Table 1.
Strains used in this work.
Fig 1.
Development of hybridomas secreting mAbs against E. coli O157 and O145.
(A-B) Analysis of test bleed sera titers by glyco-iELISA of the mice immunized with heat-killed E. coli O157 (A) or E. coli O145 (B) strains to select the mouse destined to fusion and generation of hybridomas. (C-D) Comparison of test bleed and final bleed sera titers of the mice used for fusion by O157-AcrA (C) or O145-AcrA (D) glyco-iELISAs. In (C) and (D) data represents mean±SD of two sample replicates.
Fig 2.
Specificity of O157 and O145 mAbs towards STEC strains by iELISA.
iELISA of representative STEC strains of serogroups O157, O145, O121, O111, O104, O103, O45 and O26 (A-B), and iELISA of B. abortus 2308, S. Urbana, Y. enterocolitica O9 and E. coli O157 (C) or E. coli O145 (D), using O157 1E10, 3F10 and 10G2 mAbs (A,C) or O145 2H6, 4C8 and 4E6 mAbs (B,D). In (A-B) non-pathogenic E. coli DH5α strain was used as a negative control, and in (C) O9 M84 mAb was used as B. abortus and Y. enterocolitica O9 positive control. Each point of the curve represents the mean±SD of three sample replicates.
Fig 3.
Immunoblot analysis of E. coli strains and recombinant glycoproteins with O157 and O145 mAbs.
Immunoblot using mAbs O157 1E10, 3F10 and 10G2 (A) or mAbs O145 2H6, 4C8 and 4E6 (B). Non-pathogenic E. coli DH5α strain and non-glycosylated AcrA were loaded as controls. SDS-PAGE analysis of E.coli O157 and O145 strains, and purified O157-AcrA and O145-AcrA glycoproteins by Coomassie brilliant blue staining (C). The positions of the molecular mass standards are indicated on the left.
Fig 4.
Relative affinity of mAbs towards O157-AcrA and O145-AcrA by glyco-iELISA.
Standard curves of O157-AcrA (A) or O145-AcrA (B) detected by glyco-iELISA with the use of mAbs O157 1E10, 3F10 and 10G2 (3:8 hybridoma supernatant dilution) (A) or mAbs O145 2H6, 4C8 and 4E6 (1:2 hybridoma supernatant dilution) (B). Each point of the curve represents the mean±SD of two sample replicates. IC50 values of the mAbs are indicated.
Fig 5.
Surface staining of O157 and O145 STEC strains with O157 and O145 mAbs.
(A-B) Binding of O157 3F10 and O145 4C8 mAbs to E.coli O157 (A) or E.coli O145 (B) heat-killed bacteria assessed by flow cytometry. An irrelevant isotype-matched murine mAb or only secondary Ab were used as controls. (C-D) Immunostaining of E.coli O157 and O145 with O157 3F10 (C) and O145 4C8 (D) mAbs, visualized by confocal microscopy. Scale bar, 3 μm.
Fig 6.
Agglutination assay of STEC strains with O157 and O145 mAbs.
Detection of O157 (A) and O145 (B) antigens in representative STEC strains of serogroups O157, O145, O121, O111, O104, O103, O45 and O26 by agglutination assays using O157 3F10 mAb or O157 mouse antisera (A), or O145 4C8 mAb or O145 mouse antisera (B). AS: antisera.