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[Screening, expression and characterization of anti-human CD20 nanobodies]
Yanning Li1, Guangqi Li1, Hongxia Wang1
1Department of Medical Laboratory, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China.
Abstract:
Objective To screen the sequence of nanobodies against human CD20, and obtain anti-CD20-human IgG Fc nanobodies with high affinity and specificity. Methods Based on the naive phage display library, 4 rounds of liquid affinity screening were performed using biotinylated CD20 antigen as the target, and positive clones were identified by ELISA. Prokaryotic expression vector CD20-IgG Fc/pCZN1 was constructed and transformed into E.coli Arctic Express, and the expression of the recombinant protein was induced by IPTG at low temperature and purified by Ni column. The purified product was identified by ELISA and Western blot analysis. Results The specific CD20 nanobody showed good repeatability and hydrophilicity. The purity of anti-CD20-human IgG Fc nanobodies was higher than 85%. ELISA indicated that anti-CD20-human IgG Fc nanobodies had high affinity with CD20 antigen, and Western blot analysis demonstrated they could specifically recognize CD20 antigen. Conclusion The sequence of anti-CD20 nanobody was successfully obtained using the naive phage nanobody library. The purified anti-CD20-human IgG Fc nanobody has high affinity and specificity.
Insights
Researchers screened nanobodies targeting human CD20, successfully obtaining high-affinity and specific anti-CD20-human IgG Fc nanobodies. This advancement offers potential for targeted therapies and diagnostics.
Area of Science:
- Immunotechnology
- Biochemistry
- Molecular Biology
Background:
- The human CD20 antigen is a significant target in B-cell malignancies.
- Development of high-affinity and specific antibodies is crucial for targeted therapies.
Purpose of the Study:
- To screen and obtain nanobodies with high affinity and specificity against human CD20.
- To produce and characterize anti-CD20-human IgG Fc nanobodies.
Main Methods:
- Utilized a naive phage display library for four rounds of liquid affinity screening against biotinylated CD20.
- Constructed a prokaryotic expression vector for recombinant protein expression in E. coli.
- Purified the nanobodies using Ni column chromatography and validated via ELISA and Western blot.
Main Results:
- Identified specific CD20 nanobodies exhibiting good repeatability and hydrophilicity.
- Achieved a purity of anti-CD20-human IgG Fc nanobodies exceeding 85%.
- ELISA and Western blot confirmed high affinity and specific recognition of the CD20 antigen.
Conclusions:
- Successfully obtained the sequence of an anti-CD20 nanobody using a naive phage nanobody library.
- The purified anti-CD20-human IgG Fc nanobody demonstrates high affinity and specificity for the CD20 antigen.
