Quantification of MBL Isoforms in Plasma and Tissue

MBL isoforms were analyzed after sample incubation on mannan-coated (10 µg/mL) plates prepared according to a procedure reported previously.^{18 (link)} Plates were incubated for 1 h at 37 °C with EDTA-plasma diluted in barbital-buffered saline (BBS; 4 mm barbital, 145 mm NaCl, 2 mm CaCl₂, 1 mm MgCl₂ (pH 7.4)) to a final plasma concentration of 2.5% or with cortical homogenates (ipsilateral and contralateral to the lesion) diluted in BBS to a final concentration of 4 mg/mL total protein. Samples were then incubated for 1 h 30 min at room temperature (RT) with anti-MBL-A or anti-MBL-C antibodies (both 1 µg/mL, Hycult, # HM1035 and #HM1038, respectively). After washing, plates were incubated with an HRP-labeled goat anti-mouse IgG antibody (Santa Cruz, CA, USA), diluted to 0.4 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of TMB substrate solution (TMB Substrate Kit, code 34021, Thermo Scientific, MA, USA; diluted 1:1 in H₂O₂ solution). The reaction was terminated by adding 100 μL of 2 m H₂SO₄, and the absorbance at 450 nm was measured using an Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).