KR20070099899A - Polyether antibiotics-carrier protein conjugates and process for preparing the same - Google Patents

Abstract

A polyether antibiotics-carrier protein conjugate is provided to be used as an antigen for producing a monoclonal antibody specifically bound to the polyether antibiotics, thereby allowing the obtained monoclonal antibody to be used for rapidly and precisely detecting a very small amount of the polyether antibiotics contained in a mixture such as an animal feed. A method for preparing a polyether antibiotics-carrier protein conjugate comprises a step of: reacting a compound obtained by reacting a polyether antibiotics having a carboxyl group with a compound having a carbodiimide group with a carrier protein having an amine nucleophilic group; or reacting a polyether antibiotics having a carboxyl group with a mixture anhydride in the presence of a solvent and then reacting it with a carrier protein having an amine nucleophilic group, wherein the polyether antibiotics are selected from the group consisting of monensin, salinomycin, laidlomycin, narasin, lasalocid and maduramicin and the carrier protein is selected from the group consisting of bovine serum albumin(BSA, ovalbumin and keyhole limpet hemocyanin( KLH)).

Description

Polyether antibiotics-carrier protein conjugates and process for preparing the same

1 is a view showing a result of conjugate formation using the Kaiser test.

Figure 2 is a photograph showing the antimicrobial activity of the polyether antibiotic-carrier protein conjugate prepared by one embodiment of the present invention.

Figure 3 is a photograph showing the SDS-PAGE results of monensin-BSA conjugates prepared by another embodiment of the present invention.

4 is a photograph showing the results of SDS-PAGE of various conjugates prepared by another embodiment of the present invention.

The present invention is a polyether antibiotic-carrier protein conjugate, which covalently binds a polyether antibiotic and a carrier protein to prevent coccidial disease in poultry and improves ruminant feed efficiency, and functions as a full antigen. It relates to a method and its use.

Animal antibiotics used to treat disease in animals or to promote animal growth have been mainly used for penicillins, tetracyclines, erythromycin, bacitracin and streptomycin. However, in order to prevent the rapid discovery of human resistance to antibiotics due to excessive use of antibiotics, the use of these antibiotics has begun to be regulated worldwide. In recent years, peptide antibiotics such as thiostrepton and polyether-based The use of antibiotics is on the rise. They are mainly biosynthesized from soil actinomycetes, and their current commercial use is used as feed additives for the prevention of coccidial disease in livestock, especially poultry, and for promoting growth of ruminants.

However, residual drugs and metabolites of polyether antibiotics are present in animal feces, and they can flow into groundwater and surface water, causing contamination in cropland, and excessive use is a problem. In addition, low concentrations of antibiotics increase the drug resistance of microorganisms, and the presence of antibiotics in groundwater or surface water can cause serious concerns in terms of environmental health. Therefore, regulations on these animal feed antibiotics are currently being considered in Europe.

Until now, efforts have been made to develop a technique for measuring the concentration of antibiotics in a mixture such as animal feed, but at present, the measurement is mainly performed through instrumental analysis such as HPLC. However, this method is difficult to analyze and takes a long time to analyze. Therefore, a simple and efficient high-sensitivity analysis method is required to replace it.

The present inventors have made intensive studies for the development of a system capable of detecting a specific antibiotic from an animal feed additive in a fast and highly accurate manner.As a result, the conjugate of a polyether antibiotic and a carrier protein prepared as described below It has been found that the gate can efficiently perform this purpose function, and have completed the present invention.

Accordingly, an object of the present invention is to provide a conjugate of a polyether antibiotic and a carrier protein, a method for producing the same, and a use thereof, in particular a kit for detecting or diagnosing a polyether antibiotic using the same.

In one aspect, the present invention provides a conjugate of a polyether antibiotic and a carrier protein and a method of producing the same.

In a further aspect, the present invention provides a use of a conjugate of the polyether antibiotic-carrier protein, in particular a kit and diagnostic method for diagnosing or detecting a polyether antibiotic using the same.

The present invention has led to the development of a system that can rapidly and precisely measure the presence and concentration of specific antibiotics using antigen-antibody reactions. This method has been mainly used to measure the concentration of antibiotics in mixtures such as animal feed. It is significant that high sensitivity analysis is possible for the presence and content of polyether antibiotics in the mixture such as animal feed by escaping from instrumental analysis such as HPLC.

As used herein, the term "polyether antibiotics" is a compound containing a plurality of oxygen-containing five-membered ring and six-membered ring in the molecule, and has one carboxyl group. define. They have a large ring structure of the whole molecule, contain metal ions therein, and high selectivity to metal ions (Tetrahed. Lett., 1973: 4955-4958, 1973).

Until recently, about 150 kinds of polyether compounds are known and exhibit various biological activities such as antibiotic effects by affecting ion permeability of cell membranes (Chemistry & Biology, 10: 431-441, 2003). Representative compounds include, but are not limited to, monensin, salinomycin, laidlomycin, narasin, rasarocid, mauramycin, and the like. Can be mentioned.

Polyether antibiotics are incomplete antigens (hapten) that do not induce antibody formation when administered directly to experimental animals because of their small molecular weight of 600-700 daltons. Therefore, in order to give antigenicity to a polyether antibiotic, it is necessary to covalently bond the polyether antibiotic to a material having a large molecular weight such as a protein to make a complex (conjugate).

The polyether antibiotics that can be used in the present invention are not particularly limited, and any polyether antibiotics as described above may be applied. In the embodiment of the present invention, as a representative example, monensin, laidlomycin, and salinomycin were used. The polyether antibiotic has a structure of the following Chemical Formulas 1-3.

As the carrier protein used in the present invention, any protein having a relatively high molecular weight and good antigenicity can be used in the present invention without particular limitation. In the examples of the present invention, bovine serum albumin (BSA), ovalbumin, and keyhole limpet hemocyanin (KLH) were used as representative materials.

Of course, in the present invention, nine polyether antibiotic-carrier conjugates were prepared by selecting the most frequently used ones among a large number of polyether antibiotic and protein carrier candidates. However, since these are presented as representative examples in the present invention, the present invention is not limited to them, and also similar compounds of polyether antibiotics and carrier proteins to which the same reaction mechanism is applied without any difficulty in view of the nature of the present invention. Naturally, it can be applied.

The polyether antibiotic-carrier conjugate prepared according to the present invention is used as an antigen for producing monoclonal antibodies that specifically bind to polyether antibiotics, and the produced monoclonal antibodies are contained in samples such as animal feed. It can be used to quickly and precisely detect trace amounts of polyether based antibiotics.

Hereinafter, the method for producing the conjugate according to the present invention will be described in more detail, but the present invention can be variously modified on the basis of this principle, and it is obvious that such modifications also fall within the scope of the present invention.

Method for preparing a conjugate of a polyether antibiotic and a carrier protein according to the present invention

(a) a compound obtained by reacting a polyether antibiotic having a carboxyl group with a compound containing a carbodiimide group and a carrier protein containing an amine nucleophilic group, or

(b) a polyether antibiotic having a carboxyl group is reacted with a mixed anhydride in the presence of a solvent followed by a carrier protein containing an amine nucleophilic group.

As a method for producing a conjugate of a polyether antibiotic and a carrier protein according to the present invention, a method using a carbodiimide group-containing compound such as EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide] And mixed anhydride method can be used.

First, a description using a carbodiimide (eg, EDC) can be summarized as shown in Scheme 1 below.

To explain the reaction mechanism, since polyether antibiotics have a carboxyl group, carbodiimide can be used to covalently bond with a carrier protein. Thus, the carboxyl group in the polyether antibiotic reacts with the carbon in the carbodiimide group to give compound (1), followed by ovalbamine (OVA), bovine serum albumin (BSA), and keyhole limpet hemoshi The amine nucleophilic group in the carrier protein, such as keyhole limpet hemocyanin (KLH), reacts with the carboxyl group of the compound (1) and the carbodiimide (e.g., EDC) is separated, such as BSA, OVA, KLH, etc. The carrier and the polyether are conjugated (conjugated).

Specifically, for example, the polyether compound is dissolved in a solvent such as DMSO, and the carrier protein is dissolved in a small amount of distilled water separately, and then, a solvent such as DMSO is added thereto, and the two solutions are combined for about 3-10 minutes at room temperature. React. Carbodiimide group-containing compound was added thereto to react the mixtures at room temperature overnight, and after 2 hours of reaction, the degree of free amine was confirmed by color using Kaiser test, and reacted until the color became light. After completion of the reaction, DMSO is removed and the conjugate is concentrated. In the above reaction mechanism, specific reaction conditions such as reaction time, temperature, humidity, and pH are obvious in the art.

In addition, the conjugate preparation method using a mixed anhydride method is shown in the following scheme 2.

This approach is characterized by the use of mixed anhydrides (eg isobutyl chloroformate, tributylamine, etc.) instead of using carbodiimide group compounds. For example, each of the polyether antibiotics containing a carboxyl group is dissolved in a solvent such as dimethylformamide. Each of isobutyl chloroformate and tributylamine was added thereto, followed by stirring at 4 ° C. for about 30 minutes. At this time, sonication is performed to increase solubility. Drop the respective carrier proteins (BSA, OVA, KLH) diluted in the solution and stir for 2-5 hours in an ice-water bath The reaction mixtures were dialyzed with PBS buffer Residual free antibiotics are removed and lyophilized Similarly, specific reaction conditions such as reaction time, temperature, humidity, pH, etc. in the reaction mechanism are apparent in the art.

(Example)

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. However, these are described to aid the understanding of the present invention and do not limit the content and scope of the invention.

Example 1 Preparation of Conjugates

Each of nonensin, laidlomycin, and salinomycin was reduced to about 15 mg in 6 ml of each DMSO. Separately, 5 mg of BSA, OVA and KLH were each dissolved in 200 μl of distilled water, and then 800 μl of DMSO was added thereto. Then, the two corresponding solutions were mixed and reacted at room temperature for 5 minutes. 250 μl of EDC (10 mg / ml in DMSO) was added to BSA and OVA and 125 μl to KLH. The mixtures were reacted overnight at room temperature, and after 2 hours of reaction, the degree of free amine was determined by color using a Kaiser test, followed by reaction until the color became light. After the reaction was completed, the concentration of the conjugate was concentrated by removing DMSO while converting into PBS (phosphate buffered saline) buffer using centricon. The conjugate concentration obtained after the preparation reaction was BSA conjugate: about 3.54 mg / 1.7 mL, OVA conjugate: about 2.26 mg / 1.7 mL, KLH conjugate: about 1.37 mg / 1.7 mL.

Test Example 1: Confirmation of Conjugation Formation of Polyether Antibiotic and Carrier Protein

Formation of the conjugate prepared in Example 1 was confirmed by Kaiser Test (E. Kaiser et al. Anal. Biochem .: 34, 595, 1975). The entirety of this document is cited as part of the disclosure of the present invention. The results are shown in FIG. 1 is a view showing a result of measuring the amount of conjugate using a Kaiser test. As can be seen from the results of Figure 1, it was confirmed that the color of the carrier protein decreases as the primary amine (primary amine) of the carrier protein decreases as the carrier protein binds.

Test Example 2: Antimicrobial Activity of the Conjugate

Antimicrobial activity of the conjugate of the polyether-based antibiotic-carrier protein prepared in Example 1 is a methicillin-resistant strain stored in Staphylococcus aureus 693E, Chosun University College of Pharmacy, and other known methicillin-resistant strains. May be used as the growth inhibition ring (inhibition zone) for. In other words, a transparent disk was placed on a nutritious agar plate coated with Staphylococcus aureus and placed on a paper disk moistened with various concentrations of antibiotic-protein conjugate solution. The results are shown in FIG. Figure 2 is a photograph showing the antimicrobial activity of the polyether antibiotic-carrier protein conjugate. A is monensin-BSA (20 μg / ml), B is monensin-BSA (50 μg / ml), C is monensin-BSA (100 μg / ml) and D is BSA (500 μg / ml) It was. As shown in the results of FIG. This indicates that in all cases the conjugates were certainly produced and act as antigens.

Example 2: Preparation of Conjugates by Use of Mixed Anhydrides

100 mg of each monensin, liedomycin, salinomycin was added to 3 ml of each dimethylformamide and stirred for about 2 hours. 20 μl of isobutyl chloroformate and 72 μl of tributylamine were added to the stirred solution, followed by sonication for about 2 hours to increase solubility. BSA, OVA, and KLH (20 mg / 7 mL distilled water) were added to each of the solutions, followed by stirring for about 3 hours in an ice bath. The reaction mixtures were dialyzed with PBS buffer to remove residual free antibiotic and lyophilized. The dry weight of the conjugate obtained after the reaction was BSA conjugate: about 1.5 mg, OVA conjugate: about 1.3 mg, KLH conjugate: about 1.7 mg.

Test Example 3: Confirmation of Conjugation of Polyether Antibiotic and Carrier Protein

Formation of the conjugate prepared in Example 2 was confirmed by performing polyacrylamide gel electrophoresis (SDS-PAGE) according to the method described in the literature (Nature, 227: 680-685, 1970). At this time, a 10% polyacrylamide gel was used, and an appropriate amount of the sample was added thereto, followed by electrophoresis at 150 V for 2 hours, and then stained and decolorized to observe the protein. The results are shown in FIGS. 3 and 4. Figure 3 is a photograph showing the SDS-PAGE results of monensin-BSA conjugates prepared by the mixed anhydride method. The lyophilized monensin-conjugate was dissolved in distilled water and centrifuged to separate the supernatant (spt) and the precipitate (ppt). Broad marker (bio-rad), 2. BSA (1 mg / ml), 3. BSA (2 mg / ml), 4. monensin + BSA (1 mg), 5. monensin + BSA (5 mg), 6. Monensin + BSA (10 mg), 7. Monensin + BSA (1 mg), spt, 8. Monensin + BSA (10 mg), spt, 9. Monensin + BSA (1 mg), ppt, 10. Monensin + BSA (10mg): ppt.

4 is a photograph showing the results of SDS-PAGE of other conjugates prepared by the mixed anhydride method. (1.broad marker (bio-rad), 2. Ova (mg / ml), 3. monensin + ova, 4. Laid + ova, 5. Sal + ova, 6. blank, 7. BSA ( mg / ml), 8. monensin + BSA, 9. Laid + BSA, 10. Sal + BSA). As can be seen from the results of FIGS. 3 and 4, the antibiotic and the carrier protein were combined to increase the molecular weight of the carrier protein. Thus, it can be seen that various conjugates were prepared by a mixed anhydride mode.

The polyether antibiotic-carrier conjugate prepared according to the present invention can be used as an antigen for monoclonal antibody production which specifically binds to polyether antibiotics, and the obtained monoclonal antibody can be used to specifically bind to polyether antibiotics. It can be used to quickly and precisely detect trace polyether antibiotics in mixtures such as feeds.

Claims (3)

A conjugate of a polyether based antibiotic containing a carboxyl group and a carrier protein containing a nucleophilic amine group. The method of claim 1, wherein the polyether-based antibiotics include monensin, salinomycin, laidlomycin, narasin, rasarocid and mauramycin. And a carrier protein is selected from the group consisting of bovine serum albumin (BSA), ovalbumin and keyhole limpet hemocyanin (KLH). (a) a compound obtained by reacting a polyether antibiotic having a carboxyl group with a compound containing a carbodiimide group and a carrier protein containing an amine nucleophilic group, or (b) preparing a conjugate of a polyether based antibiotic and a carrier protein, wherein the polyether based antibiotic having a carboxyl group is reacted with a mixed anhydride in the presence of a solvent, followed by a carrier protein containing an amine nucleophilic group. How to.

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