SU1681255A1 - Method for determining plasminogen content in biofluid - Google Patents

Abstract

The method relates to medical biochemistry and can be used in clinical laboratory practice in the diagnosis and treatment of diseases associated with the pathology of the hemostatic system. The purpose of the invention is the acceleration of the method. To achieve the goal, the biological fluid is diluted with 0.05 M phosphate buffer, then on a lysine-agarose sorbent in a sorbent to biological fluid ratio (1-2): 1, elution is carried out successively with 0.1 M sodium phosphate buffer, pH 7, 4 and 0.01-0.1 M solution of-aminocaproic acid in the indicated buffer, after which the amount of plasminogen in the eluate is determined spectrophotometrically. The method also eliminates the dependence on the species specificity of the object of study. 1 tab. w e

Description

The invention relates to medical biochemistry and can be used in clinical and laboratory practice in the diagnosis and treatment of diseases associated with the pathology of the hemostasis system.

The purpose of the invention is the acceleration of the method and the elimination of dependence on the species specificity of the object of study.

The method is illustrated by the following examples.

PRI me R 1. The reliability of the proposed method is assessed by comparatively determining the amount of plasminogen in the model mixture.

Plasmin-free plasma with a volume of 2 ml is added with a known amount of plasminogen (see table). A column filled with 2 ml of lysine-agarose (immobilized lysine concentration of 1-3 mM) is applied with 2 ml of citrate plasma diluted 2 times with 0.05 M sodium phosphate buffer. Plasma proteins elute with 0.1 M sodium phosphate buffer to zero absorption at 280 nm. The biospecific bound plasminogen is eluted with a 0.1 M solution of α-aminocaproic acid in 0.05 M sodium phosphate buffer, pH 7.4, to zero absorption of the solution at 280 nm. The rate of increase and elution is 60 ml / h. The concentration of plasminogen in plasma is calculated by the formula

DE V2 Plasminogen, mg / ml

ten,

17.0 Vi

where LE E280 - Ez20 is the difference of extinction values measured at 280 and 320 nm (the subtraction of extinction at 320 nm is an amendment to turbidity); 17.0 - extinction coefficient

Lv

E 280 for plasminogen in a weakly alkaline medium at a wavelength of 280 nm;

About 00

io ate ate

Vi is the volume of plasma taken for analysis; V2 is the volume of the spectrophotometric solution.

In our case: AE 0.114; Vi 2 ml;

/ 2 4 ML,

Plasminogen concentration 0.114 -4

17 -2

10 0.135 mg / ml plasma.

As can be seen from the table, all plasminogen introduced into plasma is biospecifically bound to the affinity sorbent and then fully eluted with α-aminocaproic acid under the proposed conditions.

PRI mme R 2. The determination and calculation was carried out as in Example 1, but 1 ml of plasma was taken for determination, and a 0.01 M solution of α-amino-caproic acid in 0.05 M Na was taken for elution of plasminogen. phosphate buffer.

Vi - 1 ml; V2 3 ml; DE 0.076.

Concentration 0.076 3

plasminogen

17 1

10 0.135 mg / ml plasma. In addition, the content of plasminogen is determined in other biological fluids.

Example 3. 2 ml of synovial fluid of a patient with rheumatoid arthritis was diluted 2 times with 0.05 M sodium phosphate buffer, applied to a column filled with 2 ml lysine-agarose, washed with 0.1 M sodium phosphate buffer, pH 7.4 . Elution of plasminogen was carried out with a 0.1 M solution of α-aminocaproic acid in 0.05 M sodium phosphate buffer, pH 7.1, then spectrophotometrized as in examples 1 and 2.

At the same time DE 0.062; Vi 2 ml; V2 4 ml.

Plasminogen Concentration

° 72-24 10 ° 073 MG / ML

The concentration of plasminogen in synbial fluid of 16 patients with rheumatoid arthritis (during the exacerbation) was 0.0668 ± 0.0079 mg / ml.

Example 4. 1 ml of mouse citrate plasma (pool of 5 individuals) was diluted 2-fold with 0.05 M sodium phosphate buffer, pH 7.4, and applied to a column filled with 2 ml of lysine-agarose. The elution is carried out in two steps: first washing, and then bound plasminogen is eluted with a 0.01 M solution of e-aminocaproic acid (Example 1).

At the same time DE 0.055; Vi 1 ml; V2 2 ml.

Concentration 0.055 2

17 1

plasminogen 10 0.065 mg / ml.

Example 5. 2 ml of citrate plasma

porcine blood is diluted 2 times with 0.05 M sodium phosphate buffer pH 7.4, and applied to a column filled with 2 ml of lysine-agarose. Subsequent elution operations are carried out as described in Example 1.

In this case, DE 0.102; Vi 2 ml; V2 4 ml.

Plasminogen Concentration

 0,102-4

17-2

10 0.120 mg / ml.

Example 6. 2 ml of a dog's citrate blood plasma was diluted 2 times with 0.05 M sodium phosphate buffer, pH 7.4. Subsequent elution operations are carried out as described in Example 1.

At the same time DE 0.063; Vi 2 ml; Chg 3 ml.

Plasminogen Concentration

0.063 3, pps, -17. „X 10 0.056 mg / ml.

To prove the validity and accuracy of the new method, the results obtained are compared with data from literature sources: the amount of plasminogen in the blood plasma is normally 1.5-2jU.M or 0.158 mg / ml on average according to the literature; 0.120 mg / ml, and in 12 patients with rheumatoid arthritis in plasma, 0.172 and synovial fluid 0.065 mg / ml when determined by immunochemical method. The results obtained according to the proposed method are in plasma

for a healthy person, 0.135 ± 0.0037 mg / ml, for patients with rheumatoid arthritis - 0.171 ± 0.115, for patients with Crohn's disease, it ranges from 0.080-0.120 mg / ml; in synovial fluid, the amount of plasminogen in 16 patients with rheumatoid arthritis during the exacerbation period is 0.0668 ± 0.0079 mg / ml.

Using a new method for determining plasminogen allows significantly

reduce the time of analysis (1 h compared to 24 h in the prototype method). At the same time, the accuracy of the method is kept fairly high, which corresponds to the literature data and the results of the experimental

analysis and clinical studies.

The use of the method also allows the determination of plasminogen in various animals, regardless of the species specificity.

Claims (1)

Invention Formula The method of determining the plasminogen content in a biological fluid by separating and detecting the product, which is such that, in order to speed up the process and eliminate the dependence on the species specificity of the object of study, the biological fluid is diluted in two times 0.05 M phosphate buffer, elution was carried out on a sorbent lysine-agarose in the ratio sorbent: biological fluid (1-2): 1.0.1 M sodium phosphate buffer pH 7.4 and 0.01-0.1 M solution Ј-aminocaproic acid in 0.05 M sodium phosphate buffer and plasminogen spectrophotometrically in the eluate.