Phillips A O, Steadman R, Donovan K D, Williams J D
Institute of Nephrology, Cardiff Royal Infirmary, U.K.
Int J Biochem Cell Biol. 1995 Feb;27(2):207-13. doi: 10.1016/1357-2725(94)00077-o.
Previous studies which examined Transforming Growth Factor beta 1 (TGF-beta 1) generation have relied on the identification of TGF-beta 1 mRNA or measurement of TGF-beta 1 by bioassay. Quantitation of TGF-beta 1 message alone however is inadequate since the regulation of TGF-beta 1 synthesis is often post-transcriptional. TGF-beta 1 is poorly immunogenic, and sensitive and specific immunoassays for this peptide have proved difficult to develop. Bioassays depend on stimulation or inhibition of cell proliferation in a TGF-beta 1 dependent manner, and are very rigid in their requirements for optimal performance. The aims of this work was therefore to develop a sensitive and reproducible immunoassay for TGF-beta 1. Microtitre plates were coated with human recombinant TGF-beta 1, unbound protein was discarded from the wells prior to blocking with bovine serum albumin. Chicken anti-human TGF-beta 1 antibody was incubated with the test solution overnight at 4 degrees C and then added to the coated wells. Bound antibody was detected with alkaline phosphatase conjugated anti-chicken antibody. The assay is sensitive to 0.2 ng/ml with a range to 100 ng/ml. The assay detects the mature form of human recombinant TGF-beta 1, natural platelet extracted TGF-beta 1, and TGF-beta 1 derived from human monocytes stimulated with Phorbol myristate acetate (PMA). Active TGF-beta 1 is measured directly and latent TGF-beta 1 can be measured indirectly following acid activation of samples. Inter-assay precision ranged from 4.3 to 9.6%, (coefficient of variation, %CV) and intraassay precision ranged from 2.8 to 8.6% (CV).(ABSTRACT TRUNCATED AT 250 WORDS)
以往研究转化生长因子β1（TGF-β1）生成情况时，依赖于TGF-β1 mRNA的鉴定或通过生物测定法来测量TGF-β1。然而，仅对TGF-β1信息进行定量是不够的，因为TGF-β1合成的调控通常发生在转录后。TGF-β1免疫原性较差，事实证明，针对该肽开发灵敏且特异的免疫测定法颇具难度。生物测定法依赖于以TGF-β1依赖的方式刺激或抑制细胞增殖，并且对最佳性能的要求非常严格。因此，这项工作的目的是开发一种灵敏且可重复的TGF-β1免疫测定法。用重组人TGF-β1包被微量滴定板，在用牛血清白蛋白封闭之前，将未结合的蛋白质从孔中弃去。鸡抗人TGF-β1抗体与测试溶液在4℃下孵育过夜，然后加入包被好的孔中。用碱性磷酸酶偶联的抗鸡抗体检测结合的抗体。该测定法对0.2 ng/ml敏感，范围为100 ng/ml。该测定法可检测重组人TGF-β1的成熟形式、天然血小板提取的TGF-β1以及佛波酯肉豆蔻酸酯乙酸盐（PMA）刺激的人单核细胞来源的TGF-β1。可直接测量活性TGF-β1，对样品进行酸激活后可间接测量潜伏性TGF-β1。批间精密度范围为4.3%至9.6%（变异系数，%CV），批内精密度范围为2.8%至8.6%（CV）。（摘要截选至250字）
