Anti m13 hrp mab

Individual phages were subjected to phage ELISA to investigate cross reactivity with other closely related members of the membrane-type matrix metalloproteinase subgroup, MT2-MMP and MT3-MMP. For this purpose, selected individual colonies of E. coli TG1 harboring phagemids were grown in 2 × YT medium overnight at 30°C in a 96-well plate (Nunclon, Roskilde). Then, the overnight culture for each clone was diluted 100-fold into fresh 2xYT medium and incubated at 37°C for 2 h. The cultures were infected with ∼10⁹ PFU of VCS-M13 (KmR; Stratagene, San Diego, CA) helper phages and grown overnight at 30°C. Finally, the culture supernatant of each well was used for ELISA. MaxiSorp 96-well plates (Nunc, Roskilde) were coated with CAT-MT1-MMP, MT2-MMP [NS0-derived fragment from Glu47 to Pro565 (Arg128Pro; Arg129Gly), R&D Systems, Minneapolis, MN] or MT3-MMP [E. coli-derived fragment from Ala32 to Gly291 (Ile152Asn); R&D Systems, Minneapolis, MN] at 4°C overnight. Bovine serum albumin (BSA, Roche, Basilea) was used as a negative control for detection. The supernatants containing the corresponding phages were applied to the plates and bound phages were detected with an anti-M13-HRP mAb (GE Healthcare, Uppsala) and o-phenylenediamine (OPD, Sigma, Saint Louis, MO) as substrate. The OD490 was determined using a microplate reader (iMark ELISA plate reader, Bio-Rad, Hercules, CA).

ELISA conditions were based on those described previously [57 (link)]. Briefly, 96-well immunoplates (Maxisorp, Nunc) were coated for 120 min at RT with purified antigens (as indicated) diluted in PBS at a concentration of 5 µg/ml. Bovine serum albumin (BSA, Roche) was used as a negative control for detection. Phabs, culture supernatants or purified Nb-HlyA fusions were added to the wells at the indicated dilutions. For detection of bound Phabs, an anti-M13-HRP mAb (GE Healthcare) was added (1:5000). For detection of Nb-HlyA fusions, anti-E-tag mAb (1:2000; Phadia) and anti-mouse IgG-POD (1:2000; Sigma), as secondary antibody, were added. The reaction was developed with o-phenylenediamine (OPD, Sigma) and H₂O₂ (Sigma) and the OD490 was determined using a microplate reader (iMark ELISA plate reader, Bio-Rad).