Measurement of the noncomplexed free fraction of tissue inhibitor of metalloproteinases 1 in plasma by immunoassay.
Holten-Andersen MN., Christensen IJ., Nielsen HJ., Lilja H., Murphy G., Jensen V., Brünner N., Piironen T.
BACKGROUND: We previously found differences in total concentrations of tissue inhibitor of metalloproteinases 1 (TIMP-1) in plasma from donors and cancer patients. Because TIMP-1 can exist in more than one molecular form, a new immunoassay to specifically detect free TIMP-1 was developed and concentrations were determined in plasma from healthy donors and colorectal cancer (CRC) patients. METHODS: We established and validated an immunoassay for the specific measurement of free TIMP-1 that uses a polyclonal anti-TIMP-1 antibody for capture and a monoclonal anti-TIMP-1 antibody that binds only free TIMP-1 for detection of antigen. Plasma samples from healthy donors and CRC patients were assayed for free TIMP-1. Total TIMP-1 was measured by our previously published assay. RESULTS: The mean (SD) concentrations of free TIMP-1 were similar in citrate [55.5 (11.5) microg/L] and EDTA plasma [58.9 (13.3) microg/L] from 76 donors (r(2) = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) microg/L] to total TIMP-1 [83.8 (19.8) microg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r(2) = 0.19; total, r(2) = 0.27; P <0.0001), increasing 50% over an age span of 45 years. Free and total TIMP-1 were significantly increased in CRC patients (P <0.0001), whereas the ratio of free to total TIMP-1 (mean, 0.58) was significantly lower than in donors. CONCLUSIONS: Most of the TIMP-1 in donor plasma is present in its free form, and free TIMP-1 increases with age. Free and total TIMP-1 are increased in CRC patient plasma, but the ratio of free to total TIMP-1 is significantly lower in these patients than in donors.