KR20130121443A - Sulfamethazine-specific monoclonal antibody and hybridoma cell producing the same - Google Patents

Abstract

The present invention relates to a monoclonal antibody binding specifically to sulfamethazine, hydridoma cells producing the same, a kit for detecting the sulfamethazine including the monoclonal antibody, and a method for detecting the sulfamethazine using the monoclonal antibody. The monoclonal antibody according to the present invention only recognizes the sulfamethazine in the sample selectively so that the sulfamethazine which is a kind of synthetic antibiotics remaining in livestock products or dairy products is able to be detected quickly.

Description

Sulphamethazine-specific monoclonal antibody and hybridoma cell producing the same

The present invention provides a monoclonal antibody that specifically binds sulfamethazine, a hybridoma cell producing the same, a sulfamethazine detection kit comprising the monoclonal antibody, and a sulfamethazine using the monoclonal antibody. It is about how to.

Recently, as the demand for livestock products and dairy foods increases gradually, the size of the breeding continues to grow. The domestic livestock industry is using dense multi-headed breeding using a small area, and the use of antibiotics and synthetic antimicrobial agents is gradually increasing to prevent, treat, and promote growth of bacterial diseases in livestock. Misuse and abuse of these antibiotics and synthetic antimicrobial agents poses a risk of accumulation and retention in meat, milk and eggs, and can lead to a variety of problems, including metastasis and initiation of antibiotic-resistant bacteria in the person who consumes them. Can be. Therefore, in Korea, we have established a policy to protect the public health by defining the types of antibiotics that can be used in the food industry and the maximum residual limit (Kim CH, Baick SC, Moon JW 1997, J. Food Hyg.Safety 12). : 71-77).

Sulfamethazine (SMZ) is a synthetic antimicrobial agent used to treat and prevent infectious diseases in animals and to promote growth. Unlike other antibiotics, sulfamethazine hardly decomposes when heated, and its absorption is fast and excretion is slow, causing accumulation in tissues and being absorbed into the human body, causing antibiotic-resistant bacteria, abnormal hematopoietic function, and cancer. Many side effects, including inhibition of immune formation, arthritis, and crystaluria, are known (Park JH, Lee MH 1990, Korean J. Anim. Sci 32: 715-717; Bhanu PR, Prithipal S, Lorelei M, Brock T, Nikolai S). , David A 1991, J. Assoc.Off.Anal.Chem 74: 43-46). In Korea, sulfamethazine content is defined as 0.1 ppm for meat and 0.01 ppm for milk (KFDA.2002 Korean Food Code.Korea Food and Drug Administration.Seoul, Korea).

Gas chromatography, gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) are used as quantitative methods for detecting sulfamethazine, and bioassay and TLC are used as qualitative methods. Law is used (Kim CH, Baick SC, Moon JW 1997, J. Food Hyg.Safety 12: 71-77). These methods use expensive equipment and require a long time for pretreatment and analysis, which makes it difficult to analyze a large amount of samples. Milan F, Vladimir K, anping D, Steven C 1999, Food Agric. Immunol 11: 339-349; Unruh J, Schwartz DP, Barford RA 1993, J. AOAC Int 76: 335-341). On the other hand, the enzyme-linked immunosorbent assay (ELISA) using the specific reaction of antigen-antibody has the advantage that the detection sensitivity and specificity are very high, can be analyzed at low cost, and multiple samples can be analyzed simultaneously.

Therefore, the present inventors endeavored to develop monoclonal antibodies capable of detecting sulfamethazine, thereby completing the present invention by identifying hybridoma cells that produce sulfamethazine-specific monoclonal antibodies among sulfa antibiotics. It was.

It is an object of the present invention to provide a monoclonal antibody that specifically binds sulfamethazine.

Still another object of the present invention is to provide a hybridoma cell producing the monoclonal antibody.

Still another object of the present invention is to provide a sulfamethazine detection kit comprising the monoclonal antibody.

Still another object of the present invention is to provide a method for detecting sulfamethazine using the monoclonal antibody.

In order to achieve the above object, the present invention provides a monoclonal antibody that specifically binds sulfamethazine.

The present invention also provides a hybridoma cell producing the monoclonal antibody.

The present invention also provides a kit for sulfamethazine detection comprising the monoclonal antibody.

The present invention also provides a method for detecting sulfamethazine using the monoclonal antibody.

The monoclonal antibody according to the present invention can selectively detect only sulfamethazine in a sample, and can quickly detect sulfamethazine, which is a kind of synthetic antimicrobial agent remaining in livestock products or dairy products.

1 is a chemical structural formula of 14 sulfa antibiotics.
Figure 2 is a diagram showing the manufacturing process of monoclonal antibodies that specifically bind to sulfamethazine.
Figure 3 is a diagram showing hybridoma cells producing monoclonal antibodies that specifically bind sulfamethazine.
4 is a diagram showing an ELISA result for evaluating the specificity of monoclonal antibodies specifically binding to sulfamethazine.

Hereinafter, the present invention will be described in more detail.

The present invention provides monoclonal antibodies that specifically bind sulfamethazine and hybridoma cells producing the same.

As used herein, the term “monoclonal antibody” is a term known in the art and means a highly specific antibody directed against a single antigenic site. Typically, unlike polyclonal antibodies that include 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 another advantage of not being contaminated by other immunoglobulins because they are synthesized by fusion culture. Such monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Mislstein (1976) European Journal of Immunology 6: 511-519) or phage antibody libraries (Clackson et al, Nature, 352: 624-628, 1991 et al.) Technology. In general, hybridoma cells that secrete monoclonal antibodies are made by fusing cancer cells with immune cells from immunologically suitable host animals such as mice injected with antigenic proteins. These two populations of cell fusion are fused using methods known in the art, such as polyethylene glycol, and the antibody producing cells are propagated by standard culture methods. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antibody specific for the antigen are cultured in large quantities in vitro or in vivo.

As used herein, the term "hybridoma" refers to a cell obtained by fusing B lymphocytes that produce antibodies and myeloma cancer cells. Myeloma cells are transformed cells, which have the advantage of being cultivated for a long time in a culture dish, so that a large amount of monoclonal antibodies can be easily obtained by separating the myeloma hybridomas that produce only one antibody.

The monoclonal antibody produced by the hybridoma of the present invention may be used in a cell culture medium containing the antibody without purification, but in order to obtain the best results according to a method well known in the art to which the present invention belongs, For example, 95% or more). Such purification techniques may include, for example, gel electrophoresis, dialysis, salt precipitation, chromatography, and the like.

The monoclonal antibodies of the present invention can specifically bind to sulfamethazine-bovine serum albumin conjugates, and can be used to quickly detect sulfamethazine present in samples such as livestock products and dairy products.

The sulfamethazine has a molecular weight of 278.32 Kda, which is not recognized by the immune system and must be combined with a carrier protein to impart antigenicity. As a carrier for imparting antigenicity, bovine serum albumin may be preferably used.

The present invention also provides a kit for sulfamethazine detection comprising the monoclonal antibody.

Kits of the invention, as well as monoclonal antibodies of the invention, as long as it can selectively recognize sulfamethazine, other antibodies, such as polyclonal antibodies, chimeric-antibodies, humanized-antibodies, monoclonal antibodies But also fragments of monoclonal antibodies can be used together.

 Kits of the invention may also include tools and reagents for use in immunological assays. Such tools and reagents include carriers, labeling substances capable of producing detectable signals, solubilizers, detergents and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Such carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS; Insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex, other papers, glass, metals , Agarose and combinations thereof.

Assay systems for use in the kits of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography test strips, radiosegmented immunoassay devices, flowthrough devices, and the like. .

In addition,

(a) contacting the monoclonal antibody with a sample; And

(b) confirming the formation of the antigen-antibody complex produced by combining sulfamethazine with the monoclonal antibody; and provides a method for detecting sulfamethazine.

As used herein, the term "sample" refers to tissues, cells, whole blood, serum, plasma, cerebrospinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), biological samples such as cell culture supernatants or supernatants, dairy products, and dairy products. And the like, but are not limited thereto. The biological sample may be collected from various animals such as humans, rabbits, pigs, mice, and the like, but is not limited thereto.

As used herein, the term “antigen-antibody complex” refers to a combination of sulfamethazine and an antibody that recognizes it to identify the presence or absence of sulfamethazine in a sample. Preferably, said antigen-antibody complex is a complex of sulfamethazine and a monoclonal antibody according to the invention.

To confirm the formation of the antigen-antibody complex, enzyme immunoassay (ELISA), Western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, immune Cytochemistry, radioimmunoassay (RIA), immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion assay), complement fixation assay (Complement Fixation Assay), protein chips (Protein Chip) or kits, etc., It is not limited to this. The enzyme immunosorbent adsorption (ELISA) method includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, and indirectly using a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid support. ELISA, direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support. Various ELISA methods include an indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . The enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase And GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase, β-latamase and the like. It is not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. The luminous material includes acridinium ester, luciferin, luciferase and the like, but is not limited thereto. The microparticles include colloidal gold, colored latex, and the like, but are not limited thereto. The redox molecule includes ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ^{2 +} , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. The radioisotope includes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re and the like, but is not limited thereto. Do not.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One. Sulfamethazine  Production of antigens

Sulfamethazine is one of 14 sulfa antibiotics with sulfonamide functional groups (FIG. 1). Since sulfamethazine is not recognized by the immune system alone and cannot act as an antigen alone, sulfamethazine was combined with a carrier protein to impart antigenicity. Bovine serum albumin was used as a carrier for imparting such antigenicity, and a combination of sulfamethazine and bovine serum albumin was prepared as an antigen. In addition, sulfa antibiotics are similar in structure, so even if monoclonal antibodies specific to sulfamethazine cannot exclude the possibility of binding to other sulfa antibiotics, the combination of other sulfa antibiotics and bovine serum albumin Prepared by antigen and compared.

More specifically, 10 mg each of various types of sulfa antibiotics including sulfamethazine and 20 mg of bovine serum albumin were mixed well in 1.5 ml of a solution of phosphate buffer and dioxane 2: 1, followed by incubation at room temperature. The reaction was carried out at 200 rpm for 10 minutes. 2 ml of 0.2% glutaraldehyde (0.2% glutaraldehyde in PBS) was slowly added to the reaction solution, reacted at 200 rpm in an incubator at room temperature for 1 hour, and 1 ml of 1M glycine (1M glycine in PBS) was added thereto. After further reacted for 1 hour. The mixture was dialyzed in phosphate buffer at 4 ° C. for 5 days, and the phosphate buffer was changed daily. Sulfamethazine-bovine serum albumin conjugates on dialysis were protein quantified.

Example  2: To sulfamethazine  To produce monoclonal antibodies against Hybridoma  Production of cells

2-1: induction of antibody-producing cells

The process for producing hybridoma cells producing monoclonal antibodies against sulfamethazine is shown in schematic diagram in FIG. 2.

More specifically, in order to use the sulfamethazine-bovine serum albumin conjugate prepared in Example 1 as an antigen, after diluting to a concentration of 0.2 mg / ml with physiological saline, the same amount of complete Freund antigen adjuvant The emulsion was prepared by mixing with (complete Freund's adjuvant). The emulsion was injected 0.5 ml each into the abdominal cavity of six 6-week old female BALB / C mice. The emulsion was used only for the first dose, and from the second dose, the sulfamethazine-bovine serum albumin conjugate was mixed with an incomplete Freund's adjuvant and injected into the abdominal cavity of the mouse three times at two-week intervals. Antibody production was induced. Five days before cell fusion, 0.1 mg / 100 μl of antigen was injected again into the tail vein of the mouse.

2-2: Hybridoma  Production of cells

Infusion of 10 ml of 11.3% sucrose solution into the abdominal cavity of 6-week-old mice one day before cell fusion, and carefully shake the peritoneum so as not to be contaminated to collect the injected solution again. The harvested cells (used as feeder cells) were washed using serum-free medium twice for 5 minutes at 1500 rpm. The washed feeder cells were suspended in 100 ml of cell fusion medium (10% fetal bovine serum RPMI1640 medium, 1% penicillin-streptomycin, 1X HAT-hypoxanthine-aminopterin-thymidine), 96 100 μl per well was dispensed into well plates and incubated in a 37 ° C., 5% carbon dioxide incubator. After 1 day, spleens were obtained by sacrifice of the mice subjected to challenge, placed in a petri dish containing serum-free medium, finely divided to separate cells one by one, and then placed in 50 ml of serum-free medium. . In addition, SP2 / O cells, which are cancer cell lines to be used for cell fusion, were placed in a 50 ml tube and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded and the splenocytes suspended to single cell level were placed in 50 ml of serum-free medium and passed through a 0.45 μm mesh into a 50 ml tube containing SP2 / O cells. After removing the mesh and centrifuged for 5 minutes at 1500 rpm, the supernatant was discarded and centrifuged again at 1500 rpm for 5 minutes. The serum-free medium remaining in the 50 ml tube was removed using a pipette, and then injected with 1 ml PEG (polyethylene glycol) solution for 1 minute in a 37 ° C. water bath, followed by stirring. After 1 minute, the tube was shaken with a little serum-free medium prepared at 37 ℃. Serum-free medium was added up to 40 ml, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, suspended in 100 ml of cell fusion media prepared at 37 ° C, and the feeder cells were prepared the day before. 100 μl was dispensed into 96 well plates, and then cultured in a 37 ° C. carbon dioxide incubator for 7 to 14 days. A 96 well plate in which the hybridoma cells were dispensed is shown in FIG. 3.

2-3: Hybridoma  Screening and Isolation of Cells

Obtained each of the hybridoma culture supernatants for 14 kinds of conjugates of the sulfonamide-based antibiotic bovine serum albumin (BSA) obtained in Example 2-2, and enzyme immunoassay (ELISA, enzyme Selected by linked immunosorbent assay. More specifically, the antigen prepared in Example 1 was dispensed 100 μl per well into a 96 well immune plate and stored at 37 ° C. for 1 hour. After 1 hour the water in the plate was removed and dried in a 37 ° C. thermostat for 1 hour 40 minutes. Blocking buffer (blocking buffer, 5% Skim milk in PBS) was then dispensed 200 μl per well and stored for 1 hour 30 minutes in a 37 ℃ thermostat. Thereafter, 100 μl of the culture supernatant obtained in Example 2-2 was dispensed per well and stored at 37 ° C. for 1 hour. The culture supernatant remaining on the plate was shaken off, and 200 μl of 0.05% PBST (0.05% Tween-20 in PBS) was aliquoted per well, followed by three times of shaking after standing for 5 minutes. Thereafter, the anti-mouse IgG HRP (horse raddish peroxidase) prepared in advance was diluted 1: 10000 in phosphate buffer solution, and 100 μl was dispensed per well, and then stored at 37 ° C. for 40 minutes. Remove the remaining antibody on the plate, and again dispense 200 μl of 0.05% PBST (phosphate buffer with 0.05% Tween-20) per well, shake for 5 minutes, and shake off, and repeat OPD (o- Positive hybridoma cells were isolated by color reaction using phenylenediamine dihydrochloride) as a color substrate. The results are shown in Fig.

As shown in FIG. 4, it was confirmed that SMZ31A6 hybridoma cells exhibited specific reaction with sulfamethazine.

Example  3: To sulfamethazine  Monoclonal Antibody Production and Isolation

Mouse intraperitoneal cultures were prepared using SMZ31A6 hybridoma cells producing monoclonal antibodies that specifically bind to sulfamethazine identified in Example 2, and antibodies were isolated therefrom. First, in order to obtain antibody ascites, the cells producing the respective antibodies were injected into the abdominal cavity of the treated BALB / C mice, which had been previously administered intraperitoneally with pristane. After confirming that the abdomen of the mouse was swollen after 2-3 weeks, the ascites of each mouse was extracted using a syringe. The ascites solution was purified using a protein G affinity column (GE health care, HiTrap protein G HP). The protein G affinity column was homogenized by passing a double volume of wash buffer (wash buffer, 20 mM NaH ₂ PO ₄ , pH 7.0) at a rate of 1 ml / min, followed by ascites. After passage through the ascites solution, the bound antibody was separated by elution buffer (elution buffer, 100 mM glycine HCl, pH 2.7), and finally, purification of the monoclonal antibody was completed.

Claims (7)

Monoclonal antibody that specifically binds sulfamethazine.
The monoclonal antibody according to claim 1, wherein the sulfamethazine is a conjugate with bovine serum albumin.
A hybridoma cell producing the monoclonal antibody of claim 1.
Sulfamethazine detection kit comprising the monoclonal antibody of claim 1.
(a) contacting a sample with a monoclonal antibody that specifically binds sulfamethazine; And
(b) confirming the formation of the antigen-antibody complex formed by combining sulfamethazine with the monoclonal antibody.
The method according to claim 5, wherein in step (a), the sample is any one or more selected from the group consisting of tissue, cells, whole blood, serum, plasma, cerebrospinal fluid, cell culture supernatant, animal products and dairy products, sulfamethazine How to detect.
The method according to claim 5, wherein the formation of the antigen-antibody complex in step (b) is performed by enzyme-linked immunoassay (ELISA), Western blotting, Western fluorescence, immunofluorescence, immunohistochemistry staining, Flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, immunodiffusion assay, complement fixation assay and protein chip Method for detecting sulfamethazine, characterized in that made by any one selected from the group consisting of.

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