WO2011146463A2

WO2011146463A2 - Enhancing endotoxin detection

Info

Publication number: WO2011146463A2
Application number: PCT/US2011/036783
Authority: WO
Inventors: Michael G. Pepe; Milton Keith Champion
Original assignee: BioDtech, Inc.
Priority date: 2010-05-17
Filing date: 2011-05-17
Publication date: 2011-11-24
Also published as: WO2011146463A3

Abstract

Provided herein are methods for detecting endotoxin in a sample or medical device. Kits for detecting endotoxin in a sample or medical device are provided.

Description

Enhancing Endotoxin Detection

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Application No. 61/345,482 filed May 17, 2010, which is hereby incorporated herein by reference in its entirety.

BACKGROUND

Endotoxin, also known as lipopolysaccharide (LPS), is an integral component of the gram-negative bacterial cell membrane and is responsible for many, if not all, of the toxic effects that occur during gram-negative bacterial sepsis. LPS is a mixture of glycolipids consisting of a variable polysaccharide domain covalently bound to a conserved glucosamine -based

phospholipid known as lipid A. LPS directly stimulates host monocytes and macrophages to secrete a wide array of inflammatory cytokines that include tumor necrosis factor-I (TNF-I), interleukins-1 (IL-1), and interleukin-8 (IL-8). Excessive release of these cytokines by host macrophages contributes to organ failure and death that occur after episodes of gram-negative bacterial sepsis.

SUMMARY

Provided herein are methods for detecting endotoxin in a biological sample or a medical device. For example provided herein is a method of detecting endotoxin in a biological sample or medical device containing heparin comprising: contacting the biological sample or medical device with heparinase; and detecting endotoxin. Also provided herein are kits for detecting endotoxin in a biological sample or medical device. The kits comprise heparinase. The details of one or more aspects are set forth in the accompanying description below. Other features, objects and advantages will be apparent from the description and from the claims.

DETAILED DESCRIPTION

Since endotoxin contains a negative charge, a lipid and a carbohydrate, many proteins can interfere with the ability of assays to properly measure endotoxin. Specifically, the presence of heparin can cause variation in the standard assays, resulting in false positives and negatives, as well as affecting accuracy. As described in the examples below, treatment of such samples, including serum and blood samples, with heparinase digests or degrades the heparin in the sample without affecting endotoxin, thus, increasing the accuracy of endotoxin detection assays. Provided herein are improved methods and kits for detecting endotoxin in a sample or medical device. For example, provided herein is a method of detecting endotoxin in a biological sample or medical device containing heparin comprising: contacting the biological sample or medical device with heparinase; and detecting endotoxin. Optionally, the method further comprises contacting the biological sample or medical device with an active acidic protease prior to contacting the biological sample or medical device with heparinase. Optionally, the method further comprises inactivating the acidic protease prior to contacting the sample with heparinase, but after contacting the biological sample or medical device with an active protease.

In the methods further comprising contacting the biological sample or medical device with an active acidic protease, the biological sample or medical device can be contacted with an active acidic protease at a pH of about less than 7, less than 6 or at a pH of about 5. Therefore, the pH can be about 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 4.9, 4.8, 4.7, 4.6, 4.5 or about 5.0.

Furthermore, in the methods comprising contacting the biological sample or medical device with an active acidic protease, the method can further comprise inactivating the acidic protease after contacting the sample with the active acidic protease. Optionally, the acidic protease is inactivated by a pH of about 7.0.

As used herein, a biological sample subjected to testing is a sample derived from a subject such as a mammal or human and includes, but is not limited to, any biological fluid, including a bodily fluid. Examples of bodily fluids include, but are not limited to, whole blood, plasma, serum, urine, saliva, tissue infiltrate, pleural effusions, lung lavage fluid, and the like. The biological fluid includes a cell culture medium or supernatant of cultured cells. For example, the sample can be a blood sample or a serum sample. Optionally, the sample is a liquid sample, such as water or other agents used, for example, in research or clinical laboratories or hospitals. Optionally, the sample is obtained from a surface, for example, in a hospital, for analysis in the provided methods. For example, a sample can be obtained from a device used in a hospital, clinical or laboratory setting and analyzed for the presence of endotoxin. Optionally, the sample is diluted in solution prior to analysis. Optionally, the sample to be tested comprises heparin or a heparin derivative that interferes with endotoxin assays. Thus, for example, the sample comprises heparin or heparin sulfate that inhibits standard endotoxin assays.

As used herein, a "medical device" can be, but is not limited to a tube, a stent, a hemodialysis membrane, a filter, a mesh, a bandage or any other product that comes in physical contact with a patient during diagnosis, prevention, therapy or surgery. A medical device can also be an instrument or a component of an instrument utilized during diagnosis, prevention, therapy or surgery.

As used herein, acid, acidic, aspartic or aspartic acid proteases refer to proteases active at low pH. For example, the protease is active at a pH from about 0.0 to about 6.0 or any pH between 0.0 and 6.0, inclusive. Such proteases are inactive at a pH of about 6.0 to about 14.0. As used herein, an inactive acidic protease refers to a protease without protealytic activity (i.e., a protease that is unable to cleave an amino acid sequence such as a polypeptide or protein). As used herein, an active acidic protease refers to a protease with protealytic activity (i.e., a protease that is able to cleave an amino acid sequence). By way of example, an active acidic protease can be inactivated by a pH of 6.5 or higher (i.e., the protease is in a solution with a pH of 6.5 or higher). The pH of a solution can be altered by addition of chemicals to a solution. For example, hydrochloric acid can be used to reduce pH and sodium hydroxide can be used to raise pH. Phosphoric acid can be used to maintain a pH of about 6.5. Optionally, a pepsin inhibitor is used to inactivate pepsin. Pepsin inhibitors include, but are not limited to, acetamidine, N- acetyl-D-phenyalanyl-L-diiodotyrosine, N-acetyl-L-phenyalanyl-D-phenylalaine, p- aminobenzamidine, benzamidine, butyamine, diazoketones, ethylamine, pepstatin, and pheny lactamidine .

Acid or acidic proteases, such as endopeptidases, are known and have been grouped into three families, namely, pepsin (Al), retropepsin (A2), and enzymes from pararetroviruses (A3). The members of families Al and A2 are known to be related to each other, while those of family A3 show some relatedness to Al and A2. Microbial acid proteases exhibit specificity against aromatic or bulky amino acid residues on both sides of the peptide bond, which is similar to pepsin, but their action is less stringent than that of pepsin. Acid proteases include microbial, fungal, viral, animal and plant acidic proteases. Microbial aspartic proteases can be broadly divided into two groups, (i) pepsin-like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and (ii) rennin-like enzymes produced by Endothia and Mucor spp (Rao et al, Microbiology and Molecular Biology 62(3):597-635 (1998); Richter et al, Biochem. J. 335:481- 90 (1998)). Examples of acidic proteases include, but are not limited to, pepsins, including pepsins A, B and C; rennin; chymosin; plasmepsin; cathepsins, such as, for example, cathepsin D and cathepsin E; human urinary acid protease; and viral proteases like HIV protease. Fungal proteases include, but are not limited to, fungal proteases derived from Neurospora oryzae, Mucor pusillus, Mucor miehei, Aspergillus niger, Rhizopus chinensis, or Endothia parasitica. Microbial proteases include, but are not limited to, yeast proteinase A, aspergillopepsinogen, rhizopuspepsin, penicillopepsin, and endothiapepsin.

Endotoxin can be detected via methods standard in the art, for example, and not to be limiting, these include gel-clot assays, turbidimetric assays, and chromogenic assays. The PyroGene^® Recombinant Factor C Endotoxin detection System (Lonza 50-658U), an example of a gel clot assay, can be utilized, as described in the Examples. It is understood that since endotoxin is an integral component of the gram-negative bacterial cell membrane, the methods set forth herein can also be utilized to detect the presence of gram negative bacteria in a biological sample or medical device.

Kits for detecting endotoxin, gram negative bacteria or lipopolysaccharide are provided. The kits comprise heparinase. The heparinase can be from any commercial or non-commercial source, and can be from any aerobic or anaerobic heparinase producing bacteria, for example, from Flavobacterium heparinum. The heparinase can also be a recombinant heparinase. The kit can also comprise an acidic protease. As discussed above, the acidic protease can be any acidic protease. For example, the acidic protease is selected from the group consisting of pepsin, rennin, chymosin, plasmepsin, cathepsin D, cathepsin E, human urinary acid protease, HIV protease, Neurospora oryzae protease, Mucor pusillus protease, Mucor miehei protease,

Aspergillus niger protease, Rhizopus chinensis protease, Endothia parasitica protease, yeast proteinase A, aspergillopepsinogen, rhizopuspepsin, penicillopepsin, and endothiapepsin.

Optionally, the kits further comprising one or more buffers, such as, for example, a heparinase buffer with a pH of about 7 or a pH that can adjusted to a pH of about 7 by one of skill in the art. An acidic protease buffer with a pH of about 5, or a pH that can be adjusted to a pH of about 5 by one of skill in the art, can also be included in the kits described herein.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation of, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that, while specific reference to each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications can be made to materials used in the method or in the steps of the method, each and every combination and permutation of the method and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, if there is a variety of additional steps that can be performed in a method, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Thus, a number of aspects have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described, it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, all combination of disclosed agents, steps and characteristics are provided even in the absence of explicit disclosure herein.

Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, this includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as

approximations, by use of the antecedent about, it will be understood that the particular value is disclosed.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of the methods and materials provided herein.

Examples

Reagents Used

• Heparinase I from Flavobacterium heparin (Sigma-Aldrich H2519, Lot 048K37951) o Prepared as a 1 mIU/μΙ stock solution in 20 mM Tris-HCl pH 7.5 + 50 mM NaCl + 4 mM CaCl₂

• Heparin from Porcine Intestinal Mucosa (Sigma-Aldrich H4784, Lot 019K1487)

• Calcium Acetate (Sigma-Aldrich C4705 , Lot 119K0064)

o Prepared as 1 M stock solution in endotoxin-free water.

• Sodium Chloride (Fisher S641 -212)

o Prepared as 5 M stock solution in endotoxin-free water. • Sodium Acetate (Alfa Aesar A 13184)

o Prepared as 1 M stock solution in endotoxin-free water.

• Alcian Blue 8GX (Sigma- Aldrich A5268, Lot 119K1528)

• PyroGene® Recombinant Factor C Endotoxin Detection System (Lonza 50-658U)

• Hydrochloric Acid (Fisher A 144-500)

• Acetic Acid (EMD B 10001 -74)

• Lipopolysaccharide from E. coli 055 :B5 (List Biologies 203)

o Prepared as 500 EU/ml stock solution in endotoxin-free water.

EXPERIMENT 1

This experiment focused on the digestion of purified Heparin using Heparinase from Flavobacterium heparin in accord with Rice et al. (1987) Biochem. J. 244: 515-522. In summary, a sample of Heparin in a defined buffer was digested with Heparinase in a 30°C water bath for 24 hours. At various times samples were collected and stored. After digestion, Heparin digestion was monitored by measuring for the digestion product (an unsaturated, non-reducing, chromophore-containing end sugar) via UV absorbance at 232 nm and visualizing the digestion products using PAGE analysis.

Sample Preparation

8 different samples/controls were prepared:

1. Heparin Digest

a. 8 mg/ml Heparin in 0.25 M sodium acetate + 2.5 mM calcium acetate pH 7.0. b. 5 μΐ of 1 mIU/μΙ Heparinase was added.

c. Incubated in 30°C water bath for 24 hours.

2. Heparin Digest + Endotoxin

a. 8 mg/ml Heparin in 0.25 M sodium acetate + 2.5 mM calcium acetate pH 7.0. b. Endotoxin to a final content of 5 EU/ml was added.

c. 5 μΐ of 1 mIU/μΙ Heparinase was added.

d. Incubated in 30°C water bath for 24 hours.

3. Incubated LPS Control

a. 5 EU/ml endotoxin in 0.25 M sodium acetate + 2.5 mM calcium acetate pH 7.0. b. Incubated in 30°C water bath for 24 hours.

4. Stored LPS Control

a. 5 EU/ml endotoxin in 0.25 M sodium acetate + 2.5 mM calcium acetate pH 7.0. b. Stored at 4°C.

5. Amgen Tube® Sample a. 10 ml endotoxin-free water was added to BD Vacutainer tubes containing 143 U Heparin

b. A sample was taken and buffered to the equivalent conditions as Reaction 1. c. 747.5 μΐ Vacutainer Sample was added to 250 μΐ 1 M sodium acetate + 2.5 μΐ 1 M calcium acetate and brought to pH 7.0 with the addition of dilute HCl.

d. 5 μΐ of 1 mIU/μΙ Heparinase was added.

e. Incubated in 30°C water bath for 24 hours.

6. Amgen Tube® Sample + Endotoxin

a. 10 ml endotoxin-free water was added to BD Vacutainer tubes containing 143 U Heparin

d. Endotoxin to a final content of 5 EU/ml was added.

e. 5 μΐ of 1 mIU/μΙ Heparinase was added.

f. Incubated in 30°C water bath for 24 hours.

7. Heparinase + Endotoxin Control

a. 5 EU/ml endotoxin in 0.25 M sodium acetate + 2.5 mM calcium acetate pH 7.0. b. 5 μΐ of 1 mIU/μΙ Heparinase was added.

c. Incubated in 30°C water bath for 24 hours.

During the incubation, various samples were collected for UV monitoring. These samples were taken at 0, 1 , 2, 4, 8, and 24 hour time points. For each sample, 10 μΐ of each digest was added to 990 μΐ 0.03 M HCl, heated to 100°C for 10 minutes to inactivate the enzyme, and frozen at -20°C.

Samples were also collected for PAGE analysis and endotoxin detection. These samples were taken at 0, 1, 2, 4, 8, and 24 hour time points. For each sample, 40 μΐ of each digest was taken, heated to 100°C for 10 minutes to inactivate the enzyme, and frozen at -20°C.

Monitoring Digestion via Absorption at 232 nm

To monitor Heparinase activity, 100 μΐ samples were tested for absorbance at 232 nm. Samples 1 and 2 were tested in duplicate. Samples 5 and 6 were tested with single samples. All digests were measured at 0, 1, 2, 4, 8, and 24 time points.

These data are included as "Heparin Digestion Samples (Amgen)/UV232 Absorbance"

Sample Layout:

1 2 3 4 5 6

B Sample 1 -0 Sample 1 -0 Sample 2-0 Sample 2-0 Sample 5-0 Sample 6-0

C Sample 1 - 1 Sample 1 - 1 Sample 2- 1 Sample 2- 1 Sample 5- 1 Sample 6- 1

D Sample 1-2 Sample 1-2 Sample 2-2 Sample 2-2 Sample 5-2 Sample 6-2

E Sample 1-4 Sample 1-4 Sample 2-4 Sample 2-4 Sample 5-4 Sample 6-4

F Sample 1-8 Sample 1-8 Sample 2-8 Sample 2-8 Sample 5-8 Sample 6-8

G Sample 1 - 24 Sample 1 - 24 Sample 2 - 24 Sample 2 - 24 Sample 5 - 24 Sample 6 - 24

As per Rice et al, the 24 hour digest was assumed to be 100% digested. This number was then used to determine the "% Digestion" at each other time point. Experiments 5 and 6 contained a negligible amount of Heparin. This was not surprising given a total amount of 143 U in the tube which was resuspended in 10 ml giving a total concentration of approximately 14.3 U/ml. As contrast, Experiments 1 and 2 contained 8 mg/ml of Heparin containing approximately 140 U/mg giving a final Heparin concentration of 1120 U/ml - nearly 80 times higher than Experiments 5 and 6.

Experiments 1 and 2 gave the following results:

l Aperiment 2

ill n m Dili s*: ill nm Dili est

0.014 7.3 0.017 17.5

0.049 25.4 0.064 66.0

0.047 24.4 0.076 78.4

0.057 29.5 0.108 111.3

0.121 62.7 0.102 105.2

W 0.193 100.0 0.097 100.0

Experiment 1 proceeded as expected. Experiment 2 never reached the total absorbance value that Experiment 1 did. This indicates that the exogenous endotoxin may be interfering with the digestion. However, the digestion that does occur happens earlier in the time course in the presence of endotoxin.

Although endotoxin may slightly inhibit the Heparinase digestion, these results demonstrate that digestion of Heparin in these conditions is possible. Testing for Endotoxin in Heparin Digests

Various digestion samples were collected, diluted 1 : 10 in endotoxin-free water and tested in the Lonza PyroGene® assay per manufacturer's specifications. In addition to testing endotoxin in each sample, each sample was tested for the ability to recover a Positive Product Control (PPC). For this, 90 μΐ of the digestion sample was mixed with 10 μΐ of 10 EU/ml LPS and tested alongside the non-PPC sample.

This data is included as "LPS Detection in Heparin Digests/60' PyroGene Incubation" Sample Layout:

* This sample was diluted 1 :10 in 10 mM MgCl₂ + 100 mM NaCl instead of water

* * This sample was diluted 1 : 100 in water instead of 1 : 10

All Sample 1 and Sample 2 results show negligible PPC recovery indicating that there is still enough Heparin or digestion by-product to inhibit the assay. The various controls show that samples not containing Heparin have acceptable PPC's. PAGE Analysis of Heparin Digests

As per Rice et ah, digestion samples were analyzed with PAGE. In the Rice et al.

manuscript, 40 μΐ of a 50% sample were run in each well. Because of smaller well sizes in the gels run for these experiments, only 17 μΐ of each 50%> sample could be loaded. The gel was run in non-reducing conditions at 500 V, 80 mA in a 10-20% acrylamide gel. In the manuscript, gel time was estimated by the migration of bromophenol blue. The gel was stopped when the dye migrated about 75% through the gel. This took approximately 6 hours. In the experiments set forth herein, the dye migrated to this point in less than 1 hour.

After electrophoresis, the gel was stained with 0.5%> Alcian Blue 8GX prepared in 2% acetic acid. The gel was destained several times in 2% acetic acid.

No Heparin or digestion products were visualized in the PAGE gel.

Summary of Experiment 1

These results show that digestion with Heparinase is possible. It also shows that monitoring the digestion process can be followed with UV absorption with high Heparin levels. However, the level of Heparin in the BD Vacutainer tubes is too low to measure with UV absorption. In addition, it was not possible to follow digestion with PAGE analysis. Last, endotoxin detection with PyroGene was not possible with the high initial Heparin concentrations. This shows that even with significant Heparin digestion, the remaining Heparin or the digestion byproducts inhibit the assay.

EXPERIMENT 2

In these experiments, samples were taken from the BD Vacutainer tubes and buffered to conditions identical to those in Experiment 1. After digestion and treatment, the samples were diluted to various concentrations and conditions and tested for endotoxin with the PyroGene assay.

Sample Preparation

A single digestion was prepared:

• 750 μΐ BD Vacutainer Sample + 250 μΐ 1 M sodium acetate + 2.5 μΐ 1 M calcium chloride

• pH to 7.4 with 5 μΐ 0.1 M HC1 These conditions exactly match those in Experiment 1. It should be noted in the final analysis that this step results in a 25% dilution of the sample. To this mixture 5 mlU of Heparinase was added and incubated for 18 hours in a 30°C water bath. After digestion, the sample was heated to 100°C for 10 minutes and frozen at -20°C for storage. The sample was diluted to various concentrations in various conditions and tested in the PyroGene assay.

These data are included as "Amgen Tubes - Digest, Mg/NaCl/60' PyroGene Incubation"

Sample Layout:

Using the PPC Control as 100%, the "% Recovery" in each condition was calculated.

There were two acceptable PPC values. This experiment was repeated with a focus on dilution in 10 mM MgC^. It also contains a control reaction in which the Heparinase enzyme was added but was not allowed to incubate.

These data are included as "Amgen Post-Digestion Analysis/60' PyroGene Incubation"

Sample Layout:

* All MgCl₂ concentrations are 10 mM.

* All NaCl concentrations are 100 mM

The results are summarized below.

These results indicate that acceptable endotoxin detection is possible at a 1 : 100 dilution in MgCl₂ after digestion.

EXPERIMENT 3

In Experiment 2 it was found that almost identical PPC recoveries with or without incubation with Heparinase. Since the control samples only had a few minutes at RT for incubation before they were heat inactivated at 100°C, it was hypothesized that the effect may be a result of buffering and/or heating and not a function of Heparin digestion.

Sample Preparation

To test this, two samples were prepared from the BD Vacutainer tubes:

• Unbuffered Samples

o 750 μΐ sample + 250 μΐ water

• Buffered Samples

o 750 μΐ sample + 250 μΐ 1 M sodium acetate + 2.5 μΐ calcium acetate, pH 7.0.

Each of these samples was diluted 1 : 10 and 1 : 100 in each of the four conditions:

1. Water

2. 10 mM MgCl₂

3. Heparinase Buffer (0.25 M sodium acetate + 2.5 mM calcium acetate, pH 7.0)

4. Heparinase Buffer + 10 mM MgCl₂ These data are included as "Amgen Buffering Tests/60' PyroGene Incubation^'

Sample Layout:

* Shaded cells in columns 4-5 indicate Buffered samples.

The results are summarized below. The PPC control values were 1.280 and 1.150 for an average of 1.215 which was used to calculate % Recovery.

These results indicate that the results are due to Heparinase and not just due to buffer/cation conditions or the heat-inactivation step.

EXPERIMENT 4

The goal of this experiment was to confirm the results from Experiment 2 and include additional control reactions to distinguish exactly what is occurring in the samples.

Sample Preparation

For these experiments a single sample was prepared:

• 1500 μΐ BD Vacutainer Sample + 500 μΐ 1 M sodium acetate + 5 μΐ 1 M calcium acetate

• Brought pH to 7.0 with 10 μΐ 0.1 M HC1

This sample was then divided and treated as detailed:

1. No Enzyme

a. 500 μΐ of the sample was heated 100°C for 10 minutes and stored at -20°C.

2. No Incubation

a. 2.5 mlU Heparinase was added to 500 μΐ of the sample and mixed for a few

minutes at RT. The sample was then heated 100°C for 10 minutes and stored at - 20°C.

3. Full Digest

a. 2.5 mlU Heparinase was added to 500 100°C μΐ of the sample, mixed, and

incubated in a 30°C water bath for 18 hours. The sample was then heated 100°C for 10 minutes and stored at -20°C.

After all samples were completed, each was diluted 1 : 10 and 1 : 100 in both endotoxin- free water and 10 mM MgCb. The samples were tested with and without a PPC in the PyroGene

These data are included as "Amgen Digestion with Controls/60' PyroGene Incubation' Sample Layout:

1 2 3 4 5 6 7

1 EU/ml 10 mM Mg lO mM Mg

PPC Control + 1 EU/ml PPC

1 EU/ml lO mM Mg

PPC Control + 1 EU/ml PPC

1 EU/ml

PPC Control

The PPC values in water were 1.625, 1.805, and 1.645 for an average of 1.692. The PPC values in 10 mM MgCl₂ were 1.664 and 1.622 for an average of 1.663. The PPC in water average of 1.692 was utilized to determine % Recovery below.

The 'Full Digest' samples show 80-118% recovery showing that such recoveries are possible with just a 1 : 10 dilution. Taking into account the 25% dilution that takes place in the buffering step means that a final endotoxin detection of 0.125 EU/ml is possible in the BD Vacutainer tubes. Of course, a higher stock concentration of sodium acetate can be utilized to remove the 25%> dilution. Based on these results, it appears that Heparinase binds and neutralizes Heparin without digestion in the presence of Mg²⁺. It appears that the 1 : 10 dilution is an approximate threshold for relieving this inhibition. - In both experiment 2 and experiment 4, a further dilution to 1 : 100 relieves most all inhibition. Taking this further, 10 mM MgCl₂ appears to have a slight inhibitory effect on the PyroGene assay (10%) suggesting that a 90% PPC recovery may be the maximum expected value. If this is so, this new protocol is very close to a full, Mg²⁺-corrected recovery.

Taken together, these results show that, for example, digesting 14 U/ml Heparin samples with 5 mlU Heparinase in the conditions outlined give acceptable PyroGene results with modest dilution in water or 10 mM MgCl₂.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting endotoxin in a biological sample or medical device containing heparin comprising:

a) contacting the biological sample or medical device with heparinase; and b) detecting endotoxin.

2. The method of claim 1, wherein the biological sample is selected from the group

consisting of plasma, blood and serum.

3. The method of claim 1, wherein the medical device is selected from the group consisting of a tube, a catheter, a stent, a hemodialysis membrane and a filter.

4. The method of claim 1, further comprising contacting the biological sample or medical device with an acidic protease prior to step (a).

5. The method of claim 4, further comprising inactivating the acidic protease prior to step (a), but after contacting the biological sample or medical device with the active acidic protease.

6. The method of claim 4, wherein the acidic protease is pepsin.

7. The method of claim 6, wherein the pepsin contacting step is performed at a pH of less than 7.

8. The method of claim 6, wherein the pepsin contacting step is performed at a pH of less than 6.

9. The method of claim 6, wherein the pepsin contacting step is performed at a pH of about 5.

10. The method of any of claims 1-9, wherein the biological sample is selected from the group consisting of plasma, blood and serum.

11. The method of any of claims 1-10, wherein the heparinase contacting step is performed at a pH of about 7.

12. A kit for detecting endotoxin in a biological sample or medical device comprising:

a) heparinase; and

b) a heparinase buffer, wherein the buffer has a pH of about 7.

13. The kit of claim 12, further comprising:

c) an acidic protease; and

d) an acidic protease buffer, wherein the buffer has a pH of about 5.

14. The kit of claim 13, wherein the acidic protease is a pepsin.