Maxisorp

Enzyme-linked immunosorbent assay plates (MaxiSorp™, Sigma-Aldrich, Saint Louis, MO) were coated overnight at 4°C with CAT-MT1-MMP or an irrelevant recombinant protein as a negative control. Wells were blocked by 3% Bovine Serum Albumin in PBS at room temperature for 1 h. Next, serial dilutions of MT1-MMP nanobodies in blocking buffer were added and incubated for 90 min at 37°C. Bound nanobodies were detected by incubation with HRP-conjugated anti-HA (clone 6E2) mouse mAb (Cell Signaling, Danvers, MA) at room temperature for 1.15 h and TMB (Sigma-aldrich, Saint Louis, MO) as substrate. The reaction was stopped by adding 1 M sulfuric acid (Sigma-Aldrich, Saint Louis, MO) and the OD450 was measured (Fluostar Omega, BMG-Labtech, Ortenberg).

First, 96-well MaxiSorp™ plates were pre-coated with 20 µg/mL poly-L-lysine (Sigma-Aldrich P4707) in 50 µL TE-buffer (10 mM Tris/HCl; 1 mM EDTA; pH 7.4) per well and incubated overnight at 4 °C. After washing with TE-buffer, 50 µL of TE-buffer containing 20 µg/mL of deoxyribonucleic acid sodium salt from calf thymus (Sigma-Aldrich, D4522-5 mg) was added per well for coating overnight at 4 °C. Further steps were carried out as described above for total IgG-ELISA.

Rabbit Poly-Arginine (9R) antibody was produced by Davids Biotechnologie GmbH (Germany) as follows. New Zealand white rabbits were immunized with newly synthesized Acetyl-RRRRRRRRR-Amide peptide and the antiserum was further purified by affinity purification. The obtained 9R antibody (Anti-R₉) was tittered by means of ELISA (enzyme linked immunosorbent assay) to determine the optimal dilution for immunocytochemical detection. Briefly, MaxiSorp (Sigma-Aldrich, MO, United States) microtiter plate was coated with the Acetyl-RRRRRRRRR-Amide peptide in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. The plate was washed three times with PBS-BSA 0.5% between every step. The remaining binding sites were blocked with PBS-BSA 0.5% for 2 h. The antiserum samples were serial diluted at a range concentration of 1/10–1/10,000 (v/v). The samples were applied in triplicate and incubated for 1 h at room temperature. HRP (horseradish peroxidase)-conjugated secondary antibody anti-rabbit (Bio-Rad) diluted in blocking buffer was incubated with the samples for 1 h at room temperature. TMB (3,3′,5,5′-tetramethylbenzidine, ThermoFisher) solution was added to each well and incubated for 30 min for signal detection. An equal volume of stopping solution (2 M H₂SO₄) was added and the completed reaction was read at 450 nm in a microplate reader (Tecan).

Humoral immune responses were assessed by indirect EnzymeLinked Immunosorbent Assay (ELISA). Total IgG, and antibody isotypes were measured from mouse serum. Briefly, 50 µg of L3 antigenic protein was dissolved in 50 mM carbonate buffer to coat Maxisorp (Millipore, Bedford, MA, USA) plates. After overnight incubation at 4 °C, plates were washed. To avoid unspecific interactions, wells were blocked with ChonBlock Buffer (Chondrex, Redmond, WA, USA) for two hours at room temperature. Serum samples were diluted at 1:50 in ChonBlock and incubated for two additional hours at room temperature. Plates were washed after incubation, and HRP-conjugated goat anti-murine IgG, or IgG1, or IgG2a antibodies (Jackson Immunoresearch, West Grove, PA, USA) were diluted in ChonBlock detection antibody dilution buffer (Chondrex, Redmond, WA, USA). Samples were incubated at room temperature for sixty minutes in secondary antibody diluted at 1:2,500. Chemiluminescence produced by the secondary antibody was measured at 450 nm using a plate reader after addition of OPD peroxidase substrate (Sigma Aldrich, St. Louis, MO, USA). The reaction was stopped using 50 µL of 3 M HCl.