RU2691413C1 - Method for determining bacterial endotoxin in biological fluids - Google Patents

Abstract

FIELD: clinical laboratory diagnostics.SUBSTANCE: invention relates to clinical laboratory diagnostics and represents a method for determining bacterial endotoxin (BE) in blood plasma and urine, characterized by that preliminary preparation of blood plasma samples involves dilution of blood plasma samples with a physiological solution in 10–100 times and their heat treatment at 45–85 °C for 30–60 minutes, and preliminary preparation of urine samples involves urine extraction using methylene chloride or 1,2-dichloroethane; after which the reaction of the samples with the LAL reagent is carried out in the following sequence: 100 mcl of treated samples containing BE are poured into sterile test tubes, and there added is 100 mcl of LAL reagent, the tubes are thermostated for 1 hour at 37 °C to terminate the reaction, 50 mcl of 50 % acetic acid is added to each tube, followed by spectrophotometric determination of BE concentration in samples, including measurement of BE concentration as to optical density of sample after LAL-test at wavelength λ=388 nm.EFFECT: invention provides reducing the number of false-positive results and increasing sensitivity when determining BE.1 cl, 2 tbl, 6 ex

Description

Technical field

The invention relates to the field of medicine and clinical laboratory diagnostics. The invention relates in more detail to a method (method) for determining the level of endotoxin based on an in vitro reaction between bacterial endotoxin (BE) and LAL-reagent in biological fluids (plasma and urine, etc.).

The level of technology

The number of known methods (methods) for the determination of bacterial endotoxins (EB) in biological fluids, for example, in the following patent documents: RF patent RU 2325645 (IPC G01N 33/48, G01N 33/579, publ. 27.05.2008 Bul. No. 15), Japanese patent JP 4761448 (IPC G01N 33/48; G01N 33/579, publ. 2011-08-31), Japanese patent application JP 2003232794 (IPC G01N 33/579, publ. 2003-08-22), Chinese patent CN 1696636 (IPC G01N 1/28, publ. 2005-11-16), international PCT application WO 2009005231 (IPC G01N 33/49, G01N 33/579, publ. 2009-01-08).

According to patent RU 2325645 METHOD FOR DETERMINING THE CONTENT bacterial endotoxins WITH TAL-test, wherein the registration form polymers coagulogy made on Protein structure forms fractals, carried out in biological fluids and medicines. The essence of the method: mix tahpleus lysate (TAL) and dilution of the test sample by drawing on a pyrogen-free surface, incubate with the lid closed for 30 ... 60 min at 37 ° C, maintain pyrogen-free surface in a thermostat with the lid open until the drops of the reagent mixture dry, register polymer formation coagulogen under the increase in the resulting protein fractals and, if present, determine the activity of endotoxin in the last dilution.

The disadvantage of this method is its low accuracy due to the subjective visual control of the test results, in contrast to the measurement of optical density as a result of chromogenic analysis used in the proposed method. In addition, the use of TAL-reagent (lysate of horseshoe crabs of the species Tachypleus tridentatus) also imposes certain restrictions on its use, due to the complexity of its acquisition in the territory of the Russian Federation (it is necessary to wait for deliveries from China), unlike LAL-reagent, which is widely and actively applied everywhere.

The method according to JP 4761448 BLOOD ENDOTOXIN MEASURING METHOD aims to quantify endotoxin in the blood, which cannot be obtained by conventional methods, and clarifies the relationship between the amount of endotoxin in the blood and the clinical condition. In the method, only red blood cells are separated and removed from whole blood, and the amount of endotoxin contained in the remaining blood particle is measured, thereby correcting the endotoxin contained in leukocytes and endotoxin contained in plasma.

The disadvantage of this method is the possibility of its use exclusively for blood analysis, in contrast to the proposed method, allowing to evaluate the content of BE in various biological fluids.

The method according to patent application JP 2003232794 METHOD FOR MEASURING ENDOTOXIN ACTIVITY is designed to accurately and quickly measure the activity of endotoxin using the LAL test (Limulus Amebocyte Lysate reagent (limulus lysate, abbreviated LAL) using a sample pretreatment that includes the steps of adding a surfactant substances in the sample, followed by ultrasonic treatment. Surfactant is an anionic substance, its concentration in the sample is less than 0.01%. Measurement of the content of bacterial endotoxin carried out by turbidimetric LAL test.

A feature of this method is a specific pretreatment, significantly different from the processing used in the proposed method.

Analogue on the preparation of blood samples for BE testing - method according to CN 1696636 METHOD FOR PREPARING BLOOD FOR TESTING ENDOTOXIN OF BACTERIA involves the preparation of blood serum, adding several times 0.1 ... 0.3% qulatong (X-100) to a small amount of blood serum , heating in a water bath, processing in an ice bath, diluting with pyrogen-free water, and mixing a small amount of diluted liquid with the same amount of Tachypleus tridentatus lysate reagent to obtain a blood product that is used to detect bacterial endotoxins.

The disadvantage of this method is the complexity of pre-treatment of samples for research, as well as the narrowness of its use only in the blood, and not in different biological fluids, as in the proposed method. In addition, also, as in patent RU 2325645, the lysate isolated from the horseshoe crab species Tachypleus tridentatus is used as a reagent, in contrast to the Limulus Polyphemus amoebocyte lysate used in our invention, which is the more common and readily available reagent.

The PCT method of WO 2009005231 METHODS FOR QUANTITATIVELY DETERMINING ENDOTOXIN (quantitative determination of endotoxin in a biological sample) includes the steps of: (a) obtaining a biological sample; (b) heat treatment of a biological sample; (c) obtaining a reaction solution by adding LAL reagent (Limulus Amebocyte Lysate reagent (abbreviated LAL) to an inactivated biological sample; and (d) measuring the optical density of the reaction solution after a certain reaction time or measuring the time when the solution reaches the specified optical density Using the invention, it is possible to determine the amount of endotoxin in a biological sample in a more accurate and convenient manner.

The main disadvantage of this method is the use of a turbidimetric LAL-test (kinetic and at the end point). Such a modification of the LAL-test requires expensive and highly specific equipment - a microplate reader to register changes in optical density caused by clouding of the samples under study. In contrast, in the proposed method, the determination of BE content is carried out using a chromogenic LAL test, which allows the use of any laboratory spectrophotometer.

DISCLOSURE OF INVENTION

The objective of the invention is to develop a more universal method suitable for the quantitative determination of BE in various types of biological fluids, including in the blood plasma and in the urine using the chromogenic LAL test to determine the BE with specific pretreatment of samples from different types of biological fluids. The advantage of the chromogenic method of analysis is the absence of the need to use expensive equipment, as well as the high sensitivity and specificity of the method.

The problem is solved by the method of determining bacterial endotoxin in biological fluids, characterized by preliminary preparation of samples for subsequent chromogenic LAL-test depending on the type of biological fluid, carrying out the reaction of the prepared samples with LAL reagent and spectrophotometric determination of the concentration of bacterial endotoxin in the samples after reaction with LAL reagent Thus, the reaction of samples with LAL-reagent includes: the introduction into sterile tubes 100 μl of the treated samples of biological endotoxin-contaminated fluids and the addition of 100 μl of LAL reagent (note: in real analyzes, pre-induced solutions with known concentrations of EB are used as a calibration curve. When analyzing a patient’s biological fluid, it is necessary to determine the BE content, it is processed by the method, described in the examples of the method. As a result, the value of optical density is determined at a wavelength of 388 nm (this wavelength corresponds to the absorption maximum of the complex of the chromogenic LAL substrate with B ), Then this value is compared with the values obtained for specific concentrations of endotoxin, and it is judged how many base elements were in a biological fluid of the patient). The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube; Spectrophotometric determination of the concentration of bacterial endotoxin in the samples includes the measurement of the concentration of BE in terms of the optical density of the sample after the LAL test at a wavelength λ = 388 nm. At the same time, in real analyzes and modeling experiments, the LAL test is always done on each sample in at least 3 replications.

Preliminary preparation of samples from blood plasma includes dilution of blood plasma samples with saline 10 ... 100 times and subsequent heat treatment at 45 ... 85 ° C for 30 ... 60 minutes. At the same time, after performing the LAL test during spectrophotometric determination of BE concentration in blood plasma samples using heat treatment and dilution, it is possible to determine the presence of BE in a solution with BE concentration at the border between 0.005 EE / ml and 0.010 EE / ml and with higher BE concentrations .

Preliminary preparation of samples from urine involves the extraction of urine using an organochlorine solvent, including but not limited to one of the following: methylene chloride, 1,2-dichloroethane. At the same time, after carrying out the LAL-test during spectrophotometric determination of the concentration of EB in urine samples using extraction with a chloro-organic solvent, it is possible to determine the presence of EB in a solution with a concentration of EB at the border between 1.00 EU / ml and 2.00 EE / ml and higher concentrations of EB.

The idea of the proposed method is that the enzyme factor C contained in the lysate of amoebocytes of the Limulus Amebocyte Lysate horseshoe crab (LAL reagent) reacts with BE, which triggers a cascade of enzymatic reactions leading to activation of the coagulating enzyme. This enzyme in turn hydrolyzes a particular chromogenic substrate to form a colored product. The signal is recorded spectrophotometrically by a change in optical density (i.e., by the intensity of color).

The advantages of the proposed method from the found analogues are as follows:

1) RU 2325645 - the proposed method is more accurate and allows you to quantify the content of bacterial endotoxin in biological fluids, thanks to the use of a chromogenic modification of the LAL-test, in contrast to the visual control used by the authors in this patent;

2) JP 761448 (B2) - the advantage of the proposed method is more versatile, as in the Japanese method, bacterial endotoxin is determined only in the blood, whereas in the proposed method, not only in blood and plasma, but also in urine, i.e. more widely applicable, universal way;

3) JP 2003232794 (A) - the method used by the authors in this patent, allows the determination of BE in specific conditions of use, significantly different from the characteristics of the proposed method.

4) CN 1696636 (A) is a close ¹ analogue of pretreatment of blood samples, which includes treatment with Triton X-100 solution, heating-cooling and dilution. The difference from the proposed method: a) other concentrations of the reagent Triton X-100; b) other cooling temperatures; and in the proposed method these are different methods: either heating and dilution, or treating Triton X-100, followed by acid-base treatment of the samples, whereas in the given patent all operations must be applied to each sample (complication), compared to the proposed method more stages on pre-treatment of blood. In addition, the definition of EB only in blood (serum) is also described.

5) WO 2009005231 (A1) - compared with this method, from the advantages of the proposed method, it can be indicated that the determination of BE content is carried out using a chromogenic LAL test, which allows using any relatively inexpensive and affordable laboratory spectrophotometer.

Examples of the implementation of the invention Examples 1, 2, 3 - for plasma samples, examples 4, 5, 6 for urine samples. Common techniques for all examples were the reaction of the prepared samples with the LAL reagent and the spectrophotometric determination of the concentration of bacterial endotoxin in the samples after the reaction with the LAL reagent.

Example 1

1. Preparation of a solution of bacterial endotoxin

The standard of bacterial endotoxin was diluted with 3.4 ml of pyrogen-free water and a concentration of 50 EU / ml was obtained.

2. Preparation of plasma

Blood samples were centrifuged at 1200 g for 10 minutes. Separated plasma was collected in sterile tubes 10 × 75 mm.

3. Plasma pollution

Plasma samples were contaminated with a solution of bacterial endotoxin (50 EE / ml) to a concentration of the latter, respectively: 5, 0.5, 0.05, 0.005, 0.0025 EE / ml.

4. Sample processing

A) In sterile test tubes, 0.9 ml of physiological sodium chloride solution was added to 0.1 ml of plasma. Thus, the blood was diluted 10 times.

B) The tubes were thermostated at 45 ° C for 60 minutes (they are not closed during thermostating).

5. Conducting the reaction with LAL-reagent

100 μl of the treated samples contaminated to certain concentrations of endotoxin were poured into sterile tubes, and 100 μl of LAL reagent was added to the same. There was also a reference sample: these are 100 μl of non-contaminated plasma and 100 μl of LAL reagent. All concentrations and reference sample were prepared in 3 replicates. The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube.

6. Spectrophotometric determination of BE concentration in samples.

The concentration of endotoxin was measured in terms of optical density at a wavelength of λ = 388 nm.

7. Results of the analysis

Negative control tubes (unpolluted plasma) after an hour of heat setting at 37 ° C, as well as tubes containing 0.005; 0,0025 EE / ml remained transparent. In tubes containing a bacterial endotoxin at a concentration of 5.00; 0.50. 0.05, 0.01 U / ml solution turned yellow. The optical density for each concentration at λ = 388 nm was determined as the average of three replications (Table 1).

Thus, in the blood plasma using heat treatment and dilution, it is possible to determine the presence of EB in a solution with an EB concentration at the boundary between 0.005 EE / ml and 0.010 EE / ml and with higher concentrations of EB.

Example 2

1. Preparation of a solution of bacterial endotoxin

The standard of bacterial endotoxin was diluted with 3.4 ml of pyrogen-free water and a concentration of 50 EU / ml was obtained.

2. Preparation of plasma

Blood samples were centrifuged at 1200g for 10 minutes. Separated plasma was collected in sterile tubes 10 × 75 mm.

3. Plasma pollution:

Plasma samples were contaminated with a solution of bacterial endotoxin (50 EE / ml) to a concentration of the latter, respectively: 5, 0.5.0.05, 0.005, 0.0025 EE / ml.

4. Sample processing

A) In sterile test tubes, 4.9 ml of physiological sodium chloride solution was added to 0.1 ml of plasma. Thus, the blood was diluted 50 times.

B) The tubes were thermostated at 60 ° C for 30 minutes.

5. Conducting the reaction with LAL-reagent

100 μl of the treated samples contaminated to certain concentrations of endotoxin were poured into sterile tubes, and 100 μl of LAL reagent was added to the same. There was also a reference sample: these are 100 μl of non-contaminated plasma and 100 μl of LAL reagent. All concentrations and reference sample were prepared in 3 replicates. The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube.

6. Spectrophotometric determination of BE concentration in samples.

The concentration of endotoxin was measured in terms of optical density at a wavelength of λ = 388 nm.

7. The results of the analysis are similar to the results of the analysis of Example 1.

Example 3

1. Preparation of a solution of bacterial endotoxin

The standard of bacterial endotoxin was diluted with 3.4 ml of pyrogen-free water and a concentration of 50 EU / ml was obtained.

2. Preparation of plasma

Blood samples were centrifuged at 1200g for 10 minutes. Separated plasma was collected in sterile tubes 10 × 75 mm.

3. Plasma pollution

Plasma samples were contaminated with a solution of bacterial endotoxin (50 EE / ml) to a concentration of the latter, respectively: 5, 0.5, 0.05, 0.005, 0.0025 EE / ml.

4. Sample processing

A) In sterile test tubes, 9.9 ml of physiological sodium chloride solution was added to 0.1 ml of plasma. Thus, the blood was diluted 100 times.

B) The tubes were thermostated at 85 ° C for 60 minutes.

5. Conducting the reaction with LAL-reagent

100 μl of the treated samples contaminated to certain concentrations of endotoxin were poured into sterile tubes, and 100 μl of LAL reagent was added to the same. There was also a reference sample: these are 100 μl of non-contaminated plasma and 100 μl of LAL reagent. All concentrations and reference sample were prepared in 3 replicates. The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube.

6. Spectrophotometric determination of BE concentration in samples Endotoxin concentration was measured in terms of optical density at a wavelength λ = 388 nm.

7. The results of the analysis are similar to the results of the analysis of Example 1.

Example 4

1. Preparation of the bacterial endotoxin solution:

The standard of bacterial endotoxin was diluted with 3.4 ml of pyrogen-free water and a concentration of 50 EU / ml was obtained.

2. Contamination of urine:

Urine samples were contaminated with a solution of bacterial endotoxin (50 EE / ml) to a concentration of the latter, respectively: 5.0; 2.0; 1.0; 0.5; 0.1; 0.05 U / ml

3. Processing samples.

A) In sterile test tubes, 0.25 ml of chloroform was added to 1 ml of urine and the mixture was stirred on a shaker for 4 hours.

B) The contents of the tubes were centrifuged at 4000 rpm for 10 minutes. The middle cloudy layer was selected and used for the LAL test.

4. Conducting the reaction with LAL-reagent:

100 μl of the treated samples contaminated to certain concentrations of endotoxin were poured into sterile tubes, and 100 μl of LAL reagent was added to the same. There was also a reference sample: these are 100 μl of unpolluted plasma and 100 μl of LAL reagent. All concentrations and reference sample were prepared in 3 replicates. The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube.

5. Spectrophotometric determination of the concentration of BE in the samples The concentration of endotoxin was measured in terms of optical density at a wavelength of λ = 388 nm.

6. Results of analysis

Negative control tubes (unpolluted urine) after an hour of heat setting at 37 ° C, as well as tubes containing 0.5; 0.1; 0.05 U / ml remained transparent. In test tubes containing bacterial endotoxin at a concentration of 5.0; 2.0; 1.0 EU / ml solution turned yellow. The optical density for each concentration at λ = 388 nm was determined as the average value of three replications (Table 2).

Thus, in the urine using extraction with a chloro-organic solvent it is possible to determine the presence of BE in a solution with a concentration of BE at the border between 1.00 EE / ml and 2.00 EE / ml and with higher concentrations of EB.

Example 5

1. Preparation of the bacterial endotoxin solution:

The standard of bacterial endotoxin was diluted with 3.4 ml of pyrogen-free water and a concentration of 50 EU / ml was obtained.

2. Contamination of urine:

Urine samples were contaminated with a solution of bacterial endotoxin (50 EE / ml) to a concentration of the latter, respectively: 5.0; 2.0; 1.0; 0.5; 0.1; 0.05 U / ml

3. Processing samples.

A) In sterile test tubes, 0.25 ml of methylene chloride was added to 1 ml of urine and the mixture was mixed on a shaker with a shaker for 4 hours.

B) The contents of the tubes were centrifuged at 4000 rpm for 10 minutes. The middle cloudy layer was selected and used for the LAL test.

4. Conducting the reaction with LAL-reagent:

100 μl of the treated samples contaminated to certain concentrations of endotoxin were poured into sterile tubes, and 100 μl of LAL reagent was added to the same. There was also a reference sample: these are 100 μl of non-contaminated plasma and 100 μl of LAL reagent. All concentrations and reference sample were prepared in 3 replicates. The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube.

5. Spectrophotometric determination of the concentration of BE in the samples The concentration of endotoxin was measured in terms of optical density at a wavelength of λ = 388 nm.

6. The results of the analysis are similar to the results of the analysis of Example 4.

Example 6

1. Preparation of the bacterial endotoxin solution:

The standard of bacterial endotoxin was diluted with 3.4 ml of pyrogen-free water and a concentration of 50 EU / ml was obtained.

2. Contamination of urine:

Urine samples were contaminated with a solution of bacterial endotoxin (50 EE / ml) to a concentration of the latter, respectively: 5.0; 2.0; 1.0; 0.5; 0.1; 0.05 U / ml

3. Processing samples.

A) In sterile test tubes, 0.3 ml of 1,2-dichloroethane was added to 1 ml of urine and the mixture was mixed on a shaker with a shaker for 4 hours.

B) The contents of the tubes were centrifuged at 4000 rpm for 10 minutes. A medium turbid layer was selected and used for the LAL test.

4. Conducting the reaction with LAL-reagent:

100 μl of the treated samples contaminated to certain concentrations of endotoxin were poured into sterile tubes, and 100 μl of LAL reagent was added to the same. There was also a reference sample: these are 100 μl of non-contaminated plasma and 100 μl of LAL reagent. All concentrations and reference sample were prepared in 3 replicates. The tubes were thermostated for 1 hour at 37 ° C. To stop the reaction, 50 μl of 50% acetic acid was added to each tube.

5. Spectrophotometric determination of the concentration of BE in the samples The concentration of endotoxin was measured in terms of optical density at a wavelength of λ = 388 nm.

6. The results of the analysis are similar to the results of the analysis of Example 4.

The proposed method has been developed and tested in the framework of the work under the Agreement No. 14.577.21.0165 between the Ministry of Education and Science of the Russian Federation (Government customer) and MGTU. N.E. Bauman.

Claims (1)

The method of determining bacterial endotoxin (BE) in plasma and urine, including preliminary preparation of samples, depending on the type of biological fluid, the reaction of the prepared samples with LAL reagent and spectrophotometric determination of BE concentration in samples after reaction with LAL reagent, characterized in preliminary preparation of plasma samples includes dilution of plasma samples with saline 10-100 times and their heat treatment at 45-85 ° C for 30-60 minutes, and the preliminary Single preparation of urine samples involves extraction urine using methylene chloride or 1,2-dichloroethane; after which the reaction of the samples with LAL-reagent is carried out in the following sequence: 100 μl of the processed samples containing BE are poured into sterile tubes, and 100 μl of LAL-reagent are added thereto, the tubes are incubated for 1 hour at 37 ° C, to stop the reaction in 50 μl of 50% acetic acid is added to each tube, followed by spectrophotometric determination of the concentration of EB in the samples, including measuring the concentration of EB in terms of the optical density of the sample after the LAL test at a wavelength λ = 388 nm.