We developed a sandwich enzyme-linked imrnuiiosorbent assay (ELISA) to quantify serum levels of human thrombopoietin (TPO) by using monoclonal and polyclonal antibodies. The. ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intraand inter-assay variation were from 3.0 10 4.9 % and from 5.9 to 6.1 %. respectively, This quantitative limit of the ELISA was 0.09 f moVml m scrum, which represents 3.0 pg of a fulllength TPO peptide in I ml scrum. The quantitative limit was lower than the normal level. The dose-response curves of serum samples from healthy volunteers and patients with hematopoieüc disorders were parallel to the standard curves. The ELISA did no! cross-react with a variety of Wood components and seven other cytokines including rhIL-3. rhlL-6. rhlL-7. rhG-CSF. rhGMCSF, rhEPO and rhSCF. The addition of extracellular domain of (he human c-MpI that may coexist in the blood samples had only a minimal effect on the ELISA. The estimated TPO levels m normal donors and in patients with hematopoietic disorders using the ELISA were as follows. The serum TPO concentrations from 29 normal males aged 24-38 (mean, 31 years) and 21 females aged 20-35 (mean, 26 years) were 0.79±0.35 <mean±SD; range 0.33 10 1.72) and 0.70±0.26 fmol/ml (range 0.33 to 1.33), respectively. No statistical difference in the serum TPO levels between males and females was noted (P-0.710). m addition, there was no statistically significant correlation between the serum TPO levels and platelet counts in the normal individuals. The serum TPO levels in the patients with idiopathic thromboeylopenic purpura (n-5, î.70±0.51 fmol/ml) were higher than the normal level. The patients with plastic anemia (n-7, 18.53± 12.37 fmol/ml) and acute lymphocytic leukemia (n-5, I0.36±5.57 fmoi/ml) had significantly higher serum TPO levels than the normal. Interestingly, in patients with essential thrombocythemia whose platelet counts were increased, the TPO lvcls(n-6, 2.80+L55 fmol/ml) were not decreased but elevated above normal. The established ELISA specific to TPO should prove useful to measure the TPO concentration in serum samples.
|出版ステータス||Published - 1996|
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