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A novel method for in situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM.

Authors
  • Duan, Zhi
  • Brüggemann, Dagmar Adeline
  • Siegumfeldt, Henrik
Type
Published Article
Journal
Journal of Agricultural and Food Chemistry
Publisher
American Chemical Society
Publication Date
Nov 11, 2009
Volume
57
Issue
21
Pages
10049–10054
Identifiers
DOI: 10.1021/jf901026m
PMID: 19831397
Source
Medline
License
Unknown

Abstract

A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward three small synthetic peptides of the alpha(s1)-casein sequence. These peptides traverse enzymatic cleavage sites of casein during cheese ripening. The specificity of the generated anti-peptide antibodies was determined by ELISA and Western blot. Finally, an immunofluorescent labeling protocol was successfully developed for the detection of scFvs binding to different alpha(s1)-casein fragments inside a cheese matrix by CLSM. To our knowledge, this is the first demonstrated immunofluorescent labeling method for in situ analysis of proteolysis phenomena in the cheese matrix. Additionally, this technique offers a high potential to study in situ dynamic spatial changes of target components in complex food systems.

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