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Immunoassays | Prototype

Erenna® TIMP-2 Immunoassay Prototype Assay (Cat# 03-0046-AA)

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Introduction

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

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® TIMP-2 Immunoassay Prototype Assay1
Lower Limit of Detection 0.01 pg/mL
Lower Limit of Quantification2 0.6 pg/mL
Upper Limit of Quantification 312 pg/mL
Low-end CV% Range 5 - 8%
Low-end CV% Average 6%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 0.001 μL

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


FIGURE 1. [TIMP-2] in EDTA plasma from 14 healthy donors, with median and interquartile range. The Erenna® TIMP-2 Immunoassay Evaluation Reagent Kit reliably quantifies TIMP-2 in healthy subjects, who have a median [TIMP-2] of 107 ng/mL that is well above the detection limit of 0.01 pg/mL.

FIGURE 2. Erenna® 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

Tissue Inhibitor Metalloproteinase 2 (TIMP-2) is an important member of the TIMP protein family, which suppresses tissue remodeling of the extracellular matrix (ECM) contributed by matrix metalloproteinases (MMPs). Since the breakdown of ECM leads to tumor progression, TIMPs have received attention for their tumor suppressing abilities and are thought to be negative regulators of metastasis. Out of the four members in the mammalian TIMP family, TIMP-2 is a distinctive member in that it can also suppress angiogenesis via MMP-independent mechanisms. Together, the TIMPs are important regulators for cell growth, apoptosis and differentiation, and they participate in pathological development of several chronic diseases such as psoriasis, rheumatoid arthritis and cancer.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Metalloproteinase inhibitor 2
UniProtKB/Swiss-Prot ID: P16035
Protein-protein interaction database: P16035
Alternative Names: CSC-21K, Tissue inhibitor of metalloproteinases 2
Gene Names: TIMP2
Synonym Gene Names:
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:
Molecular Function: metal ion binding, metalloendopeptidase inhibitor activity

* Source Info from www.uniprot.org.


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