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. 2022 Jan 26;12(2):313.
doi: 10.3390/diagnostics12020313.

Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma

Yukihiro Nishikawa  1 , Shiyo Nishida  1 , Keiko Kuroda  1 , Hirokazu Kashiwagi  2   3 , Yoshiaki Tomiyama  3 , Masataka Kuwana  4
Affiliations

Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma

Yukihiro Nishikawa et al. Diagnostics (Basel). .

Abstract

Plasma thrombopoietin (TPO) measurements help distinguish between different types of thrombocytopenia but are not feasible in routine clinical practice. We developed a fully automated quantitative chemiluminescent enzyme immunoassay (CLEIA) for measuring TPO (TPO-CLEIA), which is a one-step sandwich-type assay. This assay utilizes a mouse monoclonal capture antibody, which has the neutralizing epitope of the interaction between TPO and the TPO receptor, and a newly generated rabbit monoclonal detector antibody. In analytical performance studies, this assay showed good linearity over the measuring range and high sensitivity. The limit of quantification (LoQ) of this assay was 3.4 pg/mL; low TPO concentration values of almost all healthy individuals exceeded the LoQ value. In clinical validation studies, TPO levels obtained from patients with aplastic anemia (AA) significantly increased, whereas those of patients with immune thrombocytopenia (ITP) were normal or slightly increased. The cutoff value for TPO-CLEIA corresponding to the previously reported values was useful for distinguishing between ITP and AA. These results suggest that TPO-CLEIA can quantify human plasma TPO levels with high accuracy and sensitivity and has the potential to facilitate routine clinical measurement of TPO in patients with various types of thrombocytopenia.

Keywords: aplastic anemia; automated; chemiluminescent; immune thrombocytopenia; immunoassay; thrombocytopenia; thrombopoietin.

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Conflict of interest statement

Y.N., S.N., and K.K. are employees of Medical & Biological Laboratories (MBL). M.K.: grants and speaking fees from MBL. This study was conducted as a collaboration between the authors’ institutes and MBL.

Figures

Figure 1
Figure 1
Schematic representation of TPO measurement using a one-step sandwich method. AB, antibody; Ag, antigen; ALP, alkaline phosphatase; TPO, thrombopoietin.
Figure 2
Figure 2
Comparison of purity levels between the isolated rhTPO-His and rhTPO as in-house standard material and traceability system diagram using these materials. (A) The samples in lanes 1–3 were subjected to SDS-PAGE. Lane 1, prestained molecular mass markers (Bio-Rad) of indicated size (kDa); 2, rhTPO as in-house standard material; 3, isolated rhTPO-His. Note: 1 µg of total protein was loaded in lanes 2–3. (B) Traceability of thrombopoietin (TPO) concentration was ensured by the new CLEIA assay using BSA standard and the Bradford protein assay. BSA, bovine serum albumin; CLEIA, chemiluminescent enzyme immunoassay; rhTPO, recombinant human thrombopoietin.
Figure 3
Figure 3
Evaluation of linearity and high-dose hook effect. (A) Relationship between expected TPO concentrations calculated from dilution ratio and the observed concentrations in three samples. (B) Relationship between expected TPO concentrations and observed high TPO concentrations beyond the dynamic range. TPO, thrombopoietin.
Figure 4
Figure 4
Results of plasma thrombopoietin functional sensitivity and analytical sensitivity studies. (A) The concentration, 3.4 pg/mL, was set as the lowest concentration that showed a total precision of 15% CV. (B) The concentration, 1.3 pg/mL, was set as the minimum value in the dilution panels where the mean + 2SD value on the low side did not exceed the mean-2SD value on the high side. CV, coefficient of variation; TPO, thrombopoietin.
Figure 5
Figure 5
Clinical validation of TPO-CLEIA in study participants. (A) Plasma thrombopoietin (TPO) levels in healthy controls, patients with immune thrombocytopenia (ITP), aplastic anemia (AA). (B) Distribution of TPO concentrations in healthy controls.
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