SU1439508A1 - Method of analysis of adesine triphosphate - Google Patents

Abstract

The invention relates to the immunoassay of biologically active substances and can be used in biochemistry, molecular biology and the production of phosphorus-labeled 32 (33) adenosine triphosphates. The purpose of the invention is to increase the accuracy of the method by increasing its sensitivity. Animals are immunized with a derivative conjugate of 5,5-diadenosine tetraphosphate with bovine serum albumin. The resulting antiserum is incubated with the analyzed sample in the presence of -y-p ATP, bound and free ATP are separated by adsorption on activated charcoal, coated with dextran. The amount of ATP in the sample is determined using a calibration graph based on reference 9 samples with a known ATP content. The sensitivity of the method is 5-1 O mol / ml ATP. (L

Description

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This invention relates to the field of lunoanalysis of biologically active substances and can be used in biochemistry, molecular biology in the production of labeled with phosphorus-32 (33 adenosine triphosphates.

The aim of the invention is to improve the accuracy of the method by increasing the sensitivity of the method.

Example 1. Preparation of bovine serum albumin conjugate antigen.

8-Vg ADP and hexamethylenediamine ADP are obtained as follows

500 mg of the disodium salt of ADP in 10 ml of III Na-acetate buffer (pH 4.3) are treated with an excess of Br for 40 min. An excess of Br2 is extracted with chloroform, and an aqueous solution is treated with 2 mg of sodium metabisulphite to remove the AcTciTKOB Br of an aqueous solution. The product is precipitated by adding 3-4 volumes of ethyl alcohol at -20 ° C. The precipitate was filtered off and washed with O5U1a with a danol, dissolved in 10 ml of water and 2 g of hexamethylenediamine added. The mixture is heated at 60-70 ° for 2.5-3 hours. To precipitate from an excess of hexamethylenediamine, the product is precipitated with alcohol. The precipitate is washed by centrifugation, dissolved in water and chromatographed on DEAE-Sephadex A-25 in ammonium bicarbonate buffer solution, pH 7.5.

In order to obtain a modified 5, 5 -diadenosine tetraphosphate (PAP4A), the hexamethythiandiamine derivative of ADP is introduced into condensation with activated ADP. 0.06 mol of the triethylammonium salt of 5 -ADPA is dried by evaporation of Zc ml of anhydrous acetonitrileJ dissolved in 1.2 ml of a mixture of dimethylsulfoxide – ethyl alcohol (1: 1 by volume) and 0.3 mmol of 4-dimethylamicopyridine, 0.24 mmol of triphenylphosphine, 0.24 mmol of dipyridyl disulfide (PyS) i The mixture is held at room temperature for 20 minutes (mixture I) The yield of 4-dimethylaminophenyl-indium derivative is determined by those on Kieselgel GO, F2f4 previously diluted with an aliquot of the reaction mixture 10% - a solution of piperidine in ethanol and analyzing the resulting Ychy piperidide, Rf ADP-0.05, Rf piperidide .CF-0.3.

0 5 o

Q j

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Separately dry 0.077 mmol of the triethylammonium salt of 8- (b-aminohexamethylene) -aminoadenosine-5-diphosphate by adding and evaporating 3x1 ml of acetonitrile, dissolve in 800 µl of anhydrous dimethyl sulfoxide, transfer to mixture I, evaporate ethyl alcohol and 10 ml of water-dimethylsulfoxide mixture (1: 1) is added to the mixture and applied to a column with PEI-cellulose HCO-form, (mm), the column is washed with 50 ml of water- dimethyl sulfoxide (I :), 50 ml of water, and the product is isolated in a concentration gradient of TEAB from 0 to 0.5 M {volume of solution of 800 ml, the speed of etiotia 40 M.T1 / H, volume of fractions 15 ml). The yield of the product is 65% relative to the starting ADP. The resulting product by the time of elution of the ion exchange resin, resistance to alkaline phosphatase, the ratio of base phosphate (1: 2) and spectral characteristics in UV light (

 290,267 im, 236 im -g-jeO

 0.58) corresponds to the hexamethylenediamine derivative Aru A.

Conjugation of the obtained compound with BCA was carried out according to a known method (10). To 2 ml of 0.2 M Na-phosphate buffer pH 7.5, containing 20 mg of BCA and 20 mg of derivative Ap4. A, 1 ml of a 0.2% solution of glutaraldehyde B water is added dropwise. . The addition of dialdehyde is carried out in ice lane, and then the reaction mixture is incubated at room temperature for 2 hours. Unreacted glutaraldehyde is combined by adding an excess of carboxylic acid. The conjugate is purified from low molecular weight impurities by dialysis. The content of EAp4A in the conjugate composition is determined spectrophotometrically. The epitopic number of the conjugate is 25-27 diadenosine tetraphosphate molecules per BCA molecule.

Example 2. Obtaining antisera and its characteristics.

Rabbits weighing 1.5-2.0 kg are used for immunization. The antigen - conjugate of BCA with 5, 5 -diadenosine tetraphosphate {BCA-K - Ap4A) in an amount of 1 mg is dissolved in 0.5 ml of physiological saline and nepefl, and mixed with 0.5 ml of Freund's adjuvant

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formation of a homogeneous emulsion. The resulting preparation is administered subcutaneously at several points of the rabbit's back. The first injection is performed using Freund's complete adjuvant. After .2,4 and 8 weeks. repeated injections are carried out using Freund's incomplete adjuvant. Immunization with animals transfer well. After 9-10 days, after the fourth injection, blood is taken totally and an anti-short is obtained in a known way.

The antiserum titer, expressed as the concentration of the binding sites of the Q antigen, and the antibody affinity for the antigen as the association constant K ,,, is determined in the binding experiment -j. - Pj ATP antibodies from dilute antibloss by presenting the data obtained in Scatchard coordinates. The reaction is carried out in a buffer solution containing 50 mM Tris-HCl, 4 mM EDTA (pH8.2) To the same amount of antiserum (final dilution 1: i 20) various amounts of y-3ipj dtf of known molar radioactivity are added and the volume in all samples is adjusted to 100 µl with a buffer solution. The mixture is incubated at A C for one hour. Separation of complexed with antibodies and free ATP is carried out using a suspension of activated carbon coated with dextran. After centrifugation (2 min., 6000 g), the supernatant radioactivity is determined and the amount of ATP that is incorporated into the antigen-antibody complex is calculated.

Example 3. Determining the amount of ATP.

All reagents are prepared in a buffer solution containing 50 mM Tris-HCl and 4 mM EDTA (pH 8.2) y, ATP of high molar radioactivity (V6000 Ci / mmol) is added to samples containing known increasing amounts of non-radioactive ATP (5 , 10, 15, 20 and 25x10 mol). In a parallel series, samples contain unknown amounts of ATP. One of the samples does not contain the intended ATP (the sample is not comparable). The volume of all samples is adjusted to 150 µl with buffer solution, taking into account the anti-shortening (dilution 1: 120), which is added to all samples as a last resort. Samples are mixed by shaking and incubated at 40 ° C in a te9508

2 hours. Then all the samples, except for the comparison of the comparison, are added with 50 µl of a suspension of activated carbon coated with dextran, mixed and centrifuged at 6000 g for 10 minutes. 75 µl of the supernatant from each tube was dissolved in 1 ml of water and counted on a Mark-1J scintillation counter.

The proposed method for determining ATP is simple to perform. For performing a series of analyzes, only a few μCi of phosphorus-32 is required (33). This method can be carried out in any biochemical laboratory. Due to the high sensitivity of the method, the required. For analyzing the amount of phosphorus-32 is so small

0 which, combined with a short half-life, makes the work safe for the health of the personnel and excludes radioactive contamination of the laboratory. Currently established

5 is a regular industrial grade of P ATP of high molar activity, and, therefore, this label is widely available.

The method will be used before

In total, for technological control of product quality, to determine the molar radioactivity of high specific activity nucleoside triphosphates. Previously, to determine this characteristic of the production of isotopic synthesis, at least a few mCi of P NTF was required. Regularly conducting such analyzes is expensive and requires special measures to protect personnel from exposure.

In the field of biochemical analysis, the proposed method makes it possible to measure the content of this biologically important nucleotide in scale.

45 single cell and its intracellular distribution.

According to the proposed method, the sensitivity of the analysis is determined by Q as% 10 mol / sample, and by the known 3 1 O mol / sample. The specificity, determined by the value of 50% inhibition and antibodies with.-ATP, according to the proposed method is determined 1.6%, and according to the known - O, 3%.

Claims (1)

Invention Formula Method of determining adenochintri-phosphate 5 including imgunizac-oo 514395086 bovine serum, covalently linked to sensitivity and specificity with bovine serum albumin wound, a dioimmune assay, as blood sampling, the use of anti-helper-hapten for immunization is used ki incubating it with analyzer-8- (6-aminohexyl) -5,5-diadenosine my breakdown in the presence of labyrintetraphosphate, as labeled adenosine triphosphate analogue, adenosine triphosphate analogue section, bound and free adeno-zypol tc (j) - 32 (33) P adenosyntrizine triphosphate and the determination of coli phosphate and separation of bound and adenosine triphosphate by radio-free adenosine phosphate and zinc phosphate and zinc-phosphate and zinc-phosphate adenosine triphosphate; by adsorption on the act that, in order to increase the angle covered with the dextra method, by increasing the nom.