T4661

Active MMP1 was purified as described in a previous publication [39 (link)]. Briefly, the cDNA sequence of MMP1 was optimized for expression in E. coli and inserted into pET-21b (+) vector between NdeI (N-terminal) and HindIII (C-terminal) restriction sites. The plasmid was transformed into Rosetta (DE3) pLysS competent cells (Novagen, 70956). The cells were cultured in Luria Broth media (Sigma, L3022) to reach the optical density, OD₆₀₀ = 0.1 (pH 7.0) at 37°C at 250 rpm in presence of chloramphenicol and ampicillin at the final concentrations of 34 μg/μl and 100 μg/μl respectively. The cells were induced for 5 hr with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and harvested after centrifugation. 1 g of the centrifuged cells was reconstituted in 7 ml of lysis buffer (pH 9.0) containing 50 mM Tris base (Sigma, T4661), 100 mM NaCl (Sigma, S9888), 200 μM ZnCl₂ (Sigma, 208086), 400 μM CaCl₂ (Sigma, 746495), freshly prepared 1% Triton X-100 (Sigma, T8787), 0.1 mg/ml trypsin (Worthington, TPCK-treated and irradiated, LS003750), and 1 mg/ml lysozyme (Sigma, L6878). The reconstituted cells were incubated for 18 hr at 37°C at 250 rpm and centrifuged to collect the supernatant, followed by centrifugation using 30 kD cut-off filters. MMP1 was quantified using Bradford assay and analyzed by SDS PAGE.

The O-PAD for ELISA (O-ELISA) was designed on Whatman qualitative filter paper No. 1 and patterned using a wax printer (Xerox Phaser 8650N color printer, Norwalk, CT, USA). Four wings were fashioned to house ELISA reagent loading zones for pre-loading, and the central area was designed and reserved as a testing zone for sample loading. After heating at 105 °C for 3 min, the melted wax wicked through the paper to create hydrophobic barrier wells in the paper. Reagents were then pre-loaded into reagent zone barrier wells. These reagents included the following: (1) 2 µL of blocking buffer (5% (w/v) bovine serum albumin (BSA) (Sigma, SI-A7906, St. Louis, MO, USA) in PBS (Corning, 21-040-CM, Corning, NY, USA); (2) 2 µL solution of alkaline phosphatase (ALP)-conjugated detection antibody (Cell signaling, #7054, Danvers, MA, USA) (20 µg/mL, 0.01% (v/v) Tween-20 (Sigma, P9416, St. Louis, MO, USA); and (3) 2 µL BCIP/NBT substrate (13.4 mM BCIP (Sigma, B6274), 9 mM NBT (Sigma, N5514), 25 mM MgCl₂ (Sigma, M8266), 500 mM NaCl (Sigma, S7653) in 500 mM Tris buffer pH 9.5 (Sigma, T4661). After loading each reagent, the device was placed under ambient conditions for 5 to 10 min until the reagents dried.