SU1603280A1 - Method of investigating apo-b-lipoproteins in blood serum - Google Patents

Abstract

The invention relates to the field of biology and medicine, namely biological and medical chemistry, and can be used for diagnostics. The purpose of the invention is to improve the accuracy of the study by increasing the number of protein fractions identified in the composition of apo-B-lipoproteins, and preserving the nativeity of the apo-proteins while at the same time simplifying the method by reducing the number of labor-intensive operations. The method consists in the simultaneous electrophoresis of two samples of native serum and a serum sample, from which the apo-B-lipoproteins were previously removed, then stained for the electrophoregram protein of the sample devoid of apo-B-lipoproteins, and one sample of the native serum and carbohydrates second sample of native serum. Electrophoregrams stained for protein are compared and apo-B-lipoproteins are detected. Electrophoregrams of native serum stained for protein and carbohydrates are compared, and the loading of the apo-B-lipoprotein fractions with carbohydrates is determined. The method allows revealing the relationship between carbohydrate and protein components in apo-B-lipoproteins in different forms of pathology.

Description

The invention relates to biology and medicine, namely to biological and medical chemistry, and can be used for diagnostics.

The purpose of the invention is to improve the accuracy of the method by increasing the number of protein fractions identified in the composition of apo-B-lipoproteins (LP), and preserving the nativeity of the apoproteins while simultaneously simplifying the method by reducing the number of labor-intensive operations.

The method is carried out as follows.

Simultaneously, electrophoresis was performed on two samples of the natitive serum and a serum sample, from which apo-B lipoproteins were previously removed. Then, they are stained for the electrophoresis protein of the sample lacking apo-B- lipoproteins and one sample of native serum and the second sample of native serum for carbohydrates. Electrophoreograms stained for protein are compared and apo-B-lipoproteins are detected. Electrophotograms of native serum stained for protein and carbohydrates are compared, and the loading is determined.

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fractions of apo-B-lipoprotein carbohydrates.

Example. CO, 5 ml of serum (plasma) of blood are added 0, OA ml of heparim (1000 IU / ml) and 0.05 ml of a solution of manganese chloride (2 mol / l). After a half-hour exposure of the sample in an ice bath and its subsequent centrifugation for 30 minutes at 3000 g, a 0.05 m supernatant liquid (i.e., serum devoid of apo-B-containing lipoproteins) is pipetted and produced Electrophoretic study of the spectrum of proteins fractionated in polyacrylamide gel columns.

The method of disc electrophoretic fractionation of proteins (glycproteins) of serum and of the supernatant that does not contain apo-B-LP.

. Reagents. Source reagents: 1 n. HC1, TRIS) tetramethylethylenediamine (TEMED), methylene bisacrylamide (BIS or IBA) I ammonium persulfate} riboflavin sucrose, acetic acid ice on ;; 1 n. KOH solution; 1 mol / l solution of orthophosphoric acid.

The scheme for obtaining the initial solution for the polymerization of the gel.

Solution 1: pH 8.9; 1 N.SN - CV ml TRIS - 36.6 g; TEMED - 0.23 ML, - water - up to 100 ml.

Solution 2: acrylamide - 30.0 g; BIS - 0.8 G} water - up to 100 ml.

Solution 3: ammonium persulphate (ammonium persulphate) - 0.1 g, water - up to 100 ml (the solution should be stored no more than 7 days in the refrigerator).

Solution k: pH 6.8, 1 N.HC1 - 8 ml TRIS - 5.98 g; orthophosphoric acid solution - 25.6 ml; water - up to 100 ml.

Solution 5: acrylamide - 10, O g, BIS - 2.5 g, water - up to 100. ml.

Solution 6: riboflavin - 0.004 g; water - up to 100 ml.

Solution 7: sucrose - 40.0 g; water - up to 100 ml.

Solutions can be stored for several weeks (up to 4 months in dark bottles in the refrigerator at).

A scheme for the preparation of a fine-porous separating gel with an acrylamide concentration of kQ g / l; pH 8.9 (mixture 1 W): 1 volume of solution 1, 2 volumes of solution 2 {1 volume of bidistilled water, pH 8.9. An equal volume of solution 3 is added to the mixture.

In the end, the scheme takes the following form: 1 volume of solution 1-, 2 volumes of solution 2, 1 volume of bidistilled water, pH 8.9, solution volume 3.

Scheme for preparing a large pore concentrating gel: 1 volume of solution 2; 2 volumes of solution 5; 1 volume of solution 6; k solution volume 7.

The basic solution for electrode buffer preparation: TRIS - 6.0 ri glycine - g, distilled water - up to 1000 ml, pH 8.3. Before use, dilute 10 times.

Dye solution preparation scheme: bromophenol blue - 0.001 g; distilled water - up to 100 ml.

The dye solution preparation procedure - fixative: amido-black 10 V - 1.0 g; Acetic acid solution with a concentration of 10 g / 100 ml - up to 100 ml.

The preservative solution - the differentiator is a solution of acetic acid concentration of 7 g / 100 ml. Equipment. To carry out an analytical disc electrophoresis in PAG, domestic instruments are used for this purpose: PEFA - 1, some other and devices of the Hungarian company Reanal (model No. b9).

The progress of the method. The reagents stored in the refrigerator are removed before testing and kept for some time at room temperature to warm them. Standard glass tubes with polished ends are fixed in a special stand according to the instructions attached to the device. Next, a solution of a finely porous gel with a pH of 8.9 is prepared. The main solutions (1 volume of mixture 1 and 1 volume of solution 3) are thoroughly mixed in a small flask. In order to prevent the formation of air bubbles in the Polymerization Process of the gel, which significantly interfere with the separation process, it is desirable to remove air from the basic solutions, for which you can use a water-jet or other suction connected to the jar.

2 ml of a solution of a finely porous gel are pipetted into glass tubes and are blind in order to prevent air bubbles from falling into them. 0.2-0.3 ml of distilled water or diluted buffer is layered on the surface of this solution. The tubes are illuminated with a fluorescent lamp to accelerate the polymerization. After 30–0 minutes, the process is completed, as can be judged by the weak heating of the glass tubes, as well as by the formation of a well-defined border between the polyacrylamide gel and the aqueous phase. With a quick movement of the hand, gently spray off the layered gel with the gel (it can be removed from the tubes with the help of filter paper).

When preparing a solution of large-pore (top) gel, suck the air out of the | . The solution is not necessary, however, it is desirable to protect the preparing mixture from the influence of strong light (because of its sensitivity to riboflavin). The glass tubes are rinsed with 0.1-0.2 ml of monomer solution: (to completely remove the other components) and 0.2 ml of monomer solution used to prepare the large-pore concentrating gel is pipetted into each column. Then, 0.1-0.2 ml of bidistilled water is layered in a known manner.

The gel is polymerized using a photosensitive riboflavin catalyst. Therefore, gel gels have around a 250-500 W ultraviolet lamp or another source of bright light:

But the completion of the polymerization of the upper layer (which takes about half an hour) the applied liquid is drained from the large-pore gel, and the gel tubes are rinsed with a solution of the large-pore monomer previously prepared for analysis.

The formed (0.2 ml volume) disc in contact with the finely porous gel produces a concentrating effect.

0.01 ml of the analyzed serum and are introduced into the column (tube). layer the electrode buffer, diluted 10 times. If the sample is taken together with a large-pore monomer, it is desirable to polymerize it under ultraviolet illumination. When used as a diluent of biological fluid

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the monomer solution with sucrose (plant, thief 7) polymerization is not required. The upper part of the apparatus is joined to the lower buffer tank, which is prefilled with 10 times diluted buffer solution. Worsening electrical-contact air bubbles are removed from the ends of the tubes that enter the buffer solution of the lower reservoir by repeated but gentle shaking of the solution or by other means (for example, a syringe with a needle filled with electrode pulp). Carefully pour in the top tank ten times diluted buffer solution. The camera is preferably placed at the time of electrophoresis in the refrigerator.

During the first 15 minutes, the electrophoresis is carried out with a current not exceeding 2 mA per each individual tube (for this, a voltage must be applied). Then the current should be increased to 5 mA per tube. The current is passed until the colored ring of the buffer reaches a level that is 5 mm from the bottom edge of the column along the Q liacrylamide gel (which takes 1 - 1.5 hours). After electrophoresis is complete, the gel tubes are removed from the upper reservoir. The gel from the glass tubes can be easily removed by moving the steel needle in between.

between the gel and the tube under a layer of distilled water. The extracted gel is immersed for 20-30 minutes in a fixative solution, & carried in test tubes. After dyeing, the gels are electrophoretically provided with a special device.

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To reveal the glycoproteins, the columns of polyacrylamide gel are fixed overnight in an aqueous solution of isopropanol (250 g / l), then washed for 2 hours with distilled water and immersed for 50 minutes in a solution of iodic acid with a concentration of 10 g / l prepared in 0.2 mol / l solution

sodium acetate. After repeated washing with distilled water (for 1 h), the columns of polyacrylamide gels are placed for 50 minutes in a Schiff's reagent. Then the gel columns are treated for 30 minutes (preferably 3 times for 10 minutes) with freshly prepared 5 g / l sodium metabisulfite solution and washed with distilled

water Gels are stored in a solution of acetic acid at a concentration of 70 g / l.

Telegrams of proteins and glycoproteins of the same serum sample (plasma) are scanned on a microdensitometer. Densitograms of disc electrophoregrams are processed quantitatively (by finding the peak areas that make up the individual protein and glycoprotein fractions, their summation and determining the relative quantitative content) J after having carried out the qualitative identification of proteins and glycoproteins.

The obtained densitograms of diskelectrophoregrams of three samples of the same serum (plasma) of blood, two of which constitute the spectrum of proteins of native serum (plasma) and serum (plasma). Deprived of apo-B-LP, and one-third - the spectrum of glycoproteins of native serum (plasma), comparing, paying attention to the proteins of native (whole) serum or plasma that disappeared (compared to serum spectra lacking apo-B-LP) (they are apo-B-lipopro, theins) and on the change in the ratio between the protein and carbohydrate components in the identified protein zones.

When comparing the disc electrophoregram of plasma supernatant proteins and the disc electrophoregram of plasma proteins, one can see that the plasma supernatant of a number of protein fractions found in the blood plasma between the start line and the transferrin is absent in the spectrum of proteins.

The deisitograms of the corresponding disk electrophoregram of plasma supernatant proteins lacking apo-B-LP and the plasma itself give a more complete picture of this.

Consequently, using the proposed method of obtaining, apo-B-LP, it becomes possible not only to determine their place in the electrophoretic disk

protein spectrum, but also to judge whether the change between the carbohydrate and protein components in the apo-B-LP in different forms of pathology (if that is the case).

The use of the invention allows to increase the accuracy of the study by increasing the number of protein fractions identified in the composition of apo-B-liprproteins, as well as by reducing the measurement error twofold, to simplify the method by reducing labor-intensive operations (1.3 times) and research time c) | preserve the origin of the studied apobies.

Form ula invention

The method for the study of apr-B-lipoproteins in serum, including the precipitation of apo-B-lipoproteins and the identification of apoproteins by polyacrylamide gel electrophoresis, is so that, in order to improve the accuracy of the method by an increase in the number of protein fractions identified in the composition of apo-B-lipoproteins, and preservation of the origin of apoproteins while simplifying the process by reducing the number of labor-intensive operations, while electrophoresis is carried out

two samples of native serum and a serum sample, from which apo-B-lipoproteins were previously removed, are then stained for an electrophoregram protein of a sample lacking a-B-lipoproteins,

and one sample of native serum and the second sample of native serum for carbohydrates, apo-B-lipoproteins are revealed by comparing electrophorems stained for protein, and the load of apo-B fractions is compared by electrophoregram of native serum stained for protein and carbohydrates - lipoprotein carbohydrates.

Claims (1)

Claim A method for the study of apo-B-lipoproteins in blood serum, including the deposition of apo-B-lipoproteins and the identification of apoproteins by polyacrylamide gel disc electrophoresis, based on the fact that, in order to increase the accuracy of the method due to increasing the number of protein fractions identified in the composition of apo-B-lipoproteins, and preserving the nativeness of apoproteins while simplifying the method by reducing the number of time-consuming operations, simultaneously conduct electrophoresis of two samples of native serum and a serum sample, from which apo-B-lipoproteins are variably removed, then electrophoregrams of a sample lacking apo-B-lipoproteins and one native serum sample and carbohydrates are sampled on a protein of a second serum, by comparison of electrophoregrams stained for protein, apo-Vlipoproteins are detected, and compared electrophoregrams of native serum stained for protein and carbohydrates determine the loading of fractions of apo-B-lipoproteins with carbohydrates.