Intended Use
The Human TGF-beta 1 Immunoassay is a 4.5 hour solid phase ELISA designed to measure TGF-beta 1 in acid activated cell culture supernates, serum, plasma, and urine. It contains recombinant human TGF-beta 1 expressed by CHO cells and has been shown to quantitate the recombinant factor accurately. Results obtained using natural TGF-beta 1 showed linear curves that were parallel to the standard curves obtained using the recombinant kit standards. These results indicate that this kit will provide accurate quantitation for both recombinant and natural human TGF-beta 1.
Contents of Kit
1. TGF-β1 Microplate: 1 plate
96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody specific for TGF-β1.
Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of zipseal. May be stored for up to 1 month at 2-8 °C.*
2. TGF-β1 Standard: 2 vials
Recombinant human TGF-β1 in a buffered protein base with preservatives; lyophilized. Refer to the vial label for reconstitution volume.
Discard after use. Use a new Standard for each assay.
3. TGF-β1 Conjugate: 1 vial, 12.5 mL/vial of a polyclonal antibody specific for TGF-β1 conjugated to horseradish peroxidase with preservatives.
4. Assay Diluent CD2-32: 1 vial, 12.5 mL/vial of a buffered protein base with preservatives. For cell culture supernate/urine samples.
5. Assay Diluent CD2-84: 1 vial, 12.5 mL/vial of a buffered protein base with preservatives. For serum/plasma samples
6. Calibrator Diluent CD6-64 Concentrate: 1 vial, 21 mL/vial of a concentrated buffered animal serum with preservatives. May contain a precipitate. Mix well before and during use. Use diluted 1:4 in this assay.
7. Wash Buffer Concentrate: 1 vial, 21 mL/vial of a 25-fold concentrated solution of buffered surfactant with preservatives. May turn yellow over time.
8. Color Reagent A: 1 vial, 12 mL/vial of stabilized hydrogen peroxide.
9. Color Reagent B: 1 vial, 12 mL/vial of stabilized chromogen (tetramethylbenzidine).
10. Stop Solution: 1 vial, 23 mL/vial of a diluted hydrochloric acid solution.
May be stored for up to 1 month at 2-8 °C.*
11. Plate Sealers: 4 strips, Adhesive strips.
* Provided this is within the expiration date of the kit.
General Description
Human TGF-β1 is a 25 kDa, disulfide-linked homodimeric protein involved in a number of key developmental, immunologic, and homeostatic processes (1-4). The molecule is synthesized as a 390 amino acid (aa) precursor that contains a 23 aa signal sequence, a 255 aa pro-region, and a 112 aa mature segment. Processing of the molecule is complex, and it is generally secreted as a latent form (5). Prior to release, the prepro-form is cleaved of its signal sequence, followed by glycosylation of its pro-region. The glycosylation process includes the unusual attachment of mannose-6 phosphate residues. This is followed by furin convertase-mediated cleavage of the prohormone, creating an 80 kDa disulfide-linked proregion (termed LAP for latency-associated protein), plus a 25 kDa disulfidelinked mature segment (termed TGF-β1) (6-8). These two independent disulfide-linked polypeptides associate in a non-covalent interaction that renders TGF-β1 inactive. Although direct secretion of this 80K:25K complex can occur, it does so inefficiently. To facilitate secretion plus extracellular storage, a third 200 kDa component termed LTBP is covalently-linked to the N-terminus of one of the two LAP polypeptide chains. This promotes secretion and subsequent storage within the extracellular matrix (9, 10). After secretion, TGF-β1, via LTBP, covalently links to ECM. This complex is later cleaved by proteases and released, exposing mannose residues on LAP. It is postulated that exposed LAP mannose residues now are able to bind to cell surface IGF-II R, where dissociative events disrupt the LAP-TGF-β1 complex. This results in the release of active, homodimeric TGF-β1 (7, 10). Mature mouse TGF-β1 shares 100% aa sequence identity with rat and cotton rat TGF-β1 (11, 12), 99% aa identity with human, canine, and porcine TGF-β1 (13, 14, 15), and 97% aa identity with guinea pig TGF-β1 (16). Relative to mouse TGF-β2 and β3, mature mouse TGF-β1 shares 72% and 78% aa sequence identity, respectively (17, 18). The traditional high-affinity receptor for TGF-β1 is a heteromeric complex consisting of transmembrane serine/threonine kinases. Two types are involved; a constitutively phosphorylated, ligand-binding 80 kDa glycoprotein termed TβRII and a signal transducing, non-ligand-binding 55 kDa glycoprotein termed TβRI/ALK-5 (19-22). It is suggested that TGF-β1first binds TβRII, which then initiates a crossphosphorylation of TβRI, culminating in signal transduction. There is also a third TGF-β receptor termed TβRIII, which can be either the 250 kDa proteoglycan named betaglycan, or the 180 kDa glycoprotein termed endoglin/CD105 (23, 24). It has been proposed that TβRIII captures TGF-β and "passes" it to TβRII (20). This is perhaps true for betaglycan but not endoglin. Endoglin does not bind TGF-β by itself; only within the context of TβRII ligand binding. Evidence suggests that rather than "passing" on ligand, endoglin may actually enter the receptor complex and modulate TGF-β downstream signaling (25, 26).
Finally, and although ALK-5 has traditionally been assumed to be the only type I signaling receptor for TGF-β1, it is also possible that ALK-1 may serve as a condition-dependent, type I TGF-β receptor (27). TGF-β1 has a wide range of activities. During an immune response, TGF-β1 impacts antibody production by preferentially induceing IgA production in both mouse and human (28). It also regulates dendritic cell chemotaxis by altering the expression of chemokine receptors (29). Finally, it can downmodulate an inflammatory response by dampening macrophage activity and proinflammatory secretion (30). During wound healing, TGF-β1 is released from activated platelets. This local source of TGF-β1 has marked stimulatory effects on fibroblasts, where it induces matrix synthesis; on monocytes, where it induces proinflammatory mediator and growth factor secretion; and on keratinocytes, where it may promote keratinocyte proliferation by downmodulating its own signaling pathway (31). Finally, during development, TGF-β1 may play a role in endochondral ossification, and its absence results in severely defective yolk sac vasculogenesis and hematopoiesis (32, 33).
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