TWI309239B - Monoclonal antibody specific to enrofloxacin, hybridoma producing same and kit comprising same - Google Patents

Description

1309239 IX. Description of the Invention: [Technical Field] The present invention relates to a monoclonal antibody specific for Enrofloxacin, which secretes a fusion cell cell strain producing the monoclonal antibody, including the single A kit of strain antibodies, and a method for detecting enrofloxacin residues using the monoclonal antibody. [Previous technique] Enrofloxacin (ENR), also known as enflurazine, is a kind of fluoroquinolone antibiotic, which is a broad-spectrum antibacterial agent. The alpha unit of the bacterial DNA gyrase is broken to inhibit DNA replication. Since DNA gyrase belongs to the enzyme of topoisomerases, which is the supercoiling of the negative DNA strand, enrofloxacin can make the double strands of DNA unable to dissociate. Inhibition of DNA synthesis. With the special mechanism of action of enrofloxacin bamboo, it can effectively inhibit Gram positive bacteria, negative bacteria, Brucella, chlamydia and mold. Enrofloxacin and its analogues are commonly used in chickens and cattle in the early stages of biotin, and later abused to cause poultry to pass on (four) pathogens, such as the common eampylQbacteria that cause food poisoning. Fluoride produces resistance. These antibiotics are produced in the digestive tract of poultry and are transported and slaughtered. The scorpion is detected in the import and export of new fish in many countries. The residue of the star. Because 'European and Sand Star’s residual period in animals is quite

TBH-P060020-TW 5 1309239 'Long, if enrofloxacin remains in meat, it is bound to cause danger to human health. Its sputum action includes intestinal obstruction, highly sensitive reaction and crystallization of urine. Therefore, the international trend - bacteriostatic agents used in humans should be avoided in animal husbandry, so as to avoid the problem of bacterial resistance caused by drug residues. At present, in many countries, the ban on such antibiotics has been ordered. According to the “Regulations on the Use of Aquatic Animal Drugs” by the Agricultural Committee of the Executive Yuan, enrofloxacin is used in animals (such as cattle and pigs) and not in aquatic animals ( Among the nine species including squid, squid, squid, rainbow trout, black scorpion, yellowfin, squid, grass shrimp and spotted shrimp, according to the Department of Health's "Animal Residues for Animals", in aquatic products No residual enrofloxacin may be detected. The method for detecting enrofloxacin can be divided into three major categories: test strip reagent rapid test 埤 (thin_layer chr〇mat〇graphy, TLC) enzyme linked immunosorbent assay (ELISA), High performance liquid chromatography (HPLC). 1. Test strip reagent rapid test method: The basic principle is to analyze the specific immunochemical reaction of antigen (Ag) and antibody (antibody, Ab). The whole reaction mechanism is to let the sample-test and the antibody-colloidal gold complex in the reaction micropore compete for the reaction. The capillary phenomenon of the re-use test piece (the lower end is covered with white glass fiber) is developed, and whether it is carried out in the central interpretation area. Interpretation of the residual situation of rofefloxacin. This test kit is specific for the detection of enrofloxacin. Its detection sensitivity is 15 ppb ° 2. Enzyme-linked immunosorbent assay: The principle of ELISA is also the specificity of the combination of antigen and antibody 'plus enzyme Coloring (or glory)

6 TBH-P060020-TW 1309239 light) reaction to show the presence or absence of a specific protein. Qualitative screening and quantitative analysis of enviral residues in aquatic products, livestock, poultry, meat, eggs and dairy products can be performed using competitive enzyme combined immunosorbent assays. Take the product of the current commercially available Euro-Diagnostica B.V. (Netherlands)-enrofloxacin enzyme combined with immunosorbent assay kit as an example. It takes only 2 hours to analyze 1 sample and 1.5 hours of reagent reaction time. The detection sensitivity is 3 ppb 0 3. High-performance liquid chromatography: In the method, the sample is first obtained by extracting and purifying the previous processing steps, and then referring to the animal in the CNS-14684 food. Test method for drug residues _ norflixidine carboxylic acid, virgin fluoroquine carboxylic acid, enflurequinone carboxylic acid test (Standards of Quality and Commitment, Ministry of Economic Affairs, 2002) and the Department of Health of the Executive Yuan (Guardian Drugs Word No. 0910049817 No.) Method for the determination of animal drug residues in foods - Quinoa. Multiple residue analysis method - Using a high performance liquid chromatography with a photodiode array detector and a fluorescent detector ( The fluorescene detector can be used in tandem to identify the slaves of livestock and poultry meat and aquatic products. The detection sensitivity of this detection method is 20 ppb. Using the test and ship speed test to detect Enzafloxacin, although it has the advantage of being fast and simple, 'but because this test can only be used for detection, the sample is not, and Envifloxacin m will be used for money control. Enro's breath is born in the middle of the bay. The use of 〒 〒 液相 液相 液相 检测 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 由于 〒 由于 由于 〒 〒 〒 〒 〒 〒 〒 〒 〒 Liquid homeolysis method, enzyme combined with immunosorbent assay has: a) pure heterogeneity, can ^

TBH-P060020-TW 7 1309239 Enrofloxacin is confirmed to have low cross-reactivity with other fluoroquinone antibiotics; b) High sensitivity: the lowest detection concentration of enrofloxacin is 0.3PPb, the highest sensitivity of real samples讦3 ppb ; c) Sample diversity: for example, squid can detect carp body, sputum serum, fish, feed, etc.; d) easy to handle the sample: do not use organic solution extraction 'just use the kit The diluted solution is provided for processing; e) rapid detection: the entire detection process takes only about 20 minutes; and the design is simple: the kit is convenient to carry, and only simple equipment such as an electronic scale small centrifuge is required. Therefore, the present invention develops a monoclonal antibody against enrofloxacin, a fusion tumor cell strain secreting the monoclonal antibody, and a detection kit based on enzyme-binding immunosorbent assay for rapid, simple and high-sensitivity detection. . SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to provide a fusion tumor cell line which secretes a monoclonal antibody specific for Enro I sand star. Another object of the present invention is to provide a monoclonal antibody specific for enrofloxacin which is secreted by the above-mentioned fusion tumor cell line. A further object of the present invention is to provide a test kit that is highly specific for enrofloxacin. Still another object of the present invention is to provide a use of the monoclonal antibody, the fusion tumor cell line and the ferrule to detect the residual amount of erofloxacin. In order to achieve the above object, the present invention is prepared by a conventional technique-cell fusion method, wherein a myeloma cell and a sputum cell secreting a monoclonal antibody against enrofloxacin are added with a cell fusion agent polyethylene glycol (p〇). Lyethyiene

The two cells were fused by means of 8 TBH-P060020-TW 1309239 glycol, PEG) to prepare a B cell fusion tumor which is specific for enrofloxacin. Subsequent to hypoxanthine-aminopterin-thymidine (HAT)

The medium was screened for the fusion of the tumor cell line, and the specificity of the antibody in the culture medium of the fusion tumor cell line was analyzed by indirect enzyme-binding immunosorbent assay and competitive enzyme-binding immunosorbent assay. A single fusion cell line (7002-9.2) specific for enrofloxacin will be screened and injected into the peritoneal cavity of mice to produce ascites; then the protein purification technique will be used to recover the antibody from ascites. A direct competitive ELISA kit to detect the amount of enrofloxacin remaining in the sample. The specificity and sensitivity of immunoassays depend on the nature of the antibody. To produce antibodies with specificity and affinity, it has a great relationship with the design and preparation of immunogens. Especially the molecular weight is less than 1 耳 耳

The molecule of Ualt〇n), such as enrofloxacin (molecular weight 359.4), is a small hapten due to its small size, which means that it does not stimulate the immune system to produce antibodies when it is present alone. The half-k original binds to a large molecular weight immunogenic protein, and assists the hapten to produce immunity by the immunogenicity of the carrier protein. Among them, II-protein can be derived from one of the groups consisting of ovalbumin, bovine serum albumin, globulin, blood dust, and iron protein. It can be seen that the combination of small molecule god carrier and preparation process Video _ The quality and specificity of the antibody. In this particular aspect of the _k, a long time, the use of enrofloxacin and f

9 TBH-P060020-TW 1309239 Albumin (OVA) carrier egg conjugates were used as immunogens to generate monoclonal antibodies, the specificity of which is described in the following examples. The method for detecting enrofloxacin residues in a sample according to the present invention is a direct competition enzyme-binding immunosorbent method, and the analysis method and reaction conditions are based on conventional techniques (Antibodies: A Laboratory Manual; Ed. Harlow & Day id Lane, 1988). First, the monoclonal antibody against enrofloxacin is adhered to the surface of a solid support. When tested, different concentrations of enrofloxacin standard and sample to be tested, and connected signals> (such as radioactive elements) Enrofloxacin complexes, fluorescent agents, luminescent markers and enzymes are placed in the reaction wells. Since enrofloxacin competes with the enrofloxacin-signal complex (traCer) for a limited anti-enfloxacin antibody, the signal produced by the known standard concentration of enrofloxacin is strong and weak. An enrofloxacin inhibition standard curve was established to detect the presence and content of enrofloxacin in the sample. Therefore, another aspect of the present invention relates to the preparation of a test kit using the monoclonal antibody against enrofloxacin, wherein the enrofloxacin test kit and the group are direct competitive enzyme-binding immunoassay kits, Contains: (1) solid phase support '(2) monoclonal antibodies against enrofloxacin, (7) qualifiers of Enro gas, and (4) en, roflufloxacin signal complex, and (7) coloring matter. Wherein, the solid phase support may be a microtiter plate (at a (10) price), a micro-beads, a small dropper, and a paper, which are made of a material suitable for protein=疋. Polyvinyl chloride, polystyrene, nitrocellulose, Nylon and other methods, &, #成的群―; In addition, the solid support can also be iron-containing magnetic micro-琏. The signal can use, for example, a radioactive element

10 TBH-P060020-TW l^239 Element, fluorescent substance, luminescent label and biological luminescent label and chemical 々 1, version, '; photo-marker can be 素, alkali '___, P · galactose * yeast . The oxygen enzymology test group of Enrofloxacin invented by the present invention has a high specificity - and is for the detection of enrofloxacin. The characteristics can be applied to the above-mentioned other purposes, methods, characteristics, and advantages of enrofloxacin. More: 瞧, the following is a detailed description of the following: The present invention may be embodied in various forms, and the drawings and the following description are preferred embodiments of the invention, and are disclosed herein. The present invention is not intended to limit the invention to the specific embodiments shown and/or described. First, the English abbreviated nouns appearing in this embodiment are defined as follows: BSA : b〇vine serum albumin DMF . Dimethylformamide DMSO : dimethyl sulfoxide EDC : carbodiimide (1-ethyl-3-(3- Dimethylaminopropyl) -carbodiimide) ENR : enrofloxacin

11 TBH-P060020-TW 1309239 FCS : fetal calf serum HAT. hypoxanthine-aminopterin-thymidine HRPO . horseradish peroxidase (horseradishperoxidase) KLH. hemocyanin (NHhole.) N-hydroxysuccinimide OVA : _ albumin (ovaibumin)

PB buffer: phosphate buffer PBS. phosphate buffer saline

Tg: thyroglobulin TMB: tetramethytbenzidine Example 1 Preparation of a combination of enrofloxacin hapten-carrier protein Please refer to Figure 1 for the preparation of the present invention. Schematic diagram of the steps of the combination of enrofloxacin hapten-carrier protein. First, prepare sputum (ENR72 mg + NHS 28 mg + EDC 57 mg, dissolved in 10 ml of DMF) and β solution (OVA 80 mg dissolved in 16 ml of 0.1 MPB buffer (pH 8.0)), and slowly add solution A. In the solution B, the reaction was stirred at room temperature for about several hours. The reaction mixture was concentrated in a 30 K concentrator' and the solution was replaced with pBs; followed by filtration to remove impurities, and finally the volume of the solution was adjusted to 8 ml in PBS 'measured OD 280 nm value' and known The concentration of the protein is compared to the protein concentration (in mg/ml), which is the ENR hapten-OVA carrier protein conjugate' for immunization.

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11309239 Example 2_Preparation of fusion tumor cell line A combination of enrofloxacin-hemocyanin (ENR_KLH), wherein the molar ratio of ENR/KLH is 1:2, and the combination is combined with Freund's ( The Freunds adjuvant was mixed at a ratio of 1:1 (v/v), and the mixed emulsion was then injected subcutaneously into BALB/C mice (each using loopgENR-KLH for a total of one immunization). At the third week, the spleen cells were mixed with the murine myeloma cell line F(ATCC CRL 1646), mixed in a ratio of 5:3, and then fused with 50% polyethylene glycol-1500 (PEG-1500). . After the fusion, the cells were suspended in HAT medium, diluted with F0 cells to a number of 1 χ 5 cells, and then seeded into a 96-well culture plate (〇. 2 mi/well). After 10 days, the cells were cultured with an ELISA plate to which the enolfloxacin-ovalbumin conjugate (ENR_〇VA) was adsorbed. A fusion cell line with specificity to enrofloxacin was selected and monocultured by limiting dilution method. A single cell fusion strain 7002-9.2 (registered number BCRC 960268) was selected by this method. The monoclonal antibody of the fusion tumor cell strain was examined and found to be an IgG1 heavy chain and a Kappa light chain. The fusion tumor cell line (in 1% DMSO and 90% FCS) can be stored in liquid nitrogen and can be cultured using standard mammalian cell culture techniques. Example 3: Direct competition type EUsa kit for detecting enrofloxacin drug residues Sha Xing single 妷 Hang 艚餹 锄

The monoclonal antibody secreted by the fusion tumor cell line 7002-9.2 was dissolved in a sodium carbonate buffer (pH = 9.6) and diluted to 1 μ§/ηι1. Take 1 inch into a well and place in a 96-well polystyrene microplate, at 4. Underarm cultivation overnight. pBsT

P060020-TW 13 1309239 ^0.05% Tween 20 in coffee) Rinse the microplate 3 times, then pat dry; then, add 250 μl blocking solution (1.5% skim milk + 3% trehalose) to each well. (trehalose) in PBS buffer solution, ρΗ7·2±〇2), blocked at room temperature for 2 to 3 hours. Subsequently, the boring disc is flipped dry to remove the liquid remaining on the microplate, so that the microplate is fully dried up to the drying chamber (25, 20.7 / ^) for about 3 to 4 hours, and dried for use. .愚维虱沙盖-过乳也氲酶合体_ Prepare ENR2 mg'NHs 2 mg and EDC 4 mg into 丨mlDMp and stir at room temperature for about 1 hour. Take 2 mg of 〇 2 mg dissolved in 2 ml of pB buffer solution (pH 8.0) and mix the two evenly. After reacting for one hour, dialysis in 1× PBS at 4C for about 4 hours, this is the ENR enzyme combination, and Stored in _2 (rC. Among them, 1×PBS was taken 20 ml 20 times PBS, added to a beaker of 2 ml, and then 380 ml of pure water was added, and the homologous sentence was given. The reaction formula of _ oxidized sulphin complex is shown in Figure 2. Fraunhofer standard solution f偌 Enfolfloxacin standard 25_0 mg (INC standard) is diluted to 250 ml with decyl alcohol, shake After pouring into a 250 ml brown serum bottle, the concentration of enrofloxacin was 100 ppm (1000 pg/ml). Enrofloxacin was diluted 100 ppm to ippm with i times phosphate buffered saline (PBS). 1, ppb, 4.8 ppb, 2.4 ppb, 1.2 ppb, 〇·6 ppb, 0.3 ppb and 〇ppb. Among them, 恩ppb's enrofloxacin standard solution is 1 times pBS.

TBH-P060020-TW 14 1309239 2.4, 4 : The single antibody antibody _ cell strain 7_.2 secreted antibody antibody in the ship, plus 8 of the weishafloxacin-hydrogenase enzyme complex (diluted 1G Leave it in the dark for 40 minutes, then place the anti-storage on the mouth and then pour off the reaction solution, fill the micropores with PBST (0.05〇/〇, 20 weights), then pour off; After washing 3 times,

拍干' Add 100 pL of hydrogen peroxide to the ΤΜβ, and let it stand at room temperature in the dark. After the knife is knives, add 50 μ:ί 2N HC1 to stop the reaction, and then measure the value of 〇D 45〇/65〇. It can be suppressed, green, as shown in Figure 3 of the standard, en, and roflufloxacin standard. Table 1 below shows the absorbance of enrofloxacin standard solution, the coefficient of variation of five replicates (coefficient 0fvariati〇n, Cv), and the percent inhibition (B/B〇°/.). It can be seen that the cv value of each concentration is less than 4%, indicating that the ELISA kit for detecting enrofloxacin has high reproducibility. The percent inhibition represents the relative sensitivity of the antibody to a particular substance and is calculated as: (absorbance of standard solution / absorbance of zero standard solution) X 100%. From the percent inhibition of enrofloxacin, the lowest detectable concentration of the test kit was 0.3 ppb. Table 1: Absorbance of enrofloxacin standard solution concentration, coefficient of variation and inhibition percentage of 5 repeated tests (B/B〇°/〇), ENR concentration OD 450/650 SD CV% B/B〇% Ppb 2, 3 4 5 Average ^ 0 2.666 2.700 2.643 2.529 2.658 2.639 0.065 2.5 100 0.3 2.209 2.308 2.309 2.202 2.268 2.259 0.052 2.3 86 0.6 1.878 1.839 1.834 1.889 1.871 1.862 0.024 1.3 71 1.2 1.414 1.407 1.432 1.382 1.407 1.408 0.018 1.3 53 2.4 0.912 0.932 0.928 0.897 0.905 0.915 0.015 1.6 35 4.8 0.526 0.495 0.515 0.485 -Γ5~1 0.486 0.501 —^ 0.018 -TBH-POi 3.6 ro〇2CFTw 19 1309239 The following table shows the specific cross-reactivity test for nine quinolones As a result, it was revealed from the results that the specificity of the ELISA kit for detecting enrofloxacin of the present invention for enrofloxacin drugs was as high as 99 〇 / or more. Table 2: Cross-Reactivity test results for the following quinolones. Compound cross-reaction (%) Enrofloxacin 100 % ------- Ofloxacin ) Γ% Norfloxacin 0.5 % ----- % Ciprofloxacin 0.5 % ------ Sarafloxacin 0.1 % Danofoloxacin 0.1 % ------ Nalidixic acid <0.1 % --- Oxolinic acid : <0.1 % ---- Flurequify -Following 0.1% ~~ -----

While the present invention has been described in its preferred embodiments, it is not intended to limit the scope of the invention, and various modifications and changes can be made without departing from the spirit and scope of the invention. As explained above, various modifications and variations can be made without departing from the spirit of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims. *''' 16

TBH-P060020-TW 1309239 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the preparation steps of the enrofloxacin hapten-carrier protein conjugate according to the present invention. The first diagram uses a hapten technique to attach a long chain to enrofloxacin; the lb diagram uses a biochemical synthesis technique to attach an immunogenic carrier to the long chain of enrofloxacin. Protein, which becomes an antigen-immunogen. Fig. 2 is a view showing the steps of preparing the enrofloxacin-hydrogenase complex of the present invention.

Figure 3 is a graph showing the inhibition curves of the enrofloxacin standard of the direct competition type ELISA of the present invention. [Main component symbol description]

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Claims (1)

1309239 r年〆月丨1曰Revised replacement page I i Announcement rhyme 10. Patent application scope: 1. A fusion tumor cell line 7002-9.2 (registered number BCRC which can secrete anti-enrofloxacin) The antibody of the strain, wherein the monoclonal antibody is an IgG1 heavy chain and a Kappa light chain. 2. The fusion tumor cell line according to claim 1, wherein the fusion tumor cell line is produced by a bone tumor cell and an anti-enro A fusion cell strain according to the invention of claim 1, wherein the B cell line is obtained from an animal immunized with enrofloxacin and a carrier protein conjugate. 4. The fusion tumor cell strain according to claim 2, wherein the osteosarcoma cell line is a mouse myeloma cell FO cell strain. Lu 5. The fusion tumor cell according to claim 4 </ RTI> wherein the carrier protein is selected from the group consisting of ovalbumin, bovine serum albumin, globulin, hemocyanin and iron protein. 6. The fusion tumor cell line according to claim 2, wherein Fusion tumor system A cell strain obtained by cell fusion using polyethylene glycol. 7. A method for detecting enrofloxacin residues in a sample, which comprises the following 18 P060020-TW 1309239, the following steps: (1) Providing a monoclonal antibody specific for enrofloxacin, wherein the early strain resistance system is secreted by the fusion tumor cell line 7002-9.2 (registered number BCRC 960268); (2) providing a signal Generating a tool that operably binds to enrofloxacin to produce a signal; (3) adhering the monoclonal antibody to a support tool to form an antibody-support conjugate; (4) competing the test sample or enrofloxacin standard with the enrofloxacin-signal conjugate and immunologically binding to the antibody-supporting conjugate; and (5) measuring the signal generated by the signal generating tool Signal. 8. The method of claim 7, wherein the signal generating means is one selected from the group consisting of a radioactive element, a fluorescent substance, a luminescent label, and an enzyme. 9. The method of claim 8, wherein the luminescent marker is selected from the group consisting of a bioluminescent label or a chemically luminescent label. —10. According to claim 8 The method wherein the enzyme is one selected from the group consisting of hydrogen peroxide enzyme, alkaline phosphonate, and β-galactosidase. </ RTI> <RTIgt; Magnetic beads containing iron. 12. The method of claim 7, wherein the support tool is comprised of a suitable proteinaceous material selected from the group consisting of polyethylene, polystyrene, nitrocellulose, and nylon. The method according to claim 7, wherein the fusion tumor cell line is produced by fusion of a bone tumor cell with a B cell producing a monoclonal antibody against enrofloxacin. A kit for detecting enrofloxacin, which comprises a monoclonal antibody specific for enrofloxacin secreted by a fusion tumor cell line 7002-9.2 (Accession No. BCRC 960268). • 15. The kit according to claim 14 of the scope of the patent application, wherein the kit is a direct competitive enzyme-binding immunoassay kit. 16. The kit of claim 14, wherein the kit comprises: (1) a solid support; (2) a monoclonal antibody specific for enrofloxacin; (3) Standard for roflufloxacin; (4) Enrofloxacin-signal complex; 20 P060020-TW 1309239 々 々 修正 替换 替换 丨 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 17 17 The kit, wherein the solid phase support is a microtiter plate, a microsphere and is composed of a suitable protein fixing material selected from the group consisting of polyethylene, polystyrene, nitrocellulose, and Cailong. 18. The kit of claim 16 wherein the signal is selected from the group consisting of radioactive elements, fluorescent materials, luminescent markers, and enzymes. 19. The kit of claim 18, wherein the luminescent marker is selected from the group consisting of a bioluminescent label or a chemical luminescent label. 20. The kit of claim 18, wherein the enzyme is selected from the group consisting of hydrogen peroxide enzyme, alkaline phosphonate, and beta-galactosidase. 21 P060020-TW