Horseradish peroxidase conjugated goat antimouse secondary antibody

In order to confirm the alteration in COMP expression, as identified by label-free mass spectrometry, immunoblot analysis was employed. Electrophoretic separation of proteins from the extracts pre enzymatic digestion was performed using gradient polyacrylamide gels (Criterion XT Pre-cast 4–12% Bis-Tris gel, 26 well) and a prestained molecular weight marker (Precision Plus Protein Standard All Blue, Biorad) followed by wet transfer at 100 V for 70 min at 4°/C to Immobilon-P PVDF membrane (Millipore, Billerica, USA) in a Transblot Cell from Bio-Rad Laboratories (Hemel-Hempstead, Hertfordshire, UK). Membranes were blocked for 1 hour at room temperature with Rotiblock (Carl-Roth, Karlsruhe, Germany) prior to incubation with the primary antibody (anti-COMP, ab74524, abcam UK) at a dilution of 1:1000 in Rotiblock, overnight at 4°C with gentle agitation. Membranes were subsequently washed twice with PBS containing 0.05% Tween 10 min each time, followed by incubation for 1 h with polyclonal horseradish peroxidase-conjugated goat-anti-mouse secondary antibody (P0447, Dako, Ely, UK) at a dilution of 1:10,000 in Rotiblock. Visualisation of antibody-labelled protein bands on washed membranes was achieved using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK) according to the manufacturer’s guidelines.

Nunc MaxiSorp 96-well plates (Invitrogen, Carlsbad, CA, USA) were coated with rabbit anti-human C4d neoepitope-specific antibody [8 (link)]. After quenching with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween, pH 7.5) supplemented with 3% fish gelatin (Norland Products, Cranbury, NJ, USA), patient or control plasma and pooled plasma from healthy volunteers (lacking C4d) supplemented with Escherichia coli-expressed C4d standard in serial dilutions were diluted to 4% in PBS with 0.02% Tween-20 and 0.02 M disodium ethylenediaminetetraacetate dihydrate and added to the plate. Detection was achieved using mouse anti-human C4d antibody (catalogue number 253; Quidel, San Diego, CA, USA) followed by horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Dako, Carpinteria, CA, USA). Plates were developed using o-phenylenediamine dihydrochloride as a substrate, and absorbance was measured at 490 nm using a Varian Cary 50 microplate reader (Agilent Technologies, Santa Clara, CA, USA). The lowest detection limit was 5.6 μg/L. Values below the detection limit were set to 0.001 mg/L for statistical calculations. The inter-assay coefficient of variation was 16.7%, and the intra-assay coefficient of variation was 13.2%.