RU2169367C1 - Method for determining endotoxin activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves mixing Limulus polyphemus lisate and the diluted sample under test, with polymer causing coagulum production detected in the last dilution. The polymer causing coagulum production is identified by examining protein fractals structure after drying the mixture. Mixing Limulus lisate and the diluted sample under test is carried out by placing 2-8 mcl of Limulus lisate solution and the diluted sample under test onto plastic surface. The plastic surface with reagents placed thereon is covered with cover and incubated at 37 C during 30 min. It is uncovered and kept in the thermostat until reagent mixture drops dry. The polymer causing coagulum production is recognized by detecting structure of protein fractals produced. Endotoxin being available, the fractals as specific crystal-shaped structures are observed. Otherwise, when no endotoxin being found or negative control being the case, smooth field with rare intrusions of rhombic salt crystals is observed in the peripheral part of the field. EFFECT: enhanced sensitivity in determining endotoxin activity. 4 cl, 4 dwg, 1 tbl

Description

 The invention relates to medicine, in particular to pharmacy and clinical laboratory diagnostics.

 A known method for determining the amount of endotoxin (patent N 7015474 B4 Japan, G 01 N 33/579), in which, when conducting a study in a solution where the reaction between endotoxin and a lysate of Japanese horseshoe crab amoebocytes occurs, add a water-soluble polysaccharide, the structural component of which is β - 1 , 3-glycan and / or a water-soluble derivative of this polysaccharide, in an amount of 100 ng / ml - 100 mg / ml. In this way, a trace amount of endotoxin in a sample is determined.

 A known method for measuring the concentration of endotoxin (patent N 0350273 B1 EPO, G 01 N 33/579), in which the measurement is carried out by changing the optical density due to the reaction of the AL solution with endotoxin.

A known method for determining the amount of endotoxin (Sitnikov A.G., Travina L.A., Bagirova V.P. LAL Test. Modern approaches to determining pyrogenicity. - M., 1997, 96 pp.) By mixing the limulus lysate and diluting the subject sample with subsequent registration of the result of the analysis of the formation of the coagulogen polymer in the last dilution. Moreover, the prepared samples of the negative control, standard dilutions of endotoxin and dilutions of the drug are introduced into test tubes for staging a gel thrombus test of 0.1 ml each. The resulting reaction mixture (in a volume of 0.2 ml) must be gently mixed for 2 to 3 seconds, and the tube should be placed in a water bath or dry-air incubator. The incubation time is (60 ± 2) min at a temperature of 37 ^o C ± 1 ^o C. Each tube is slowly and gently turned upside down and the presence of gel on the bottom of the tube that does not break when the tube is turned over determines the concentration of endotoxin in this sample, which is considered equal to or greater than the sensitivity of the LAL reagent.

 This method is characterized by insufficient sensitivity (not more than 0.001 EU / ml), a high consumption of reagents for the reaction.

 The objective of the invention is to increase the sensitivity of the method and reduce the consumption of reagents for the reaction.

 This is achieved by the fact that according to the method for determining the activity of endotoxin by mixing the limulus lysate and diluting the test sample with subsequent registration of the formation of the coagulogen polymer in the last dilution, the formation of the coagulogen polymer is recorded according to the structure of the protein fractals formed after the mixture is dried.

This is also achieved by the fact that according to the method for determining the activity of endotoxin by mixing the limulus lysate and diluting the test sample with the subsequent registration of the formation of the coagulogen polymer in the last dilution, mixing the limulus lysate and diluting the test sample is carried out by applying 2-8 μl of the solution of the limulus lysate and diluting the test subject solution, the plastic surface with the applied reagents is closed with a lid and incubated for 30 min at 37 ^o C, then open and kept in a thermostat until the droplets of the mixture of reagents dry, registration of the formation of the coagulogen polymer is carried out according to the structure of the protein fractals formed, and in the presence of endotoxin, fractals are specific crystal-like structures, and in the absence of endotoxin or in the negative control, an even field with rare interspersed edges is observed fields of rhombic crystals of salts.

 In addition, in the method for determining the activity of endotoxin, mixing is performed on a pyrogen-free plastic surface.

 In FIG. Figures 1–3 show images of protein fractals in the presence of endotoxin.

 In FIG. 4 shows an image of protein fractals in the absence of endotoxin.

 The method is as follows.

According to option 1. On the pyrogen-free plastic surface (Petri dish, ELISA plate), apply the micro amount (drop of 6 μl) of solutions of the limulus lysate and dilute the test solution using varipipettes with pyrogen-free tips. The plastic with the applied reagents is incubated for 30 min at 37 ^o C, after which the Petri dish (or ELISA plate) is opened and kept in a thermostat until the drops of the reagent mixture dry. Then, in the presence of endotoxin, fractals are formed - specific crystal-like structures (Figs. 1, 2, 3), and in the absence of endotoxin or in the negative control (pyrogen-free water), the drop dries out in the form of an even field with rare interspersed salt field rhombic crystals sometimes present in test samples (Fig. 4). It is necessary to use a research surface for mixing from a material in which the probability of adsorption of endotoxins is minimized, which also applies to varipipets. Working dilution of the limus lysate is prepared according to the recommendations of the manufacturer (Sigma Technical Bulletin N 210. Previous Revision November 1993. Revised January 1994). Standard dilutions of endotoxin are prepared according to the recommendations given in the collection Sitnikov A.G., Travina L.A., Bagirova V.P. LAL-Test. Modern approaches to the determination of pyrogenicity.

 The implementation of the method according to option 2 is described by the following example. Determination of the activity of endotoxin in serum (plasma) of blood by the formation of fractals.

Serum sample preparation. Blood sampling from a patient is carried out with a pyrogen-free syringe, transferred to a pyrogen-free (glass or plastic) tube, kept in an thermostat for 30 minutes at 37 ^o C for complete coagulation and retraction of the clot. 10 μl of serum is transferred into a test tube with 90 μl of endotoxin-free distilled water and heated for 2-3 minutes at 100 ^° C to denature the serum proteins and release the bound protein and lipoproteins of endotoxin (Sitnikov A.G., Travina L. A., Bagirova VP LAL Test. Modern approaches to the determination of pyrogenicity). All operations are carried out under conditions that prevent endotoxin from entering the test medium and the reagents used.

Preparation of standard dilutions of endotoxin. On a pyrogen-free polystyrene surface at a distance of 1 cm from each other, 6 μl of a working dilution of the limus lysate are applied with a varipette according to the number of test samples and standard dilutions of endotoxin. Working dilution of the limus lysate is prepared according to the recommendations of the manufacturer (Sigma Technical Bulletin N 210. Previous Revision November 1993. Revised January 1994.). On top of the drops of lysate, 6 μl of the dilutions of the samples are applied, closed with a lid to prevent drying and placed in a sterile thermostat 37 ^o C for 30 minutes

 Accounting for the result. After 30 minutes of incubation, the plastic surface is opened and left in the same thermostat for a period until the reagent mixture dries, which averages 25-35 minutes. The result is taken into account visually under magnification x10 - x20. With a positive reaction, the dried spots of the mixture of reagents are in the form of "winter glass", "spruce branch", "snowflakes". Take into account the last dilution, where a similar reaction is still encountered.

 The table shows the results of the reaction with the above dilutions of endotoxin and 3 serum samples from healthy donors and patients with viral hepatitis C.

According to the results given in the table, the used batch of limulus lysate in the proposed formulation of the test has a sensitivity of 0.0001 EU / ml. Corresponding samples of the studied samples had the following endotoxin activities:
Hepatitis 1 - 0.0001 • 80 = 0.008 EU / ml.

 Hepatitis 2 - 0.0001 • 800 = 0.08 EU / ml.

 Hepatitis 3 - 0.0001 • 10 = 0.001 EU / ml.

 Donor 1 - 0.0001 • 10 = <0.0005 EU / ml.

 Donor 2 - 0.0001 • 10 = 0.001 EU / ml.

 Donor 3 - 0.0001 • 20 = 0.002 EU / ml.

 In the above example, volumes of limulus lysate and dilution of the test sample of 6 μl were used. Similar results were obtained for volumes of 2-8 μl.

 In the above example, the proposed method provides a sensitivity of 0.0001 EU / ml, reduces the time of setting and evaluating the result to one hour, reduces the consumption of limus lysate by 50-100 times (depending on the number of dilutions needed), does not require additional reagents and special equipment and Available for any clinical and analytical pharmaceutical laboratory.

 Thus, the proposed method for determining the activity of endotoxin increases the sensitivity of the determination of activity and reduces the consumption of reagents for the reaction. In addition, it is available for any clinical and analytical pharmaceutical laboratory, as it does not require special expensive equipment and additional reagents.

Claims (4)

 1. A method for determining the activity of endotoxin by mixing the limulus lysate and diluting the test sample with subsequent registration of the formation of the coagulogen polymer in the last dilution, characterized in that registration of the formation of the coagulogen polymer is performed by increasing the protein fractals formed after the mixture is dried and, in the presence of fractals, the endotoxin activity is determined in last dilution.  2. The method for determining the activity of endotoxin according to claim 1, characterized in that the mixing is carried out on a pyrogen-free plastic surface. 3. A method for determining the activity of endotoxin by mixing the limulus lysate and diluting the test sample with subsequent registration of the formation of the coagulogen polymer in the last dilution, characterized in that mixing the limulus lysate and diluting the test sample is performed by applying 2-8 μl of the limulus lysate solutions and diluting the test subject on a plastic surface solution, the plastic surface with the applied reagents is closed with a lid and incubated for 30 min at 37 ^o C, then open and incubated in t thermostat until the droplets of the mixture of reagents dry, the formation of the coagulogen polymer is recorded under magnification of the formed protein fractals and, in the presence of fractals, the activity of endotoxin in the last dilution is determined.  4. The method for determining the activity of endotoxin according to claim 4, characterized in that the mixing is carried out on a pyrogen-free plastic surface.

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