Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients
2.2. Construction of Rabbit Immune Antibody Library
2.3. Anti-TPO Antibody Screening Using a Phage Display
2.4. Conversion of Anti-TPO Rabbit Monoclonal IgG from Phage Antibodies
2.5. Preparation of Alkaline Phosphatase (ALP)-Labeled Anti-TPO Antibody
2.6. Recombinant Human TPO with a C-Terminal 6× His Tag (rhTPO-His) Antigen
2.7. Measurement of Plasma TPO Concentrations
2.8. Characterization of In-House Standard Material and Concentration Assignment
2.9. Evaluation Methods for TPO-CLEIA
2.10. Statistical Analysis
3. Results
3.1. Characterization of Recombinant Human TPO Antigen
3.2. Analytical Performance of TPO-CLEIA
3.3. Clinical Validation in the Study Participants
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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High-Titer TPO Plasma Samples from AA Patients | TPO-CLEIA Value (pg/mL) | Multiplied by the Coefficient * | TPO-ELISA Value (pg/mL) | % Difference |
---|---|---|---|---|
No. 1 | 294.8 | 1263.7 | 1242.9 | 2.0 |
No. 2 | 200.1 | 857.7 | 896.1 | −0.4 |
No. 3 | 446.3 | 1912.8 | 2083.6 | −0.8 |
No. 4 | 479.6 | 2055.4 | 2155.3 | −0.5 |
No. 5 | 503.6 | 2158.5 | 2142.6 | 1.0 |
10-fold dilution of No. 5 | 52.7 | 225.9 | 240.1 | −5.9 |
20-fold dilution of No. 5 | 26.6 | 114.0 | 120.0 | −5.0 |
40-fold dilution of No. 5 | 12.8 | 54.6 | 58.6 | −6.7 |
80-fold dilution of No. 5 | 6.2 | 26.4 | 18.5 | 42.7 |
TPO Level of Samples | Precision | Intra-Assay Precision | Interassay Precision | ||||
---|---|---|---|---|---|---|---|
Reagent Lot | Lot.1 | Lot.2 | Lot.3 | Lot.1 | Lot.2 | Lot.3 | |
Low level | Mean (pg/mL) | 52.2 | 50.7 | 52.2 | 46.8 | 45.9 | 47.1 |
SD (pg/mL) | 1.3 | 1.3 | 0.9 | 1.3 | 1.5 | 1.1 | |
CV | 2.5% | 2.5% | 1.8% | 2.9% | 3.2% | 2.3% | |
Middle level | Mean (pg/mL) | 127.3 | 121.2 | 126.1 | 108.2 | 106.7 | 106.8 |
SD (pg/mL) | 3.1 | 5.7 | 1.0 | 4.6 | 2.0 | 1.4 | |
CV | 2.4% | 4.7% | 0.8% | 4.3% | 1.9% | 1.3% | |
High level | Mean (pg/mL) | 326.0 | 314.3 | 331.5 | 309.2 | 288.6 | 286.7 |
SD (pg/mL) | 6.3 | 12.3 | 8.2 | 5.6 | 6.7 | 16.9 | |
CV | 1.9% | 3.9% | 2.5% | 1.8% | 2.3% | 5.9% |
TPO Level of Samples | Unspiked Samples (pg/mL) | Spiked TPO (pg/mL) | Observed (pg/mL) | Recovery |
---|---|---|---|---|
Low level | 41.8 | 49.3 | 87.9 | 93.5% |
137 | 181 | 101.6% | ||
333.9 | 349.6 | 92.2% | ||
Middle level | 148.1 | 49.3 | 194.9 | 95.0% |
137 | 282.6 | 98.2% | ||
333.9 | 437.9 | 86.8% | ||
High level | 398.4 | 49.3 | 450.6 | 106.0% |
137 | 539.3 | 102.9% | ||
333.9 | 719.4 | 96.2% |
Interfering Substances | Concentration | % Difference from Control | ||
---|---|---|---|---|
TPO Level of Samples | ||||
Low | Middle | High | ||
Hemoglobin | 500 mg/dL | 2.3 | 0.7 | 4.0 |
Free bilirubin | 20 mg/dL | −2.1 | 2.1 | −8.0 |
Conjugated bilirubin | 20 mg/dL | −2.3 | −0.4 | −2.6 |
Chyle material | 1360 FTU | −2.7 | 0.7 | −0.9 |
Rheumatoid factor | 500 IU/mL | 1.2 | 0.4 | −2.4 |
High concentration of IgG | 500 mg/dL | −6.5 | 0.2 | −6.9 |
Sample Type | % Difference from Control | Average Difference, % | |
---|---|---|---|
Spiked TPO (pg/mL) | |||
50 pg/mL, % | 200 pg/mL, % | ||
EDTA plasma-1 | 2.3 | −6.0 | −0.9 |
EDTA plasma-2 | 11.6 | −3.3 | |
EDTA plasma-3 | −1.6 | −5.9 | |
EDTA plasma-4 | 4.4 | −1.1 | |
EDTA plasma-5 | −1.4 | −7.7 | |
Heparin plasma-1 | 10.5 | −1.1 | 2.2 |
Heparin plasma-2 | 11.7 | 1.6 | |
Heparin plasma-3 | −6.8 | −6.1 | |
Heparin plasma-4 | 8.7 | 0.7 | |
Heparin plasma-5 | 6.1 | −3.1 | |
Citrate plasma-1 | 1.1 | −8.0 | −0.7 |
Citrate plasma-2 | 7.9 | −4.2 | |
Citrate plasma-3 | 1.8 | −5.9 | |
Citrate plasma-4 | 10.5 | −0.9 | |
Citrate plasma-5 | −2.1 | −6.7 |
Principle of Measurement | CLEIA | ELISA |
---|---|---|
Trade name | Under consideration | Human Thrombopoietin Quantikine ELISA Kit |
Manufacturer | Medical & Biological Laboratories Co., Ltd. | R & D Systems |
Capture antibody | Monoclonal | Monoclonal |
Detection antibody | Monoclonal | Polyclonal |
Operation | Fully automated | Hand method |
Measurement time | 20 min | 4.5 h |
Sample volume | 40 μL | 200 μL |
LoQ | 14.6 * (3.4) pg/mL | 40.7 pg/mL |
LoD | 5.6 * (1.3) pg/mL | 21.4 pg/mL |
Upper limit of measurement | 3000 * (700) pg/mL | 2000 pg/mL |
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Nishikawa, Y.; Nishida, S.; Kuroda, K.; Kashiwagi, H.; Tomiyama, Y.; Kuwana, M. Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma. Diagnostics 2022, 12, 313. https://doi.org/10.3390/diagnostics12020313
Nishikawa Y, Nishida S, Kuroda K, Kashiwagi H, Tomiyama Y, Kuwana M. Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma. Diagnostics. 2022; 12(2):313. https://doi.org/10.3390/diagnostics12020313
Chicago/Turabian StyleNishikawa, Yukihiro, Shiyo Nishida, Keiko Kuroda, Hirokazu Kashiwagi, Yoshiaki Tomiyama, and Masataka Kuwana. 2022. "Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma" Diagnostics 12, no. 2: 313. https://doi.org/10.3390/diagnostics12020313