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Immunoassays | Prototype

Erenna® MMP-2/TIMP-2 Immunoassay Prototype Assay (Cat# 03-0045-AA)

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Introduction

The Erenna® MMP-2/TIMP-2 Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure MMP-2/TIMP-2 complex in plasma and serum samples. A capture antibody specific for MMP-2/TIMP-2 complex has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the MMP-2/TIMP-2 complex present in the sample binds to the capture antibody on the coated MP. Unbound molecules are washed away during the subsequent buffer exchange and wash steps. Fluor-labeled detection antibody is added to each well and incubated. This detection antibody recognizes and binds to MMP-2/TIMP-2 complex that has been captured onto the MP. During the following wash step the MPs are transferred to a clean plate. Elution buffer is then added and incubated. The elution buffer dissociates the bound protein sandwich from the MP surface, releasing the labeled antibodies. These antibodies are separated during transfer to a final microplate. The plate is loaded into the Erenna System where the labeled molecules are detected and counted. The number of fluor-labeled detection antibodies counted is directly proportional to the amount of MMP-2/TIMP-2 complex present in the sample when captured. The amount of MMP-2/TIMP-2 complex in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® MMP-2/TIMP-2 Immunoassay Prototype Assay1
Lower Limit of Detection 0.8 pg/mL
Lower Limit of Quantification2 3.9 pg/mL
Upper Limit of Quantification 1000 pg/mL
Low-end CV% Range 7 - 16%
Low-end CV% Average 11%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 0.01 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery
3 based upon median [MMP-2/TIMP-2 complex] in a healthy reference


FIGURE 1. [MMP-2/TIMP-2] in EDTA plasma from 14 healthy donors, with median and interquartile range. The Erenna® MMP-2/TIMP-2 Immunoassay Evaluation Reagent Kit reliably quantifies MMP-2/TIMP-2 in healthy subjects, who have a median [MMP-2/TIMP-2] of 57 ng/mL that is well above the detection limit of 0.8 pg/mL.

FIGURE 2. Erenna® MMP-2/TIMP-2 Immunoassay Evaluation Reagent Kit low-end standard curve signal (left) and curve fit (right).

Representative data shown for demonstration purposes only. Individual results may vary depending upon samples tested and protocol used.

Biology and Disease

Interactions between Matrix Metalloproteinase-2 (MMP-2) and its endogenous tissue inhibitor molecule TIMP-2 have important effects on tumor invasion and spreading. While MMP-2 accelerates the breakdown of extracellular matrix attributing to metastasis, TIMP-2 negatively regulates this activity by forming a complex that blocks access to the MMP-2 catalytic site. The ratio between the number of activated MMP molecules and free TIMP molecules thus determines the extent of vasculature remodeling. The prognostic relevance of measuring circulating levels of MMP-2/TIMP-2 complex to the degree of risk in developing prostate cancer, malignant lymphoma, and plaque instability for cardiovascular disease is currently being examined.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: 72 kDa type IV collagenase complex with Metalloproteinase inhibitor 2
UniProtKB/Swiss-Prot ID:
Protein-protein interaction database:
Alternative Names: 72 kDa gelatinase, Gelatinase A, Matrix metalloproteinase-2, TBE-1, CSC-21K, Tissue inhibitor of metalloproteinases 2
Gene Names: MMP2, TIMP2
Synonym Gene Names: CLG4A,
Function: Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them by binding to their catalytic zinc cofactor. Known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, MMP-15, MMP-16 and MMP-19.
Biological Processes: angiogenesis, collagen catabolic process, proteolysis,
Molecular Function: metalloendopeptidase activity, protein binding, zinc ion binding, metal ion binding, metalloendopeptidase inhibitor activity

* Source Info from www.uniprot.org.


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