WO2009005231A1

WO2009005231A1 - Methods for quantitatively determining endotoxin

Info

Publication number: WO2009005231A1
Application number: PCT/KR2008/003484
Authority: WO
Inventors: Kye Shin Park
Original assignee: Diatech Korea Co., Ltd.
Priority date: 2007-07-02
Filing date: 2008-06-19
Publication date: 2009-01-08
Also published as: KR20090002570A; KR100888788B1

Abstract

The present invention relates to a method for quantitatively determining endotoxin in a biological sample, which comprises the steps of: (a) preparing a biological sample; (b) treating the biological sample with a heat inactivation; (c) providing a reactant through adding LAL reagent (Limulus Amebocyte Lysate reagent) into the biologically heat-inactivated sample; and (d) measuring the OD (optical density) value of the reactant after a particular reaction time or measuring the time when the reactant reaches a predetermined OD value. This invention may determine the amount of the endotoxin in the biological sample in more accurate and convenient manner.

Description

METHODS FOR QUANTITATIVELY DETERMINING ENDOTOXIN

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method and a kit for quantitatively determining endotoxin in a biological sample.

BACKGROUND OF TECHNIQUE

Sepsis is one of mortality causes generated occasionally in chemotherapies and surgical operations. Sepsis is caused by endotoxin called lipopolysaccharide (LPS). Endotoxin triggers the cascade of events causing organ failure, irreversible shock and death. Endotoxin is bound to membrane protein or receptor and induces the formation of various mediators, e.g., oxygen-free radical, nitric oxide, arachidonic acid metabolites, tromboxane, prostacyclin, platelet activating factor, interleukin-8 (IL-8), leukotriene B4, cytokine, IL-I, TNF-α (tumor necrosis factor-α) and protease, through activation of diverse cells, e.g., endothelial cell, neutrophil, monocyte and macrophage, resulting in endotoxin-inducing organ injury and septic shock (Akarasereenont et al., Eur. J. Pharmacol., 273:121-128(1995); Brigham et al., Am. Rev. Respir. Dis., 133:913-927(1986); Morrison, Ann. Rev. Med., 38:417- 432(1987); Williams et al., Surgery, 112:270-277(1992); Wright, Current Opinion in Immunol., 83-91(1991)).

There has been intensively developed diverse methods for detecting endotoxin because of the severity of these endotoxin-induced diseases.

For instance, Korean Pat. No. 255260 discloses an analytic solution containing LAL reagent (Limulus Amebocyte Lysate reagent) and aprotinin for endotoxin assay. Korean Pat. No. 539096 discloses a method for detecting endotoxin using lab-on-a- chip. Korean Pat. Publication No. 2005-0027223 discloses a detection method of endotoxin by use of bacteriophage tail protein. U.S. Pat. No. 6,908,742 discloses the method for detection of endotoxin using Al adenosine receptor.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have made intensive studies to measure an amount of endotoxin in a biological sample, particular blood, by means of more accurate and more convenient method. As results, we have discovered that the amount of endotoxin could be detected much more accurately and conveniently by pretreating the biological sample with a heat inactivation and then measuring OD (optical density) values by use of a LAL reagent (particularly, by measuring OD values via a kinetic turbidimetric assay).

Accordingly, it is an object of this invention to provide a method for quantitatively determining endotoxin in a biological sample. It is another object of this invention to provide a kit for quantitatively determining endotoxin in a biological sample.

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

In one aspect of this invention, there is provided a method for quantitatively determining endotoxin in a biological sample, which comprises the steps of: (a) preparing the biological sample;

(b) subjecting the biological sample to a heat inactivation;

(c) preparing a reaction solution by adding a LAL reagent (Limulus Amebocyte Lysate reagent) into the heat-inactivated biological sample; and (d) measuring an OD (optical density) value of the reaction solution after a particular reaction time or measuring the time when the reaction solution reaches a predetermined OD value.

In another aspect of this invention, there is provided a kit for quantitatively determining endotoxin in a biological sample, which comprises: (a) a heating device to heat-inactivate the biological sample; (b) a LAL reagent (Limulus Amebocyte Lysate reagent); (c) a microplate in which the heat-inactivated biological sample and the LAL reagent are added; (d) a microplate reader for measuring OD (optical density) values of a reaction product in the microplate; and (e) endotoxin solutions with predetermined concentrations. The present inventors have made intensive studies to measure an amount of endotoxin in a biological sample, particular blood, by means of more accurate and more convenient method. As results, we have discovered that the amount of endotoxin could be detected much more accurately and conveniently by pretreating the biological sample with a heat inactivation and then measuring OD (optical density) values by use of a LAL reagent (particularly, by measuring OD values via a kinetic turbidimetric assay).

The term "endotoxin" as used herein means a toxic substance causing Gram- negative bacteria sepsis as a component of Gram-negative bacteria membrane, known as lipopolysaccharide (LPS). The instant invention may be applied to various biological samples, for example, blood, lymph, bone marrow fluid, saliva, urine, feces, ascites and amniotic fluid, preferably blood. More preferably, the present method may be used to measure the amount of endotoxin in plasma. It is one of the most striking features of this invention to perform the heat inactivation prior to incubating the biological sample with the LAL reagent. The heat inactivation is responsible for removal of activities of interference substances to inhibit reactions with the LAL reagent in the biological sample, resulting in successful reactions between biological sample-contained endotoxin and the LAL reagent. The great improvement in analytic accuracy of endotoxin with the help of heat inactivation is demonstrated in Hg. 2.

The heat inactivation treatment is performed by heating a biological liquid sample. The heat inactivation performs at preferably 60-90⁰C, more preferably at 65-8O⁰C and most preferably at 70-80⁰C. The treatment time of the heat inactivation is 1-60 min, preferably 5-50 min, more preferably 8-40 min and most preferably 8-

20 min. The heat inactivation may be performed by various methods and heating devices, preferably by placing tubes containing the biological sample on a water bath, a heating block or an incubator, more preferably a water bath or a heating block.

The heat inactivation in the present invention contributes to much more excellent measuring ability for endotoxin than strong acid-inactivation method (see Example 5).

The heat-inactivated biological sample, particularly plasma per se may be used but preferably used after dilution. The dilution factor is preferably 2-20 times, more preferably 5-15 times and most preferably 9-11 times.

It is preferable to use a buffer than water for dilution of the biological sample, especially the plasma. In this instance, the buffering region of the buffer to be used is preferably pH 6.0-8.0, more preferably pH 6.5-8.0 and most preferably pH 7.0-7.4. Additionally, the buffer used for dilution of the biological sample comprises preferably Na⁺ and divalent cations.

The present method for analyzing endotoxin levels may be classified to a turbidimetric assay. That is to say, the present method for measuring endotoxin as a kinetic turbidimetric assay determines the amounts of endotoxin by means of a gel formation by the reaction between endotoxin present in the plasma and the LAL reagent.

The step (c) of this invention, i.e., the reaction between the biological sample and the LAL reagent may be performed in various reaction vessels, preferably in a microplate having multiple wells.

The present method for analyzing endotoxin level is in principle performed in accordance with a turbidimetric assay. Accordingly, the present invention may be carried out by two process types: (i) an endpoint turbidimetric assay; and (ii) a kinetic turbidimetric assay.

First, the endpoint turbidimetric assay is to determine the amount of endotoxin by measuring the OD (optical density) value of the reaction solution after a particular reaction time.

Secondly, the kinetic turbidimetric assay is the method for determining the amount of endotoxin by measuring the time when the reaction solution comprising the biological sample and the LAL reagent reaches a predetermined OD value.

According to a preferred embodiment, the step (d) of the present invention is carried out by the kinetic turbidimetric assay. In the experimental results of the present inventors, the kinetic turbidimetric assay may measure endotoxin more accurate than the endpoint turbidimetric assay.

In compliance with the kinetic turbidimetric assay, the step (d) is preformed by measuring the time reaching preferably 0.03-0.159 (more preferably 0.03-0.08, much more preferably 0.03-0.07 and most preferably 0.04-0.06) of OD values at

300-420 nm wavelength (more preferably 320-400 nm and most preferably 330-400 nm).

According to a preferred embodiment, the method of the instant invention further comprises a step of preparing a standard curve for using endotoxin solution of predetermined concentrations. The amount of endotoxin in the biological sample may be determined by incorporating OD values or the time reaching the OD value measured for the biological sample into the prepared standard curve.

According to a preferred embodiment, the kit of the invention further comprises a solution for diluting the biological sample. The buffering region of the buffer to dilute the biological sample ranges preferably from pH 6.0 to pH 8.0, more preferably pH 6.5-8.0 and most preferably pH 7.0-7.4. The buffer used for dilution of the biological sample comprises preferably Na⁺ and divalent cations.

This invention can determine the amount of endotoxin in the biological sample in more accurate and more convenient manner.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents the schematic diagram for dividing the samples into a 96- well plate according to a particular example of this invention.

Fig. 2 is a histogram showing the result of the recovery rate for control standard endotoxin (CSE) measured according to the present invention.

Fig. 3 represents a standard curve for endotoxin according to the present invention. The x-axis represents the log values of EU and the y-axis the log values for the period of time (second) when the value of OD_340nm reaches 0.05.

Fig. 4 is a graph comparing the results for using PCA (perchloric acid) strong acid treatment as inactivation method with the experimental results of this invention.

EXAMPLES EXAMPLE 1: Preparation of Plasma Sample from Blood 3-3.5 mL of blood was mixed thoroughly with heparin in a sodium heparin tube (BD Vaccutainer Co.), from which 1.5 mL blood was collected by pipet and transferred into a 2.0 mL eppendorf tube (e-tube). To isolate plasma from blood, the 2.0 mL e-tube was centrifuged at 6,000 rpm for 3 min using a table-top microcentrifuge (Micro-12, HanilSC Co.) and each 100 μL of the supernatant (plasma) was transferred into 5 mL polystyrene tubes (Falcon Co.). The tubes containing 100 μL plasma were diluted 10 times by adding 900 μL BDlOO buffer (Charles River ENDOSAFE Co., pH 7.0, 0.05% (v/v) phosphate ester surfactant solution) (100 μL plasma plus 900 BDlOO buffer). The ten-fold diluted plasma was heat-inactivated for 10 min at a 75°C incubation bath. Subsequently, the plasma was slow cooled on ice for 10 min. After cooling of the plasma, the amount of endotoxin was measured using a 96-well plate. The plasma was stored at -20⁰C (or -7O⁰C) before measurements unless it was analyzed immediately. The activity of endotoxin was retained even after one week storage. The 65% activity of endotoxin was persisted after one month storage.

EXAMPLE 2: Preparation of the Standard Solution

5, 0.5, 0.05 and 0.005 EU/mL solutions were prepared using 50 EU (endotoxin unit)/mL CSE (control standard endotoxin, Charles River ENDOSAFE Co.) and LRW (LAL reagent water, Charles River ENDOSAFE Co.): © 5 EU/mL: 200 μL 50 EU/mL + 1800 μL LRW; © 0.5 EU/mL: 200 μL 5 EU/mL + 1800 μL LRW; (D 0.05 EU/mL: 200 μL 0.5 EU/mL + 1800 μL LRW; and ® 0.005 EU/mL: 200 μL 0.05 EU/mL + 1800 μL LRW.

EXAMPLE 3: Quantitative Measurement of Endotoxin

100 μL aliquots of each CSE were added into two wells and 100 μL of LRW for background value was also introduced into two wells. The plasma sample was placed into four wells at 100 μl_ per well. To measure a positive control, 10 μl_ aliquots of 5 EU/mL CSE was added into two of four wells containing the plasma samples. The positive control was used to examine whether the interference with the measurement by substances in the plasma samples occurs or not. Where the plasma samples do not contain substances to induce the interference, a recovery rate of the positive control will be 100% (the experimental data were considered experimentally meaningful values where the recovery rate is between 50% and 200%).

CSE, LRW and the plasma sample were incubated at 37°C after adding each 100 μL LAL reagent (Limulus Amebocyte Lysate reagent, Charles River ENDOSAFE Co.). The bubbles within wells were checked (The bubbles generated errors for the analysis of measurement values. The bubbles in wells must be eliminated by a clean needle or tip) and then 96-well plate was measured by an endotoxin- measuring apparatus equipped with a microplate reader (Bio-Tek Co.), finally analyzing the measurement values.

The method for dividing the plasma samples into 96-well plates was described more detail as follows (Fig. 1). Two plasma samples per person (Sample 1: Sl & Sl-I, Sample 2: S2 & S2-1, Sample 3: S3 & S3-1, Sample 4: S4 & S4-1, Sample 5: S5 & S5-1) were prepared and two measurement sets per plasma sample were prepared. The positive control, a group in which CSE is spiked at a final concentration of 0.5 EU/mL, was prepared for each plasma sample. Fig. 1 represents the manner for dividing the samples into the 96-well plate.

The present method for measuring endotoxin as a kinetic turbidimetric assay determines the amounts of endotoxin by means of a gel formation by the reaction between endotoxin present in the plasma and the LAL reagent. The times when the OD (optical density) value reaches 0.05 at 340 nm wavelength were measured. The time measured for the plasma sample was converted into the amount of endotoxin in the plasma using a standard curve obtained from CSE (control standard endotoxin).

Figure 2 indicates the recovery rates of the positive control measured by the present invention. The present method showed 98.01 ± 12.55% (SD) of the recovery rate, exhibiting close to 100% recovery rate. However, no heat inactivation for 10 min at 75°C resulted in measurement values of no more than 0.005 EU/mL, suggesting a positive control of 1.0 EU/mL was hardly retrieved due to strong interference of proteins present in blood (or plasma). Accordingly, it could be appreciated that the method of the present invention is the most applausible approach to minimize the interference occurrence between proteins present in blood (or plasma) and a LAL reagent.

Figure 3 is a standard curve obtained according to this invention. The x-axis represents the log values of EU and the y-axis the log values of the period of time (second) when the value of OD_340Pm reaches 0.05.

Tables Ia and Ib described below represent the experimental results for 20 healthy persons and 20 patients having gastrointestinal disease. TABLE Ia.

As demonstrated in Tables, the present invention exhibits excellent recovery rates of the positive control spike to enable the quantification of endotoxin with much higher accuracy. Relatively low amounts of endotoxin were measured in healthy human plasma samples but relatively high in plasma samples of patients having gastrointestinal disease. EXAMPLE 4: Analysis of Approximate Dilution Solution and Dilution Factor

The groups containing spiked CSE for verifying suitable dilution solution and factors were prepared using LRW (LAL reagent water, Charles River ENDOSAFE Co.) or BDlOO buffer at a final concentration of 1.0 EU/mL and the amount of endotoxin was measured as described in Example 3. The experimental results were shown in Table 2. TABLE 2.

In Table 2, LRW refers to distilled water and BDlOO is a buffer (pH 7.0) containing Na⁺ and divalent cations.

The recovery rate of endotoxin diluted by distilled water was low but that of endotoxin was relatively high in samples diluted by BDlOO buffer. In addition, the most preferably relative amount of endotoxin {i.e., 100% recovery rate) was exhibited in a ten-fold diluted sample using BDlOO buffer.

EXAMPLE 5: Comparative Evaluation with Other Inactivation Methods

One of other methods for inactivating interferences in the plasma is to treat the plasma with a strong acid. The heat inactivation of the present invention was compared with the use of PCA (perchloric acid) as a strong acid.

The preparation of the plasma sample was as follows: 3-3.5 mL of blood was mixed thoroughly with heparin in a sodium heparin tube (BD Vaccutainer Co.), from which 1.5 mL blood was collected by pipet and transferred into a 2.0 mL eppendorf tube (e-tube). To isolate plasma from blood, the 2.0 mL e-tube was centrifuged for 3 min using table-top microcentrifuge (Micro-12, HanilSC Co.) and 200 μL of the supernatant (plasma) was well mixed with 0.4 mL PCA (1.9%). The mixing supernatant was centrifuged for 1 min using table-top microcentrifuge. 0.2 mL of the supernatant was collected and then treated with 0.2 mL NaOH (0.2 N), finally preparing an inactivated plasma sample. The measurement of endotoxin levels was performed as described in Example 3.

The experimental result is represented in Hg. 4. The storage time in Fig. 4 is the period of time to keep plasma samples at -20⁰C after preparation. As shown in Figure 4, the recovery rate of endotoxin in this invention came up to about 90%, but that of endotoxin in PCA method was only about 20%. Accordingly, it would be appreciated that the present invention is a more preferable method for measuring endotoxin in the plasma.

As described above, the present invention provides a method and a kit for quantitatively determining endotoxin in a biological sample. This invention may determine the amount of endotoxin in a biological sample in more accurate and convenient manner.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A method for quantitatively determining endotoxin in a biological sample, which comprises the steps of:

(a) preparing the biological sample; (b) subjecting the biological sample to a heat inactivation;

(c) preparing a reaction solution by adding a LAL reagent (Limulus Amebocyte Lysate reagent) into the heat-inactivated biological sample; and

(d) measuring an OD (optical density) value of the reaction solution after a particular reaction time or measuring the time when the reaction solution reaches a predetermined OD value.

2. The method according to claim 1, wherein the biological sample is blood.

3. The method according to claim 2, wherein the biological sample is plasma.

4. The method according to claim 1, wherein the heat inactivation performs at 60- 90⁰C.

5. The method according to claim 1, wherein the heat inactivation is carried out by placing a tube comprising the biological sample on a heating device.

6. The method according to claim 1, wherein the step (c) performs in a microplate.

7. The method according to claim 1, wherein the step (d) performs by measuring the time when the reaction solution of the step (c) reaches the predetermined OD value.

8. The method according to claim 7, wherein the step (d) performs by measuring the time when the OD value reaches 0.03-0.15 at 300-420 nm wavelength.

9. The method according to claim 1, wherein the biological sample in the step (a) is diluted by 9-11 folds.

10. The method according to claim 1, wherein the method further comprises the step of preparing a standard curve using endotoxin solutions with predetermined concentrations.

11. A kit for quantitatively determining endotoxin in a biological sample, which comprises: (a) a heating device to heat-inactivate the biological sample; (b) a LAL reagent (Limulus Amebocyte Lysate reagent); (c) a microplate in which the heat- inactivated biological sample and the LAL reagent are added; (d) a microplate reader for measuring OD (optical density) values of a reaction product in the microplate; and (e) endotoxin solutions with predetermined concentrations.

12. The kit according to claim 11, wherein the biological sample is blood.

13. The kit according to claim 11, wherein the biological sample is plasma.

14. The kit according to claim 11, wherein the kit further comprises a solution for diluting the biological sample.