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Immunoassays | Prototype

Erenna® MMP-2 Immunoassay Prototype Assay (Cat# 03-0044-AA)

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Introduction

The Erenna® MMP-2 Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure MMP-2 in plasma and serum samples. A capture antibody specific for MMP-2 has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the MMP-2 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 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 present in the sample when captured. The amount of MMP-2 in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® MMP-2 Immunoassay Prototype Assay1
Lower Limit of Detection 0.4 pg/mL
Lower Limit of Quantification2 7.8 pg/mL
Upper Limit of Quantification 1000 pg/mL
Low-end CV% Range 2 - 9%
Low-end CV% Average 6%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 0.1 μL

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


FIGURE 1. [MMP-2] in EDTA plasma from 14 healthy donors, with median and interquartile range. The Erenna® MMP-2 Immunoassay Evaluation Reagent Kit reliably quantifies MMP-2 in healthy subjects, who have median [MMP-2] of 30 ng/mL that is well above the detection limit of 0.4 pg/mL.

FIGURE 2. Erenna® MMP-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

Matrix Metalloproteinase-2 (MMP-2) is part of a family of zinc and calcium-dependent endopeptidases that are induced by cytokines such as IL-1β, TNF-α and EGF. As with most MMPs, MMP-2 is secreted in the proenzymatic form and must be cleaved for activation. Active MMP-2 is responsible for the breakdown of the extracellular matrix, and is involved in tissue remodeling under normal physiological and diseased conditions. Because of its role in tumor infiltration and progression, cancer cells that produce MMP-2 have been shown to have an increased potency toward metastasis. Thus, molecules with MMP-2 suppressing activity, such as the MMP-2 tissue inhibitor TIMP-2, are investigated for their ability to combat epithelial cancer.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: 72 kDa type IV collagenase
UniProtKB/Swiss-Prot ID: P08253
Protein-protein interaction database: P08253
Alternative Names: 72 kDa gelatinase, Gelatinase A, Matrix metalloproteinase-2, TBE-1
Gene Names: MMP2
Synonym Gene Names: CLG4A
Function: Ubiquitinous metalloproteinase that is involved in diverse functions such as remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. As well as degrading extracellular matrix proteins, can also act on several nonmatrix proteins such as big endothelial 1 and beta-type CGRP promoting vasoconstriction. Also cleaves KISS at a Gly-|-Leu bond. Appears to have a role in myocardial cell death pathways. Contributes to myocardial oxidative stress by regulating the activity of GSK3beta. Cleaves GSK3beta in vitro.
Biological Processes: angiogenesis, collagen catabolic process, proteolysis
Molecular Function: metalloendopeptidase activity, protein binding, zinc ion binding

* Source Info from www.uniprot.org.


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