Journal
ANALYTICAL CHEMISTRY
Volume 88, Issue 24, Pages 12371-12379Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.6b03614
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Funding
- National Research Program for Biopharmaceuticals
- Ministry of Science and Technology, Taipei, Taiwan [MOST 105-2325-B-037-002, MOST 105-2325-B-037-001, MOST 105-2325-B-041-001, MOST 105-2314-B-037-010-MY3]
- Kaohsiung Medical University [KMU-TP103C00, KMU-TP104C00, KMU-TP105C00, KMU-TP104D06, D08-00005]
- KMU [KMU-DT105003]
- Grant of Biosignature in Colorectal Cancers, Academia Sinica, Taiwan [BM10501010045]
- Health and Welfare Surcharge of Tobacco Products [MOHW105-TDU-B-212-134007]
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Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG(5K)-NH2 and mPEG(5K)-NH2) or PEG-conjugated small molecules (mPEG(5K)-biotin and mPEG(5K)-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL(-1) levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL(-1). All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum dot quantification down to 7 pM. These results suggest that the combination of 293T/15-2b cells and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules, and PEGylated proteins, whereas the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles. The optimized anti-PEG cell-based sandwich ELISA can provide a sensitive, precise, and convenient tool for the quantification of a range of PEGylated molecules.
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