ADAM12, BioAssay™ ELISA Kit
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Human ADAM12 (a disintegrin and metalloproteinase domain-containing protein 12) belongs to the ADAM metalloproteinase family. It is a single polypeptide chain composed of multiple domains, including the signal peptide, propeptide, metalloproteinase, disintegrin, cystein-rich, and EGF-like domains, as well as the transmembrane segment and cytoplasmic tail. Human ADAM12 has two alternatively transcribed forms: a membrane-anchored long form of 110kD and a 90kD shorter secreted form lacking the transmembrane domain and the cytoplasmic tail (1-3). ADAM12 is initially synthesized as an inactive enzyme. Its latency requires the coordination of a cysteine residue in the pro domain with the Zn2+ in the active site of the catalytic domain. It is then activated by a furin-like protease in the trans-Golgi apparatus through cleavage of the pro domain before it is transported to the cell surface and secreted as the active form (4, 5). Upon activation, the pro domain remains non-covalently attached to the mature enzyme (6). Endogenous inhibitors for ADAM12 include TIMP-3 and a2-macroglobulin (7). The soluble form of ADAM12 is detectable in various body fluids, such as serum and urine (8-10). Like other metalloproteinases, ADAM12 possesses gelatinase activity. It can cleave extracellular matrix proteins, such as gelatin, type IV collagen, and fibronectin (10). Thus, ADAM12 may play important roles in cell adhesion and extracellular matrix degradation. Over-expression of ADAM12 has been observed in various types of cancer. In mouse prostate cancer, ADAM12 is highly expressed in the carcinoma-associated stroma, and it is required for tumor progression (11). In a mouse breast cancer model, ADAM12 accelerates tumor progression through increasing the apoptotic sensitivity of normal stromal cells while rendering tumor cells more resistant to apoptosis (12, 13). Additionally, it has been reported that in breast cancer patients, ADAM12 has significantly increased amounts in the urine compared to healthy controls and higher levels of urinary ADAM12 seem to correlate with more aggressive phenotypes and more advanced stages (10).
ADAM12 may also participate in cell signaling. It has been shown that it can facilitate the phosphorylation of Smad2 and the stabilization of the TGF-b receptor type II (14). It is likely that ADAM12 contributes to TGF-b signaling via a mechanism independent of its protease activity. Additionally, the cytoplasmic tail of ADAM12 can directly interact with a number of cell signaling molecules, such as a-actinin, c-Src, and PI-3 kinase (15-17). Substrates for ADAM12 also include IGFBP-3 and IGFBP-5 (8). ADAM12 is abundantly expressed in the placenta. During pregnancy, its serum levels steadily increase, which correlate with the elevation of IGFBP-3 proteolysis. Thus, it has been postulated that ADAM12 may be the enzyme that is responsible for the cleavage of IGFBP-3 during pregnancy (8). Abnormal ADAM12 serum levels in pregnant women are associated with a number of neonatal and pregnancy-related disorders, such as Down's syndrome, chromosome 18 trisomy, intra-uterine fetal growth restriction, and preeclampsia (9, 18-21).
The Human ADAM12 immunoassay is a 4.5 hour solid phase ELISA designed to measure human ADAM12 in cell culture supernates, serum, plasma, and urine. It contains CHO cell-expressed recombinant human ADAM12 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural ADAM12 showed linear curves that were parallel to the standard curves obtained using the kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring ADAM12.
Serum/Plasma/Urine-Samples from apparently healthy volunteers were evaluated for the presence of ADAM12 in this assay. No medical histories were available for the donors used in this study.
Cell Culture Supernates-U-87 MG human glioblastoma/astrocytoma cells were cultured in MEM media supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100u/ml penicillin, 1 mM sodium pyruvate, and 100mg/ml streptomycin sulfate until confluent. An aliquot of the cell culture supernate was removed, assayed for levels of natural human ADAM12, and measured 0.164ng/ml.
Twenty assays were evaluated and the minimum detectable dose (MDD) of ADAM12 ranged from 0.016-0.076ng/ml. The mean MDD was 0.030ng/ml.
The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Recognizes recombinant and natural human ADAM12. The factors listed below were prepared at 100ng/ml in Calibrator Diluent and assayed for cross-reactivity. Preparations of the following factors at 100ng/ml in a mid-range recombinant human ADAM12 control were assayed for interference. No significant cross-reactivity or interference was observed.
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human ADAM12 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any ADAM12 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for human ADAM12 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of ADAM12 bound in the initial step. The color development is stopped and the intensity of the color is measured.
Mean (ng/ml): 1.39, 3.57, 6.84
Standard Deviation: 0.057, 0.131, 0.235
CV (%): 4.1, 3.7, 3.4
Mean (ng/ml): 1.43, 3.62, 6.74
Standard Deviation: 0.079, 0.145, 0.299
CV (%): 5.5, 4.0, 4.4
ADAM12 Microplate 1x96 well polystyrene microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against human ADAM12.
ADAM12 Conjugate 1x21ml of a monoclonal antibody against human ADAM12 conjugated to horseradish peroxidase with preservatives.
ADAM12 Standard 3x(100 ng/vial) of recombinant human ADAM12 in a buffered protein solution with preservatives; lyophilized.
Assay Diluent RD1X 1x12ml of a buffered protein solution with preservatives. Calibrator Diluent RD5-26 Concentrate 1x21ml of a buffered protein solution with preservatives.
Wash Buffer Concentrate 1x21ml of a 25-fold concentrated solution of a buffered surfactant with preservatives.
Color Reagent A 1x12.5ml of stabilized hydrogen peroxide.
Color Reagent B 1x12.5ml of stabilized chromogen (tetramethylbenzidine).
Stop Solution 1x6ml of a 2 N sulfuric acid.
Storage and Stability:
Store components at -20°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.