KR20230149702A - Monoclonal Antibody Specifically Binding to Flunixin and Kit for Detecting Flunixin - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to flunixin and a kit for detecting flunixin using the same. Using the monoclonal antibody of the present invention, flunixin remaining in concentrated marine products can be quickly and accurately detected.

Description

Monoclonal antibody specifically binding to flunixin and its use {Monoclonal Antibody Specifically Binding to Flunixin and Kit for Detecting Flunixin}

The present invention relates to a monoclonal antibody that specifically binds to flunixin and a kit for detecting flunixin using the same.

Flunixin is a non-steroidal anti-inflammatory drug with an anti-inflammatory effect. It is used for the treatment and pain relief of fever and inflammation caused by respiratory diseases in cattle and pigs, relief of pain and inflammation related to musculoskeletal disorders in horses, and relief of visceral pain related to colic. It is mainly used for etc. With the increased demand for meat and animal protein intake, the scale of the livestock industry has increased, and the use of anti-inflammatory agents, antibacterial and antibiotic substances for systematic management, disease prevention and treatment, and hygiene has become inevitable. However, during this process, some antibiotics may accumulate in the bodies of livestock, and anti-inflammatory, antibacterial, and antibiotic substances remaining in the bodies of livestock may return to consumers through agricultural and livestock products such as meat, milk, eggs, and fish. In particular, unlike crops, veterinary drugs are administered directly to animals through injections, drinking water, or feed to treat diseases and promote growth, so they accumulate and remain in edible parts used as food or in specific organs during biochemical metabolic processes. Residues exist not only as direct parent compounds, but also as metabolites that have undergone biochemical metabolism and are mainly introduced into the human body through food. In this way, anti-inflammatory agents and antibacterial and antibiotic substances remaining in agricultural, livestock and marine products are absorbed by humans, causing disturbance of homeostasis in the body, causing drug-specific reactions (allergic reactions in hypersensitive individuals), inhibiting the therapeutic effect of patients receiving drug treatment, or causing side effects and substance-resistant bacteria (indirectly). through induction of resistant strains of bacteria).

In Korea, policies are being implemented to protect public health by legislating the types of veterinary drugs and maximum residue limits that can be used in the Food Code. Livestock and marine products are inspected upon shipment or after slaughter to ensure that the residue limits are exceeded. If so, shipment and sale are prohibited. In the case of flunixin, the residue tolerance standards are set differently for each food, such as 0.02 mg/kg in milk and 0.05 mg/kg in pork muscle.

In order to minimize consumer distrust in safe food, there is an urgent need to develop a simple test method that can easily and conveniently check the presence of anti-inflammatory, antibacterial, and antibiotic substances in agricultural, livestock, and marine products such as meat, milk, and eggs.

Republic of Korea Patent No. 10-1459577 (registered on November 3, 2014)

The present inventors have made extensive research efforts to develop a monoclonal antibody that specifically binds to flunixin and a composition and kit for detecting flunixin using the same in order to quickly detect flunixin remaining in concentrated and marine products. As a result, the present invention was completed by establishing a monoclonal antibody that specifically binds to flunixin and a hybridoma cell line producing the same.

Therefore, the purpose of the present invention is to provide a monoclonal antibody that specifically binds to flunixin.

Another object of the present invention is to provide a hybridoma cell line that produces a monoclonal antibody that specifically binds to flunixin.

Another object of the present invention is to provide a composition and kit for detecting flunixin containing a monoclonal antibody that specifically binds to flunixin.

Another object of the present invention is to provide a method for detecting flunixin.

According to one aspect of the present invention, the present invention provides a monoclonal antibody that specifically binds to Flunixin.

In this specification, "Flunixin" is a non-steroidal anti-inflammatory drug with an inflammation-inhibiting effect, for treatment and analgesic of fever and inflammation caused by respiratory disease in cattle and pigs, relief of pain and inflammation related to musculoskeletal disorders in horses, and colic. It is mainly used for alleviating visceral pain.

As used herein, the term “antibody” refers to a wide range of proteins or fragments thereof that specifically bind to an antigen, also known as an immunoglobulin, and more specifically, a complete antibody. In addition, it includes an antigen binding fragment of an antibody.

A complete antibody has a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and is subclassed as gamma1 (γ1), gamma2 (γ2), and gamma3 (γ3). ), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term “antigen binding fragment” refers to a fragment that possesses an antigen-binding function and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab (fragment antigen binding) has one antigen binding site with a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are known in the art. A two-chain Fv (two-chain Fv) connects the heavy chain variable region and the light chain variable region by a non-covalent bond, and a single-chain Fv (single-chin variable fragment, scFv) generally connects the heavy chain variable region and the short chain variable region through a peptide linker. The domains can be covalently linked or linked directly at the C-terminus to form a dimer-like structure, such as double-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), or It can be produced through genetic recombination technology.

As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3 comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen, and a full-length heavy chain thereof. It means all fragments. Also, as used herein, the term "light chain" refers to both a full-length light chain and fragments thereof including the variable region domain VL and the constant region domain CL containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

As used herein, the term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain having four conserved framework regions (FR) and three hypervariable regions (HVR). ) includes. (Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., page 91 (2007)).

As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (H-CDR1, H-CDR2, and H-CDR3) and light chain (L-CDR1, L-CDR2, and L-CDR3) each contain three CDRs. CDRs provide key contact residues for an antibody to bind to an antigen or epitope.

As used herein, the term “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. FRs in variable domains generally consist of four FR domains FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences generally appear in the VH in the following order:

FRH1 (Framework region 1 of Heavy chain)-HCDR1 (complementarity determining region 1 of Heavy chain)-FRH2-HCDR2-FRH3-HCDR3-FRH4;

Additionally, HVR and FR sequences typically appear in the VL (or Vk) in the following order:

FRL1(Framework region 1 of Light chain)-LCDR1(complementarity determining region 1 of Light chain)-FRL2-LCDR2-FRL3-LCDR3-FRL4.

Also, as used herein, the term "human antibody" or "humanized antibody" refers to an antibody produced by a human or human cell, or a non-human antibody that utilizes human antibody repertoires or other human antibody coding sequences. It possesses an amino acid sequence corresponding to the amino acid sequence of the antibody from which it was derived.

As used herein, the term “monoclonal antibody” is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Monoclonal antibodies are also called single-cell antibodies, monoclonal antibodies, or monoclonal antibodies. Unlike polyclonal antibodies, which typically contain different antibodies directed against different epitopes (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical methods using antigen-antibody combinations, and because they are produced by hybridoma culture, they have another advantage of not being contaminated by other immunoglobulins.

The monoclonal antibodies produced by the above-mentioned hybridomas can be used without purification, but to obtain the best results, they are purified to high purity (e.g., 95% or more) according to methods well known in the technical field to which the present invention pertains. It is desirable to do so. These purification techniques include, for example, gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, etc., for separation from the culture medium or ascites fluid.

As used herein, the term “specifically binds” or the like means that a monoclonal antibody forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 ^-6 M or less (e.g., a smaller K _D indicates tighter binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site on a molecule (e.g., an antibody or monoclonal antibody) and its binding partner (e.g., an antigen). it means. Unless otherwise specified, “binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity _of a molecule Affinity can be measured by routine methods known in the art, including those described herein.

In one embodiment of the present invention, the flunixin is bound to KLH (Keyhole Limpets Hemocyanin) or BSA (Bovine Serum Albumin). Flunixin has a molecular weight of 392.461 g/mol, and is not antigenic on its own, so it cannot be used with a carrier protein with a large molecular weight such as KLH (Keyhole Limpets Hemocyanin) or BSA (Bovine Serum Albumin). Antibodies are developed using conjugates combined with carrier proteins as antigens.

In one embodiment of the present invention, the monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line of the embodiment of the present invention. More specifically, the monoclonal antibody was produced by the hybridoma cell line 46C9 with accession number KCTC15069BP.

In one embodiment of the present invention, the monoclonal antibody specifically binds to 5-Hydroxy Flunixin.

As used herein, “5-Hydroxy Flunixin” is the primary metabolite of flunixin. The monoclonal antibody of the present invention cross-reacts with 5-hydroxy flunixin and can quickly detect flunixin and 5-hydroxy flunixin remaining in concentrated marine products.

According to another aspect of the present invention, the present invention provides hybridoma cell line 46C9 with accession number KCTC15069BP, which produces a monoclonal antibody that specifically binds to flunixin.

According to another aspect of the present invention, the present invention provides a composition for detecting flunixin comprising a monoclonal antibody that specifically binds to flunixin.

The composition contains a monoclonal antibody that specifically binds to flunixin, produced by the hybridoma cell line, and detects an antigen-antibody complex formed against flunixin using the antibody to immunologically detect flunixin. It can be detected.

As described above, the monoclonal antibody of the present invention exhibits specific immunoreactivity to flunixin and can easily detect flunixin. Therefore, when applied as an antibody for flunixin, flunixin remaining in concentrated marine products can be detected. It can be a kit.

According to another aspect of the present invention, the present invention provides a kit for detecting flunixin comprising a monoclonal antibody that specifically binds to flunixin.

As used herein, the term "antigen-antibody complex" refers to a complex of flunixin in a sample and the monoclonal antibody of the present invention that recognizes it, and such antigen-antibody complex can be prepared by colorimetric methods, electrochemical methods ( It can be detected by any method selected from the group consisting of electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment, and scintillation counting method. It may be possible, but it is not limited to this.

The composition for detecting flunixin of the present invention may further include additives that can be included in conventional compositions for detecting residual drugs, such as auxiliary substances for maintaining antibody activity and confirming antigen-antibody binding.

The composition for detecting flunixin of the present invention includes tools for collecting samples of test subjects or applying diagnostic reagents, reagents or devices for confirming antigen-antibody binding, and other antibodies to confirm the presence or absence of flunixin from biological samples. Tools, devices, or reagents that can be included in a typical diagnostic composition may be further included.

Tools/reagents that may be included in the diagnostic composition include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, etc. If the labeling substance is an enzyme, it may include a substrate that can measure enzyme activity and a reaction stopper. Suitable 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, It may be polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, polymers such as magnetic particles made by plating metal on latex, other paper, glass, metal, agarose, and combinations thereof.

According to another aspect of the present invention, the present invention provides a method for detecting flunixin, comprising the step of detecting a flunixin-antibody complex formed by contacting a sample sample with a monoclonal antibody that specifically binds to flunixin. .

In one embodiment of the present invention, the monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line 46C9 of the embodiment of the present invention.

In one embodiment of the present invention, various labeling substances can be used to detect the complex of flunixin and the antibody. Specific examples may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes, but are not limited thereto.

Preferred enzymes used as the detection labeling substance include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, or β-lactamase, and preferred fluorescent substances include quantum dots, Fluorescein, Eu ³⁺ , Eu ³⁺ chelate or cryptate, preferred ligands include biotin derivatives, preferred luminescent substances include acridinium ester or isoluminol derivatives, and preferred microparticles include colloidal gold. or colored latex, and preferred radioactive isotopes include, but are not limited to, ⁵⁷ Co, ³ H, ¹²⁵ I or ¹²⁵ I-Bolton Hunter's reagent.

The method for detecting the antigen-antibody complex is preferably using enzyme-linked immunosorbent assay (ELISA), but is not limited thereto. Enzyme immunosorbent methods include direct ELISA using an antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support, and Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, a label that recognizes this antibody after reacting with another antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support It includes various ELISA methods, such as indirect sandwich ELISA using secondary antibodies.

The monoclonal antibody can be conjugated with a detection label, and if it is not conjugated with a detection label, these monoclonal antibodies or fragments thereof can be captured and confirmed by processing another antibody having a detection label. You can.

In this specification, the term "sample sample" refers to an object on which analysis is to be performed on the basis that it contains or is presumed to contain flunixin, and includes liquid, soil, air, food such as meat, raw milk, eggs, waste, animal tissue, and cells. , may be collected from any one or more selected from the group consisting of whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, feces, or urine, but is not limited thereto. The animals include, but are not limited to, livestock such as horses, pigs, cows, and dogs, and poultry such as chickens, ducks, and turkeys. Additionally, the animal tissues include tissues such as mucosa, skin, integument, hair, scales, eyeballs, tongue, cheeks, hooves, beak, snout, feet, hands, mouth, nipples, ears, and nose.

According to another aspect of the present invention, the present invention provides a kit for detecting flunixin containing the monoclonal antibody.

In one embodiment of the present invention, the kit for detecting flunixin may be for ELISA method. Specifically, a substrate comprising a solid-phase support on which an antigen is fixed or conjugated to the surface; And it may include a portion containing the above-mentioned monoclonal antibody.

The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide, but is not limited thereto.

In one embodiment of the present invention, the portion containing the above-mentioned monoclonal antibody may be prepared by pretreatment with antibody-colloidal gold.

In one embodiment of the present invention, the kit for detecting flunixin may further include a part that absorbs unreacted substances in the sample after completion of the immune response.

In one embodiment of the present invention, the flunixin detection kit may further include a part that absorbs the sample.

In one embodiment of the present invention, the kit for detecting flunixin may be a lateral flow assy kit, but is not limited thereto.

The kit for detecting flunixin of the present invention is an invention that uses the composition for detecting flunixin and the method for detecting flunixin of the present invention described above, and content overlapping with these is omitted to avoid complexity of the present specification.

The present invention relates to a monoclonal antibody specific for flunixin and a kit for detecting flunixin using the same. Using the monoclonal antibody of the present invention, flunixin remaining in concentrated marine products can be quickly and accurately detected.

Figure 1 shows flunixin-KLH conjugate and flunixin-BSA conjugate prepared to develop a hybridoma cell line that produces an antibody that specifically binds to flunixin. It represents.
Figure 2 shows the procedure for developing a hybridoma cell line that produces a monoclonal antibody that specifically binds to flunixin.
Figure 3 shows the results of confirming the serum titer of mice immunized with flunixin-KLH conjugate.
Figure 4 shows a schematic diagram of a competitive enzyme-linked immunosorbent assay (Competitive ELISA) used to test the cross-reactivity of the monoclonal antibody of the present invention with other anti-inflammatory agents and antibacterial and antibiotic substances.
Figure 5 shows the cross-reaction test results of the monoclonal antibody of the present invention with other anti-inflammatory agents.
Figure 6 shows the cross-reaction test results of the monoclonal antibody of the present invention with other antibacterial and antibiotic substances.
Figure 7 shows a schematic diagram of the process of detecting flunixin using the monoclonal antibody and immunochromatography method of the present invention.
Figure 8 shows the configuration of the lateral flow assy kit for detecting flunixin of the present invention.
Figure 9 shows the results of determining the detection level of flunixin (detection limit 0.5 ppb).
Figure 10 shows the cross-reaction test results of the flunixin detection kit of the present invention with non-steroidal anti-inflammatory drugs.
Figure 11 shows the cross-reaction test results of the flunixin detection kit of the present invention with a steroidal anti-inflammatory agent.
Figure 12 shows the cross-reaction test results of the flunixin detection kit of the present invention with other antibacterial and antibiotic substances.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Preparation of antigens

Substances with a small molecular weight, such as Flunixin, are not antigenic on their own, so carriers with a large molecular weight, such as KLH (Keyhole Limpets Hemocyanin) or BSA (Bovine Serum Albumin), are used. Antibodies are developed by immunizing with a conjugate combined with a carrier protein. To develop a hybridoma cell line that produces an antibody that specifically binds to flunixin, flunixin-KLH conjugate and flunixin-BSA conjugate were prepared (Figure One).

Example 2: Mouse immunization and production of monoclonal antibodies

A hybridoma cell line producing a monoclonal antibody that specifically binds to flunixin was developed according to the procedure shown in Figure 2. As shown in Figure 2, in order to obtain immunized mice required for the development of hybridoma cell lines, the flunixin-KLH conjugate prepared in Example 1 was used as an immunogen and inoculated into 6-week-old mice. After the first boost vaccination, blood was collected to test the ELISA serum titer, and after the second boost vaccination, blood was collected to test the ELISA serum titer. Afterwards, mouse spleen cells and myeloma cells were fused and screened, and the final hybridoma cell line 46C9 was cultured through primary cloning and screening and secondary cloning and screening.

Serum titers of mice immunized with flunixin-KLH conjugate were confirmed. The results are shown in Figure 3. As shown in Figure 3, it was confirmed that an antibody that specifically binds to flunixin was successfully formed.

Example 3: Cross-reactivity test

Cross-reactivity with other anti-inflammatory agents and antibacterial/antibiotic substances was confirmed using competitive enzyme-linked immunosorbent assay (Competitive ELISA) (Figure 4). The anti-inflammatory drugs used in the cross-reaction test were Phenylbutazone, Meloxicam, Dexamethasone, Prednisolone, Tolfenamic acid, Aminopyrine, Triamcinolone, and Phena. It was Phenacetin, and the antibacterial and antibiotic substances used in the cross-reaction test were ampicillin, Enrofloxacin, Oxytetracycline, Streptomycin, Sulfadimethoxine, They were Spiramycin and Chloramphenicol.

The degree of cross-reaction was expressed as Inhibition %, indicating the degree to which absorbance was suppressed compared to the negative control. The calculation formula for Inhibition % is as follows.

Inhibition % = (1-sample OD/Negative OD) x 100%

As a result of the cross-reaction, as shown in Figures 5 and 6, it was confirmed that the developed monoclonal antibody specifically binds to flunixin and does not react with other types of anti-inflammatory drugs, antibacterial and antibiotic substances.

Example 4: Construction and performance evaluation of a lateral flow assy kit using a monoclonal antibody that specifically binds to flunixin

Example 4-1: Construction of a lateral flow assy kit using a monoclonal antibody that specifically binds to flunixin

A lateral flow assy kit that can be tested immediately on site was produced using the monoclonal antibody produced in Example 2. The kit uses an immunochromatographic method, and a schematic diagram of the detection process is shown in Figure 7.

The kit includes i) a sample pad, ii) an antibody-gold pad, iii) a nitrocellulose membrane containing a test line and a control line, and iv) It includes four parts consisting of an absorbent pad, and was manufactured through pretreatment and assembly (FIG. 8).

i) The sample pad is a part that absorbs the sample to be measured, and a pad made of cellulose material was used.

ii) Dispense Flunixin-BSA conjugate to the test line of the nitrocellulose membrane and goat anti-mouse IgG to the control line, 25 It was prepared by drying at ℃ for 24 hours.

iii) Antibody-gold pad is the part where antigen-antibody immune reaction occurs between flunixin remaining in raw milk, meat, blood, eggs, etc. and monoclonal antibodies. Antibody-gold pad is placed on the polyester pad as follows: It was prepared by pretreatment of colloidal gold. Colloidal gold with a particle size of about 40 nm was prepared by reducing gold chloride by adding Na-citrate to the gold chloride solution. 5 μg/ml of purified monoclonal antibody was added to the prepared gold colloid and then shaken for 1 hour at room temperature. When the reaction was completed, the gold particles were washed and quantified by measuring the absorbance at 520 nm using an absorbance meter. Then, a preservative was added and the gold particles were uniformly absorbed into polyester fibers and dried at a relative humidity of 20% or less for more than 4 hours.

iv) The moisture absorption pad is a part that absorbs unreacted substances in the sample after completion of the immune reaction and thus allows the sample solution containing the test substance to move by capillary action, and was attached to the top of the nitrocellulose membrane.

Lamination was performed by attaching the prepared components of i) to iv) by overlapping them on a plastic card. Remove the double tape portion of the plastic card with the membrane coating completed, place the antibody-gold pad on the bottom of the antibody-coated nitrocellulose membrane so that it overlaps 2 mm, and the sample pad on the nitrocellulose pad so that it overlaps 2 mm on the bottom of the antibody-gold pad. Moisture absorption pads were placed in order with an overlap of 2 mm on top of the membrane, and an assembly process was performed in which each component was overlapped and attached to fit the internal strip structure of the test kit. I gently pressed the assembled card again with my hand to ensure there were no gaps.

In the kit of this example, as shown in FIGS. 9 to 12 of the example to be described later, the control line is displayed darker than the test line in the case of flunixin positive, and the test line is displayed darker than the control line in the case of flunixin negative. If it is difficult to read with the naked eye, it is read using a reader. If the ratio is more than 1, it is read as positive, and if the ratio is less than 1, it is read as negative.

Example 4-2: Performance evaluation of the prepared flunixin detection kit

1) Test method, result interpretation, and determination of detection limit

A test was conducted to evaluate the performance of the manufactured flunixin detection kit. 150 ㎕ of raw milk was added to the kit without pretreatment. For meat, the broth was diluted 1:1 with the dilution solution, and then 120 ㎕ was instilled into the kit. Egg yolk and egg white were mixed well, diluted 1:1 with dilution solution, and 120 ㎕ was instilled into the kit. The kit containing each sample was reacted at 40°C for 10 minutes and then read using a reader. If the ratio was more than 1, it was read as positive, and if it was less than 1, it was read as negative.

By spiking each concentration and testing repeatedly, the minimum concentration that reads 100% positive was determined as the detection limit. The limit of detection (LOD) of flunixin in raw milk and egg samples was determined to be 20 ppb (Table 1 and Figure 9).

reagent Concentration (ppb) average signal Average Ratio Number of positives/Number of tests positivity rate Flunixin 10 74.20 1.03 15/30 50% 20 46.19 1.98 30/30 100% 30 20.29 2.00 30/30 100%

2) Check cross-reaction

It was tested whether the manufactured flunixin detection kit cross-reacts with non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, and antibacterial/antibiotic substances.

As shown in Table 2 and Figure 10, the manufactured kit had a cross-reaction with 5-Hydroxy Flunixin, a metabolite of flunixin, but did not contain meloxicam and tolfenamic acid ( There is no cross-reaction with non-steroidal anti-inflammatory drugs such as tolfenamic acid, so it can be confirmed that flunixin or 5-hydroxy flunixin, a metabolite of flunixin, can be specifically detected. there was.

anti-inflammatory Concentration (ppb) average signal Average Ratio Number of positives/Number of tests positivity rate phenacetin
(Phenacetin) 100 147.30 0.00 0/10 0% 5-Hydroxyflunixin
(5-Hydroxyflunixin) 20 50.17 0.20 10/10 100% Metamizole
(Metamizole) 100 158.95 0.00 0/10 0% Acetanilide
(acetanilide) 100 156.72 0.00 0/10 0% Phenylbutazone
(Phnylbutazone) 100 156.64 0.00 0/10 0% Meloxicam
(Meloxicam) 100 155.04 0.00 0/10 0% aminopyrine
(Aminopyrine) 100 146.32 0.00 0/10 0% antipyrine
(antipyrine) 100 152.03 0.00 0/10 0% Tolpenamic acid
(Tolfenamic acid) 100 149.50 0.00 0/10 0%

As shown in Table 3 and Figure 11 below, it was confirmed that flunixin could be specifically detected as there was no cross-reaction with the steroidal anti-inflammatory drugs Prednisone, Triamcinolone, and Dexamethasone.

anti-inflammatory Concentration (ppb) average signal Average Ratio Number of positives/Number of tests positivity rate prednisone
(Prednisone) 100 143.58 0.00 0/10 0% triamcinolone
(Triamcinolone) 100 143.87 2.00 0/10 0% dexamethasone
(Dexamethasone) 100 148.97 1.98 0/10 0%

In addition, as shown in Table 4 and Figure 12 below, representative antibacterial and antibiotic substances used as veterinary medicines include Oxytetracycline, Enrofloxacin, Sulfamethazine, and Ampicillin. It was confirmed that flunixin could be specifically detected because there was no cross-reaction.

Antibacterial/antibiotic substances Concentration (ppb) average signal Average Ratio Number of positives/Number of tests positivity rate Oxytetracycline
(Oxytetracycline) 100 149.94 0.00 0/10 0% Enrofloxacin
(Enrofloxacin) 100 155.81 0.00 0/10 0% Sulfamethazine
(Sulfamethazine) 100 153.79 0.00 0/10 0% Ampicillin 100 151.83 0.00 0/10 0%

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15069BP 20220901

Claims (7)

A monoclonal antibody that specifically binds to Flunixin.
The monoclonal antibody according to claim 1, which is produced by hybridoma cell line 46C9 with accession number KCTC15069BP.
The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically binds to 5-Hydroxy Flunixin.
Hybridoma cell line 46C9 with accession number KCTC15069BP producing the monoclonal antibody of paragraph 1.
A composition for detecting flunixin comprising the monoclonal antibody of claim 1.
A kit for detecting flunixin containing the monoclonal antibody of claim 1.
A method for detecting flunixin comprising the step of detecting a flunixin-antibody complex formed by contacting the monoclonal antibody of claim 1 with a sample.