Active and acid-activatable TGF-beta in human sera, platelets and plasma.
Assays which measure active and latent forms of transforming growth factor beta (TGF-beta) separately in human serum and plasma are required to investigate the biological role of TGF-beta in a variety of human diseases. We have developed an enzyme-linked immunosorbent assay (ELISA) using two polyclonal antibodies against TGF-beta which rapidly determines the amount of active plus acid-activatable, latent TGF-beta forms ((a+l)TGF-beta) present in human serum and plasma in the range 4 pmol/l to 2000 pmol/l. To measure active TGF-beta alone, we have developed a second ELISA using the extracellular domain of the TGF-beta type II receptor as the capture reagent which detects active TGF-beta in serum and plasma samples in the range 20 pmol/l to 4000 pmol/l. Both assays detect TGF-beta 1 and TGF-beta 3 with similar sensitivity, are > 10-fold less sensitive to TGF-beta 2 and are not affected by a range of other peptide growth factors. The mean (a+l)TGF-beta present in human serum was 330 pmol/l but the range was very large (< 4 pmol/l to 1400 pmol/l). The mean active TGF-beta present was 230 pmol/l (range < 20 pmol/l to 1400 pmol/l) and the proportion of the (a+l)TGF-beta present which was active [a/(a+l)] varied from < 10% to 100%. The concentration of (a+l)TGF-beta and the proportion of TGF-beta which was active were very similar in the serum and platelet-poor plasma prepared from the same whole blood sample. The clot formed during serum preparation retained all of the TGF-beta which was detected by the (a+l)TGF-beta ELISA in the corresponding platelet releasate, although the PDGF in platelets was released into the serum. In contrast, platelet-poor plasma contained no detectable PDGF demonstrating that the (a+l)TGF-beta assayed in the plasma was not due to platelet degranulation after bleeding. Serum active TGF-beta and (a+l)TGF-beta concentrations therefore provide a reliable estimate of these forms of TGF-beta present in plasma.
Grainger
D J
DJ
Department of Biochemistry, University of Cambridge, UK.
Mosedale
D E
DE
Metcalfe
J C
JC
Weissberg
P L
PL
Kemp
P R
PR
D000328:Adult; D001483:Base Sequence; D001792:Blood Platelets / Q000737:chemistry*; D001798:Blood Proteins / Q000378:metabolism; D003429:Cross Reactions; D017077:Culture Media, Conditioned; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000032:analysis; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D016212:Transforming Growth Factor beta / Q000097:blood*
D001798:Blood Proteins; D017077:Culture Media, Conditioned; D010982:Platelet-Derived Growth Factor; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta