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

TY - JOUR

T1 - A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

AU - Duan, Zhi

AU - Brüggemann, Dagmar Adeline

AU - Siegumfeldt, Henrik

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1021/jf901026m

DO - 10.1021/jf901026m

M3 - Journal article

C2 - 19831397

SN - 0021-8561

VL - 57

SP - 10049

EP - 10054

JO - Journal of Agricultural and Food Chemistry

JF - Journal of Agricultural and Food Chemistry

IS - 21

ER -