Imark elisa plate reader

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).

Nunc Maxisorp ELISA plates (Nalgene Nunc) were coated overnight at 4 °C with MuHV-4 virions (10⁶ PFU ml⁻¹ of carbonate buffer pH 9.5 containing 0.1% Triton X-100), or SEA (10 µg mL⁻¹ in carbonate buffer) before being incubated for 1 h in wash/blocking buffer (0.1% Tween-20 and 3% BSA in PBS) at RT. Plates were then incubated with mouse sera (serial dilutions) in wash/blocking buffer for 2 h at RT. Detection was performed using alkaline phosphatase-conjugated anti-mouse IgG1 or IgG2a (BD Biosciences) in wash/blocking buffer for 1 h at RT. Chromogenic reaction was performed using p-Nitrophenylphosphate (Sigma) and stopped in NaOH 1 M before absorbance was read at 405 nm using a iMark ELISA plate reader (Biorad).

Nitric oxide (NO), an important physiological messenger and effector molecule in immunological systems [42 (link)], was determined by a previously described method using the Griess reagent System [43 (link)]. Briefly, the cell free suspension of the joint extract was prepared in 0.05M Tris-HCl buffer, pH-7.6 and then 50 μl of experimental sample was added in 96 well ELISA plate in duplicate followed by addition of 100 μl of Griess reagent (0.04g/ml) (Sigma, U.S.A.). Samples were incubated at room temperature for 10 minutes in dark. The reading was taken at 540nm on iMark Elisa Plate Reader (BioRad, U.S.A.). The level of NO (μM/ mg protein) in unknown sample was measured by comparing with standard curve prepared with sodium nitrite (Fisher Scientific, U.S.A.).