Conjugate oxytetracycline with bovine serum albumin for immunochemical detection method oxytetracycline

 

(57) Abstract:

The invention relates to the field of organic chemistry. The object of the invention is a conjugate of oxytetracycline with bovine serum albumin (BSA) for immunochemical method of determination of tetracycline in physiological fluids and tissues of animals. The conjugate is obtained by reaction of oxytetracycline and formaldehyde with BSA in aqueous medium or in a mixture of water with methanol. Conjugate and obtained on the basis of a specific rabbit serum used for immunochemical determination of oxytetracycline. table 2.

The invention relates to the field of organic chemistry, in particular to the conjugate oxytetracycline with bovine serum albumin (BSA) for immunochemical detection method oxytetracycline. Immunochemical method for the determination of oxytetracycline in tissues and body fluids of animals have not been developed. Preferred for immunochemical method for the determination of oxytetracycline in tissues and fiziologicheskii liquids animals would be its conjugate with BSA, but sources of information on this issue was not found.

The purpose of the invention, the conjugate of oxytetracycline with bovine serum albumin (BSA) is in the tissues and fiziologicheskii liquids animals immunochemical method, obtained by reaction of oxytetracycline and formaldehyde with BSA in aqueous medium or in a mixture of water with methanol.

The conjugate obtained by the method based on the reaction of manniche [1]. To do this, oxytetracycline (1) condense with bovine serum albumin (BSA) using formaldehyde at 20°C in the aquatic environment.

The invention is illustrated by the following examples.

Example 1.

To a solution containing 150 mg of BSA in 8 cm3water, add 2 cm 3 n sodium acetate solution and adjusted the pH to 6.7 with 5% caustic soda.

To the resulting solution was added sequentially a solution of 50 mg of oxytetracycline hydrochloride (1) 12 cm3water and 2 cm3a 7.5% solution of formaldehyde, pH again adjusted to 6.7. The reaction mixture is stirred for 2 h in a closed container on a magnetic stirrer at room temperature, after which cialiswhat in running tap water for 3 days and centrifuged for 30 min at 15 000 R/min

The supernatant determine the amount of protein by the method of Lowry and the amount of bound oxytetracycline (1) by the spectrophotometric method, then the conjugate solution lyophilizer.

Receive 125 mg K0 nm (acetate buffer pH 7.4).

The conjugate contains an average of 7.8 molecules oxytetracycline (1) 1 mol of BSA.

The IR spectrum of the conjugate (2) found bands that are characteristic of the hapten (1).

IR-spectrum (KBR), cm-1: 3379, 3311, 3108, 1672, 1616, 1584, 1535, 1359, 1233,1004,950 and 676.

Example 2.

Condensation oxytetracycline (1) with BSA carried out as described in example 1, in water and in mixtures of water with dioxane and water with methanol under different reaction conditions and the composition of the reaction mixtures.

In a molar ratio of oxytetracycline and formaldehyde used in a large excess relative to the BSA.

Conditions for obtaining conjugate oxytetracycline with BSA and its characteristics are presented in table 1.

From table 1 it follows that increasing the amount of formaldehyde in the reaction mixture is immaterial, however, increasing the concentration of oxytetracycline in it useful. The use of dioxane affects the results of the reaction, and methanol, in contrast, gives a positive effect.

The positive impact of the dilution of the reaction mixture with water. The change in pH from 6.5 to 8.0 and the reaction time from 0.5 to 24 h has little effect on the results.

The maximum concentration of protein in the conjugate to one mol of BSA, equal to 10.

Example 3.

Get rabbit anticigarette specific to oxytetracycline (1), by immunization of three rabbits (males) weight 2-3 kg conjugate oxytetracycline with BSA (2) obtained in example 1, in an amount of 0.1 mg per animal. 0.1 mg before the introduction of conjugate diluted in 1.5 cm3sterile isotonic and emuleret 1.5 cm3complete adjuvant's adjuvant. The antigen is injected subcutaneously in 30-40 points in the region of the back. 4 weeks after immunization are reimmunization animals aqueous solution of the antigen without adjuvant. After 1 week of reimmunization in rabbits away the blood from the marginal vein of the ear and get the serum.

The resulting serum is mixed with glycerine in equal proportions, Packed on 1 cm3in tubes of the type Eppendorf and stored at minus 20°C for 2 years.

The titer of the antisera to the conjugate oxytetracycline with BSA (2) obtained in example 1, in an indirect solid-phase ELISA equal to 12 800.

Example 4.

Obtained according to example 3 of the immune rabbit serum at a dilution of 1:12 800 is used to determine the number of oxytetracycline (1) in its solutions with known concentrations (1,0; 10,0 and 100 ng/cm33), which compete for the sites of antibody binding to solid-phase antigen in ELISA. Not bound peroxidase antibodies are removed by washing. The amount of bound peroxidase specific antibodies is determined using secondary (antivitamin) antibodies labeled with horseradish peroxidase and detected by the substrate solution, containing the Chromogen - orthophenylphenol and hydrogen peroxide. In the reaction of hydrogen peroxide with horseradish peroxidase in molecules bound peroxidase peroxidase conjugate Chromogen stain solution in a yellow-orange color. The intensity of this color is measured on the vertical spectrophotometer at 492 nm (OD492). The intensity of the color (O.D.) is inversely proportional to the concentration of oxytetracycline in the samples.

Calculate the ratios of antibody binding as the ratio of the average OD of the control (serum) and experience (serum+solution of oxytetracycline):

The results are presented in table 2.

Thus, the resulting serum is lysocline in solution is 0.001 ág/cm3. Conjugate (2) can be used for immunochemical detection method oxytetracycline (1) in physiological fluids and tissues of animals.

The conjugate of the antibiotic oxytetracycline with bovine serum albumin (BSA) for immunochemical method for determination of oxytetracycline General formula

where n=2-10,

obtained by reaction of oxytetracycline and formaldehyde with bovine serum albumin in the presence of water or mixtures of water with methanol.



 

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