Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex
Abstract
:1. Introduction
2. Results and Discussion
2.1. Immunization and Immune Response
2.2. Hybridoma Screening
2.3. Antibody Specificity
2.4. Description of ELISA and KinExA
2.5. Optimization of Reagent Concentrations for Development of ELISA
2.6. Optimization of KinExA Conditions and Its Running
2.7. Validation of ELISA and KinExA
2.7.1. Calibration Curves and Detection Limits
2.7.2. Precision and Recovery
2.8. Comparison of ELISA and KinExA with ICP-AES
2.9. Comparative Evaluation of ELISA and KinExA
3. Experimental
3.1. Instruments
3.2. Materials
3.3. Preparation of Copper-EDTA-Protein Conjugates
3.4. Immunization and Cell Fusion
3.5. Non-Competitive and Competitive ELISA for Screening
3.6. Analysis of Cu(II) by ELISA
3.7. Analysis of Cu(II) by KinExA
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Metal Ions | IC50 (ng/mL) a | Cross Reactivity (%) b |
---|---|---|
Cu | 4.7 c | 100 c |
Zn | 73.4 | 6.4 |
Ni | 184.2 | 2.6 |
Hg | 467.8 | 1.0 |
Cd | ND d | ND |
Pb | ND | ND |
Mn | ND | ND |
Mg | ND | ND |
Fe | ND | ND |
Ca | ND | ND |
Parameter/Condition | Optimum Value |
---|---|
ELISA | |
Coating Cu(II)-EDTA-BSA conc. (μg/mL) | 0.5 |
Coating buffer | HBS |
Coating time (h)/temperature (°C) | 2/37 |
BSA concentration for blocking (%, w/v) | 3 |
Blocking with BSA: time (min)/temperature (°C) | 1/37 |
Concentration of EDTA (mM) | 10 |
8D66 antibody concentration (μg/mL) | 0.25 |
IgG-HRP concentration (μg/mL) | 1 |
Binding of IgG-HRP: time (h)/temperature (°C) | 1/37 |
TMB: time (min)/temperature (°C) | 15/25 |
Reagent for stopping the color reaction (M) | HCl (2) |
Measuring wavelength (nm) | 450 |
KinExA | |
Coated Cu(II)-EDTA-BSA (µg/mL) | 1 |
Volume of beads suspension (µL) | 583 |
Flow rate of beads suspension (mL/min) | 1 |
Time of beads drawing (s) | 35 |
Concentration of 8D66 antibody (μg/mL) | 0.5 |
Volume of Cu(II sample (µL) | 500 |
Time of samples drawing (s) | 120 |
Concentration of IgG-FITC (µg/mL) | 0.25 |
Volume of IgG-FITC (µL) | 500 |
Flow rate (mL/min) | 0.25 |
ELISA (n = 5) | KinExA (n = 3) | ||||
---|---|---|---|---|---|
Concentration (ng/mL) | Intra-Assay | Inter-Assay | Concentration (ng/mL) | Intra-Assay | Inter-Assay |
0.5 | 5.4 a | 6.1 | 0.1 | 4.2 | 6.2 |
2 | 3.5 | 4.3 | 0.5 | 3.2 | 3.8 |
20 | 6.2 | 6.8 | 2 | 4.7 | 5.6 |
ELISA | KinExA | ||
---|---|---|---|
Added (ng/mL) | Recovery a (% ± CV) | Added (ng/mL) | Recovery a (% ± CV) |
1.25 | 105.2 ± 5.4 | 0.25 | 103.5 ± 6.2 |
2.5 | 96.4 ± 4.2 | 0.5 | 102.6 ± 5.3 |
5 | 101.8 ± 4.1 | 1 | 96.7 ± 4.9 |
10 | 98.6 ± 5.9 | 2 | 102.5 ± 4.8 |
20 | 102.6 ± 6.2 | 4 | 97.8 ± 4.2 |
Average | 100.9 ± 3.5 | 100.6 ± 3.1 |
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Darwish, I.A.; Wang, Z.; Darling, R.J. Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex. Molecules 2023, 28, 7017. https://doi.org/10.3390/molecules28207017
Darwish IA, Wang Z, Darling RJ. Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex. Molecules. 2023; 28(20):7017. https://doi.org/10.3390/molecules28207017
Chicago/Turabian StyleDarwish, Ibrahim A., Zongzhi Wang, and Ryhan J. Darling. 2023. "Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex" Molecules 28, no. 20: 7017. https://doi.org/10.3390/molecules28207017
APA StyleDarwish, I. A., Wang, Z., & Darling, R. J. (2023). Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex. Molecules, 28(20), 7017. https://doi.org/10.3390/molecules28207017