Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods.
OBJECTIVE
In the past, one of the major problems in gluten analysis has been the unavailability of an efficient, universal, extraction procedure of gliadins - the alcohol-soluble proteins of gluten - from both heat processed and unprocessed products. This study was designed to develop a universal, extraction procedure capable of extracting the totality of gliadins from both unprocessed and heat processed foods for coeliac patients.


METHODS
A simple quantitative extraction solution containing 250 mM 2-mercaptoethanol and 2 M guanidine hydrochloride ('cocktail'), was developed to extract gliadins from heated foods.


RESULTS
The diluted reducing and disaggregating agents reaching the micro plate at low concentration do not affect the ELISA system based on the R5 monoclonal antibody. The recovery of gliadins extracted by the cocktail from spiked samples was nearly complete, with an average mean value of 95.5%, which is clearly superior to 44.4% obtained with conventional 60% aqueous ethanol. The cocktail always yielded either slightly similar or higher values than 60% aqueous ethanol depending on the type of foods: 1.1-fold in unheated foods, 1.4-fold in wheat starches and 3.0-fold in heated foods. False positives or negatives were never observed using the cocktail solution.


CONCLUSIONS
We present a general complete gliadin extraction procedure based on reducing and disaggregating agents for both heated and unheated foods as a crucial tool for gliadin analysis. The new extraction solution is used for corresponding proteins from rye (secalins) and barley (hordeins). The cocktail was employed as the extraction method in the international ring trial evaluation of sandwich R5-ELISA as proposed by the Codex Alimentarius and organized by the Working Group on Prolamin Analysis and Toxicity.
García
Enrique
E
Unidad de Gluten, Centro Nacional de Biotecnología, Madrid, Spain.
Llorente
Mercedes
M
Hernando
Alberto
A
Kieffer
Rolf
R
Wieser
Herbert
H
Méndez
Enrique
E
D015153:Blotting, Western; D002446:Celiac Disease / Q000178:diet therapy; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000431:Ethanol; D005504:Food Analysis / Q000379:methods*; D005511:Food Handling / Q000379:methods; D005903:Gliadin / Q000032:analysis / Q000302:isolation & purification*; D019791:Guanidine; D001467:Hordeum / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D008623:Mercaptoethanol; D010084:Oxidation-Reduction; D012434:Secale / Q000737:chemistry; D013696:Temperature
D000431:Ethanol; D008623:Mercaptoethanol; D005903:Gliadin; D019791:Guanidine