Dot blot chemiluminescence assay for studying food protein binding to small intestinal brush border membranes in vitro.
Interactions of food proteins with the apical membrane of small intestinal epithelial cells can influence enterocytic antigen handling. For studying these interactions in vitro, isolated brush border membrane vesicles are a widely accepted model. In order to improve measurement of food protein binding, we developed a sensitive dot blot chemiluminescence assay. This assay comprises immobilization of membrane vesicles on nitrocellulose, detection of bound biotinylated food proteins by a peroxidase-catalyzed chemiluminescence reaction, and densitometric quantitation of signal intensities. By using this assay, saturation of brush border membrane binding of food proteins (gliadin peptides, alpha-casein, beta-lactoglobulin, ovalbumin) was demonstrated. Inhibition studies indicated components of specific membrane binding of gliadin peptides, alpha-casein and beta-lactoglobulin, whereas aggregation tendency of ovalbumin interfered with inhibition experiments. Maximal binding intensities of gliadin peptides (22.2 +/- 1.2 densitometric units (d.u.)/microgram membrane protein), alpha-casein (27.9 +/- 1.7 d.u./microgram) and ovalbumin (21.3 +/- 1.6 d.u./microgram) were comparable to sugar-specific lectin binding (range from 23.4 to 35.1 d.u./microgram), in contrast to significantly less binding of beta-lactoglobulin (6.8 +/- 0.6 d.u./microgram). The dot blot chemiluminescence assay is appropriate for characterizing interactions between food proteins and brush border membranes. Its sensitivity makes investigation of pathological membrane alterations possible. Besides, it might be useful for any studies defining ligand-membrane interactions.
Bolte
G
G
University Children's Hospital, Tuebingen, Germany.
Knauss
M
M
Metzdorf
I
I
Stern
M
M
D000818:Animals; D002364:Caseins / Q000378:metabolism; D004044:Dietary Proteins / Q000378:metabolism*; D005260:Female; D005903:Gliadin / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism* / Q000648:ultrastructure*; D007782:Lactoglobulins / Q000378:metabolism; D008163:Luminescent Measurements; D008871:Microvilli / Q000378:metabolism; D010047:Ovalbumin / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
D002364:Caseins; D004044:Dietary Proteins; D007782:Lactoglobulins; D010047:Ovalbumin; D005903:Gliadin