Molecular distribution of elastase between its two main inhibitors: direct quantitation of elastase-α₂-macroglobulin complex with a novel ELISA technique

Abstract

A novel enzyme-linked immunosorbent assay for the quantitation of elastase linked to α₂-macroglobulin (α₂M) (elastase-a₂M complex, EMC) in body fluids is presented. The assay has a lower detection limit of 1.5 ng bound elastase per ml. The critical factor allowing immunological detection of α₂M-bound elastase is the addition of phenyl methyl sulphonyl fluoride (PMSF) to the assay buffer. The assay has been used to quantitate EMC in plasma and serum of healthy volunteers, and assess the distribution of elastase between its two main inhibitors, α₂M and a_rproteinase inhibitor (API). EMC levels in serum samples from volunteers were significantly higher than in plasma (26.9 vs 21.9 ng/ml, p=0.05, Student's t-test for paired samples). The API-bound elastase levels were 70.2 and 170.1 ng/ml in plasma and serum respectively (statistically significantly higher in serum, p<0.0001).

The ratio of macroglobulin to a_rproteinase-bound elastase levels was 0.333 in plasma and 0.168 in serum (p<0.0001). As α₂M bound elastase retains some proteolytic activity, direct measurement of circulating levels in inflammatory conditions should be of interest.

Key Words:

• α₂-macroglobulin
• α₁proteinase inhibitor
• elastase-α₂-macroglobulin complex
• elastase-α₁ -protein inhibitor complex
• ELISA
• phenyl methyl sulphonyl fluoride (PMSF)