Development of a sensitive and reliable ELISA for quantification of wheat germ agglutinin.
Among others, lectin-mediated drug delivery is currently discussed as a promising strategy towards improved bioavailability of biotech drugs. For quantitative determination of the lectin from wheat germ (WGA), a sandwich ELISA relying on capture of the lectin by pig gastric mucin coated wells and detection of bound WGA by a lectin specific first antibody followed by peroxidase-labelled second antibody was elaborated. The stepwise optimised protocol allows quantification over the range from 10 to 1000 ng/ml WGA with a coefficient of determination of 0.9991. The day to day variation was +/-0.09 OD at 500 ng/ml WGA. Additionally, the presented ELISA-protocol allows determination of WGA in serum with the same sensitivity and reliability as in buffer. Lectins with different carbohydrate specificity such as those from jack beans and peanuts exhibited no cross-reactivity. Among the lectins with the same carbohydrate specificity that from potatoes interfered with the assay, whereas that from tomatoes was not recognised by the first antibody. Since the potato lectin is fully degraded in the intestine, no cross-reactivity with WGA is expected in serum samples. Following on from these results, the absorption rate of WGA in biologically active form might be determined as a basis for further steps towards improved drug delivery systems.
Güll
Iris
I
Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna, Austria.
Wirth
Michael
M
Gabor
Franz
F
D000818:Animals; D000906:Antibodies / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005752:Gastric Mucins / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000276:immunology; D010969:Plastics / Q000737:chemistry; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013552:Swine; D013997:Time Factors; D014909:Wheat Germ Agglutinins / Q000032:analysis* / Q000097:blood / Q000737:chemistry
D000906:Antibodies; D005752:Gastric Mucins; D037102:Lectins; D010969:Plastics; D014909:Wheat Germ Agglutinins; D012710:Serum Albumin, Bovine; D006861:Hydrogen Peroxide