The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) based on AlphaLISA® technology. We describe two procedures: (i) one approach is used to analyze MMP-9-TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii) the second approach is used to analyze native or endogenous MMP-9-TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9-TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9-TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9-TIMP-1 complexes in circulating blood as biofluid.
CITATION STYLE
Pulido-Olmo, H., Rodríguez-Sánchez, E., Navarro-García, J. A., Barderas, M. G., álvarez-Llamas, G., Segura, J., … Ruiz-Hurtado, G. (2017). Rapid, automated, and specific immunoassay to directly measure matrix metalloproteinase-9-tissue inhibitor of metalloproteinase-1 interactions in human plasma using AlphaLISA technology: A new alternative to classical ELISA. Frontiers in Immunology, 8(JUL). https://doi.org/10.3389/fimmu.2017.00853
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