Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals.
To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.
Beaugrand
Johnny
J
Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium.
Gebruers
Kurt
K
Ververken
Cedric
C
Fierens
Ellen
E
Dornez
Emmie
E
Goddeeris
Bruno M
BM
Delcour
Jan A
JA
Courtin
Christophe M
CM
D002352:Carrier Proteins / Q000032:analysis; D002523:Edible Grain / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D010940:Plant Proteins / Q000032:analysis*; D014908:Triticum / Q000737:chemistry*
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D010940:Plant Proteins; C422246:TAXI I protein, Triticum aestivum; C422247:TAXI II protein, Triticum aestivum; C485063:XIP-I protein, Triticum aestivum; D043364:Endo-1,4-beta Xylanases