Grainger D J, Mosedale D E, Metcalfe J C, Weissberg P L, Kemp P R
Department of Biochemistry, University of Cambridge, UK.
Clin Chim Acta. 1995 Feb 28;235(1):11-31. doi: 10.1016/0009-8981(94)05995-4.
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.
为了研究转化生长因子β（TGF-β）在多种人类疾病中的生物学作用，需要能够分别检测人血清和血浆中活性和潜伏形式的TGF-β的检测方法。我们开发了一种酶联免疫吸附测定（ELISA），使用两种抗TGF-β的多克隆抗体，可快速测定人血清和血浆中活性加酸可激活的潜伏TGF-β形式（（a + l）TGF-β）的量，范围为4 pmol/l至2000 pmol/l。为了单独测量活性TGF-β，我们开发了第二种ELISA，使用II型TGF-β受体的细胞外结构域作为捕获试剂，可检测血清和血浆样品中20 pmol/l至4000 pmol/l范围内的活性TGF-β。两种检测方法对TGF-β1和TGF-β3的检测灵敏度相似，对TGF-β2的灵敏度低10倍以上，且不受一系列其他肽生长因子的影响。人血清中（a + l）TGF-β的平均含量为330 pmol/l，但范围非常大（<4 pmol/l至1400 pmol/l）。活性TGF-β的平均含量为230 pmol/l（范围<20 pmol/l至1400 pmol/l），活性TGF-β占（a + l）TGF-β的比例[a/(a + l)]从<10%到100%不等。在由相同全血样品制备的血清和少血小板血浆中，（a + l）TGF-β的浓度和活性TGF-β的比例非常相似。血清制备过程中形成的凝块保留了（a + l）TGF-β ELISA在相应血小板释放物中检测到的所有TGF-β，尽管血小板中的血小板衍生生长因子（PDGF）释放到了血清中。相比之下，少血小板血浆中未检测到PDGF，这表明血浆中检测到的（a + l）TGF-β不是出血后血小板脱颗粒所致。因此，血清活性TGF-β和（a + l）TGF-β浓度提供了血浆中这些形式的TGF-β的可靠估计值。
