Human Total MMP-3 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human Total MMP-3 Immunoassay is a 4.5 hour solid phase immunoassay designed to measure total MMP-3 (pro- and active MMP-3) in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant human Pro-MMP-3 and antibodies raised against the recombinant factor. Both antibodies also recognize recombinant human active MMP-3. Natural human MMP-3 showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards, indicating that this kit can be used to determine relative levels of natural human MMP-3.

Preparation and Storage

Background: MMP-3

Matrix metalloproteinases (MMPs), also called matrixins, constitute a family of zinc and calcium dependent endopeptidases that function in the breakdown of extracellular matrix (ECM). They play an important role in many normal physiological processes such as embryonic development, morphogenesis, reproduction and tissue remodeling (1). They also participate in many pathological processes such as arthritis, cancer and cardiovascular disease (2). While the amounts of newly synthesized MMPs are regulated mainly at the levels of transcription, the proteolytic activities of existing MMPs are controlled through both the activation of proenzymes or zymogens and the inhibition of active enzymes by endogenous inhibitors, alpha -macroglobulins and tissue inhibitors of metalloproteinases (TIMPs). 

MMP-3 (also referred to as stromelysin-1) may be expressed in fibroblasts, chondrocytes, endothelial cells, macrophages, vascular smooth muscle cells, osteoblasts, and keratinocytes in response to appropriate stimuli (3). Various agents regulate its biosynthesis. Inflammatory cytokines such as IL-1 and TNF-alpha, epidermal growth factor, platelet-derived growth factor, phorbol and oncogenic cellular transformation are the inductive agents. In comparison, retinoic acid, glucocorticoids, estrogen, progesterone and TGF-beta suppress MMP-3 synthesis. 

MMP-3 is secreted from the cells as a proenzyme. The proenzyme has been shown to stimulate plasminogen activation (4). The N-terminal pro-domain contains the cysteine switch motif conserved in MMPs that maintains MMP-3 in the latent state (5). Activation of the proenzyme results in the removal of the pro-domain. MMP-3 activation can be achieved in vitro by proteases such as itself, chyrotrypsin, neutrophil elastase and plasma kallikrein, and by mercury compounds (3). The resulting active enzyme consists of a catalytic domain with a zinc-binding motif conserved in metzincins (6,7). A short hinge peptide links the catalytic domain to the C-terminal hemopexin-like domain. The active MMP-3 is capable of cleaving types III, IV, IX and X collagen, aggrecan, fibronectin, laminin, IGFBP-3, serpins, and IL-1 beta. The active enzyme also activates proMMP-1, -8, -9, and -13. Therefore, it is suggested that MMP-3 may participate in physiological matrix turnover and pathological destruction of the tissue. For example, MMP-3 is required for the generation of a macrophage chemoattractant in a model of herniated disc resorption (8).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.