Complementary Strategy Enhancing Broad-Specificity for Multiplexed Immunoassay of Adulterant Sulfonylureas in Functional Food
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Instruments
2.3. Buffers and Solutions
2.4. Conjugation
2.4.1. Hapten 1-OVA/BSA
2.4.2. Hapten 2-OVA/BSA
2.4.3. Hapten 3-OVA
2.4.4. Hapten 4-OVA/BSA
2.5. Antibody Generation
2.6. ELISA Procedure
2.7. Specificity
2.8. Sample Preparation
2.9. Matrix Effect
2.10. Recovery and Confirmation
2.11. Analysis of Blind Samples
3. Results
3.1. Antibody Production
3.1.1. Hapten Design
3.1.2. Conjugate Preparation
3.1.3. ELISA Optimization
3.1.4. Broad-Specificity
3.2. Matrix Effect
3.3. Recovery and Confirmation
3.4. Analysis of Blind Samples
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Name | General Structural | R1 a | R2 b | Sulfonylureas | R1 | R2 |
---|---|---|---|---|---|---|
Glyburide | Tolbutamide | |||||
Glipizide | Carbutamide | |||||
Glimepiride | Gliclazide | |||||
Gliquidone | Tolazamide | |||||
Acetohexamide | Glibornuride | |||||
Chlorpropamide | ||||||
Hapten | Hapten 1 | Hapten 2 | ||||
Hapten 3 | Hapten 4 |
Name | H1-Ab | H2-Ab | ||||||
---|---|---|---|---|---|---|---|---|
LOD a nmol/μL | Dynamic Working Range (nmol/μL) | IC50 b nmol/μL | CR c % | LOD nmol/μL | Dynamic Working Range nmol/μL | IC50 nmol/μL | CR % | |
Glimepiride | 0.1 | 0.6–102.5 | 7.1 | 100.0 | 1672.4 | - | 17,228.5 | 0.1 d (0.04) e |
Glipizide | 0.7 | 1.6–45.8 | 8.5 | 83.6 | 1823.3 | - | >10,000 | <0.01 |
Glyburide | 0.4 | 1.6–96.4 | 12.1 | 58.7 | 3788.8 | - | >10,000 | <0.01 |
Gliquidone | 0.04 | 1.1–190.7 | 14.8 | 48.3 | 2455.7 | - | >10,000 | <0.01 |
Acetohexamide | 2.1 | 5.3–161.9 | 29.4 | 24.2 | 23.5 | - | 4173.0 | 0.6 (0.2) |
Tolazamide | 7.7 | 19.9–530.5 | 103.1 | 6.9 | 2.2 | 11.2–3221.6 | 189.5 | 12.3 (3.8) |
Tolbutamide | 32.2 | 81.7–1947.8 | 398.7 | 1.8 | 1.5 | 4.4–127.2 | 23.3 | 100 (30.6) |
Carbutamide | 66.3 | 161.1–3331.6 | 732.3 | 1.0 | 15.8 | 62.7–6911.3 | 658.6 | 3.5 (1.1) |
Gliclazide | 37.7 | 115.3–5224.3 | 775.8 | 0.9 | 3.1 | 13.9–2347.8 | 179.6 | 13.0 (4.0) |
Chlorpropamide | 80.2 | 267.8–16,444.3 | 2098.7 | 0.3 | 0.7 | 6.9–163.7 | 33.6 | 69.3 (21.2) |
Glibornuride | 228.1 | - | >10,000 | <0.01 | 9.8 | 52.4–15,498.0 | 901.0 | 2.6 (0.8) |
Repaglinide | - | - | >10,000 | <0.01 | - | - | >10,000 | <0.01 |
Rosiglitazone | - | - | >10,000 | <0.01 | - | - | >10,000 | <0.01 |
Phenformin | - | - | >10,000 | <0.01 | - | - | >10,000 | <0.01 |
Metformin hydrochloride | - | - | >10,000 | <0.01 | - | - | >10,000 | <0.01 |
Name | Spiked Level (μg/kg) | ELISA | LC-MS/MS | R2 b | ||||
---|---|---|---|---|---|---|---|---|
Observed Level (μg/kg) | Average Recovery % | CV a % | Observed Level (μg/kg) | Average Recovery % | CV % | |||
Glipizide | 320 | 259.5 | 81.1 | 10.9 | 244.3 | 76.4 | 1.0 | 0.995 |
1600 | 1272.0 | 79.5 | 7.9 | 1543.5 | 96.5 | 0.8 | ||
4800 | 4434.7 | 92.4 | 4.1 | 4701.4 | 98.0 | 2.4 | ||
Glimepiride | 320 | 318.8 | 99.6 | 10.8 | 253.5 | 79.2 | 9.4 | 0.994 |
1600 | 1548.8 | 96.8 | 18.5 | 1340.3 | 83.8 | 3.1 | ||
6400 | 5526.3 | 86.3 | 14.2 | 5745.4 | 89.8 | 1.6 | ||
Gliquidone | 640 | 703.8 | 110.0 | 12.4 | 647.8 | 101.2 | 0.4 | 0.995 |
3200 | 3092.0 | 96.6 | 5.7 | 3366.3 | 105.2 | 2.1 | ||
12,800 | 11,472.8 | 89.6 | 7.4 | 12,304.1 | 96.1 | 1.8 | ||
Glyburide | 320 | 301.9 | 94.3 | 6.8 | 267.4 | 83.6 | 10.4 | 0.978 |
1600 | 1554.4 | 97.1 | 6.2 | 1409.5 | 88.1 | 1.3 | ||
6400 | 5621.5 | 87.8 | 3.7 | 6363.2 | 99.4 | 2.8 | ||
Tolbutamide | 320 | 319.2 | 99.7 | 10.4 | 315.9 | 98.7 | 5.8 | 0.999 |
1600 | 1550.3 | 96.9 | 9.0 | 1722.5 | 107.7 | 0.9 | ||
6400 | 6494.9 | 101.5 | 6.5 | 6539.4 | 102.2 | 0.7 | ||
Gliclazide | 3200 | 2534.1 | 79.2 | 18.1 | 3013.1 | 94.2 | 2.7 | 0.994 |
16,000 | 16,034.9 | 100.2 | 6.8 | 14,888.0 | 93.1 | 1.1 | ||
64,000 | 67,399.1 | 105.3 | 4.1 | 71,709.9 | 112.1 | 3.8 |
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Li, Z.; Xie, H.; Fu, T.; Li, Y.; Shen, X.; Li, X.; Lei, Y.; Yao, X.; Koidis, A.; Liu, Y.; et al. Complementary Strategy Enhancing Broad-Specificity for Multiplexed Immunoassay of Adulterant Sulfonylureas in Functional Food. Biosensors 2022, 12, 591. https://doi.org/10.3390/bios12080591
Li Z, Xie H, Fu T, Li Y, Shen X, Li X, Lei Y, Yao X, Koidis A, Liu Y, et al. Complementary Strategy Enhancing Broad-Specificity for Multiplexed Immunoassay of Adulterant Sulfonylureas in Functional Food. Biosensors. 2022; 12(8):591. https://doi.org/10.3390/bios12080591
Chicago/Turabian StyleLi, Zhaodong, Haihuan Xie, Tingdan Fu, Yingying Li, Xing Shen, Xiangmei Li, Yi Lei, Xiaojun Yao, Anastasios Koidis, Yingju Liu, and et al. 2022. "Complementary Strategy Enhancing Broad-Specificity for Multiplexed Immunoassay of Adulterant Sulfonylureas in Functional Food" Biosensors 12, no. 8: 591. https://doi.org/10.3390/bios12080591