Human TGF-beta 2 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human TGF-beta 2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure activated TGF-beta 2 in cell culture supernates, serum, and plasma. The Quantikine Human TGF-beta 2 Immunoassay contains CHO cell-derived recombinant human TGF-beta 2 and antibodies raised against the recombinant protein. The assay has been shown to quantitate the recombinant factor accurately. Results obtained using human TGF-beta 2 showed linear curves that were parallel to the standard curves obtained using the recombinant TGF-beta 2 kit standards. These results indicate that this kit can be used to determine relative mass values for natural human TGF-beta 2.

Preparation and Storage

Background: TGF-beta 2

TGF-beta 2 is synthesized as a prepro-cytokine with a 19 amino acid (aa) signal sequence, a 283 aa pro-region, and a 112 aa mature segment (1-5). It dimerizes with formation of disulfide bonds between the 'pro' regions and disulfide bonds between the 'mature' regions. The mature region is 71% and 80% identical with human TGF-beta 1 and TGF-beta 3 (6, 7) and 97% identical with the corresponding mouse protein (8). After proteolytic cleavage of the disulfide-linked mature region, it remains hydrogen-bonded to the disulfide-linked prosegments (LAP or latencyassociated protein) (1, 2, 9). If secreted in this form, LAP keeps TGF-beta 2 in an inactive state until dissociation, caused by proteases, glycosidases, or extreme pH (2, 9). In many types of cells, an additional protein, latent TGF-beta binding protein (LTBP), is covalently linked to the LAP homodimer prior to secretion. LTBP, a 130 kDa cysteine-rich glycoprotein, creates a 235 kDa large latent complex that is secreted, most likely binding to the extracellular matrix (1, 9-11). The latency components are believed to act as natural antagonists of TGF-beta activity, to target TGF-beta to distinct tissues, and to maintain a reservoir of TGF-beta (1, 2, 12). On release from latency, active homodimeric TGF-beta can bind to cell-surface receptors or to other proteins, such as alpha ₂-macroglobulin (13). 

The signal transducing receptor for TGF-beta 2 is a heterotetrameric complex of two type I signal-transducing receptors (53 kDa; TGF-beta RI) and two type II ligand-binding receptors (75-85 kDa; TGF-beta RII) (14-19). The binding of TGF-beta 2 appears to initially involve a type III TGF-beta receptor, either 300 kDa betaglycan (20) or 180 kDa endoglin (14, 21), which then "hands off" to TGF-beta RII. 

TGF-beta 2 is expressed by a variety of cells, including osteoclasts, thymic epithelium, keratinocytes, hepatocytes, chief cells of the stomach, satellite cells, skeletal muscle cells, prostatic epithelium, bronchial epithelium, neurons and astrocytes, fibroblasts and visceral smooth muscle, and macrophages (14-19). TGF-beta 2 has marked cross-species bioactivity (e.g., human TGF-beta 2 is active on mouse cells (33), while porcine TGF-beta 2 is active on rabbit cells (34)). TGF-beta 2 has four fundamental activities: it is a growth inhibitor for most types of cells; it enhances the deposition of extracellular matrix; it is immunosuppressive, suppressing APC expression of both IL-12 and CD40L while upregulating IL-10 secretion; and during fetal development, it is expressed in discrete areas, such as epithelium, myocardium, cartilage and bone of extremities and in the nervous system, suggesting specific functions (1, 35-37)

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.