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Dual fluorescence confocal imaging of the accessibility and binding of F(ab′)2 to an EBA resin with various immobilised antigen densities

Paper ID Volume ID Publish Year Pages File Format Full-Text
35983 45117 2007 8 PDF Available
Title
Dual fluorescence confocal imaging of the accessibility and binding of F(ab′)2 to an EBA resin with various immobilised antigen densities
Abstract

Dual fluorescence confocal laser scanning microscopy has been used to visualise the binding of a fluorescently labelled polyclonal ovine anti-fluorescein F(ab′)2 antibody to immobilised fluorescein. The fluorescent ligand was immobilised on a Streamline quartz base agarose matrix; a resin used industrially for expanded bed chromatography, using two different fluorescein initial concentrations in order to obtain two batches of immunogen-affinity adsorbent with different immobilised ligand densities. The fluorescein specific F(ab′)2 were purified from anti-fluorescein serum pepsin digest by adsorption on immobilised antigen chromatographic resin, followed by conjugation to the fluorescent probe Alexa Fluor 660. The dual fluorescence signals from the immobilised antigen and the immuno-specific F(ab′)2 were used to map the progressive depth of the bound F(ab′)2 layer within individual adsorbent beads. In addition, the labelled anti-fluorescein F(ab′)2 was diluted to identical antigen binding activity concentrations in crude serum digest and in blank buffer and the resulting fluorescence intensity profiles were comparatively assessed for any detectable differences in binding patterns that might be caused by processing the more complex mixture of crude serum digests. It was observed that the relative immobilised ligand utilisation was higher when using the immuno-adsorbent with lower immobilised antigen density. Furthermore, the progression of the adsorbed F(ab′)2 front inside the immuno-adsorbent beads displayed closer agreement with the postulates of the shrinking core mechanism (SCM) when the immuno-adsorbent with lower immobilised antigen was used. The confocal images did not reveal any differences between the depth of the adsorption fronts of crude serum digest and pre-purified F(ab′)2 samples.

Keywords
Confocal scanning laser microscopy; F(ab′)2; Fluorescein; Immuno-affinity chromatography; Adsorbent; CSLM
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Dual fluorescence confocal imaging of the accessibility and binding of F(ab′)2 to an EBA resin with various immobilised antigen densities
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 42, Issue 5, May 2007, Pages 812–819
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us