KR20180031375A - A monoclonal antibody for distinguishing honey, a method for distinguishing honey using the same, and a composition thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody for distinguishing honey, a method for distinguishing honey by using the same, and a composition thereof. According to the present invention, native honey and beekeeping honey can be immunologically distinguished through ELISA, an immunoblotting method and the like by using hybridoma cell lines and an antibody, which produces the hybridoma cell lines wherein the hybridoma cell lines generate antibodies, which recognize only native honey, and antibodies, which recognize only beekeeping honey.

Description

[0001] The present invention relates to a monoclonal antibody for distinguishing honey, a method for distinguishing honey using the same,

The present invention relates to a monoclonal antibody for distinguishing honey, a method for distinguishing honey using the same, and a composition thereof.

The native honey is a honey bee gathering flower honey and storing it in a honeycomb, and after the honey has not added any other materials such as pollen, royal jelly, sugar, sweetener and the like.

The nature and condition of the honey must have a distinctive color and flavor and be viscous and can be determined according to the source. The content of native honey is 21.0% or less, 0.5% or less of water insoluble, 40.0 meq / kg or less of acidity, 65.0% or more of sucrose and 7.0% or less of sucrose, 80.0 mg / kg of HMF (hydroxymethylfurfural) , No tar coloring and artificial sweeteners should be detected, and the isomerized sugar should be negative.

Specification of honey is mainly done in spring and is carried out in the rainy season. Specification (bean) for bees can include sugar water, honey, and pollen. Honey, propolis, flowerpot, honeycomb are the things a person receives as a by-product from bees. In this method, sugar bees and honey are given to the bees as a specimen. The bees collect them, use them for food, leave the honeycomb, or keep them in the room after the larva is born.

However, it is difficult for the general public to distinguish between the specially preserved honey and the ordinary honey that has been collected from nature. Therefore, it is not clear whether the products sold on the market are genuine honey or fake honey.

In general, fake honey or artificially produced honey is made from monosaccharide by adding invertase from sugar + yeast origin. This is mainly used as an extender.

On the other hand, the specification honey is a monosaccharide produced by feeding sugar to bees, and is a honey recognized as food in food circulation. It is not honey that honey bees have obtained from flowers, but it can also be used as an extender.

However, in order to clearly distinguish the real honey from the scientific one, it is necessary to make comparative analysis using expensive equipments. It is a great waste to spend the expensive examination fee and several days to buy a bottle of honey.

In other words, in order to identify whether a person is a native honey, it is necessary to examine whether the native honey product to be purchased has the above-mentioned ingredient contents. In addition to a comparative analysis using expensive equipment, It is a reality that there are problems such as expensive examination fee to spend and long time to buy.

In addition, there are problems that the same results can not be obtained even when the test is performed because the numerical values are different depending on the climatic condition, the soil problem of the soil, the collection method of the individual, the amount of sunshine, and so on.

In addition, there is a need to distinguish native honey with low production (expensive domestic honey: 2.4 ~ 2.4 million won) and honey bee honey (2.4 ~ 400 ~ 50,000 won).

[Prior Patent Literature]

Korean Patent Publication No. 10-2010-0110431

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a monoclonal antibody for distinguishing honey.

Another object of the present invention is to provide a novel method for distinguishing honey.

Another object of the present invention is to provide a novel composition for distinguishing honey.

In order to accomplish the above object, the present invention provides a monoclonal antibody that specifically recognizes native honey produced by a hybridoma of Accession No. KCLRF-BP-00371.

The present invention also provides a monoclonal antibody that specifically recognizes bee honey produced by a hybridoma of Accession No. KCLRF-BP-00372.

The present invention also provides a hybridoma cell of Accession No. KCLRF-BP-00371 producing the above monoclonal antibody of the present invention.

The present invention also provides a hybridoma cell of Accession No. KCLRF-BP-00372 producing the above monoclonal antibody of the present invention.

The present invention also provides a method for distinguishing honey from a monoclonal antibody of the present invention by contacting it with a honey sample to examine the presence of the honey and the antibody complex.

In one embodiment of the present invention, the honey is preferably native or bee honey, but is not limited thereto.

The present invention also provides a composition for distinguishing honey comprising the above monoclonal antibody of the present invention as an active ingredient.

The present invention also provides a method for producing a monoclonal antibody that specifically recognizes native honey by culturing a hybridoma of Accession No. KCLRF-BP-00371.

The present invention also provides a method for producing a monoclonal antibody that specifically recognizes bee honey by culturing a hybridoma of Accession No. KCLRF-BP-00372.

Hereinafter, the present invention will be described.

The present invention develops hybridoma cell lines that produce antibodies that recognize only native honey and antibodies that recognize only bee honey, and immunologically distinguish native and bee honey from ELISA and immunoblotting methods using antibodies produced by them .

Unlike polyclonal antibodies, which typically contain different antibodies directed against different epitopes, monoclonal antibodies are directed against a single determinant on the antigen.

Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and have other advantages that are not contaminated by other immunoglobulins because they are synthesized by fusion culture.

The monoclonal antibody of the present invention may be prepared by a hybridoma method (see Kohler and Mislstein (1976) European Journal of Immunology 6: 511-519) or a phage antibody library (Clackson et al, Nature, 352: 624-628, 1991, etc.) techniques. Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells and cancer cell lines from immunologically appropriate host animals, such as mice injected with antigen proteins. The cell fusion of these two groups is fused using a method known in the art such as polyethylene glycol, and the antibody producing cells are proliferated by a standard culture method. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antigen-specific antibody are cultured in vitro or in vivo.

The term " hybridoma " as used herein refers to a cell made by artificially fusing two different kinds of cells. Hybridoma cells are produced by fusing two or more homologous cells or xenogeneic cells using a substance that causes cell fusion such as polyethylene glycol or a virus of a specific species. By forming a hybridoma cell, different functions of different cells can be obtained It can be integrated into one cell.

Such hybridoma cells can be produced by fusing cancer cells that normally proliferate even in vitro, and large cells having a specific differentiation trait extracted from a living body. A typical hybridoma is a lymphocyte hybridoma, which is a hybridoma cell in which a myeloma cell and a B cell are fused, a T cell that is a tumor cell, a hybridoma cell that is a tumor cell thereof, a T cell that produces a lymphocaine (a physiologically active substance) Hybridoma cells that are tumor cells thereof, etc. It is obvious that the hybridoma cell line of the present invention can be fused and cultured under optimal conditions for effectively producing the monoclonal antibody of the present invention according to the selection of a person skilled in the art.

In a preferred embodiment of the present invention, the hybridoma cells of the present invention were deposited on September 5, 2016 at the Korean Cell Line Research Foundation, Yeongeon-dong, Jongro-gu, Seoul, Korea (accession numbers KCLRFBP00371BP and KCLRFBP00372BP, BP-00371 'and " KCLRF-BP-00372 ").

The monoclonal antibody produced by the hybridoma cells of the present invention may be used as a cell culture solution without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma cells of the present invention may be purified in high purity (for example, 95% or more). Such purification techniques may include, for example, gel electrophoresis, dialysis, salt precipitation, chromatography, and the like.

As can be seen from the present invention, hybridoma cell lines that produce antibodies recognizing only native honey of the present invention and antibodies that recognize only honey bee honey of the present invention and antibodies produced by them are used for ELISA and immunoblotting, The bee honey can be immunologically distinguished.

FIG. 1 is a picture of a selected hybridoma cell line. As a primary antibody, a culture solution of a hybridoma developed using native honey and honey bee as an antigen was used.
A. TY1, TY5, TY6, and TY7 are hybridoma cell lines that produce antibodies that recognize both loci and bee. TY2, TY3, and TY4 were thought to be hybridoma cell lines that produce antibodies that recognize only bee, but they do not appear to develop on subsequent ELISA, suggesting that the antibody is splitting in the wash step.
B. Five hybridoma cell lines (T1 to T5) producing antibodies that specifically recognize native honey
C. Six hybridoma cell lines (Y1-Y6) that produce antibodies specifically recognizing the bee.
FIG. 2 is a diagram related to antibody development. FIG. 2 is a graph showing the antibody against Honey TY cell line using ELISA and western blot.
A. The Honey TY cell line was coated with the primary antibody and the antigen with native honey and bee honey. The antibody was diluted 10-fold to 5-fold, and the limit of Honey TY antibody was observed. B. Similarly to ELISA, native honey and bee honey were used as the antigen, and the culture solution of Honey TY cell line was used as the primary antibody.
FIG. 3 is a diagram related to antibody development. FIG. 3 shows a honey-specific antibody. The antibody of Honey T cell line was verified using ELISA and western blot.
A. Honey T cell line was coated with primary antibody and antigen with native honey and bee honey. The antibody was diluted 10 times to 25600 times and the limit of Honey T antibody was observed. B. As in ELISA, native honey and bee honey were used as antigens, and the culture medium of Honey T cell line was used as the primary antibody.
FIG. 4 is a diagram related to antibody development. FIG. 4 shows the honey bee specific antibody. The antibody of Honey Y cell line was verified using ELISA and western blot.
A. The Honey Y cell line was coated with the primary antibody and the antigen with native honey and bee honey, and the antibody was diluted 10-fold to 25600-fold to reveal the limit of Honey Y antibody. B. As in ELISA, native honey and bee honey were used as antigens, and culture solution of Honey Y cell line was used as primary antibody.
FIG. 5 is a diagram related to Isotyping in relation to antibody characteristics. In FIG. 5, TY Ab of Honey TY, T Ab of Honey T, and Y Ab of Honey Y both have IgG1 as the heavy chain and kappa chain as the light chain have.
FIG. 6 is a picture of an ELISA relating to the analysis of native bee-mixed honey. The mixture of native honey and bee honey was coated with an antigen and assayed using three antibodies.
In case of TY Ab, it shows almost constant regardless of the ratio of native honey and bee. In the case of T Ab, the absorbance decreases linearly with the ratio of native antigen. Y Ab is not linear, but the absorbance decreases as the bee rate decreases.
FIG. 7 is a diagram related to immunoblotting with respect to the analysis of native honeycomb mixed honey, wherein honey mixed with native honey and bee honey was analyzed by immunoblotting. TY Ab showed almost the same band regardless of the ratio of native honey and bee, and T Ab and Y Ab showed that the band became thicker as the ratio of native honey and bee respectively increased.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example 1: Development of monoclonal antibody

Domestic bean honey was dialyzed against distilled water and centrifuged (10000g, 10 minutes) to remove the pollen. The supernatant was used as a sample for making monoclonal antibodies by lyophilization. 100ug of the dialyzed sample of native and bee honey was mixed with complete adjuvant and injected into the abdominal cavity of female Balb / c mouse. Two weeks later, the same amount of sample is mixed with the incomplete adjuvant and injected twice more. One week after the last injection, the mouse is sacrificed and the spleen is fused with sp2 / 0. After that, only hybridoma producing antibody recognizing honey as an antigen was selected through ELISA, and a hybridoma having high affinity to honey was selected. Selected hybridomas produced single colonies and monoclone.

Example 2: ELISA

Carbonate coating buffer (0.05M sodium carbonate buffer, pH 9.6) is mixed with 100 μl of the antigen. The plate is then incubated at 4 ° C overnight and washed three times with PBS-T. Block with 1% BSA for 1 hour and wash three times. The supernatant of the hybridoma was used as the primary antibody, diluted with PBS-T, reacted for 1 hour and washed three times. The secondary antibody (goat anti-mouse IgG HRP) is also reacted for 1 hour and then washed three times. Add the ABTS solution containing 0.03% H2O2 and incubate at 37 ° C for 20 minutes. The absorbance at 405 nm is observed.

Example 3: Western blot

Samples are dialyzed against antigen and subjected to SDS-PAGE. Transfer the gel protein to the membrane via transfer, and block with 1% skim milk diluted in TBS-T for 1 hour. After blocking, wash with TBS-T three times for 10 minutes and allow the primary antibody to react for 1 hour. As the primary antibody, the supernatant of the hybridoma is diluted in TBS-T. Wash three times and allow the secondary antibody to react for 1 hour. After applying the secondary antibody and washing three times, ecl is sprayed onto the membrane and developed on the film in the dark room.

Example 4: Isotyping

The Pierce Rapid ELISA Mouse mAb Isotyping kit from Thermo SCIENTIFIC was used.

Add 50 μl of the supernatant diluted in TBS to 8 wells per well, add 50 μl of Goat Anti-Mouse IgG + IgA + IgM HRP mixture, and incubate at 37 ° C for 1 hour. Wash three times with wash buffer and add 75 ul of TMB to each well. After about 1 minute, the antibody is blue when it has the corresponding isotype. After 5 ~ 15 minutes, put stop solution and change to yellow and absorbance is measured at 450 nm.

The results of the above embodiment will be described below.

Antibody development

Hybridoma was obtained by fusion of spleen of mouse injected with antigen into peritoneal cavity and sp2 / 0 of mouse myeloma. A hybridoma cell line was selected which produces an antibody recognizing the honey in the hybridoma obtained by the ELISA method as an antigen. Four hybridoma cell lines (TY1, TY5, TY6, TY7) were selected to produce antibodies that recognize both native and bean-like antibodies (Figure 1.A). TY2 ~ TY4 was a hybridoma cell line that produced an antibody recognizing the bee antigen. However, the antibody did not develop in the subsequent ELISA, suggesting that the antibody splattered during the wash step or primary antibody insertion in the adjacent well.

Five hybridoma cell lines T1 ~ T5 (Figure 1B) and 6 hybridoma cell lines (Y1 ~ Y6) producing antibodies that specifically recognize native honey were selected (Figure 1.C).

These cell lines were single-colonized to mono-clone. Hybridoma cell lines that produce antibodies with high affinity to antigens were selected using ELISA and western blot. Hybridoma cell lines that produced antibodies recognizing both native and bean-like antibodies showed the highest affinity of TY5 and specificity of native honey The affinity of Y1 was the highest in the hybridoma cell line producing the antibody and in the hybridoma cell line producing the antibody specifically recognizing the bee. TY5, T2 and Y1 named Honey TY, Honey T and Honey Y, respectively, and deposited on September 5, 2016.

  Honey TY was verified by ELISA. The dialyzed honey was coated with the antigen and the culture of Honey TY was used as the primary antibody. When diluted 10 to 5 times and ELISA was performed, both native and amphoteric antigens were developed. The color development was maintained until the 1250-fold dilution, and the color was lengthened according to the dilution ratio at the dilution more than that (Figure 2.A). Western blot also validated Honey TY antibodies. As the antigen, native honey and bee honey were used, and both native honey and bee honey were recognized as shown in Figure 1.B (Figure 2.B).

Antibodies to Honey T and Y were also tested by ELISA and western blot. The native honey and bee honey were coated with the same amount of antigen, and the culture medium of Honey T and Y was diluted 10 times to 25600 times and used as the primary antibody. The antibody of Honey T did not recognize bee honey as an antigen but specifically developed only in native honey and the color became more and more tender as the dilution ratio increased (Fig. 3. A). Honey Y also did not recognize native honey, but only specially developed in a well coated with bee honey. Western blot results were similar to ELISA (Fig. 4.A). When Honey T was used as the primary antibody, specific bands appeared only in the native honey (Fig. 3.B). When Honey Y was used as the primary antibody, the band was specifically detected only in the place where bee honey was used as the antigen (Fig. 4B).

Antibody characteristics: Isotyping

TY Ab, Hon T antibody, T Ab, and Honey Y antibody were identified using a kit. Both antibodies have an IgG1 heavy chain and a light chain kappa chain. The absorbance is different, however, because the amount of antibody produced by each hybridoma cell line is different from the amount of cells.

Analysis of native bee mixed honey

Because there are some honeys which are mixed with native honey and bee honey, mixed honey and bee were mixed at a certain ratio to make mixed honey, and analyzed by ELISA and Immunoblotting,

 Analysis of the mixed honey by ELISA (Figure 6) showed that TY Ab, an antibody of Honey TY that recognizes both native and beekeeping, showed almost constant absorbance regardless of the ratio of native and bee honey. T Ab, an antibody of Honey T, which recognizes only native honey, decreases linearly as the ratio of native honey decreases, and Y Ab, an antibody of Honey Y that specifically recognizes only bee honey, Although not diminished, the absorbance decreases as the proportion of bee honey decreases.

Mixed honey was analyzed by immunoblotting (Figure 7). TY Ab showed almost the same band regardless of the ratio of native honey and bee, and T Ab and Y Ab showed that the band became thicker as the ratio of native honey and bee respectively increased.

Korea Cell Line Research Foundation KCLRFBP00371BP 20160905 Korea Cell Line Research Foundation KCLRFBP00372BP 20160905

Claims (12)

A monoclonal antibody that specifically recognizes native honey produced by a hybridoma of Accession No. KCLRF-BP-00371. A monoclonal antibody that specifically recognizes bee honey produced by a hybridoma of Accession No. KCLRF-BP-00372. A hybridoma cell of Accession No. KCLRF-BP-00371 producing the monoclonal antibody of claim 1. A hybridoma cell of Accession No. KCLRF-BP-00372 producing the monoclonal antibody of claim 2. Contacting the monoclonal antibody of claims 1 and 2 with a honey sample to determine the presence of the honey and said antibody and honey antigen complex. 6. The method of claim 5, wherein the honey is native or bee honey. The method according to claim 5, wherein the complex is distinguished by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, hue binding, or chemiluminescent substance binding method. A composition for distinguishing honey comprising the monoclonal antibody of claims 1 and 2 as an active ingredient. 9. The composition of claim 8, wherein the honey is native or bee honey. 9. The method according to claim 8, wherein the discrimination is performed by detecting the antibody and the honey antigen complex by a radioimmunoassay (RIA), an enzyme linked immunosorbent assay (ELISA), an immunofluorescence assay, a hue binding assay, . A method for producing a monoclonal antibody that specifically recognizes native honey by culturing a hybridoma of Accession No. KCLRF-BP-00371. A hybridoma of Accession No. KCLRF-BP-00372 is cultured to produce a monoclonal antibody that specifically recognizes bee honey.

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