Fig. 4

Secretion of Nb-HlyA fusions and antigen binding of representative clones. Representative clones of the identified VHH sequences against Int280, EspA and TirM were secreted as Nb-HlyA fusions. a Western blot developed with anti-E-tag-mAb of supernatants from induced cultures of the indicated clones showing a major protein band of ca. 42–45 kDa corresponding to Nb-HlyA fusions. The image shown is overexposed to visualize clearly protein bands of clones with lower expression levels. b ELISA of culture supernatants containing secreted Nb-HlyA fusions of the indicated clones against Int280 (top), EspA (middle) and TirM (bottom) developed with anti-E-tag mAb-POD. The represented OD490 values are the average from triplicate culture supernatants of each clone. Background signals to BSA are subtracted from the values obtained against the specific antigen (Int280, EspA, TirM)