Related Experiment Video
Updated: Aug 29, 2026
Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
Published on: May 4, 2012
Single-chain antibody fragment-based adsorbent for the extracorporeal removal of beta2-microglobulin
Eric A Grovender1, Brenda Kellogg, Jasleen Singh
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Background:
Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of beta2-microglobulin (beta2-m). Removal of beta2-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to beta2-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution.
Methods:
An antihuman beta2-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent beta2-m in serum.
Results:
The displayed scFv possessed a nanomolar affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed beta2-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human beta2-m.
Conclusion:
Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.
Insights
Engineered antibody fragments (scFvs) show high affinity for beta2-microglobulin, offering a novel approach to remove this protein during dialysis. This method may reduce the loss of essential proteins associated with current therapies.
Area of Science:
- Biotechnology
- Immunology
- Nephrology
Background:
- Dialysis-related amyloidosis (DRA) is a complication of end-stage renal disease (ESRD) linked to beta2-microglobulin (beta2-m) accumulation.
- Current therapies for beta2-m removal are nonspecific, leading to the loss of vital proteins.
- Single-chain variable region (scFv) antibody fragments offer advantages over whole antibodies for targeted protein removal due to their size and genetic definition.
Purpose of the Study:
- To engineer and characterize a novel immunoadsorbent using scFv fragments for specific beta2-microglobulin removal.
- To evaluate the binding affinity and capacity of the scFv-based immunoadsorbent.
Main Methods:
- Constructed an anti-human beta2-m scFv from the BBM.1 hybridoma using yeast display.
- Quantified binding affinity via flow cytometry and measured immunoadsorbent capacity using fluorescent beta2-m in serum.
- Expressed soluble scFv, purified it, and immobilized it onto agarose beads.
Main Results:
- The scFv demonstrated nanomolar binding affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L).
- The immunoadsorbent achieved an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel.
- Adsorbed beta2-m to antibody mass ratio was 70% higher than reported for whole antibodies, with preliminary specificity confirmed.
Conclusions:
- Recombinant DNA technology successfully created an scFv-based immunoadsorbent.
- Immobilized scFvs could minimize valuable protein loss during hemodialysis.
- This approach facilitates the removal of large macromolecules exceeding membrane cut-offs.
More Related Videos
09:22Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum
Published on: April 13, 2018
14:43Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications
Published on: September 23, 2013