Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments
Abstract
:1. Introduction
2. Material and Methods
2.1. SPR Instruments and Software
2.2. Consumables, Sensor Chips, Reagents and Buffers
2.3. Antigens
2.4. Antibodies
2.5. Functionalizing the Sensor
2.6. Interaction Analysis via SPR
2.7. Blocking
2.8. Comparing and Ranking the Epitope Accessibility of Antibody Pairs
2.9. Evaluating the SPR Data
3. Theory and Calculations
3.1. Sandwich-Assay Setup
- (1)
- Capturing
- (2)
- Target-Enrichment
- (3)
- Binding-kinetics of the 2nd binder
3.2. Target-Enrichment Step
3.3. Simulating the Targets’ Enrichment Time
4. Results and Discussion
4.1. In Silico-Determined Assay Settings Successfully Proven In Vitro
4.2. Proved Technical Reproducibility of Antibody Kinetics with Native GDF15 from Donors of Different Sources
4.3. Small Study Reveals Comparable Kinetic Constants for Native GDF15 from Different Sources
4.4. Kinetic Constants for Antibodies Binding to Native Neurofilament Light Chain from Patients’ Cerebrospinal Fluid (CSF)
4.5. Empirical Relation for Offered Amount of Native NFL and Its Surface Density
4.6. Identifying Antibodies Binding to Native NFL from CSF
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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GDF15 Origin | ka [M−1 s−1] | SE (ka) [%] | kd [s−1] | SE (kd) [%] | t/2diss [min] | KD [nM] | Rmax [RU] | U-Value |
---|---|---|---|---|---|---|---|---|
recombinant | 3.3 × 105 | 0.8 | 3.4 × 10−2 | 0.6 | <1 | 103 | 37.4 | 4 |
recombinant | 3.2 × 105 | 0.9 | 3.3 × 10−2 | 0.7 | <1 | 104 | 39.7 | 4 |
native, healthy | 3.3 × 105 | 2.3 | 2.8 × 10−2 | 1.9 | <1 | 83 | 7.6 | 12 |
native, healthy | 2.6 × 105 | 3.2 | 2.4 × 10−2 | 2.5 | <1 | 94 | 7.9 | 15 |
native, oncological | 4.4 × 105 | 2.0 | 4.3 × 10−2 | 1.6 | <1 | 98 | 8.8 | 7 |
native, oncological | 2.8 × 105 | 1.3 | 3.1 × 10−2 | 1.1 | <1 | 111 | 8.6 | 5 |
native, cardiovascular | 4.8 × 105 | 0.7 | 3.7 × 10−2 | 0.6 | <1 | 78 | 28.4 | 3 |
native, cardiovascular | 3.7 × 105 | 1.3 | 3.5 × 10−2 | 1.2 | <1 | 96 | 33.9 | 4 |
native, pregnant | 4.8 × 105 | 0.7 | 3.7 × 10−2 | 0.6 | <1 | 78 | 28.4 | 3 |
native, pregnant | 3.7 × 105 | 1.3 | 3.5 × 10−2 | 1.2 | <1 | 96 | 33.9 | 4 |
2nd Fab | 1st Ab | Biomarker | ka [M−1 s−1] | ±SE (ka) [M−1 s−1] | kd [s−1] | ±SE (kd) [s−1] | t/2diss [min] | KD [M] | Rmax [RU] | U-Value |
---|---|---|---|---|---|---|---|---|---|---|
A | B | recombinant NFL | 1.2 × 106 | 3.9 × 103 | 9.2 × 10−3 | 2.5 × 10−5 | 1 | 8.0 × 10−9 | 24 | 2 |
A | B | native NFL from CSF | 8.4 × 105 | 1.0 × 104 | 6.8 × 10−3 | 6.5 × 10−5 | 2 | 8.0 × 10−9 | 10 | 7 |
B | J | recombinant NFL | 3.2 × 106 | 6.9 × 103 | 1.0 × 10−5 | 2.8 × 10−8 | 1155 | 3.2 × 10−12 | 18 | 95 |
B | J | native NFL from CSF | 2.3 × 106 | 1.2 × 104 | 3.6 × 10−5 | 5.3 × 10−7 | 322 | 1.5 × 10−11 | 6 | 5 |
C | B | recombinant NFL | 3.6 × 106 | 9.7 × 103 | 1.0 × 10−5 | 2.7 × 10−7 | 1155 | 2.8 × 10−12 | 29 | 12 |
C | B | native NFL from CSF | 2.9 × 106 | 1.1 × 104 | 1.1 × 10−5 | 2.4 × 10−7 | 1058 | 3.8 × 10−12 | 13 | 9 |
C | A | native NFL from CSF | 2.1 × 106 | 2.9 × 103 | 1.0 × 10−5 | 1.5 × 10−7 | 1155 | 4.7 × 10−12 | 15 | 9 |
I | B | recombinant NFL | 8.4 × 105 | 3.8 × 103 | 1.7 × 10−4 | 5.1 × 10−7 | 67 | 1.8 × 10−10 | 46 | 2 |
I | B | native NFL from CSF | n.b. | n.b. | n.b. | n.b. | n.b. | n.b. |
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Jucknischke, U.; Friebe, S.; Rehle, M.; Quast, L.; Schmidt, S.H. Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments. Biosensors 2023, 13, 1030. https://doi.org/10.3390/bios13121030
Jucknischke U, Friebe S, Rehle M, Quast L, Schmidt SH. Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments. Biosensors. 2023; 13(12):1030. https://doi.org/10.3390/bios13121030
Chicago/Turabian StyleJucknischke, Ute, Sebastian Friebe, Markus Rehle, Laura Quast, and Sven H. Schmidt. 2023. "Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments" Biosensors 13, no. 12: 1030. https://doi.org/10.3390/bios13121030
APA StyleJucknischke, U., Friebe, S., Rehle, M., Quast, L., & Schmidt, S. H. (2023). Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments. Biosensors, 13(12), 1030. https://doi.org/10.3390/bios13121030