Schematic outline of the Proximity Ligation Assay (PLA) technical procedure. A) Schematic outline of the Proximity Ligation Assay (PLA) technical procedure. PLA probes directed against MMP-9 and TIMP-1 are incubated with the plasma sample allowing a binding of antibodies to target epitopes. Enzymatic ligations of the two oligonucleotide strands can be carried out only when the two PLA probes are in proximity, due to complex formation between MMP-9 and TIMP-1. Forming a PCR amplicon the antibody to antigen binding is now converted into a DNA strand, which can be amplified and later detected by real-time qPCR. B) Principle and sequential design of the MMP-9:TIMP-1 proximity probes. Each polyclonal antibody (MMP-9 and TIMP-1) has been divided into two pools, with one pool conjugated to the 5’ oligonucleotide (5’end) and the other pool conjugated to the 3’oligonucleotide (3’end). When mixing MMP-9 (3’end) probe with TIMP-1 (5’end) probe and plasma, the two probes will come into proximity, if their target antigens form a complex. The connector oligonucleotide is then used to connect the two oligonucleotide arms. Adding a ligase, the two arms will be ligated together, now forming the template of a PCR amplicon. The flanking 20 base pairs of the conjugated oligonucleotide represent the unique primer specific sequence, while the central part represents a universal sequence matching the connector oligonucleotide. The sequences of the specific primers are illustrated in the lower section of the figure. Rew: reverse primer, Frw: forward primer.