shiga Toxins; D014281:Trihexosylceramides; C018549:globotriaosylceramide