Mouse/Rat/Canine/Porcine TGF-beta 2 Quantikine ELISA Kit Summary

Product Summary

The Quantikine® Mouse/Rat/Canine/Porcine TGF-β2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure TGF-β2 in mouse, rat, canine, or porcine cell culture supernates, cell lysates, serum, and plasma. It contains CHO cell-expressed recombinant mouse mature TGF-β2 and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant mouse TGF-β2. Results obtained using natural mouse, rat, canine, or porcine TGF-β2 showed dose response curves that were parallel to the standard curves obtained using the Quantikine® kit standards. These results indicate that this kit can be used to determine relative mass values for natural mouse/rat/canine/porcine TGF-β2.

Preparation and Storage

Background: TGF-beta 2

TGF-beta 2 (Transforming Growth Factor beta 2) is one of three closely related mammalian members of the large TGF-beta superfamily that share a characteristic cysteine knot structure (1-7). TGF-beta 1, 2 and 3 are encoded by separate genes, but are often called isoforms. They are highly pleiotropic cytokines that are proposed to act as cellular switches regulating processes such as immune function, proliferation and epithelial-mesenchymal transition (4-8). Mammalian TGF-beta 2 is secreted as a 395 amino acid (aa) proprotein that is processed by a furin-like convertase to generate an N-terminal latency-associated peptide (LAP, ~232 aa) and a C-terminal mature TGF-beta 2 (~112 aa) that remain associated via hydrogen bonding (9-13). Serine proteases such as plasmin, matrix metalloproteases, or thrombospondin-1, along with cofactors such as certain integrins, can dissociate LAP and release active TGF-beta 2 (11-14). In many types of cells, latent TGF-beta binding protein (LTBP) is covalently linked to the LAP homodimer prior to secretion. LTBP creates a large latent complex that is secreted, but may bind and localize to the extracellular matrix (10, 11). For TGF-beta isoforms, the latency components act as natural antagonists of TGF-beta activity, target TGF-beta to distinct tissues, and maintain a reservoir of TGF-beta (1). Mature mouse and rat TGF-beta 2 share 100% aa sequence identity, and share 97% aa identity with human, porcine, canine, equine and bovine TGF-beta 2. 

TGF-beta 2 signaling begins with binding to a complex of the accessory receptor betaglycan (also known as TGF-beta RIII) and the TGF-beta RII type II ser/thr kinase receptor (15). In contrast, TGF-beta 1 and 3 have higher affinity for TGF-beta RII and do not require betaglycan (15). TGF-beta RII then phosphorylates and activates another ser/thr kinase receptor, TGF-beta RI (also called activin receptor-like kinase (ALK) -5), or alternatively, ALK-1. The whole complex phosphorylates and activates Smad proteins that regulate transcription (1, 6, 15, 16). Differences in structure of the prodomains and mature sequences of TGF-beta isoforms, and use of Smad-independent signaling pathways, allow for disparate actions observed in response to different TGF-beta isoforms and contexts (1-3, 12, 13, 15, 16). 

Although many functions are overlapping, each TGF-beta isoform has some non-redundant functions. TGF-beta 2 plays a non-redundant role in human and mouse developmental heart valve remodeling, and mice with targeted deletions of TGF-beta 2 show defects in development of the cardiac system as well as lung, craniofacial, limb, eye, ear and urogenital systems (2, 7, 17, 28). TGF-beta 2 plays a unique positive regulatory role in hematopoiesis by enhancing Flt-3 signaling in hematopoietic progenitors (19). In humans, TGF-beta isoforms, especially TGF-beta 2, are identified as key factors in the progression of malignant glioma, gastric and ovarian cancer (8, 20-22). TGF-beta isoforms, particularly TGF-beta 2, suppress macrophage cytokine production and mucosal inflammatory responses in the developing intestine (23). In turn, macrophage LRP-1 can downregulate expression of TGF-beta 2 during vascular remodeling, which suppresses neo-intima formation (24).

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.