Research Use Only

TIMP-1 ELISA

Intended Use
The TIMP-1 ELISA is an enzyme-linked immunoassay for the quantitation of total(free and complexed) tissue inhibitor of metalloproteinase-1 (TIMP-1) in human serum and plasma. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Background
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a 28-kDa protein present in most human tissues and body fluids that inhibits matrix metalloproteinases (MMPs), stimulates cell growth, and prevents apoptosis [1,2]. Numerous studies have examined the level of TIMP-1 in serum and plasma in association with disease.

Matrix metalloproteinases degrade proteins in the extracellular matrix (ECM), playing an important role in development, morphogenesis, and wound healing [3]. Their unregulated activity is implicated in a wide variety of diseases from atherosclerosis to cancer. MMPs are regulated by binding to a family of homologous proteins known as tissue inhibitors of metalloproteinases (TIMPs) [2]. TIMPs have two domains, an N-terminal inhibitory domain and a C-terminal domain, which can influence their binding affinity [4]. TIMPs and MMPs form 1:1 enzyme inhibitor complexes. The correct balance between TIMPs and MMPs is critical for ECM metabolism.

Investigators examined the level of TIMP-1 in different cancers. Holten-Anderson, et al., reported that total levels of TIMP-1 were significantly higher in patients with colonic and rectal cancer than in healthy patients or patients with inflammatory bowel disease [5]. Pellegrini, et al., compared the serum levels of TIMP-1 to the serum levels of carcinoembryonic antigens (sCEA) in patients with colorectal cancer. They found that simultaneous measurement of the two could be used as a marker of disease progression from the pre-invasive to the invasive phase [6]. Researchers have shown that expression levels of TIMP-1 in the plasma of patients with ovarian carcinoma were found to be significantly higher than that in normal patients [7]. Jung, et al., analyzed plasma concentrations of TIMP-1 in healthy males, in patients with benign prostatic hypertrophy (BPH), and in patients with prostate cancer (PCa) with and without metastatic disease. They found that TIMP-1 concentrations were significantly higher in PCa with metastases compared with controls, BPH, and PCa patients
without metastases [8]. Susskind, et al., found elevated levels of TIMP-1 in the plasma of patients with lung and breast cancer [9].

The serum levels of TIMP-1 have been investigated as a marker of chronic liver disease. Serum
TIMP-1 levels in patients with chronic liver disease showed significant correlations with the stage of disease determined by histomorphological evaluation of liver biopsies [10]. Nie, et al., compared the levels of TIMP-1 in the liver and serum of patients with cirrhosis. Immunohistochemistry showed that TIMP-1 was present in all diseased livers and not present in normal livers. This correlated with the finding that TIMP-1 was elevated in 74% of serum samples from patients with diseased livers [10].

The levels of TIMP-1 have been evaluated in a variety of other diseases. The ratio between
metalloproteinases and their inhibitors has been investigated in association with multiple sclerosis. A high serum ratio of MMP-9/TIMP-1 is linked to the activity phase of the disease [11]. Jinnin, et al., found that the serum levels of TIMP-1 in patients with mixed connective tissue disease were significantly higher than those in healthy patients [12].

The TIMP-1 ELISA is designed to provide the investigator with a convenient, accurate, and reproducible method to determine TIMP-1 levels in human serum or plasma. Clinical research, using the TIMP-1 ELISA, will help to better define the role of TIMP-1 in cancer and other diseases.

Assay Principle
The TIMP-1 ELISA is a sandwich-type immunoassay that uses a mouse monoclonal Capture Antibody and an alkaline phosphatase conjugated mouse monoclonal antibody as detector. This combination of capture and detector reagents allows detection of free TIMP-1 and TIMP-1 complexed to metalloproteinases such as MMP-9 [13]. The Capture Antibody has been immobilized on the interior surface of the microtiter plate wells. To perform the test, an appropriate volume of specimen is incubated in the wells to allow binding of the antigen by the Capture Antibody. The immobilized antigen is then exposed to the alkaline phosphatase-labeled Detector Antibody (Conjugate). Addition of Substrate to the wells allows the catalysis of a chromogen into a colored product, the intensity of which is proportional to the amount of TIMP-1 that is bound
to the plate.

Standards are provided in the kit that allow accurate, quantitative determinations of TIMP-1
in suitable samples. Using a microtiter plate reader, one can measure simultaneously the
absorbance of the colored product in the Standards and sample wells. Correlating the absorbance values of samples with the Standards allows the investigator to determine the levels of TIMP-1 in a sample. Samples may be assigned a quantitative value of TIMP-1 in ng/mL of serum or plasma.

Selected References

1. Opbroek A, Kenney MC, Brown D. Characterization of a human corneal metalloproteinase inhibitor (TIMP-1). Current Eye Research 1993;12(10):877-883.
2. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: Evolution, structure and function. Biochemica at Bioysica Acta 2000(1477):267-283.
3. Stamenkovic, I. Extracellular matrix remodelling: The role of matrix metalloproteinases. Journal of Pathology 2003;200:448-664.
4. Nagase H, Brew K. Engineering of tissue inhibitor of metalloproteinases mutants as potential therapeutics. Arthritis and Research Therapy 2003;Supplement 3:51-61.
5. Holten-Andersen MN, Christensen IJ, Nielsen HJ, et al. Total levels of tissue inhibitor of metalloproteinases 1 in plasma yield high diagnostic sensitivity and specificity in patients with colon cancer. Clinical Cancer Research 2002;8(1):156-164.
6. Pellegrini P, Contasta I, Berghella AM, et al. Simultaneous measurement of soluble carcinoembryonic antigen and the tissue inhibitor of metalloproteinase TIMP-1 serum levels for use as markers of pre-invasive to invasive colorectal cancer. Cancer Immunology Immunotherapy 2000;49(7):388-394.
7. Manenti L, Paganoni P, Floriani I, et al. Expression levels of vascular endothelial growth factor, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 in the plasma of patients with ovarian carcinoma. European Journal of Cancer 2003;(39):1948-1956.
8. Jung K, Nowak L, Lein M, et al. Matrix metalloproteinases 1 and 3, TIMP-1 and the complex of metalloproteinase-1/tissue inhibitor in plasma of patients with prostate cancer. International Journal of Cancer 1997(74):220-223.
9. Susskind H, Hymowitz M, Hong Lau Y, et al. ncreased plasma levels of MMP-9 and TIMP-1 in lung and breast cancer are altered during chest radiotherapy. International Journal of Radiation Oncology, Biology, Physics 2003;56(4):1161-1169.
10. Nie Q, Zhou Y, Xie Y. Expression and significance of tissue inhibitors of metalloproteinases-1 and -2 in serum and liver of patients with cirrhosis. Zhonghua Yi Xue Za Zhi 2001;(81):805-807.
11. Waubant E, Goodkin MD, Gee L, et al. Serum MMP-9 and TIMP-1 levels are related to MRI activity in relapsing multiple sclerosis. Neurology 1999;(53):1397.
12. Jinnin M, Ihn H, Yamane K, et al. Serum levels of tissue inhibitors of metalloproteinases in patients with mixed connective tissue disease. Clinical Experiments in Rheumatology 2002;20(4):539-542.
13. Holten-Andersen MN, Brunner N, Maimonis P, Jensen V, Murphy G, and Piironen T. Characterization of monoclonal antibodies to tissue inhibitor of metalloproteinases-1. Journal of Clinical Ligand Society 2002;25(1):87-90.