Development of an ultrasensitive enzyme immunoassay for the determination of matrix metalloproteinase-9 (MMP-9) levels in normal human cerebrospinal fluid

Determination of matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF) to study blood-brain barrier impairment and immune cell migrati on in inflammatory neurological diseases recently became a matter of major interest. Regularly, MMP-9 was determined qualitatively or semi-quantitativ ely by zymography (gelatin gel electrophoresis) or quantitatively by enzyme immunoassay (EIA). As yet, it was not possible by either method to detect MMP-9 in CSF of controls (patients without pathologically increased CSF par ameters). We developed an ultrasensitive two-side enzyme-linked immunosorbe nt assay (ELISA) which allows for the first time to measure reliably MMP-9 concentrations in CSF of controls. This ELISA uses a monoclonal as capture and a polyclonal as detector antibody. The detection limit of the assay is below 10 pg/ml and the assay range is 15-2000 pg/ml. Intra-assay precision is 2.5% for low and 3.7% for high, inter-assay precision is 11% for low and 10.7% for high values, respectively. The determination of the MMP-9 concen tration in 50 control CSF gave the following results: range, 22-146 pg/ml; median, 76 pg/ml. The measurement of native and recombinant MMP-9 was carri ed out with three commercially available ELISAs, most widely employed in MM P-9 research, and compared to the newly developed one. All ELISAs recognize recombinant MMP-9 by factors of 5-20 less sensitively than native MMP-9. ( C) 2001 Elsevier Science B.V. All rights reserved.