Antibody Screening by Microarray Technology—Direct Identification of Selective High-Affinity Clones
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents, Buffers, Materials, and Equipment
2.2. Preparation of Epoxy Slides
2.3. Sample Printing and Incubation
2.4. Design and Synthesis of Hapten–Fluorophore Conjugates
2.5. Competition Experiments
3. Results
3.1. The Coating of Microarray Slides
3.2. Antibody Printing
3.3. Design and Synthesis of Hapten–Fluorophore Conjugates
3.4. Incubation Steps of Reagents and Hybridoma Supernatants
- (a)
- The dye conjugate is monovalent, which avoids confusing avidity (multivalency) effects, which are often misinterpreted.
- (b)
- This monovalent binding restricts the signals to high-affinity antibodies. With weakly binding antibodies, the tracer (labeled antigen) is washed away. The washing duration might modulate the cutoff of the detected clones according to their off rate.
- (c)
- The tracer binding is highly reversible, which makes it possible to reuse the chip without strong regeneration steps.
3.5. Data Evaluation
3.6. Identification and Ranking of Hybridoma Clones
3.7. Competition Experiments
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Antibody | IC50 (µg/L) | Isotype | Rating | Test Result (1 mg/L) | Test Result (10 mg/L) | References |
---|---|---|---|---|---|---|
BAM-mab 01 (CBZ) * | 0.32 | IgG1 | good | + + | + + | [25,37,38] |
B3212M | 0.15 | IgG1 | good | + + | + + | [25,39,40,41,42,43,44,45] |
6C5 | 23.0 | IgG1 | poor | − − | − − | [26] |
3B3 | 1700 | IgG1 | very poor | − − | − − | [26] |
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Paul, M.; Weller, M.G. Antibody Screening by Microarray Technology—Direct Identification of Selective High-Affinity Clones. Antibodies 2020, 9, 1. https://doi.org/10.3390/antib9010001
Paul M, Weller MG. Antibody Screening by Microarray Technology—Direct Identification of Selective High-Affinity Clones. Antibodies. 2020; 9(1):1. https://doi.org/10.3390/antib9010001
Chicago/Turabian StylePaul, Martin, and Michael G. Weller. 2020. "Antibody Screening by Microarray Technology—Direct Identification of Selective High-Affinity Clones" Antibodies 9, no. 1: 1. https://doi.org/10.3390/antib9010001