Research Article: Development of a sandwich enzyme-linked immunosorbent assay for dTMP-GH fusion protein by rational immunogen selection

Date Published: July 17, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Song Wang, Mingqiang Shen, Shilei Chen, Cheng Wang, Fang Chen, Mo Chen, Gaomei Zhao, Xinze Ran, Tianmin Cheng, Yongping Su, Yang Xu, Junping Wang.


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.

Partial Text

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. Nowadays, TPO has been considered as one of the most effective cytokines to raise platelet (Bartley et al. 1994; Kuter et al. 1994; Vainchenker et al. 1998). To date, research and development on recombinant TOP drugs is still in a dilemma due to formation of neutralizing antibodies to the natural TPO (Basser et al. 2002; Li et al. 2001). In 1997, Dower et al. synthesized a TPO-mimetic peptides (TMP) consisting of 14 amino acids that could bind with the c-Mpl with high affinity to promote the proliferation and differentiation of megakaryocytes in vitro (Cwirla et al. 1997). However, because of the small molecular weight, such linear peptides were too short-lived in circulation to be applicated in vivo (Kuter 2007). Recently, several methods have been utilized to extend the half-life of TMP in vivo such as pegylation, as well as binding with Fab or Fc fragments (Broudy and Lin 2004; Frederickson et al. 2006; Kuter 2007). Nevertheless, a time lag of platelet recovery was also observed in the c-Mpl based TMP (Kuter 2002; Kuter and Begley 2002).

Pharmacokinetic analysis is essential for the research and development of protein therapeutic candidates. Thus, it is indispensable to establish methods for specific candidate detection in vivo. For the development of fusion protein, the binding of the target fragment and some kind of endogenous fragment could contribute to the extension of the circulating half-life and help to attenuate the risks of formation of neutralizing antibodies. Therefore, the specific TMP-based fusion protein could be detected from the expressed or purified product using the commercial antibodies against the part fused with TMP (Fayaz et al. 2016). In our previous study, similar detection had been carried out after the expression and purification of dTMP-GH using anti-hGH antibody (Wang et al. 2013). However, such techniques were not sufficient to some extent. For example, anti-hGH antibody could not testify the dTMP-GH and endogenous hGH. Radioactive isotope has been commonly in the development of TMP-based fusion protein (Broudy and Lin 2004). In our preliminary study, we also detect the pharmacokinetics of dTMP-GH in animals using radioactive isotope (data not shown). However, such method is not only labor-intensive and time-consuming, but also unacceptable in clinical practice on patients. Hall et al. developed ligand-binding mass spectrometry (LBMS) for the detection of TMP fusion protein AMG531 (romiplostim) (Hall et al. 2010) that composed by TMP and the human Fc fragments as a typical “peptibody”, in which those peptibodies were captured by anti-human Fc antibody for further Mass Spectrometry analysis. Such method is effective for the evaluation of AMG531 PKs (Krzyzanski et al. 2013). In order to eliminate the interference of IgG, Hall et al. developed anti-human Fc fragment with no cross reactivity to the human IgG, while intact hGH was integrated in the dTMP-GH, which could not eliminate the effects of endogenous hGH on the metabolites. In addition, MS based method involves additional requirements to the test facility. On this basis, it is urgent to develop a quantitative method for the specific detection of fusion protein in a sensitive manner.




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