Palabras clave: proteínas de unión TGF-β latente, ELISA.

The transforming growth factor-β (TGF-β) family of three related mammalian peptides exerts a multitude of effects on most cell types (1-3). Of these, the TGF-β1 isoform is the one most closely associated with immune modulation (4). The numerous biological functions of all TGF-β's require a set of post-translational modifications termed "activation." The bioactive forms of the TGF-β's are 25 kDa homodimers produced from 50 kDa monomers that dimerize to form the ca. 100 kDa TGF-β precursor. This dimeric precursor is cleaved intracellularly by furin proteases to yield the 25 kDa active TGF-β dimer, which remains associated with the remaining portion of its own pro-form, the latency-associated peptide (LAP, ca. 75 kDa). This complex is termed "latent TGF-β,"and is secreted in this form. Other proteins, such as latent TGF-β binding proteins (LTBP, which targets TGF-β's to the extracellular matrix) or α2 macroglobulin (which is associated with circulating TGF-β1) can bind to this complex, creating the so-called large latent complex (5). Latent TGF-β is activated by a process that involves dissociation and degradation of LAP by proteins (e.g. plasmin and transglutaminase), heat, chaotropic agents, acid, and oxygen and nitrogen free radicals (1;5-8). This post-translational control of TGF-β1 through activation is arguably the most potent regulatory mechanism for this cytokine (5). Once activated, TGF-β1 binds to its signaling receptor complex (type I, type II, and type III in concert) (9).

A common and commercially-available method for the detection of TGF-β1 consists of an enzyme-linked immunosorbent assay (ELISA), which detects active TGF-β1 (or total TGF-β1 after activation by transient acidification or treatment with urea (10). In our own previous studies on the activation of latent TGF-β1 by nitrogen free radicals, we had used heat as the positive control for TGF-β1 activation (8,11). This assay is based on the binding of active TGF-β1 by the immobilized specific monoclonal antibody that been pre-coated onto a microplate. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TGF-β1 is added to the wells to sandwich the TGF-β1 immobilized during the first incubation. 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 TGF-β1 bound in the initial step. Because of its simplicity, specificity and sensitivity, this method is the best option when working on a cell-free system.

As mentioned above, latent TGF-β1 can be activated by heat, acidification, alkalinization or action of chaotropic agents in vitro (5), but some discrepancies can be found in the literature regarding this process. Using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model and radioreceptor and soft agar assays for monitoring activation, it was suggested that conditioned medium may contain at least two different pools of latent TGF-β1: one pool resistant to mild acid and/or plasmin that requires strong acid or alkali treatment for activation, and a second pool activated by mild pH change and/or plasmin (12). Similarly, incubations of the latent form of TGF-β1 at extreme pH values, in 0.02% SDS or in 8 M urea, lead to activation of TGF-β1, whereas the complex was resistant to treatment with 5 M NaCl or heat (3 min at 95ºC) (13). In contrast, others have reported that thermal activation of native and recombinant latent TGF-β1 occurs over the temperature ranges of 75-100 ºC and 65-100 ºC, respectively, with complete activation after 5 min at 80ºC. Temperatures above 90 ºC result in thermal denaturation of TGF-β1 itself (14).

The present study was performed to compare the effectiveness of acidification vs. heat treatment in activating latent TGF-β1 in a cell-free system. The ELISA assay was carried out using recombinant human latent TGF-β1 and the Quantikine® mouse/rat/porcine TGF-β1 Immunoassay Kit, both from R&D Systems, Inc. (Minneapolis, MN). The samples (5000 pg/ml latent TGF-β1 in PBS) were activated by treatment with 1N HCl and then neutralized by 1.2 N NaOH/0.5 mol/L HEPES according to the instructions of the manufacturer, or by incubation at 80ºC for 5 min in a water bath. The levels of active TGF-β1 in the samples were calculated by interpolation from the standard curve as per the kit instructions. We found that while both heat and HCl lead to activation of latent TGF-β1 (Figure 1), heat treatment was significantly more effective than acidification (15% vs. nearly 10%, respectively). Interestingly, neither treatment induced full activation. We cannot explain this observation, but it should be noted that R&D Systems is the only commercial supplier of recombinant latent TGF-β1 available, and this company's instructions for the use of Quantikine® Human TGF-β1 Immunoassay (Cat. No. DB100) report 15% cross-reactivity with recombinant human latent TGF-β1.

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