Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays
Abstract
1. Introduction
2. Experimental Design
3. Results and Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Variation of Reagent Concentration (Either Antibody or Conjugate), Second Reagent Is Kept Constant | |||
---|---|---|---|
1 (Start) | 1:3 | 1:9 | |
Analyte concentration (mol/L) | 10-3 | 10-3 | 10-3 |
10-4 | 10-4 | 10-4 | |
10-5 | 10-5 | 10-5 | |
10-6 | 10-6 | 10-6 | |
10-7 | 10-7 | 10-7 | |
10-8 | 10-8 | 10-8 | |
10-9 | 10-9 | 10-9 | |
10-10 | 10-10 | 10-10 | |
10-11 | 10-11 | 10-11 | |
10-12 | 10-12 | 10-12 |
Direct, Competitive ELISA Competitor: FLAG Peptide | Conjugate HRP-FLAG (Tracer) | |||
---|---|---|---|---|
1:90,000 (Start) | 1:270,000 (1:3) | 1:810,000 (1:9) | ||
Antibody M2 | 1:90,000 (start) | 166 ± 21 nM | 139 ± 28 nM | 121 ± 30 nM |
1:270,000 (1:3) | 147 ± 23 nM | 133 ± 42 nM | 90 ± 13 nM | |
1:810,000 (1:9) | 127 ± 18 nM | 126 ± 73 nM | 90 ± 37 nM |
Indirect, Competitive ELISA Competitor: FLAG Peptide | Conjugate BSA-FLAG (Immobilized) | |||
---|---|---|---|---|
1:1,500 (Start) | 1:4,500 (1:3) | 1:15,000 (1:10) | ||
Antibody M2 | 1:30,000 (start) | 169 ± 21 nM | 146 ± 10 nM | 160 ± 16 nM |
1:90,000 (1:3) | 121 ± 16 nM | 104 ± 8 nM | 121 ± 16 nM | |
1:270,000 (1:9) | 109 ± 12 nM | 93 ± 5 nM | 93 ± 15 nM |
Method | Kd (nM) | FLAG Sequence | Conjugate | Reference |
---|---|---|---|---|
direct IA | 90 ± 37 | DYKDDDDK | HRP-CDYKDDDDK | This work |
indirect IA | 93 ± 15 | DYKDDDDK | BSA-CDYKDDDDK | This work |
FPIA | 150 | - | 5-FAM-SGSGDYKDDDDK | [63] |
SPR | 50 ± 30 | DYKDDDDK | M2 (immobilized) | [64] |
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Fischer, J.; Kaufmann, J.O.; Weller, M.G. Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays. Methods Protoc. 2024, 7, 49. https://doi.org/10.3390/mps7030049
Fischer J, Kaufmann JO, Weller MG. Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays. Methods and Protocols. 2024; 7(3):49. https://doi.org/10.3390/mps7030049
Chicago/Turabian StyleFischer, Janina, Jan Ole Kaufmann, and Michael G. Weller. 2024. "Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays" Methods and Protocols 7, no. 3: 49. https://doi.org/10.3390/mps7030049
APA StyleFischer, J., Kaufmann, J. O., & Weller, M. G. (2024). Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays. Methods and Protocols, 7(3), 49. https://doi.org/10.3390/mps7030049