Mouse Clusterin Quantikine ELISA Kit Summary

Product Summary

The Quantikine Mouse Clusterin Immunoassay is a 4.5 hour solid phase ELISA designed to measure mouse Clusterin levels in cell culture supernates, cell lysates, serum, plasma, and urine. It contains NS0-expressed recombinant mouse Clusterin and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant factor accurately. Results obtained using natural mouse Clusterin showed dose-response curves that were parallel to the standard curves obtained using the recombinant kit standards. These results indicate that this kit can be used to determine relative mass values for natural mouse Clusterin.

Preparation and Storage

Background: Clusterin

Clusterin, also known as Apolipoprotein J, Sulfated Glycoprotein 2 (SGP-2), TRPM-2, and SP-40, is a secreted multifunctional protein that was named for its ability to induce cellular clustering. It binds a wide range of molecules and may function as a chaperone of misfolded extracellular proteins. It also participates in the control of cell proliferation, apoptosis, and carcinogenesis (1, 2). Clusterin is expressed in adult testis, ovary, adrenal gland, liver, heart, brain, and in many epithelial tissues during embryonic development (3). Mouse Clusterin is synthesized as a precursor that contains two coiled coil domains, two nuclear localization signals (NLS), and one heparin binding domain (4-6). Intracellular cleavages of the precursor remove the signal peptide and generate comparably sized alpha and  beta chains which are secreted as an approximately 80 kDa N-glycosylated and disulfide-linked heterodimer (7-9). Mature mouse Clusterin shares 77% and 93% amino acid sequence identity with human and rat Clusterin, respectively. 

An alternately spliced 50 kDa isoform of mouse Clusterin (nCLU) remains intracellular and is neither glycosylated nor cleaved into alpha and beta chains (4). Cellular exposure to ionizing radiation promotes the translocation of nCLU to the nucleus where it interacts with Ku70 and promotes apoptosis (4, 10). This function contrasts with the cytoprotective effect of secreted Clusterin (11). High μg/mL concentrations of Clusterin circulate predominantly as a component of high density lipoprotein particles, and these are internalized and degraded through interactions with LRP-2/Megalin (12, 13). The ability of Clusterin to bind and neutralize non-oxidatively modified LDL reduces cytotoxicity in atherosclerotic plaques (14). The chaperone function of Clusterin also helps to reduce the accumulation of beta -amyloid fibrils and damage due to amyloid plaques in Alzheimer's disease (15). Clusterin levels are elevated in the cerebrospinal fluid of patients with Alzheimer's disease, Parkinson's disease, and multiple sclerosis (9, 16, 17) and in the urine of patients with kidney injury or bladder cancer (18-20). Clusterin is released by activated platelets at sites of vascular injury and is found in the synovial fluid of rheumatoid arthritis and osteoarthritis patients (21, 22). During human tumor progression, nCLU is downregulated while the secreted form is upregulated and may be aberrantly glycosylated (10, 23-25). Increased circulating levels of Clusterin enhance tumor aggressiveness by inhibiting apoptosis and by promoting epithelial to mesenchymal transition (26-28).

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.