TW514638B - Monoclonal antibody specific to chloramphenical, hybridoma producing same and kit comprising same - Google Patents

Abstract

The invention relates to monoclonal antibodies specific to chloramphenicol, a hybridoma producing the antibodies, a kit containing the antibodies, and the detection of chloramphenicol using the antibodies, hybirdoma, or kit.

Description

514638 A7 B7 V. Description of the invention (1) Field of the invention The present invention relates to a single antibody specific to aeromycin, a fusion tumor producing the antibody, a kit containing the single antibody, and using the antibody, fusion Tumors and kits to detect chloramphenicol residues. BACKGROUND OF THE INVENTION Chloramphenicol is a broad-spectrum antibiotic produced by Streptomyces venezuelae and can now be artificially synthesized. Chloramphenicol is fat-soluble, and can enter the cell through the cell membrane through diffusion, and bind to the nucleosome to inhibit protein synthesis. It has an inhibitory effect on most Gram-positive and negative bacteria, chlamydia, and rickettsia. Because it can effectively inhibit the growth of Salmonella and coliform bacteria and because it is cheaper, it is often used in the treatment of animals. However, long-term use of chloramphenicol in the human body may cause severe hematopoietic dysfunction, affect the formation of bone marrow and red blood cells, and cause aplastic anemia. Because chloramphenicol is stable to heat, it cannot be completely destroyed by ordinary cooking methods. Therefore, if chloramphenicol is left in meat food, it is bound to cause harm to human health. Therefore, it is necessary to detect the presence of chloramphenicol in livestock products to protect the health of consumers. China, the United States, Japan, Europe and other countries have stipulated that amycin cannot be present in animals that are human diets. However, according to the Drug and Food Inspection Bureau (* 1), A survey by livestock farmers in counties and cities found that there are many types of antibacterial substances used in livestock, among which chloramphenicol was found to be used in the treatment of ducks, dairy cows, and pigs. In addition, Lu et al. (* 2) detected high-screen areas Examination of pork shoulder meat found that 10%

47493.DOC This paper size applies to Chinese National Standard (CNS) Λ4 specification (210X 297 mm) Please read the notice on the back of the page. • Order t Printed by the Central Consumers Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 514638 Λ7 ________ B7 5. Description of the invention (2) The remaining amount is greater than 0.05PPm. Because of the serious side effects caused by long-term use of aeromycin, it is indeed necessary to screen for aerobicin residues in livestock products. Because there is no national standard gasomycin test method, microbiological method is generally used in conjunction with the legality and quantification of liquid chromatography. However, these two methods are time-consuming and the sensitivity of the microbiological method is not high enough. Neither method is suitable for mass screening. Because the immunoassay method is simple, time-saving, and sensitive, and does not require the use of radioactive substances, it is very convenient for the screening of a large number of specimens. SUMMARY OF THE INVENTION An object of the present invention is to provide a single-body antibody specific to chloramphenicol. Another object of the present invention is to provide a fusion tumor that secretes the monoclonal antibody. Another object of the present invention is to provide a kit comprising the single antibody for detecting aerobicin residue. Still another object of the present invention is to provide an application for detecting residual chloramphenicol using the single antibody, fusion tumor and kit. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economics The purpose, advantages and characteristics of the present invention will be made clearer by the following description. DETAILED DESCRIPTION OF THE INVENTION The present invention includes a fusion tumor cell line specific for aeromycin, and anti-fa and antibody fragments produced by the same. The present invention also includes a reagent set 'direct competition type enzyme immunoassay kit prepared using the antibody. The present invention also includes a method for detecting the amycin residue using the antibody and the kit. 4 74 9 3.DOC — 5 — This paper size applies to the Chinese National Standard (CNS) Λ4 specification (210X297 public holiday) ~ '----- Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 514638 A7 B7 V. Description of the invention (3) The present invention uses a known technique to prepare a B-cell fusion tumor specific to chloramphenicol by means of the fusion of B cells and osteoclast tumor cells. The method is to use a known cell fusion agent, such as polyethylene glycol (polyethylene glycol), to fuse mouse myeloma cell lines with B cells that produce anti-chloramphenicol antibodies, and then select fused tumor cell lines with HAT culture medium, and then Indirect enzyme-linked immunosorbent assay (ELISA) and competitive ELISA were used to analyze the specificity of the antibodies in the fusion tumor medium, and then a single fusion tumor cell line specific to chloramphenicol was selected. This fusion tumor cell line was then injected into the abdominal cavity of a mouse to produce ascites, and a single antibody was purified from the ascites using protein purification technology. This single antibody was then used to develop a Direct Competative ELISA Kit for the detection of chloramphenicol residues in livestock products. The specificity and sensitivity of the immunoassay depends on the characteristics of the antibody, but whether it can produce antibodies with high specificity and affinity has a lot to do with the design and preparation of the immunogen. In particular, molecules with a molecular weight of less than one thousand Daltons, such as chloramphenicol (molecular weight 323.1), are small immunogenic antibodies due to their small molecular weight, that is, they do not stimulate the immune system to produce antibodies when they exist alone. In order to generate an immune response against a small molecule hapten, it is generally necessary to combine a small molecule hapten material with a large molecular weight immunogenic carrier protein to assist the hapten to generate immunity by virtue of the immunogenicity of the carrier. Therefore, the present invention uses chloramphenicol-alpha-succinic acid and bovine serum albumin (BSA) as immunogens to generate a monoclonal antibody, and the specificity is as described in the following examples. . The direct competitive immunoassay method is to attach a monoclonal antibody against chloramphenicol to the surface of a solid support. When testing, different standard concentrations of amycin may be 47493.DOC — 6 — This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) Fill in π and fill in too, 11 514638 A7 B7 V. Description of the invention (4) The test sample is placed in the reaction together with the aeromycin complex that has signals (such as enzymes, fluorescent agents, radioactive elements, etc.) connected. Because chloramphenicol and amycin-signal complexes compete with each other for limited anti-chloramphenicol antibodies, a standard curve of aeromycin inhibition can be established based on the strength of the signal produced by a known standard concentration of amycin. The presence and amount of chloramphenicol in the sample. The direct-competitive immunoassay kit includes a solid phase support, a monoclonal antibody against chloramphenicol, a standard chloramphenicol, a chloramphenicol-signal complex, and a colorant. The solid support can be a microtiter plate, microbeads, and paper. It is composed of materials suitable for protein immobilization. Suitable materials can be polyvinylchloride, polystyrene, nitration Fiber, Nylon, etc. Signals can use, for example, fluorescent materials such as lanthanide fluorescent materials; luminescents such as biological luminescent and chemical luminescent indicators; radioactive elements; and enzymes such as catalase, transesterification Enzymes, β-galactosidase, etc. The analysis method and reaction conditions used conventional techniques (Antibodies: A Laboratory Manual; Ed. Harlow & Dayid Lane, 1988). In order to make the objects, methods, features, and principles of the present invention clearer, the following examples are described in detail with reference to the accompanying drawings, but these examples do not limit the scope of the present invention in any way. The English abbreviations in this manual are as follows: • Definition: HAT: Hypoxanthine / Aminopterin / Thymus

Tg: Bovine adenoglobulin FCS: Fetal bovine serum DMSO: Dimethylarsine 47493.DOC ~ 1 ~ This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) (Please read the notes on the back first (Install-this page)

1. 1T printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 514638 A7 B7 5. Description of the invention (Printed by the Central Standards Bureau of the Ministry of Economics PBS: Phosphate buffer solution TMB: Tetramethyl benzidine BSA : Examples of bovine serum albumin-Example 1: Production of fusion tumors chloramphenicol-α-succinic acid-bovine serum albumin conjugate (CAP-BSA) (CAP / BSA molar ratio is 40) and Freunds Adjuvant (full adjuvant for the first immunization; incomplete adjuvant for subsequent immunizations) 1: 1 (v / v) emulsion was injected subcutaneously into BALB / C mice (125 pg BSA each) for one week Once, 4 times in succession, and then fortified once every four weeks (40pg each) for a total of 5 times. Three days before cell fusion, an additional intravenous injection (without adjuvant) was added once. Three days later, Spleen cells were mixed with murine myeloma cell FO cell line (ATCC CRL 1646) at a ratio of 5: 3, and then fused with 50% polyethylene glycol 1500 (PEG-1500). After fusion, the cells were suspended Diluted into 1 X 105 FO cells in HAT medium and seeded into 96-well culture plate 0.2 ml / well). After 10 days, the cell culture supernatant was tested in an ELISA plate adsorbed with chloramphenicol-bovine thyroid protein conjugate (CAP-Tg). A fusion tumor specific to chloramphenicol was selected to Restriction dilution method was used for singulation. Using this method, single cell fusion tumors 3202-52.27 were selected. The single antibody secreted by this method was determined to be IgG2b and Kappa light bonds. This fusion tumor (containing 10% DMSO and 90% FCS) can be stored in liquid nitrogen, and can be cultured using standard mammalian cell culture techniques (RPMI 1640 with 10% FCS supplemented with 200 mM glutamine and 50 μM 2-lucyl ethanol). This fusion tumor was established on June 2, 1986.

47493.DOC This paper size applies Chinese National Standard (CNS) A4 specification (210X297mm) Please read the 5th on the back (r ^ J page t 514638 A7 _________B7 V. Description of the invention (6)-10th Patented Microbiological Depository Center of Institute of Food Industry Development, No. CCRC 960063. Example 1: Preparation method of direct competitive Elisa method good body attachment disk for detecting chlorbumin residue < 3202-52 · 27 single antibody Dilute to 5 pg / ml with carbonate buffer (pH = 96). Put 100 μl / well in a 96-well polyphenylene terephthalate microplate and incubate at 4 C overnight. Use pbs / 0.05% Tween Rinse 3 times, then pat dry. Then add 250 μΐ of PBS / 0.1% skim milk powder to each microwell, and block at room temperature for 2 hours. Then wash three times with pBS / a〇5% Tween 20, pat Prepared after the drying. The preparation method of rapamycin-hydrogenase conjugate is printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. The hydrogen peroxide enzyme (2 mg / ml) is first dissolved in pH 8.0, 50 mM phosphate buffer. In solution, place on ice, slowly add 0.25 ml DMF dropwise, and then Add 0.25 ml of chloramphenicol-〇-succinyl-N-hydroxysuccinimide (chloramphenicol-0-succinyl-N-hydroxysuccinimide ester) (lmg / ml DMF) and react at 4 ° C for 4 hours. The material was dialyzed for 4 hours with pH 7.0 phosphate buffer solution, and then stored separately at -7 ° C until use. During the test, 100 μΐ of chloramphenicol standard (0, 0.5, 1, 2, 4 ng / ml) and The drugs listed in Table 2 were added to the microwells to which antibodies 3202-52.27 were attached, and then 100 μ dysomycin-hydrogenase conjugate (diluted 2500 times) was added. After mixing, they were allowed to react at room temperature for 20 minutes. Discard the reaction solution, fill the microwells with 0.05% Tween 20-PBS, and then discard. After washing 5 times in this way, take a photo of 47493.DOC — 9 — I The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 514638 7 7 Λ Β 5. Description of the invention (7) Dry, add 100 μΐ of hydrogen peroxide enzyme substrate TiMB, react at room temperature for 10 minutes, and add 100 μΐ of 2N HC1 to stop the reaction. Determination of OD 450/650 nm radon. Table 1 shows the standard concentration of chloramphenicol and 6 weights. The test coefficient of variation (coefficient of variation, CV) and the percent inhibition. It can be seen that the CV of each concentration is less than 10%. The percentage of inhibition represents the relative sensitivity of the antibody to a specific substance, and is calculated as (1-absorbance of standard solution / zero absorbance of standard solution) X 100%). Indicates that the nine drugs listed did not produce > 20% inhibition when tested at 100 ng / ml. Table 1. Absorbance and standard of chloramphenicol at various concentrations. Coefficient of variation and inhibition percentage of repeated tests. CAP concentration 0D 450/650 SD CV% inhibition ng / ml 1 2 3 4 5 6 average 4 0.463 0.461 0.460 0.475 0.492 0.507 0.476 0.0193 4.1 76% 2 0.817 0.756 0.779 0.733 0.715 0.732 0.755 0.0375 5.0 62% 1 1.138 1.136 1.143 1.167 1.206 1.224 1.169 0.0377 3.2 42% 0.5 1.575 1.518 1.506 1.521 1.434 1.468 1.504 0.0484 3.2 25% 0 1.953 1.925 1.992 2.014 2.040 2.070 1.999 0.0540 2.7 0% Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. Table 2. The percentage of this P system is small.> 20% of the drug (100 ng / ml). Yuean Yeqingde: Ampicillin aeromycin matrix (Chloramphenicol base) 4 74 9 3.DOC—10— This paper size applies to the Chinese National Standard (CNS) A4 specification (210 × 297 mm) 514638 A 7 B7 V. Description of the invention (8) Jinlortetracycline Jianda Gentamycin Penicillin G Sulfisoxazole Sulfisoxazole Sulfamethazine ^ Tetracycline Thiamphenicol The present invention is susceptible to various modifications and variations. Specific specific embodiments thereof have been illustrated and described in detail by way of example. It should be understood, however, that it is not intended to limit the invention to the particular form disclosed, but is intended to cover all modifications, similarities, and changes within the spirit and scope of the invention as defined by the scope of the appended patent application. References V Hong Qibi et al. Drug and Food Inspection Bureau Investigation Annual Report 1: 71-138 * 2 Lu Chefeng et al. Journal of Pingtung Institute of Technology 2: 75-81. Please read 1 Italian and then fill out this page Printed by the Central Consumers Bureau of the Ministry of Economic Affairs Consumer Cooperatives This paper is printed in accordance with China National Standard (CNS) A4 (210X 297 mm) Patent Application No. 086116069 (Main Supplementary Specification) (91 Year 4 50 milk samples were prepared. The products were sold first and the lysozyme containing the fresh residue was exempted from the inspection test. It was determined to be age-reducing. The commercially available product (γ-biophann) and the test enzyme enzyme immunoassay 4 containing the silk fibroin were tested. Figure 1 shows the test results of the commercial product and the drug concentration test. The correlation coefficient between the two reached 0.951. The second is the result of the test of the product containing the single antibody of the case and the test of the concentration of the drug. The correlation coefficient between the two reaches 0 · 940. Figure 3 shows the comparison between the two reagents. The correlation number of Yu | is 0.932. This phenomenon shows The test reagents in this case can be compared with commercially available products. Figure 1. Milk test test, comparison of commercial products with spike

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Claims (1)

514638 Patent Application No. 86116069 Revised Chinese Patent Application Scope (February 1990) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 1. A fusion tumor is a cell line fused with myeloma cells and anti-chloramphenicol-producing B cells to produce a single antibody specific to aeromycin, of which The B cell line was obtained from an animal immunized with a combination of chloramphenicol and a carrier protein. The myeloma cell was a mouse-derived myeloma cell F0 cell line. The B cell line was obtained from chloroformin-α-succinic acid-bovine serum. The albumin-immunized rat is deposited in the Hsinchu City Food Industry Development Research and Development Institute under the number CCRC 960063. 2. A single-body antibody specific to aerobicin, which is produced by the fusion tumor in item 1 of the patent application. 3. A method for preparing a fusion tumor according to item 1 of the scope of the patent application, which includes fusion of myeloma cells and B cells producing anti-chloramphenicol by a B-cell fusion method, and selection of specificity for amycin A single fusion tumor cell line, in which the B cell line is obtained from an animal immunized with a conjugate of chlorazin and a carrier protein, the myeloma cell is a Tibetan myeloma cell f0 cell line, and the heart cell line is obtained from chloramphenicol Succinic acid_Bovine serum albumin immunized rats. 4. The method according to item 3 of the patent application park, wherein polyethylene glycol is used for cell fusion. 5 · A kit for the detection of chloramphenicol residues in a specimen, including a monoclonal antibody according to item 2 of the patent application. This paper size applies Chinese National Standard (CNS) A4 specification (21〇 > < 297) (please read the precautions on the back before filling in this page)-binding-order Φ 514638 Α8 Β8 C8 D8 patent application scope Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs6. According to the set of the patent application park No. 5, it is a direct competition ELISA set. 7 · The set according to item 6 of the scope of the patent application, which includes ⑴ solid phase support, ⑵ single plant anti- 鳢 according to item 2 of the patent application range, (3) qi element standard, (4) amycin- Signal complexes, and tritium pigments. 8) The set according to item 7 of the scope of patent application, wherein the solid support is a microdroplet, microsphere or paper. 9. The set according to item 7 of the scope of patent application, wherein the signal is selected from the group consisting of light answering substance, cold light marker, radioactive element and enzyme. 10. The set according to item 9 of the scope of patent application, where enzyme A ”τ ′ D 1¾ Self-Catalase, Quantitative Lecitase, and β-Galactosidase. 11. According to the set of the scope of application for patent item 9, wherein the progenitor of the depletion is selected from the group consisting of lanthanum phosphor. 12. Root ^ The set of patent application scope item 9, in which human and N ν light markers are also biological cold light markers and chemical cold light markers. 13 · —A method for detecting chloramphenicol residues in a sample, visit ^ ^ The Awan method uses the single plant anti-shedding or rooting of the scope of the patent application No. 2 & & A to request patent kits of any of 5 to 12 kits to detect the gas amycin residue in the specimen. No. -------- 0 ^ ------ 1Τ -------- (Please read the precautions on the back before filling this page) -2-This paper size is applicable to China Standard (CNS) Α4 specification (210 × 297 mm)