Abstract
dTMP-GH is a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide (dTMP) fused to human growth hormone (hGH) prepared previously by our team. It shows significant bioactivity in promoting thrombocytopoiesis, but detection of intact dTMP-GH in plasma is still a challenge due to the presence of endogenous hGH. In this study, a rabbit polyclonal antibody with high affinity to dTMP was obtained with a BSA-conjugated immunogen composed of 20 amino acids sequence spanning two TMP and the linker. A monoclonal antibody termed as 3B2 was screened out by using immunizing mice with whole dTMP-GH, which was proved to simultaneously interact with rhGH, TMP-GH, and dTMP-GH, respectively. In this study, we developed a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies (one polyclonal and one HRP-conjugated monoclonal) to quantify dTMP-GH. The polyclonal antibody and HRP-conjugated monoclonal antibody 3B2 were applied as the capture antibody and detection antibody, respectively. A good correlation between ELISA and bicinchoninic acid (BCA) assay in the quantification of diluted dTMP-GH was observed (r2 = 0.996). Meanwhile, the standard curve of this ELISA method was found in a linear relationship between 0.2 and 10 ng/mL in the presence of rabbit plasma. In vivo experiments demonstrate that the newly developed method is effective to detect dTMP-GH in rabbits, which paves the way for further pharmacokinetic evaluation.
Highlights
Thrombopenia has been commonly reported in clinical practice
Amino acid sequence corresponding to TPO-mimetic peptides (TMP) dimer of dimer of TMP (dTMP)-Growth hormone (GH) served as immunogen, and a sensitive and specific enzymelinked immunosorbent assay (ELISA) method was developed for the quantitative determination of dTMP-GH, which contributed to the measurement of dTMP-GH pharmacokinetics in vivo
The O D450 values of TMP-GH and recombinant human GH (rhGH) wells were similar to BSA, which was used as a negative control
Summary
Thrombopenia has been commonly reported in clinical practice. Thrombopoietin (TPO), a natural ligand for c-Mpl, is the major modulating factor for the formation of megalokaryocyte that plays important roles in the generation of platelets. The hGH is efficient in promoting the differentiation, especially the terminal differentiation, of human megakaryocytes through stage- and time-specific activation of extracellular signal-regulated kinase (ERK1/2) and protein kinase B (Akt) It shows a complementary and synergistic effect with the dTMP on thrombocytopoiesis (Xu et al 2014). As the anti-hGH antibody could only capture the epitope of the hGH in the fusion protein, it is still a challenge to identify whether the detected protein contains complete N-terminal TMP dimer. To solve this problem, mass spectrum is needed to detect the purified dTMP-GH, but the method is labor-intensive and timeconsuming. Amino acid sequence corresponding to TMP dimer of dTMP-GH served as immunogen, and a sensitive and specific ELISA method was developed for the quantitative determination of dTMP-GH, which contributed to the measurement of dTMP-GH pharmacokinetics in vivo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
