WO2006051145A1 - Elisa competitif de detection de gluten hydrolyse et applications - Google Patents

Elisa competitif de detection de gluten hydrolyse et applications Download PDF

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Publication number
WO2006051145A1
WO2006051145A1 PCT/ES2005/070025 ES2005070025W WO2006051145A1 WO 2006051145 A1 WO2006051145 A1 WO 2006051145A1 ES 2005070025 W ES2005070025 W ES 2005070025W WO 2006051145 A1 WO2006051145 A1 WO 2006051145A1
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WO
WIPO (PCT)
Prior art keywords
standard
hydrolyzed
gliadins
gluten
monoclonal antibody
Prior art date
Application number
PCT/ES2005/070025
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English (en)
Spanish (es)
Inventor
Enrique MÉNDEZ CORMÁN
Enrique Garcia Ortiz
Sergio FERRE LÓPEZ
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Consejo Superior De Investigaciones Científicas
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Application filed by Consejo Superior De Investigaciones Científicas filed Critical Consejo Superior De Investigaciones Científicas
Publication of WO2006051145A1 publication Critical patent/WO2006051145A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/10—Starch-containing substances, e.g. dough
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants

Definitions

  • This invention relates, in general, to the analysis of foods for celiac patients, and, in particular, relates to a method for the identification of hydrolyzed gluten in foods and raw materials, and in particular to an immunosorbent assay with bound enzymes. (ELISA) competitive, and the kits comprising said ELISA.
  • ELISA immunosorbent assay with bound enzymes.
  • gluten detection and quantification techniques are very important, both in prepared foods and in the raw materials with which they are to be prepared.
  • Gluten which is part of a group of proteins called prolamines, is made up of various proteins and is found in cereals, some of them being toxic to celiacs, such as wheat (gliadins), barley ( hordeinas) and rye (secalinas).
  • celiacs such as wheat (gliadins), barley ( hordeinas) and rye (secalinas).
  • Much of the food for celiac patients has undergone hydrolysis processes in its preparation.
  • Gluten is frequently hydrolyzed in products such as syrups, baby foods, beers, etc., and it cannot be measured accurately using the ELIS A-R5 Sandwich system (Valdes I, Garc ⁇ a E, Llórente M, Méndez E.
  • the invention faces the problem of providing new procedures and tools for the identification of gluten in food and raw materials.
  • an object of the present invention is an "equipment" ELISA kit
  • an object of the present invention is a Competitive ELISA kit, hereinafter Competitive ELISA kit of the present invention, based on the use of a single monoclonal antibody that recognizes a gluten fragment with one or more epitopes related to the gluten toxicity in patients with celiac disease, preferably the R5 monoclonal antibody (Osman AA, Uhlig HH, Valdes I, Amin M, Méndez E, Mothes T. A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins. Eur J Gastroenterol Hepatol.
  • a standard selected from the following group: a) a standard made from a set of non-hydrolyzed native gliadins, preferably the "native" EE standard or b) of a "pool” of different digestions made on a standard made from a set of native gliadins, preferably, from the EE, with different proteolytic enzymes, for the detection and quantification of prolamins (gliadins, hordeins and secalins) partially hydrolysates present as contaminants in food or raw materials, which have undergone proteolytic treatment.
  • epitopes related to gluten toxicity in patients with celiac disease refers to epitopes belonging to the following group: QQPFP, LQPFP, QLPYP, QLPTP, QQSFP, QQTFP, PQPFP, QQPYP, PQPFP (Valdes I, Garda E, Llorente M, Méndez E.
  • QQPFP Low-level gluten determination in foods using a novel sandwich enzyme- linked immunosorbent assay protocol.
  • Another specific object of the present invention is the Competitive ELISA kit of the present invention in which the monoclonal antibody is the R5 monoclonal antibody and the selected standard is a "pool" of different digestions made on the EE with different proteolytic enzymes, preferably with the enzymes belonging to the following group: pepsin, trypsin / pepsin, trypsin and chymotrypsin.
  • Another object of the present invention is the use of the Competitive ELISA kit of the present invention for the identification and quantification of hydrolyzed prolamines (gliadins, hordeins and secaline) present as contaminants in food or raw materials, which have undergone proteolytic treatment.
  • hydrolyzed prolamines gliadins, hordeins and secaline
  • those belonging to the following group those belonging to the following group: syrups, baby foods (gluten-free porridge, hydrolyzed porridge, etc.) and beers.
  • the advantages of this new Competitive ELISA R5 are: a) The use as standard of complete gliadins, preferably the European Standard (hereinafter, EE) of "native" gliadins, which is validated by the Prolamin Working Group, and recommended for use in ELISAs for the detection of gluten, it can be obtained commercially and does not require special preparation.
  • EE European Standard
  • This advantage is the most relevant in the development of this Competitive ELISA since it can universalize its use, in a reliable and reproducible way, solving the problem of finding a representative hydrolyzed standard of all types of processes that can undergo raw materials from its origin until they become part of a final product.
  • a pool of enzymatic digesters from the EE can also be used as standard in the Competitive ELISA-R5 with an efficiency similar to that of the "native", with the disadvantage that the preparation of the digested pool is more laborious and less reproducible.
  • the advantage of Competitive ELISA-R5 is that it allows quantifying in its entirety fragments of wheat or barley / rye prolamines of high or low molecular weight containing one or more epitopes, compared to the ELISA-R5 Sandwich which is limited to quantify fragments with more than two epitopes.
  • FIG. 1 SDS-PAGE (Left) and Western-Blot R5 (right) of the results of the "native" EE (Nat.) Of the digestions of the gliadin EE, with Pepsin (P), Trypsin (T), Trypsin + Pepsin (T + P), Chymotrypsin (QT), and Pool (prepared equally, P: T: T + P: QT: Native; l: l: l: l: l: l).
  • Example L- Assessment of a Competitive R5 ELISA For the design of a Competitive ELISA (Crowther JR (1995) ELISA, theory and practice. Methods in Molecular Biology, vol 42, pp 35-50. New Jersey: Humana Press Inc.) which Be able to detect and quantify small fragments of hydrolyzed prolamines present in certain foods such as syrups, beers, baby foods, etc., there are two problems that must be addressed: a) The first one is to select what type of hydrolyzed standard should be used, which It is similar to that of hydrolyzed gliadins in food, to detect hydrolyzed prolamines, and with which to perform the standard curve.
  • the EE ethanol solution is diluted to 1 mg / ml (actual, 40,000 ppm) with 60% ethanol, a 1: 800 dilution of the starting solution must be performed.
  • Those of 25 and 12.5 are prepared in serial dilutions at 1 A in 60% ethanol, and that of 5 ppm 1 / 2.5 with respect to that of 12.5.
  • ELISA-R5 Competitive 1.- Upholstery of the ELISA plate:
  • the coating solution is prepared from the EE solution (at 1 mg / ml), making a 1: 8000 dilution in coating buffer.
  • Dilution 1:25 and 1:50 if higher dilutions are necessary they can be prepared from these above by 1: 2 dilution.
  • conjugate As conjugate the monoclonal antibody R5 conjugated with peroxidase diluted 1: 166667 is used. The dilution of the conjugated antibody is prepared just before preincubation with the samples.
  • the diluted samples and conjugated antibody are mixed v / v to pre-incubate them before applying them on the ELISA plate.
  • 0.3 ml of the diluted samples and the standards are mixed with 0.3 ml of the diluted conjugate antibody 1: 166667. It is incubated at room temperature while constantly stirring in the orbital shaker at the minimum speed.
  • the final concentrations of the standards are as follows: C 1 (1.25 ⁇ g / ml) C 2 (0.31 ⁇ g / ml) C 3 (0.078 ⁇ g / ml) C 4 (0.019 ⁇ g / ml)
  • the absorbance is read on an ELISA plate reader at 450 nm.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Food Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un kit ELISA compétitif d'identification de gluten hydrolysé dans des aliments et des matières premières, et son utilisation. L'avantage de ce kit par rapport à d'autres est qu'il permet l'identification de gluten hydrolysé.
PCT/ES2005/070025 2004-03-15 2005-03-11 Elisa competitif de detection de gluten hydrolyse et applications WO2006051145A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200400636 2004-03-15
ES200400636A ES2239545B1 (es) 2004-03-15 2004-03-15 Elisa competitivo para la deteccion de gluten hidrolizado y sus aplicaciones.

Publications (1)

Publication Number Publication Date
WO2006051145A1 true WO2006051145A1 (fr) 2006-05-18

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WO (1) WO2006051145A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013045737A1 (fr) 2011-09-29 2013-04-04 Universidad De Valladolid Peptide immunogène du gluten et ses applications
WO2013178844A1 (fr) * 2012-05-31 2013-12-05 Universidad De Oviedo Aptamères spécifiques contre le gluten et procédé de détection du gluten associé

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1424345A1 (fr) * 2001-05-14 2004-06-02 Consejo Superior De Investigaciones Cientificas Procede d'extraction de gluten contenu dans des aliments ayant subi ou non un traitement thermique, compatible avec un dosage immunoenzymatique (elisa), composition et necessaires renfermant ladite composition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1424345A1 (fr) * 2001-05-14 2004-06-02 Consejo Superior De Investigaciones Cientificas Procede d'extraction de gluten contenu dans des aliments ayant subi ou non un traitement thermique, compatible avec un dosage immunoenzymatique (elisa), composition et necessaires renfermant ladite composition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BAZZIGALUPPI E. ET AL: "Comparison of Tissue Transglutaminase-Specific Antibody Assays with Established Antibody Measurements for Coeliac Disease.", JOURNAL OF AUTOIMMUNITY., vol. 12, 1999, pages 51 - 56, XP002281144 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013045737A1 (fr) 2011-09-29 2013-04-04 Universidad De Valladolid Peptide immunogène du gluten et ses applications
WO2013178844A1 (fr) * 2012-05-31 2013-12-05 Universidad De Oviedo Aptamères spécifiques contre le gluten et procédé de détection du gluten associé
ES2436861A1 (es) * 2012-05-31 2014-01-07 Universidad De Oviedo Aptámeros específicos contra el gluten y método de detección del gluten asociado

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Publication number Publication date
ES2239545B1 (es) 2006-12-16
ES2239545A1 (es) 2005-09-16

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