Conjugate oxytetracycline with bovine serum albumin for immunochemical detection method oxytetracycline
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 . 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.
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.
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.
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.
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
obtained by reaction of oxytetracycline and formaldehyde with bovine serum albumin in the presence of water or mixtures of water with methanol.
FIELD: preparative biochemistry, medicine, pharmacology.
SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.
EFFECT: improved purifying method.
8 cl, 1 tbl, 6 ex
FIELD: veterinary, in particular large-scale albumin production.
SUBSTANCE: claimed method includes four-step deposition of blood plasma protein fractions with alcohol solution, wherein before deposition said albumin binding ability (ABA) is determined by fluorescence method using cation probes. ABA value is defined as ABA = EAC/TAC.100 %, wherein EAC is effective albumin concentration, and TAC is total albumin concentration. After albumin fraction providing blood plasma ABA is determined again by fluorescence method, obtained albumin fraction is purified on carbohydrate sorbents, blood plasma ABA is determined again by fluorescence method, purified albumin is stabilized with sodium chloride up to final concentration of 10-20 %, defecated by filtration, pasteurized and finally stabilized.
EFFECT: veterinary albumin with increased detoxification effect and enhanced sorption capacity; process with decreased energy and labor consumption.
2 dwg, 1 ex
FIELD: gene engineering, in particular yeast strain modified by introducing of foreign genetic material.
SUBSTANCE: claimed strain is obtained by transforming of starting culture Pichia pastoris X-33 with two albumin structural genes with signals of yeast alpha-factor secretion, transcribed under control of 5'AOX1 promoter and transcription termination region of hepatitis G virus. Strain of present invention is useful in production of albumin-containing drugs.
EFFECT: strain for production of human recombinant albumin of increased yield.
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.
EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.
2 cl, 7 dwg
SUBSTANCE: according to the invention there is provided protein fused with albumine comprising two or more tandem-oriented polypeptides GLP-1 or its fragments containing (7-36 A8G)), nucleic acid molecules coding albumine proteins according to the investigation as well as vectors containing the specified nucleic acids, host cells transformed by vectors comprising the specified nucleic acids, methods of albumine protein preparation according to the invention and their application methods. Moreover, the present invention refers to pharmaceutical composition containing fused proteins of albumine, treatment of diseases, disorders and conditions using fused proteins of albumine according to the invention.
EFFECT: improved therapeutic polypeptide stability.
34 cl, 81 ex, 11 dwg
SUBSTANCE: invention relates to biochemistry. Disclosed is a method for synthesis of a conjugate of nona-β-(1→3)-glucoside with bovine serum albumin (BSA) using a squarate technique. Nona-β-(1→3)-glucoside is reacted with diethyl squarate first. The obtained ligand - monoamide of squaric acid - then reacts with BSA protein in a mixture of a borate buffer pH 9.0 and dimethyl sulphoxide. The ratio of the borate buffer to dimethyl sulphoxide is equal to 9:1.
EFFECT: method increases efficiency of the conjugation reaction and reduces ligand consumption by 20-25%.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, specifically to a fused protein containing a variant of rodostomin, and can be used in medicine. An ανβ3 integrin selective polypeptide consisting of an amino acid sequence SEQ ID NO:1 conjugated on the N terminal by a linker amino acid sequence containing a combination of the amino acids glycine and serine with a variant of a human serum albumin (HSA) with SEQ ID NO:4.
EFFECT: invention enables the higher therapeutic effectiveness in the diseases related to ανβ3 integrin.
12 cl, 14 dwg, 2 tbl, 7 ex
SUBSTANCE: present invention relates to biotechnology, namely to production of versions of albumin with changed half-life period in plasma in comparison with initial albumin, and can be used in medicine. Version of albumin is obtained, containing one or more substitutes relative to SEQ ID NO: 2, selected from following: 1) E492A, C, D, F, G, H, I, K, L, M, N, Q, R; 2) K500I, R; 3) N503H; 4) E505Q; 5) H510D; 6) D550E, H, I, M, N, R, S, W; 7) K573A, C, D, F, G, H, I, L, M, N, P, R, S, V, W, Y; 8) K574D, F, G, H, I, L, M, P, R, S, T, V, W, Y; 9) A578F; 10) S579C; 11) Q580I, K, M, R, V; or 12) G584D.
EFFECT: invention enables to obtain version of albumin or its fragment, capable to bind with FcRn, or chimeric polypeptide, including said version of albumin or its fragment, with longer half-life period in plasma or increased binding affinity with FcRn in comparison with initial albumin, its fragment or chimeric polypeptide.
10 cl, 32 dwg, 22 tbl, 22 ex