Immunoblotting detection method:The nitrocellulose membranes were blocked with buffer containing 5 % (w/v) skimmed milk powder, and 0.1 % (v/v) Tween 20 in phosphate buffered saline (PBS (0.01 M potassium phosphate buffer, pH 7.4, 0.13 M sodium chloride)). Nitrocellulose strips were incubated overnight with patients sera. After incubation with rabbit anti-human IgE (1:4000), biotinylated goat anti-rabbit IgG (1:6000) and streptavidin-horseradish peroxidase conjugate (1:20000), the blots were stained with 3,3',5,5'-tetramethylbenzidine-dioctylsodiumsulphosuccinate (Kinder et al. 1999)
The nitrocellulose membranes were blocked with buffer containing 5 % (v/v) skimmed milk powder, and 0.1 % (v/v) Tween 20 in phosphate buffered saline (PBS (0.01 M potassium phosphate buffer, pH 7.4, 0.13 M sodium chloride)). Nitrocellulose strips were incubated overnight with patients sera. After incubation with rabbit anti-human IgE (1:4000), biotinylated goat anti-rabbit IgG (1:6000) and streptavidin-horseradish peroxidase conjugate (1:20000), the blots were stained with 3,3',5,5'-tetramethylbenzidine-dioctylsodiumsulphosuccinate (Paschke et al. 2001) [145] [146] Protein were transferred on a nitrocellulose membrane (0.2 µm). After blotting, one strip with separated extract and one strip with molecular-mass-marker proteins were briefly stained with colloidal gold to visualize the transfer of proteins. To prevent unspecific binding, the dried membranes were treated twice with 5% (w/v) skimmed milk powder and 0.1% (v/v) Tween 20 in PBS. Subsequently nitrocellulose strips were incubated with pooled patients' sera diluted 1:14 with incubation buffer (0.3% (w/v) BSA, 0.1% (v/v) Tween 20 in PBS). The strips were then incubated with rabbit antihuman IgE (1:4000, 60 min), biotinylated goat antirabbit IgE (1:6000, 60 min), and streptavidin-horseradish peroxidase conjugate (1:20000; 20 min), respectively, and stained with 3,3',5,5'-tetramethylbenzidine (TMB) and dioctylsodiumsulfosuccinate (DONS) (Dube et al. 2004)
Immunoblotting results:IgE binding was observed to 25-50 kDa mango polypeptides together with IgE binding to a 40 kDa latex protein (Duque et al. 1999)
In another study, IgE-binding proteins with molecular masses of approximately 14, 30, 40, 43, and 67 kDa were detected in mango (Paschke et al. 2001) [145]
A thirth study showed IgE binding in 46/52 allergic individuals of 30 and 40 kDa polypeptides. In addition, IgE-binding proteins were seen at 14 kDa (8 sera), 16 kDa (6 sera), 25 kDa (6 sera ), 43 kDa (17 sera), 50 kDa (12 sera) and 67 kDa (21 sera) (Paschke et al. 2001) [146]
The allergens in extracts of the mango varieties Osteen, Eden, Tommy Atkins and Ngowe were characterized by SDS-PAGE / immunoblotting using seven sera from mango allergic patients. All sera detected the same allergens in each strain. Allergens with Mr of approx. 14, 30, 40, 43 and 67 kDa were visible in each strain. Furthermore there was no difference between the varieties in detection intensity of any allergen (Kinder et al. 1999)
IgE binding in 7/9 individuals of 30 kDa, in 6/9 of 40 kDa major allergen was observed. Specific binding was also observed for allergens with molecular masses of 67, 60, 43, 37, 20, 18, 16, 14 kDa, and above 67 kDa (Dube et al. 2004)