KR20090002570A - Methods for quantitatively determining endotoxin - Google Patents

Abstract

A method for quantitatively measuring the amount of endotoxin in a biological sample, and a kit used in the measurement method are provided to allow the amount of endotoxin in a biological sample to be determined precisely and conveniently. A method for quantitatively measuring the amount of endotoxin in a biological sample comprises the steps of preparing a biological sample; a treating the biological sample by heat inactivation; adding the Limulus Amebocyte Lysate reagent to the inactivated biological sample to obtain the reactant; and measuring the OD (optical density) of the reactant after a ceratin time or measuring the time required for reaching a certain OD value.

Description

Method for Quantitatively Determining Endotoxin

Figure 1 shows the manner in which the measurement sample is dispensed into a 96-well plate according to one specific embodiment of the present invention.

Figure 2 is a graph showing the recovery results for the positive control (CSE: control standard endotoxin) measured according to the method of the present invention.

3 is a standard curve for endotoxin prepared according to the method of the present invention. x-axis is logarithm of EU, y-axis is OD _340nm The log value for the time (seconds) that the value reaches 0.05.

4 is a graph comparing the experimental results with the case of using a perchloric acid (PCA) strong acid as the inactivation method.

The present invention relates to a quantitative measurement method and a measurement kit of endotoxin in a biological sample.

Sepsis is one of the causes of death that often occurs in medical and surgical treatment. Sepsis is caused by endotoxin called lipopolysaccharide (LPS). Endotoxin triggers a cascade of events leading to organ failure, non-recovery shock and death. Endotoxins bind to cell membrane proteins or receptors and act on a variety of cells, such as endothelial cells, Neutrophils, monosites, and macrophages, and the like to form a variety of mediators such as oxygen free lycals, nitric oxides, arachidonic acid. Leads to the formation of metabolites, thromboxanes, prostacyclins, platelet activators, interleukin-8, leukotriene B4, cytokines, IL-1, TNF-α (tumor necrosis factor) and proteases, which eventually lead to endotoxin-induced organ damage And septic shock (Akarasereenont et al. Eur . J. Pharmacol . 1995, 273: 121-128; Brigham et al. Am. Rev. Respir. Dis . 1986, 133: 913-927; Morrison Ann. Rev . Med 1987, 38: 417-432; Williams et al Surgery 1992, 112:... 270-277; Wright Current Opinion in Immunol 1991, 83-91).

Because of the severity of these endotoxin-induced diseases, various methods have been developed to detect endotoxins.

For example, Korean Patent No. 255260 discloses a reagent for an endotoxin assay, including a LAL reagent (Limulus Amebocyte Lysate reagent) and apronitine. Korean Patent No. 539096 discloses a method for detecting endotoxin using a lab-on-a-chip. Korean Patent Application Publication No. 2005-0027223 discloses a method for detecting endotoxin using a bacteriophage tail protein.

US Patent 690872 discloses a method for detecting endotoxins using A ₁ adenosine receptors.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors endeavored to develop a method that can accurately and easily measure the amount of endotoxin in a biological sample, particularly blood, resulting in thermal inactivation and pretreatment of the biological sample, and measurement and analysis of OD values using LAL reagents. The present invention has been completed by confirming that the amount of endotoxin can be measured in an improved manner in terms of accuracy and simplicity, in particular when measuring and analyzing OD values according to kinetic turbidity analysis methods.

Accordingly, it is an object of the present invention to provide a quantitative measurement method of endotoxin in a biological sample.

Another object of the present invention is to provide a quantitative measurement kit of endotoxin in a biological sample.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the invention provides a method for quantitative determination of endotoxin in a biological sample comprising the following steps:

(a) preparing a biological sample;

(b) subjecting the biological sample to heat inactivation;

(c) adding a LAL reagent (Limulus Amebocyte Lysate reagent) to the thermally inactivated biological sample to prepare a reactant; And

(d) measuring the optical density (OD) value after a predetermined time of the reactant or measuring the time to reach the constant OD value of the reactant.

According to another aspect of the present invention, the present invention relates to a heating apparatus for thermally inactivating a biological sample, (b) a Limulus Amebocyte Lysate reagent, and (c) a thermally inactivated biological sample. A microplate to which a LAL reagent is added, (d) a microplate reader for measuring the optical density (OD) value of the reaction product in the microplate, and (e) a endotoxin solution of known concentration Provided are kits for the quantitative determination of endotoxin in a sample.

The present inventors endeavored to develop a method that can accurately and easily measure the amount of endotoxin in a biological sample, particularly blood, resulting in thermal inactivation and pretreatment of the biological sample, and measurement and analysis of OD values using LAL reagents. Lower levels (particularly when measuring and analyzing OD values according to kinetic turbidity analysis methods) have shown that the amount of endotoxin can be measured in an improved manner in terms of accuracy and simplicity.

As used herein, the term “endotoxin” refers to a toxic substance that causes gram-negative bacterial sepsis as a component of a gram-negative bacterial cell membrane known as lipopolysaccharide (LPS).

The method of the present invention can be applied to a variety of biological samples such as blood, lymph, bone marrow fluid, saliva, urine, feces, ascites and amniotic fluid, but preferably to blood. More preferably, the method of the present invention is used to measure the amount of endotoxin in plasma.

One of the greatest features of the method of the present invention is the thermal inactivation treatment of the biological sample prior to reaction with the LAL reagent. This inactivation treatment serves to remove the activity of the interfering substance in the biological sample that interferes with the reaction with the LAL reagent, so that the reaction with the LAL reagent by the endotoxin in the biological sample is well performed. The improvement of the endotoxin measurement by thermal inactivation treatment is well illustrated in FIG. 2.

Thermal inactivation treatment is carried out by applying heat to a biological sample in the liquid phase. Preferably, the heat inactivation treatment is carried out at 60-90 ° C, more preferably at 65-80 ° C, most preferably at 70-80 ° C. The heat inactivation treatment time is 1-60 minutes, preferably 5-50 minutes, more preferably 8-40 minutes, and most preferably 8-20 minutes. The heat inactivation treatment can be carried out using a variety of methods and heating apparatus, and preferably, the tube containing the biological sample is water bath, heating block or incubator, more preferably water bath. Or place it in the heating block.

The heat inactivation method employed in the present invention shows a much better endotoxin measuring ability than the strong acid inactivation method (see Example 5).

Biological samples, especially plasma, to be thermally inactivated can be used as they are, but can preferably be diluted. The dilution multiple is preferably 2-20 times, more preferably 5-15 times and most preferably 9-11 times.

Dilution of biological samples, particularly plasma, preferably uses buffer rather than water. In this case, the buffering region of the buffer used is preferably pH 6.0-8.0, more preferably pH 6.5-8.0 and most preferably pH 7.0-7.4. In addition, the buffer used for dilution of the biological sample preferably comprises Na ⁺ and divalent cations.

The method for measuring the endotoxin of the present invention can be basically referred to as a turbidimetric assay method. That is, the method of measuring the amount of endotoxin present in the biological sample by using the principle that the endotoxin present in the biological sample (eg, plasma) and LAL reagent (Limulus Amebocyte Lysate reagent) react to form a gel.

Step (c) of the present invention, that is, the reaction between the biological sample and the LAL reagent can be carried out in various reaction vessels, preferably in a microplate having a plurality of wells.

The method of the present invention is basically a turbidimetric assay method, which can be carried out in two main ways: (i) an endpoint turbidimetric assay; And (ii) kinetic turbidimetric assay.

First, endpoint turbidity analysis is a method of determining the amount of endotoxin by measuring the optical density (OD) value after a certain time of the reaction consisting of a biological sample and the LAL reagent.

A second kinetic turbidity assay is a method of determining the amount of endotoxin by measuring the time to reach a constant OD value of a reactant consisting of a biological sample and a LAL reagent.

According to a preferred embodiment of the present invention, step (d) of the present invention is carried out according to the method of kinetic turbidity analysis. Our experimental results show that the kinetic turbidity analysis method enables a much more accurate endotoxin measurement than the endpoint turbidity analysis method.

According to the kinetic turbidity analysis method, when step (d) is carried out, it is preferably performed at a wavelength of 300-420 nm (more preferably at a wavelength of 320-400 nm, most preferably at 330-400 nm). It is carried out by measuring the time to reach 0.03-0.159 (more preferably 0.03-0.08, even more preferably 0.03-0.07, most preferably 0.04-0.06) OD value.

According to a preferred embodiment of the present invention, the method further comprises preparing a standard curve using a known concentration of endotoxin solution. The drawn standard curve can be used to determine the amount of endotoxin in the biological sample by substituting the OD value measured by the biological sample or the time to reach the OD value.

According to a preferred embodiment of the present invention, the kit of the present invention further comprises a buffer to dilute the biological sample. The buffering region of the buffer in which the biological sample is diluted is preferably pH 6.0-8.0, more preferably pH 6.5-8.0 and most preferably pH 7.0-7.4. In addition, the buffer used for dilution of the biological sample preferably comprises Na ⁺ and divalent cations.

The present invention makes it possible to determine the amount of endotoxin in a biological sample in a more accurate and convenient manner.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  1: Preparation of Plasma Samples from Blood

Collect 3.5 to 3.5 ml of blood into a sodium heparin tube (BD Vaccutainer), shake carefully to mix the heparin in the blood well, take 1.5 ml of blood from this tube (using a pipette) and a 2.0 ml Eppendorf tube (e- tube). In order to separate plasma from blood, the 2 ml e-tube was centrifuged at 6,000 rpm for 3 minutes with a table-top microcentrifuge (Micro-12, Hanil Science), and 100 μl of the supernatant (plasma) was added. Transfer to 5 ml polystyrene tube (BD Falcon). The tube containing 100 μl plasma was diluted 10-fold by adding 900 μl BD100 buffer (Charles River ENDOSAFE, pH 7.0, 0.05% (v / v) solution of phosphate ester surfactant) (100 μl plasma + 900 μl BD100 buffer). ).

The 10-fold diluted plasma was then heat inactivated for 10 minutes in a 75 ° C. constant temperature bath. The plasma sample was then slowly cooled on ice for 10 minutes. Once the plasma sample had cooled down, the amount of endotoxin was measured using a 96-well plate. If the plasma samples were not measured immediately, they were stored in a -20 ° C (or -70 ° C) freezer until the measurement. Endotoxin activity was maintained for up to one week. By 1 month of storage, the activity of endotoxin was up to 65%.

Example  2: Prepare Standard Solution

5, 0.5, 0.05 and 0.005 EU / mL were prepared using 50 endotoxin units (EU) / mL CSE (control standard endotoxin, Charles River ENDOSAFE) and LRW (LAL reagent water, Charles River ENDOSAFE): EU / mL: 200 μL 50 EU / mL + 1800 μL LRW; ② 0.5 EU / mL: 200 μL 5 EU / mL + 1800 μL LRW; ③ 0.05 EU / ml: 200 μl 0.5 EU / ml + 1800 μl LRW; And ④ 0.005 EU / mL: 200 μL 0.05 EU / mL + 1800 μL LRW.

Example  3: Endotoxin  Quantitative Measurement

100 μL of each CSE was dispensed into 2 wells, and 100 μL of LRW was dispensed into 2 wells as background values. Plasma samples were dispensed into 4 wells of 96-well plates at 100 μl. 10 μl of 5 EU / ml CSE was added to two wells out of four wells in which plasma samples were dispensed for the positive control measurement. Positive controls are used to determine if there are interfering substances in the plasma sample that affect the measurements. If no interfering substance is found in the plasma, the recovery of the positive control is 100% (if the recovery is within 50-200%, this test is considered valid).

100 μl of LAL reagent (Limulus Amebocyte Lysate reagent, Charles River ENDOSAFE) was added to CSE, LRW, and plasma samples, and the reaction was performed at 37 ° C. After confirming that there are bubbles in the wells (bubbles cause errors in the analysis of the measured values. Bubbles in the wells are removed using clean needles or tips), the 96-well plate is microplate reader (Bio-Tek) Into the endotoxin measuring device consisting of the measurement was started, and the measured value was calculated and analyzed.

The manner in which the measurement samples are dispensed on the 96-well plate is described in more detail as follows (FIG. 1). Two plasma samples per person (Sample 1: S1 & S1-1, Sample 2: S2 & S2-1, Sample 3: S32 & S3-1, Sample 4: S4 & S4-1, Sample 5: S5 & S5-1) was prepared and two measurement sets were made for each plasma sample. A positive control, ie a group spiking CSE to a final concentration of 0.5 EU / ml, was made for each plasma sample. 1 shows how measurement samples are dispensed into 96-well plates.

The method for measuring the endotoxin in the present invention is a kinetic turbidimetric assay, an endotoxin present in the plasma using the principle of forming a gel by reacting the endotoxin in the plasma with the LAL reagent. It is a way to measure quantity. At 340 nm, the time at which the optical density (OD) reaches 0.05 is measured. The time measured for the plasma sample is determined by the amount of endotoxin in the plasma by a standard curve made by a control standard endotoxin (CSE) which knows the endotoxin concentration.

2 shows the recovery for the positive control measured according to the method of the present invention. As a result of the experiment, the recovery rate was 98.01 ± 12.55% (SD), indicating almost 100% recovery. On the other hand, when heat inactivation was not performed at 75 ° C. for 10 minutes, a value of 0.005 EU / ml or less was measured, so that a positive control of 1.0 EU / ml was hardly recovered due to the strong interference action of proteins in blood (or plasma). Thus, the method of the present invention is an optimal method for maximizing the interference of LAL reagents with proteins present in the blood (or plasma).

3 is a standard curve obtained according to the method of the present invention. In the standard curve, the x-axis is the logarithm of the EU and the y-axis is OD _340nm The log value for the time (seconds) that the value reaches 0.05.

Tables 1a and 1b below are experimental results for 20 normal and gastrointestinal disease individuals, respectively.

sample Endotoxin amount (EU / ml) Spike recovery rate (%) One <0.05 112 2 <0.075 89 3 0.012 105 4 0.095 123 5 <0.05 96 6 <0.05 103 7 <0.05 116 8 <0.05 130 9 <0.05 125 10 <0.05 103 11 <0.05 85 12 <0.05 114 13 <0.05 108 14 <0.05 120 15 <0.05 101 16 <0.05 93 17 <0.05 98 18 <0.07 102 19 <0.05 104 20 <0.05 116

sample Endotoxin amount (EU / ml) Spike recovery rate (%) One 2.725 128 2 0.7 168 3 1.08 98 4 1.367 144 5 0.1 136 6 2.5205 84 7 7.348 81 8 0.554 111 9 0.1 101 10 0.1 115 11 1.7155 86 12 0.1 93 13 0.95 123 14 2.2795 119 15 2.9315 109 16 <0.05 115 17 1.4345 118 18 0.66 133 19 2.282 119 20 0.678 101

As can be seen from the table, according to the method of the present invention, the positive control spike recovery is excellent and accurate endotoxin measurement is possible. The amount of endotoxin was low in the plasma of normal humans, and the amount of endotoxin was relatively high in the plasma of humans with gastrointestinal diseases.

Example  4: suitable diluent  And identification of dilution drainage

A dilution drainage group spiked with CSE using LRW (LAL reagent water, Charles River ENDOSAFE) or BD100 buffer to a final concentration of 1.0 EU / mL was prepared, and the amount of endotoxin was changed in the same manner as in Example 3. Measured. The experimental results are shown in Table 2 below.

Diluent type Dilution drainage 0 2 4 6 8 10 15 20 Relative amount of spiking endotoxin (%) LRW - 15.0 24.5 29.4 50.6 52.3 58.5 72.7 BD100 - 22.0 30.4 47.8 57.9 101.6 113.5 124.3

In Table 2, LRW is distilled water, BD100 is a buffer of pH 7.0 containing Na ⁺ and divalent cations.

In Table 2, when distilled water was used, the recovery rate of endotoxin was low, whereas when BD100 buffer was used, the recovery rate of endotoxin was relatively high. In addition, the relative amount of endotoxin (i.e., 100% recovery) of the most preferred sample in 10-fold dilution is shown.

Example  5: Comparison with Other Deactivation Methods

Another way to inactivate the interference in plasma is to treat strong acids. Perchloric acid (PCA), a type of strong acid, was used to compare the thermal inactivation method of the present invention.

Plasma samples were prepared as follows: blood was collected in a sodium heparin tube (BD Vaccutainer), 3-3.5 ml, carefully shaken to allow heparin to mix well in the blood, and 1.5 ml of blood was taken from the tube (pipette). Transfer to 2.0 ml Eppendorf tubes (e-tube). To separate plasma from blood, the 2 ml e-tube was centrifuged for 3 min with a table-top microcentrifuge (Micro-12, Hanil Science), and 1.9% PCA 0.4 in 200 µl of supernatant. ㎖ was added and mixed well. The supernatant was then centrifuged for 1 minute with a table-top microcentrifuge. 0.2 mL of the supernatant was taken and treated with 0.2 mL of 0.2 N NaOH to prepare a finally inactivated plasma sample. Next, the method of measuring the endotoxin is the same as in Example 3.

The experimental results are shown in FIG. In Figure 4, the storage time is the time stored frozen at -20 ℃ after making a plasma sample. As can be seen in Figure 4, according to the present invention, the recovery rate of the endotoxin is about 90%, according to the PCA method is about 20% recovery. Thus, it can be seen that the method of the present invention is a very preferred method for detecting and measuring endotoxin in plasma.

As described in detail above, the present invention provides a method and kit for quantitative measurement of endotoxin in a biological sample. The present invention makes it possible to determine the amount of endotoxin in a biological sample in a more accurate and convenient manner.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (14)

Quantitative measurement of endotoxin in a biological sample comprising the following steps: (a) preparing a biological sample; (b) subjecting the biological sample to heat inactivation; (c) adding a LAL reagent (Limulus Amebocyte Lysate reagent) to the thermally inactivated biological sample to prepare a reactant; And (d) measuring the optical density (OD) value after a predetermined time of the reactant or measuring the time to reach the constant OD value of the reactant. The method of claim 1, wherein the biological sample is blood. The method of claim 2, wherein the biological sample is plasma. The method of claim 1 wherein the heat inactivation treatment is carried out at 60-90 ° C. The method of claim 1, wherein the thermal inactivation treatment is performed by placing a tube containing a biological sample in a heating treatment device. The method of claim 1, wherein step (c) is performed in a microplate. The method of claim 1, wherein step (d) is performed by measuring the time to reach a constant OD value of step (c) reactant. 8. The method of claim 7, wherein step (d) is performed by measuring the time to reach a 0.03-0.15 OD value at a wavelength of 300-420 nm. The method of claim 1, wherein the biological sample in step (a) is diluted 9-11 times. The method of claim 1, wherein the method further comprises the step of preparing a standard curve using a known concentration of endotoxin solution. (a) a heating treatment device for thermally inactivating a biological sample, (b) a Limulus Amebocyte Lysate reagent, (c) a microplate to which the thermally inactivated biological sample and LAL reagent are added, (d) ) A quantitative measurement kit of endotoxin in a biological sample comprising a microplate reader for measuring the optical density (OD) value of the reaction result in the microplate, and (e) an endotoxin solution of known concentration. 12. The kit of claim 11, wherein said biological sample is blood. 12. The kit of claim 11, wherein said biological sample is plasma. 12. The kit of claim 11, wherein said kit further comprises a buffer to dilute the biological sample.

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