First Immunoassay for Measuring Isoaspartate in Human Serum Albumin
Abstract
:1. Introduction
2. Results
2.1. Identification of the “IsoAsp Meter” Peptide in HSA
2.2. Comparison Results of Different ELISAs
2.3. Indirect ELISA of Deamidated HSA
2.4. Sequence Analysis of mAb Variable Region
2.5. Kinetic Analyses
2.6. Paratope Mapping
2.7. Glycosylation Effect on 1A3 Specificity
2.8. Effect of the Ig isotype
2.9. Determination of IsoAsp Levels in Healthy Plasma
3. Discussion
4. Materials and Methods
4.1. Artificial Deamidation of Proteins
4.2. Generation of mAbs against the “IsoAsp Meter” in HSA
4.3. Indirect ELISA
4.4. DNA Sequence Analysis of mAbs Variable Regions
4.5. De Novo Protein Sequencing of 1A3
4.6. Surface Plasmon Resonance (SPR)
4.7. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)
4.8. Deglycosylation of 1A3 mAb
4.9. Production of 1A3* mAb
4.10. ELISA of Blood Plasma from Healthy Donors
5. Conclusions
6. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Indirect ELISA | Indirect Sandwich ELISA | ||||
---|---|---|---|---|---|
Plate | White Polystyrene | Copper Coated | White Polystyrene | White Polystyrene | Copper-coated |
Blocking buffer | 10% Milk in PBST | ||||
Capture antibody | N/A | 15C7 anti-HSA mAb | 1A3 mAb | 1A3 mAb | |
Primary/Detection antibody | 1A3 mAb | 1A3 mAb | 1A2 mAb | 1E1 anti-HSA mAb | |
Secondary antibody | Peroxidase AffiniPure Polyclonal Goat Anti-Mouse IgG + IgM (H + L) | ||||
Detection | Chemiluminescence | Absorbance | Chemiluminescence | Chemiluminescence | Absorbance |
S/N at 6% isoAsp | 39.9 | 12.1 | 0.8 | 0.6 | 0.8 |
p value | <10−5 | <10−5 | 7.0 × 10−3 | <10−5 | <10−5 |
S/N at 0.6% isoAsp | 4.3 | 3.6 | N/A |
No. | Poition of Glycans | ∆M (kD) |
---|---|---|
a | HexNAc[4]Hex[4] | +1.461 |
b | HexNAc[4]Hex[3] | +1.298 |
c | dHex[1]HexNAc[3]Hex[3] | +1.241 |
d | dHex[1]HexNAc[4]Hex[6] | +1.931 |
e | dHex[1]HexNAc[4]Hex[4] | +1.607 |
f | dHex[1]HexNAc[4]Hex[3] | +1.445 |
g | HexNAc[2]Hex[5] | +1.216 |
h | HexNAc[4]Hex[5]NeuAc[1] | +1.914 |
Antibody-Antigen | ka (M−1 s−1) | kd (s−1) | KD (M) |
---|---|---|---|
1A3-aHSA | 2.6 ± 0.5 × 10−5 | 5.3 ± 0.4 × 10−3 | 2.1 ± 0.3 × 10−8 |
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Wang, J.; Lundström, S.L.; Seelow, S.; Rodin, S.; Meng, Z.; Astorga-Wells, J.; Jia, Q.; Zubarev, R.A. First Immunoassay for Measuring Isoaspartate in Human Serum Albumin. Molecules 2021, 26, 6709. https://doi.org/10.3390/molecules26216709
Wang J, Lundström SL, Seelow S, Rodin S, Meng Z, Astorga-Wells J, Jia Q, Zubarev RA. First Immunoassay for Measuring Isoaspartate in Human Serum Albumin. Molecules. 2021; 26(21):6709. https://doi.org/10.3390/molecules26216709
Chicago/Turabian StyleWang, Jijing, Susanna L. Lundström, Sven Seelow, Sergey Rodin, Zhaowei Meng, Juan Astorga-Wells, Qinyu Jia, and Roman A. Zubarev. 2021. "First Immunoassay for Measuring Isoaspartate in Human Serum Albumin" Molecules 26, no. 21: 6709. https://doi.org/10.3390/molecules26216709