JPH06209787A - Method of quality evaluation of durham wheat - Google Patents

Abstract

PURPOSE: To determine the quantity of non-drum grains in a durum wheat sample by an immunological reaction with a friabilin-specific monoclonal antibody.

CONSTITUTION: The friabilin content in a sample is determined by immunoassay with a friabilin-specific monoclonal antibody, and thereby the content of non- drum grains mixed in a durum wheat is determined.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

The present invention is directed to friabilin.
in), a method for quantifying non-durum kernels in a sample of durum wheat or a sample of a product containing durum wheat by immunological detection of a protein having a recognized characteristic.

Wheat is a major food source for humans worldwide. Many cultivars are known for wheat, and their characteristics determine the use of milled flour. Wheat can generally be classified as hard wheat or soft wheat. Soft wheat has a protein content of about 8-10% and is used in the manufacture of cakes, biscuits, crackers and the like. Hard wheat has a protein content of about 10-18% and is mainly used for bread making. Furthermore, durum wheat with a protein content of 10-16% is used for the production of pasta and semolina. Most of all wheat cultivated falls into the category of hard wheat or soft wheat. Durum wheat is relatively low in production and therefore expensive.

The quality of pasta produced from durum wheat flour depends on the quality of the flour. Unexpected contamination of non-durum wheat can occur during cultivation, storage and shipping. Further, durum wheat is inevitably expensive, and it is often revealed that the durum wheat is mixed with another kind of wheat or another grain in order to make a profit. Therefore, it is desirable to be able to confirm the durum wheat content in the flour sample so that the suitability of durum flour for pasta production can be evaluated.

Protein is about 0.2% of wheat starch granules
Make up. Characterization of starch granule associated proteins can be performed by polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. Ten major polypeptides are consistently detected in wheat starch granule proteins. These have a relative molecular weight Mr of about 5000, 8000, 14700, 19000.
And 5 kinds of surface proteins (su
(rface protein). Also, the molecular weight is 5
9,000, 77,000, 86,000, 95,000 and 1
49000 kinds of internal proteins (integra
1 protein). The terms "surface protein" and "internal protein" relate to the water temperature when extracting protein from starch particles with water,
Low molecular weight surface proteins extract at 20 ° C, internal proteins require higher temperatures of 50 ° C to 100 ° C.

The amount of furiavirin having a relative molecular weight of 14700 varies considerably depending on the variety of wheat. 147
The 00 polypeptide band is observed as a large band in all soft wheat, but is a relatively small band in hard wheat and the 14700 band is absent in durum wheat. The reason is that the corresponding gene is missing in the durum wheat genome. A method for screening furiavirin in wheat samples by gel electrophoresis is described in B. D. Sulaiman & W. R. Mor
Rison, Journal of Cereal S
Science 12 (1990) pages 53-61.

Furiavirin has been partially sequenced and the amino-terminal sequence of 36 amino acids corresponding to 1/3 of the molecule has been determined as follows: Ser-Gly-Pro-Trp-Met-Cys. -T
yr-Pro-Gly-Gln-Ala-Phe-Gl
n-Val-Pro-Ala-Leu-Pro-Ala
-Cys-Arg-Pro-Leu-Leu-Arg-
Leu-Gln-Cys-Asn-Gly-Ser-G
ln-Val-Pro-Glu-Ala- An object of the present invention is to detect the presence or absence of furiavirin (which is suspected to be due to the incorporation of non-durum kernels) in durum wheat samples (flour and prepared foods). To calibrate non-durum kernels.

[0007] In general, the present invention relates to the use of a friavirin-specific monoclonal antibody for detecting the presence or absence of friavirin in a durum wheat sample by an immune reaction between a friavirin protein antigen and an antibody.

More specifically, one object of the invention is to determine the content of said other non-durum kernels in a grain sample containing durum wheat and another unexpected non-durum kernels. Prepare a monoclonal antibody specific for the furiavirin protein, measure the furiavirin content in the sample by an immunoassay using the monoclonal antibody, and determine the content of the other non-durum kernels in the sample by measuring the furiavirin content in the sample. To provide a method for quality evaluation of grain samples containing durum wheat and another unexpected non-durum grain, including the step of determining.

Friavirin-specific antibodies are antibodies that have sufficient specificity to give useful assay results and that bind to native or denatured furiavirin proteins. In particular, since the antibody binds to heat-denatured furiavirin, it can be used in heat-treated grain or cereal flour samples, such as heat-denatured flour foods. However, the antibody does not bind to furavirin, which has broken disulfide bonds.

Friavirin-specific monoclonal antibodies are prepared in a conventional manner. Typically, a mammal such as a mouse is immunized with furiavirin or a furiavirin-carrier protein conjugate, usually in the presence of an adjuvant. Repeat immunization until appropriate antibody levels are obtained. Next, a hybridoma is prepared by fusing an antibody-producing splenocyte derived from a mammal with a substantially immortal cell line, and a monoclonal antibody is isolated from the hybridoma.

The inventors have found that the antigenically effective region of furiavirin is adjacent to one of the disulfide bonds. However, it is possible that there is another antigenically effective region.

[0012] To facilitate the evaluation of the immune complex, it is usually done, for example, with an enzyme label (typically horseradish peroxidase or alkaline phosphatase), a radiolabel, a fluorescent label or any other labeling system known in the art. Label the antibody.

The immunoassay is carried out by ELISA (enzym
e-linked immunosorbent as
Say) is preferable. In this assay, an antibody linked to an enzyme (eg horseradish peroxidase or alkaline phosphatase) is reacted with antigenic furavirin to form a complex. The complex is evaluated by adding an enzyme substrate such as 3,3 ', 5,5'-tetramethylbenzidine, which decomposes to give a colored product. The resulting colored product is measured spectrophotometrically.

Alternatively, the complex may be evaluated by reaction with a second monoclonal antibody specific for the first antibody.
For example, an anti-mouse IgG monoclonal antibody can be used as the second antibody. The second antibody may be labeled with a suitable label, such as an enzyme, radio label, fluorescent label, colloidal gold, or any other suitable labeling system known in the art.

In a variant, a rod-shaped or strip-shaped plastic substrate (for example a dipstick) is provided with a surface coating which binds to the proteins in the wheat sample. The substrate is then incubated with labeled antibody and the amount of complex assessed. When using an enzyme-labeled conjugate, further incubation of the dipstick with a substrate that gives rise to a color reaction allows the abundance of furavirin to be determined by the degree of color development. The surface coating preferably comprises a nitrocellulose membrane. Film thickness 0.3-0.6
It has been found that the μ nitrocellulose membrane gives particularly good results due to the brightness and vividness of the color.

The present invention also affinity the wheat protein in the sample to determine the content of said other non-durum kernels in a grain sample containing durum wheat and another unexpected non-durum kernels. The column is bound by a non-specific protein (eg, agarose coated with a non-specific protein-binding substance) to completely block the remaining reactive groups on the affinity substrate (eg, milk protein such as casein). Treatment, treating the affinity substrate with a labeled monoclonal antibody (eg antibody-enzyme conjugate) specific for the furiavirin protein and detecting the presence of the resulting furiavirin-antibody conjugate using a precipitating substrate. Provided is a method for quality evaluation of grain samples containing durum wheat and another unexpected non-durum grain.

This method does not require an expensive spectrophotometer. Examples of precipitating substrates are diaminobenzidine or preferably 4-chloro-1-naphthol.

The abundance of furavirin may be measured by a latex agglutination assay in which a monoclonal antibody is bound to latex particles. Aggregation of the latex particles occurs due to reaction with the furiavirin antigen. Similarly, hemagglutination assays may be used that bind the antibody to sheep or human red blood cells.

The present invention also provides a furiavirin-specific monoclonal antibody, which enables quantitative evaluation of the furiavirin-antibody complex, and an antibody bound to an enzyme label.

The present invention also provides a conjugate of an antibody with an insoluble substrate such as latex, colloidal gold or plastic material.

The present invention further provides a kit for an ELISA assay for the content of furavirin in a durum wheat sample, which comprises an enzyme-linked monoclonal antibody specific for furavirin.

The present invention also provides a kit for assaying furiavirin content in a durum wheat sample, which comprises a plastic substrate having a surface coating that binds to a protein in the sample, and an enzyme-bound furiavirin-specific monoclonal antibody. provide.

The present invention further provides a second antibody which is sensitized and usually labeled to a monoclonal antibody.

Non-limiting examples of the present invention are described below. TWEEN 20 in the examples is a trademark (polyoxyethylene sorbitan monolaurate).

Example 1 Preparation of Monoclonal Antibody Conjugate F7F-HRP MS Islam & WH Stimson, Let
ters in Applied Microbiol
The general procedure described in Ogy 1987, Vol. 4, 85-89 was used. Mice were immunized with a furiavirin conjugate linked to 1-ethyl-3- (3-dimethylaminoprolyl) carbodiimide hydrochloride (EDC) to hemocyanin (KLH) of keyhole limpets. 50 mice per group (N
ZB / BALB / c F1 hybrid, female, 6-8 weeks of age) was administered with conjugate (equivalent to 10 μg of furiavirin) in the first injection with Freund's complete adjuvant, and then 4 Weekly injections of conjugate (equivalent to 5 μg of furavirin) were continued for 1 year. 10
One mouse was sacrificed, but at the end of the first year, one mouse used Friavirin-bovine serum albumin.
The LISA test showed antibody activity. Kill this mouse,
Splenocytes were fused with myeloma cells using the general procedure described above. The produced monoclonal antibody was named F7F.

Two-step sodium periodate method (Boor
sma DM & Treefkerk JG
(1979) J. Immunol. Methods, V
30, 245), F7F monoclonal antibody was conjugated to horseradish peroxidase (HRP) to prepare F7F-HRP conjugate.

Constant of common wheat for durum wheat in pasta; [0027] Example 2 direct ELISA
The following procedure was used.

1.50 mg of finely ground dry pasta or flour is weighed.

2.50 mM Tris / HCl buffer Add 1 ml of extraction buffer containing 0.25% w / v sodium dodecyl sulphate (SDS) in pH 7.4.

3. The pasta sample in extraction buffer is ground with a mortar and pestle or incubated for 1 hour at room temperature. For flour samples, incubation is 10 minutes.

4. The pasta extract is centrifuged to remove insoluble particles. Then, 20 mM Tris / HCl buffer containing 150 mM NaCl was added so that attachment of the sample to the ELISA wells was prevented as little as possible.
The supernatant is diluted to 1/25 with pH 7.4 (TBS) and SD is added.
Make the S concentration less than 0.02%. If a centrifuge is not available, dilute all samples to 1/25 with TBS and
All that is required is to bring to ml and stir well before allowing insoluble material to settle.

A volume of 5.3 × 150 μl was added to the appropriate ELISA
Apply to A plate wells.

6. Plate at 37 ° C for 3 hours or 4 ° C
Incubate overnight.

The plate wells are washed 6 times with TBS buffer containing 7.05% TWEEN 20 detergent.

8. Dry the plate with a paper towel.

T containing 9.0.05% TWEEN 20
1 with 10% v / v normal sheep serum in BS buffer
F7F-HRP (antibody-horseradish peroxidase) diluted / 500 is added in an amount of 150 μl / well.

10. If the furavirin protein is present, the plates are incubated at 37 ° C for 1.5 hours to allow the protein to bind to the antibody conjugate.

11.05% v / v TWEEN
Wash plate wells 6 times with TBS containing 20 times.

12. Dry the plate with tissue paper.

13.6 mg / ml of dimethyl sulfoxide containing 3,3'5,5'-tetramethylbenzidine (TMB) and 0.1 M sodium acetate, adjusted to pH 6.0 with solid citric acid. Substrate prepared from 100 dilution stock solution was added and 5 μl of 30% hydrogen peroxide was added to 25
Add to TMB solution diluted to ml and add in volume of 150 μl / well.

14. Hold for 15 minutes at room temperature to react HRP enzyme with TMB substrate.

The reaction is stopped by adding 15.75 μl of 10% sulfuric acid.

16. Read the absorbance at 450 nm on a plate reader. Absorbance is proportional to the abundance of the furiavirin / antibody-conjugate immunogenic complex.

Results FIGS. 1 to 5 show the results of the direct ELISA method performed on various flour and pasta samples.

(A) FIG. 1 shows Flour Millin.
g and Backing Research Ass
The results of tests of 30 flour samples supplied by M.R.O.C. (F.M.B.R.A.) were used as parameters to determine the hard (strong) / soft (weak) properties of the flour. Index (Particle Size)
Index = P.S.I.).

Sample Nos. 1-10 are designated as soft (PSI> 3.8).

Samples Nos. 11-20 are designated as hard (PSI <3.8).

Sample Nos. 21-24 are designated as durum samples (PSI> 2.0).

Sample Nos. 25-28 are designated as hybrids of rye and wheat (PSI> 3.
3).

Samples 29-30 are designated as rye (PSI> 5.9).

The above samples, except for the four durum samples, showed the same high fravirin values as before. The four durum samples always showed negative furiabilin values.

When a sample selected from the group classified as described above was evaluated by Western blotting,
The 15K protein was detected by antibody F7F in all samples except the durum sample. No 15K basic protein was detected in the durum sample.

(B) FIG. 2 shows the results for another sample tested using the direct ELISA method.

Samples 1 to 5 are flour samples obtained from FMBRA. Sample 1 is a durum flour sample.

Samples 6-11 are a group of pasta samples, Commission Commission
of the European Communities
s (Community Bureau Bureau Community Bureau
This is a standard sample group obtained from of References). These samples were prepared from pasta samples dried at elevated temperatures of 92 ° C and 78 ° C. Current methods used to determine the abundance of common wheat in pasta are based on the content of specific soluble proteins detected by gel electrophoresis. The reliability of the above method is lost because the proteins are denatured when high temperature drying is used when preparing pasta. The results obtained according to the invention show that the antibody F7F also detects the presence of furavirin in pasta standards in which high temperature drying was used.

Samples 12-19 are Rank Hov
is another pasta standard provided by is McDougall (RHM). These samples are 0-25
% Non-durum contaminants. The obtained results are considered to correlate well with the expected values.

FIG. 3 shows the results obtained from pasta standards made by R. H. M., with values plotted linearly against the content of non-durum wheat.

FIG. 4 shows the results obtained by diluting the extract to 2 mg / ml and 1 mg / ml using the sample of FIG.

FIG. 5 shows the results obtained with the pasta sample of FIG. 2 together with the percent incorporation of non-durum kernels.

Sample 108 has a non-durum incorporation rate of 15%.

This sample was processed by Western blotting. It was observed that in Samples 01 and 108 there was a faint band at 15K.

Western blotting was also performed to show total protein by staining with silver nitrate and ferric sulfate. This indicates that different types of different proteins are present in the crude extract of pasta or flour samples. Calculating the protein in the adherent samples of FIGS. 1 and 2, the value assessed by the Bradford protein assay is approximately 200 μg / ml.

In terms of percentage of total protein of wheat flour, the furiavirin content was calculated to be less than 0.2% of total protein.

[0064] The non-de Interview of pasta in a sample using Example 3 dip stick
Quantitative Assay of Lamb Wheat Since the direct ELISA test takes a long time (usually a minimum of 5 hours), another system was tested using a plastic stick with a nitrocellulose surface that binds rapidly to the pasta extract, then F7F. The antibody was incubated with horseradish peroxidase conjugate and then with the insoluble peroxidase substrate 4-chloro-1-naphthol.

Extract of semolina flour (ie durum flour) sample containing a measured amount of non-durum kernels (200 mg
/ Ml) was filtered and centrifuged and 2.5 μl of the resulting supernatant was spotted on a polypropylene strip coated with a 0.45 μ thick nitrocellulose coating. Strips were washed, incubated in F7F-HRP for 15 minutes at room temperature, washed and incubated with 4-chloro-1-naphthol.

The degree of color development was found to be proportional to the amount of non-durum contamination and was easy to visualize (the corresponding ELISA test against the semolina standard also showed a linear response).

Although this method is somewhat inferior in quantitative accuracy, it shortens the experiment time to about 1 hour.

[Brief description of drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Test results of 30 flour samples supplied by FM B.R.A. are known parameters to determine the hard / strong / soft properties of flour. It is shown together with the value of the particle size index.

FIG. 2 shows the results for another sample tested using the direct ELISA method.

FIG. 3 shows the results obtained from RHM pasta standards as values plotted linearly against the content of non-durum wheat.

FIG. 4 uses the sample of FIG. 3 to extract 2 mg / ml.
And the results obtained by diluting to 1 mg / ml are shown.

FIG. 5 shows the results obtained with the pasta sample of FIG. 2 with% incorporation of non-durum kernels.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location G01N 33/548 A 9015-2J 33/577 B 9015-2J (72) Inventor William Stamson United Kingdom , Grande-Brittagne, Milunga Gee G 62 Kue L, Fairways, Lone Park 7

Claims (16)

[Claims] 1. Use of a furiavirin-specific monoclonal antibody for detecting the content of furiavirin in a durum wheat sample by immunoreaction of a furiavirin protein antigen with an antibody. 2. A monoclonal antibody specific for a furiabilin protein is provided to determine the content of said other non-durum kernels in a grain sample containing durum wheat and another unexpected non-durum kernels. A durum wheat comprising the steps of: determining the content of furiavirin in a sample by an immunoassay using the monoclonal antibody, and determining the content of the other non-durum kernels in the sample by measuring the content of furiavirin in the sample; A method for quality evaluation of a grain sample containing another unexpected non-durum grain. 3. The method of claim 2, wherein the grain sample is a grain flour sample. 4. The method according to claim 2, wherein the grain sample is a heat-denatured flour food product. 5. The method according to claim 2, wherein the monoclonal antibody is enzyme-labeled and the immunoassay is performed by an ELISA method. 6. The method according to claim 5, wherein the enzyme label is horseradish peroxidase. 7. A plastic substrate coated with a protein-binding substance is coated with the extract of the grain sample and incubated with a second enzyme-labeled monoclonal antibody specific for the first antibody. 6. The method according to any one of 6. 8. Binding wheat protein in a sample to an affinity column to determine the content of said other non-durum grain in a grain sample containing durum wheat and another unexpected non-durum grain. The column was treated with a non-specific protein to completely block the remaining reactive groups on the affinity substrate, and the affinity substrate was treated with a labeled monoclonal antibody specific for the furiabilin protein. -A method for quality evaluation of grain samples containing durum wheat and another unforeseen non-durum grain, comprising the step of detecting the presence of antibody conjugates using a sedimenting substrate. 9. The method according to claim 8, wherein the precipitating substrate is 4-chloro-1-naphthol. 10. A Friavirin-specific monoclonal antibody. 11. An enzyme-linked furiavirin-specific monoclonal antibody. 12. A kit for an ELISA assay for the content of furiavirin in a durum wheat sample, which comprises an enzyme-linked furiavirin-specific monoclonal antibody. 13. A kit for assaying furiavirin content in a durum wheat sample, which comprises a plastic substrate having a surface coating that binds to a protein in the sample, and an enzyme-bound furiavirin-specific monoclonal antibody. 14. The kit according to claim 13, wherein the plastic substrate is rod-shaped or strip-shaped. 15. The kit according to claim 13 or 14, wherein the surface coating is nitrocellulose. 16. The nitrocellulose coating has a thickness of 0.3 to
The kit according to claim 15, wherein the kit has a size of 0.6μ.