WO2008009292A1

WO2008009292A1 - An in vitro method for detection of inflammatory contaminants

Info

Publication number: WO2008009292A1
Application number: PCT/DK2007/050101
Authority: WO
Inventors: Michael Timm; Erik Wind Hansen
Original assignee: Kobenhavns Universitet
Priority date: 2006-07-18
Filing date: 2007-07-18
Publication date: 2008-01-24

Abstract

A method for detection of one or more inflammatory contaminants in a sample, said method comprising the steps of : (i) exposing the sample to a cell derived from a myeloid-like cells in the presence of a reactive oxygen species (ROS) reporter probe, (ii) measuring the amount of ROS produced by said cells, and (iii) determining the presence of said sample by evaluation of the data obtained in step (ii).

Description

AN IN VITRO METHOD FOR DETECTION OF INFLAMMATORY CONTAMINANTS

FIELD OF INVENTION

The present invention relates to the field of biological assays. In particular, the present invention relates to an in vitro method for measuring the presence of inflammatory contaminants in a sample.

BACKGROUND

Certain chemical or biological compounds are capable of eliciting an inflammatory response. Such substances are known as inflammatory contaminants. When humans or other mammals are exposed to an inflammatory contaminant, a local and/or systemic inflammatory response can occur. A local response to an inflammatory contaminant is characterized by an activation of immune cells, normally leading to redness and swelling of surrounding tissue due to local vasodilation. The systemic response occurs when inflammatory contaminants are brought into contact with the circulatory system. Even low concentrations of inflammatory substances can result in septic shock characterized by loss of blood pressure, edema and high fever.

Inflammatory contaminants/substances/compounds include materials such as "pyrogens" or "pyrogenic" compounds which are substances capable of evoking a systemic inflammatory response characterized by fever due to the production of acute-phase proteins, IL-I, IL-6 and TNF-α. In connection with the present invention, the terms "inflammatory substances", "inflammatory compounds", "inflammatory contaminants", and "pyrogens" are used interchangeably. Microorganisms and substances originating from microorganisms are well-known pyrogens. Compounds that pose a particular risk of contamination by pyrogens include pharmaceutical products which can be inhaled, injected or infused, and medical devices such as membranes or implanted materials. Even nutrients can represent a risk of pyrogenicity. In addition to the pyrogenic nature of the product itself or by-products of its production, microbial contamination of the product can often cause pyrogenicity. This problem persists even if the product is "sterilized" by heat or chemical methods, as the main pyrogenic component of the microorganisms, endotoxin, (or cell wall lipopolysaccharide, LPS), lipoteichoic acid (LTA) and other microbial cell envelope components will remain after the microorganisms are killed. Other types of inflammatory contaminants include various organic and/or inorganic compounds that may induce inflammatory responses. Some inflammatory contaminants derived from air/water/soil samples produce local inflammatory responses of the respiratory tract upon inhalation or other local inflammatory responses.

In order to avoid a pyrogenic reaction and ensure the safety of any drug or pharmaceutical product administered parenterally, pyrogenic contamination must be monitored to identify individual lots that are contaminated with pyrogens.

Two pharmacopoeial methods, The Limulus Amebocyte Lysate (LAL) test and the Rabbit pyrogen test, are currently used routinely to monitor pyrogen contamination in mass-produced pharmaceutical products.

The rabbit pyrogen test is an in vivo test which consists of measuring the rise in body temperature evoked in rabbits by the intravenous injection of the substance to be examined. Although the rabbit pyrogen test is responsive to a wide spectrum of pyrogenic agents, including lipopolysaccharide (LPS), the rabbit test has a relatively high detection limit to LPS (100 pg LPS/ml) compared to other pyrogen tests (-0.5 pg LPS/ml for the LAL test). Also, on the other hand, the in vivo rabbit test is problematic. The immune response in terms of fever to a given stimulus varies considerably from species to species. It is an open question whether or not the rabbit is representative of other mammals, including humans, because the sensitivity to endotoxin is known to vary by a factor of 10,000 between various species. For pyrogenic components other than endotoxin, no such investigations are available. Experience however has shown that substances which have passed the rabbit pyrogen test, still can pose a potential risk of being pyrogenic in humans.

Bacterial endotoxin (lipopolysaccharide or LPS) is among one of the best described compounds causing fever. The compound originates from the bacterial envelope of Gram-negative bacteria. It was therefore thought to be generally useful to replace expensive and time-consuming rabbit experiments with a direct LAL-test for endotoxin. The LAL-test is a very sensitive in vitro test to endotoxin; however, it is insensitive to other pyrogens in relevant concentrations. Furthermore, it gives false negative results with certain products which can still stimulate monocytes to make pyrogenic cytokines. The LAL test is also altered by endotoxin binding components that are present in blood or blood components. Some of these endotoxin binding components bind to endotoxin and prevent it from being detected. These components may also affect the immune reaction with monocytes, i.e., the primary pyrogenic reaction. This interference is problematic, as testing for exogenous pyrogens in blood products is essential in order to ensure safe administration of these products in the clinical setting.

Yet another problem with LAL pyrogen tests is that they do not function properly in the presence of e.g. aluminium ions. As such constituents are usually present in vaccine formulations as a part of the adjuvant, there exist a need in the art for a reliable test system for assessing vaccine-safety.

Over the past years, several alternative assays to the Rabbit test and the LAL-test have emerged using various monocytic cell lines for in vitro detection of pyrogenic substances. Previous work based on pyrogenic challenge of the monocytic cell line Mono Mac 6 has shown to provide a sensitive and inexpensive assay for detection of various pyrogens e.g. lipopolysaccharide (LPS), lipoteichoic acid (LTA) and other bacterial components. However, the Mono Mac 6 assay has shown very low sensitivity towards fungi, yeast and endospores (Moesby et al., 2003; Moesby et al., 2000). The assay relies on the cytokine production of the monocytic cells subsequently measured in an immunoassay, consequently the time period to perform the assay normally exceeds 24 hours.

Studies have shown that LPS can be detected either in whole blood or by isolated neutrophils by utilization of the ROS production of polymorphonuclear leucocytes (PMN) as a detection platform. Utilization of human whole blood however requires repeated invasive procedures on donors since PMN's are relatively short lived. The assay is therefore unsuitable for pyrogen-testing of pharmaceuticals. Furthermore, the use of untreated human blood always represents a possible risk of disease transmittance, which one wishes to avoid.

The following documents describe in vitro methods for detection of pyrogens: US 6,696,261 describes a pyrogenicity test for use with automated immunoassay systems. The test is based on the use of monocytes characterized in that they release cytokines if exposed to pyrogens. The test sample is mixed with a monocyte-containing reagent and the amount of released cytokines (e.g. IL-6) is measured by anti-cytokine antibodies. The test is highly sensitive to LPS (same detection levels as the LAL-test) and lipooligosaccharride (LOS, from N. menigitis) endotoxin. However, there is no description or mentioning of that the test is capable of detecting pyrogens such as e.g. fungi, yeast and endospores.

US 5,891,728 describes a procedure for testing the pyrogenicity of materials, such as chemical and biological compounds based on the use of whole blood. The procedure makes use of the fact that leukocytes present in the blood release endogenous pyrogens, such as cytokines, colony stimulating factors and growth factors. The method comprises mixing the test sample with whole blood and measuring the amount of released endogenous pyrogens by antibodies directed to the endogenous pyrogen in question (e.g. IL-I, IL-6, TNF and prostaglandin E₂). The method is highly sensitive to endotoxin (e.g. 1 pg/ml), however there is no description or mentioning of detection of pyrogens such as e.g. fungi, yeast and endospores.

US 5,294,541 describes a method for continuously monitoring in real time the generation of ROS from in vitro interactions between cells, such as HL-60 cells and surfaces of material, such as those of medical devices destined for implantation.

US 2004/0053342 describes a method for determining the level of a pre-selected analyte in a sample including endotoxin and other analytes related to sepsis. The method comprises (i) incubating the test sample with an antibody specific to the analyte to form an immunocomplex, (ii) reacting the immunocomplex with an ROS-producing phagocytic cell, and (iii) measuring the amount of ROS produced by the phagocytic using chemiluminiscent or fluorogenic reagents. The preferred use of the method is measuring analytes in sample from a patient's body fluid, such as blood. However, other medical uses are suggested, e.g. a test for indicating if a particular sepsis-related antigen or cytokine is present at a level above a certain critical level. The analyte includes a wide variety of microorganisms, however, yeast cells and endospores are not mentioned. The phagocytic cell is preferably present in the sample, such as when the sample is a body fluid of a patient, however monocytees, lymphocytes or neutrophils cells such as e.g. HL-60 cells can be added to the sample. Detection limits of 20 pg/ml LPS are described.

Timm et al. 2006 describes an in vitro assay for detection of microorganisms and related substances utilizing HL-60 cells for chemiluminescence. The assay is based upon the production of reactive oxygen species (ROS) by differentiated HL-60 cells measured by luminol-enhanced chemiluminescence. The assay is capable of measuring the presence of a wide variety of microorganisms including yeast cells, however endospores are not mentioned. Several detection limits are described, e.g. LPS (100 pg/ml) and Gram-negative bacteria, S.typhimurium (10⁶ bac/ml).

As outlined above, there is an unmet need for a fast and highly sensitive in vitro method for detection of a broad range of pyrogens to replace the two existing pharmacopoeial methods, which have the drawbacks of being based on experimental animals, being time consuming (Rabbit pyrogen test) and only being sensitive to a few number of inflammatory contaminants and being limited by interference from certain substances (LAL-test).

SUMMARY OF THE INVENTION

Inflammatory contaminants are able to stimulate cells originating from the myeloid compartment that are capable of producing reactive oxygen (ROS) species such as e.g. cells derived from a pluripotent stem cell or polymorphonuclear leukocyte-like cells to produce and release these pyrogenic markers, such as superoxide (O₂ ^") and hydrogen peroxide (H₂O₂). The in vitro test of the present invention relies on the measurement of ROS as a marker for the presence of a pyrogen in a sample. In more details, the invention relates to a fast and highly sensitive method, capable of detecting a broad range of pyrogens in a sample, comprising (i) exposing the sample to be tested to cells characterized by producing reactive oxygen species (ROS) if exposed to a pyrogen, (ii) measuring the amount of reactive oxygen species (ROS) produced by said cells, and (iii) determining the presence of the pyrogen(s) in the sample by evaluation of the data obtained in step (ii).

Apart from detection of the presence of pyrogens such as bacteria and bacterial cell envelope components, the method is capable of detecting the presence of microbial cell envelope components, yeast cells, fungi and fungal spores which have proven complicated to detect in other in vitro pyrogen cell assays.

The positive detection concentrations of the method of the present invention are comparable or lower than the two existing pharmacopoeial methods. Table I below outlines the detection limits in the Rabbit pyrogen test and the LAL-test compared to the concentrations of positive detection of pyrogenic substances by the method of the present invention.

Table I Detection of pyrogens

ND: not detectable, NK: not known, i.e. no data available in the literature.

As seen from Table I, the method of the present invention is able to detect a wider range of pyrogens than the Rabbit pyrogen test and the LAL-test and importantly in concentrations that are comparable or lower than the detection limits of these applied tests.

Thus, the method of the present invention is a relevant alternative to the two existing pharmacopoeial methods, as a "stand alone" pyrogen test for testing products for human and animal use and/or environments relevant for humans for inflammatory contamination, and/or for the general presence of microbial contamination in samples such as e.g. test for sterility.

In a first aspect, the present invention thus relates to a method for detection of one or more inflammatory contaminants (such as e.g. a pyrogen) in a sample, said method comprising the steps of:

(i) exposing the sample to a cell derived from a myeloid-like cell (such as e.g. a PMN-like cell) in the presence of at least one reactive oxygen species (ROS) reporter probe, and preferably also in the presence of one or more components of the immune system,

(ii) measuring the amount of ROS produced by said cells, and

(iii) determining the presence of said one or more inflammatory contaminants in said sample by evaluation of the data obtained in step (ii).

In a second aspect, the present invention relates to a kit for detecting presence of one or more inflammatory contaminants (such as a pyrogen) in a sample, wherein the kit comprises:

(i) cells derived from myeloid cells,

(ii) one or more components of the immune system, and

(iii) at least one ROS reporter probe.

In a third aspect, the present invention relates to use of cells derived from myeloid-like cells, that have been preserved by a freezing process, for detecting presence of inflammatory compounds in a sample. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows ROS production of ATRA-differentiated HL-60 cells stimulated with zymosan in a luminol-enhanced chemiluminometric assay. The ROS production is illustrated as a function of time.

Figure 2 shows in the top graph the ROS production of zymosan-stimulated HL-60 cells differentiated with 1 μM ATRA for 4, 5, 6, 7, and 8 days, respectively. The bottom graph depicts the ROS production from non-stimulated HL-60 cells differentiated with 1 μM ATRA for 4, 5, 6, 7, and 8 days, respectively.

Figure 3 shows the ROS production of GM-CSF primed and non-primed cells quantified by luminol-enhanced chemiluminescence.

Figure 4 shows the effect of plasma in the assay buffer in respect to the stimulation with 100 μg/ml zymosan (top graph) and a non stimulated reference sample (bottom graph).

Figure 5 shows the effect of EDTA pre treatment of S. typhimurium at a concentration of 10⁴ bacteria/ml

Figure 6 shows the time frame for activation of HL-60 cells with S. typhimurium 10⁵ bacteria/ml.

Figure 7 shows the principle of qualitative determination of pyrogens in a sample by the method of the invention.

Figure 8 shows the ROS response of HL-60 cells measured by the method of the present invention to an environmental sample collected by air filtration in an outhouse.

Figure 9 shows ATRA differentiated HL-60 cells (5xlO⁵ cells/well) supplemented with 283 μM luminol and 2.5 % plasma. The volume is adjusted to 100 μl with HBSS and allowed to temperature equilibrate for 15 min at 37 ⁰C before addition of 100 μl test solution. 9. a. depict the subsequent light emission in RLL) plotted against time - - stimulation with 100 μl C. albicans 10⁶ yeasts/ml (mean ± SEM, n = 8). - - non-stimulated cells (mean ± SEM, n = 8). 9.b. depict the subsequent light emission in RLL) plotted against time - - stimulation with 100 μl LTA 10 ng/ml (mean ± SEM, n = 6). - - non-stimulated cells (mean ± SEM, n = 6). The results are displayed in relative luminescence units (RLU) and the results measured from O - 104 min after pyrogen stimulation.

Figure 10 shows ATRA differentiated HL-60 cells stimulated with S. typhimurium 10⁴-10⁶ bacteria/ml (lO.a.) or B. subtilis 10⁴-10⁶ bacteria/ml (lO.b). The results 5 display the AL)C of the RLL) values obtained from 0-104 min of 3 concentrations and one reference solution with non-stimulated cells. Each bar is the median value of eight experiments and the error bars indicate the 25^th and 75^th percentile. "*" indicate significant difference from the non-stimulated cells.

Figure 11 shows ATRA differentiated HL-60 cells stimulated with S. cerevisiae 10⁴- 10 10⁶ yeasts/ml (ll.a.) or C. albicans 10⁴-10⁶ yeasts/ml (11. b.). The results display the AL)C of the RLL) values obtained from 0-104 min of 3 concentrations and one reference solution with non-stimulated cells. Each bar is the median value of eight experiments and the error bars indicate the 25^th and 75^th percentile. "*" indicate significant difference from the non-stimulated cells.

15 Figure 12 shows ATRA differentiated HL-60 cells stimulated with LPS 10²-10⁴ pg/ml (12.a.) or LTA 10⁴-10⁶ pg/ml (12. b.). The results display the AUC of the RLU values obtained from 0-104 min of 3 concentrations and one reference solution with non-stimulated cells. Each bar is the median value of six experiments and the error bars indicate the 25^th and 75^th percentile. "*" indicate

20 significant difference from the non-stimulated cells.

Figure 13 shows ROS production of ATRA-differentiated HL-60 cells, which have been cryopreserved and which upon thawing have been stimulated with 10 ng/ml LPS and 100 ng/ml LPS, respectively. The ROS production is measured in a luminol-enhanced chemiluminometric assay and illustrated as a function of time.

25 Figure 14 shows ROS production of ATRA-differentiated HL-60 cells, which have been cryopreserved and which upon thawing have been stimulated with 100 μg/ml zymosan. The ROS production is measured in a luminol-enhanced chemiluminometric assay and illustrated as a function of time.

Figure 15 shows ROS production of ATRA-differentiated HL-60 cells, which have 30 been cultivated normally (not been cryopreserved) and ROS production of ATRA- differentiated HL-60 cells which have been cryopreserved. The cells have been stimulated with 100 μg/ml zymosan or HBSS buffer. The ROS production is measured in a luminol-enhanced chemiluminometric assay and illustrated as a function of time.

Figure 16 shows the dose dependent LPS detection of ATRA-differentiated HL-60 cells, which have been stimulated with 500 pg/ml, 100 pg/ml, 50 pg/ml, 25 pg/ml LPS and HBSS buffer (control).

Figure 17 shows the dose dependent LTA detection of ATRA-differentiated HL-60 cells, which have been stimulated with 100 μg/ml, 10 μg/ml, 1 μg/ml LTA and HBSS buffer (control).

Figure 18 shows the dose dependent peptidoglycan detection of ATRA- differentiated HL-60 cells, which have been stimulated with 10 μg/ml, 1 μg/ml, 500 ng/ml peptidoglycan and HBSS buffer (control).

Figure 19 shows the improved detection of LTA using antibodies raised against LTA in the HL-60 assay. The figure shows the response to HBSS buffer (control), LTA lng/mL, LTA lng/mL ÷antibody raised against LTA (1 : 50) preincubated 1 hr. at 37°C, and HBSS + antibody raised against LTA from S. aureus (1 : 50) preincubated 1 hr. at 37°C (control).

Figure 20 shows the response of ATRA-differentiated NB-4 cells which have been stimulated with 100 μg/ml zymosan or HBSS buffer (control).

Figure 21 shows the dose dependent LPS detection of ATRA-differentiated NB-4 cells, which have been stimulated with 10 ng/ml, 1 ng/ml, 100 pg/ml LPS and HBSS buffer (control).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to a method for detection of one or more inflammatory contaminants (such as e.g. a pyrogen) in a sample, said method comprising the steps of:

(iv) exposing the sample to cells derived from myeloid-like cells (such as e.g. a PMN like cell) preferably in the presence of one or more components of the immune system and at least one reactive oxygen species (ROS) reporter probe, (v) measuring the amount of ROS produced by said cells, and

(vi) determining the presence of said one or more inflammatory contaminants in said sample by evaluation of the data obtained in step (ii).

What is thus provided is a sensitive assay which can be used for assessing safety of various products. In addition, it appears from the Examples that the present invention can be used for quantitative and/or qualitative analysis of inflammatory compounds.

In a preferred embodiment, the immune components comprise freeze dried/lyophilized plasma components. In another embodiment, the cells are differentiated with one or more differentiating agents for a period of 2-12 days, preferably 7 days before exposing the sample to the cells.

In a particularly preferred embodiment, the cells have been preserved by a freezing process after differentiation of the cells. Preferably, the sample is exposed to the cells immediately after thawing of the cells.

In yet another preferred embodiment, the cells are pre-treated with one or more priming agents before exposure of the sample to the cells.

In a particularly preferred embodiment, the amount of ROS produced by the cells is measured by chemiluminescence or alternatively fluorescence.

Preferably, the sample is selected from the group consisting of: a pharmaceutical composition, an ingredient for a pharmaceutical composition, an infusion liquid, a biological material, and a parenteral nutrition. Alternatively, the sample is selected from the group consisting of air, soil and water.

In a second aspect, the present invention relates to a kit for detecting presence of one or more inflammatory compounds (such as e.g. a pyrogen) in a sample, wherein the kit comprises:

(iv) a cell derived from a myeloid cells, such as e.g. a PMN like cell,

(v) one or more components of the immune system, and (vi) at least one ROS reporter probe.

In a preferred embodiment, the kit comprises freeze dried plasma components. Freeze dried plasma components surprisingly result in an assay with improved sensitivity.

According to a particularly preferred embodiment, the kit according to the present invention comprises cryo preserved cells, said cells preferably being differentiated with one or more differentiating agents for a period of 2-12 days, preferably 7 days. It was otherwise expected that such cells, especially fully or partly differentiated myeloid like cells, would either not be able to survive freezing or they would not be able to function effectively in a pyrogen assay subsequent to freezing. However, the inventors have shown in the examples that the method according to the present invention works well with cells that have been preserved by freezing methods, thus opening the possibility for more efficient marketing, distribution and storage of test kits.

The kits according to the present invention furthermore comprise at least one chemiluminescent probe and/or at least one fluorescent probe.

In a final aspect, the present invention relates to use of cells derived from myeloid-like cells that have been preserved by a freezing process, for detecting the presence of inflammatory compounds in a sample.

The sample

The sample to be tested by the method of the present invention can be a gaseous, a liquid, a powdery and/or a particulate sample. Powdery and/or particulate samples typically comprise particles of a size of less than about 1 μm and up to about 5 mm in diameter. Samples according to the present invention may also be a biological material, such as e.g. cultured cells, tissues, organs, implantable biological material, etc. In a preferred embodiment, the sample is a liquid sample. In a further preferred embodiment, the sample is a gaseous sample.

The sample can be taken from all kinds of products wherein a determination of the presence of one or more pyrogens is required. In one embodiment of the invention, the sample is from a product for human or animal use. In a preferred embodiment, the product is a pharmaceutical composition, or an ingredient for a pharmaceutical composition, or an infusion liquid such as e.g. a peritoneal dialysing fluid, or a cosmetic product, or a nutrient product such as but not limited to a parenteral nutrition product, or materials for medical use. In another preferred embodiment, the sample is an environmental sample selected from air, soil or water. In a further preferred embodiment, the sample in an air sample.

Exposing the sample to the cells (step (i) of the method of the invention)

The sample to be tested for the presence of one or more pyrogens by the method of the present invention is, as a first step, exposed to cells characterized by producing reactive oxygen species (ROS) if exposed to a pyrogen. The sample can either be mixed directly with the cells or be dissolved or suspended in a liquid before mixture with the cells. The liquid for dilution of the sample is preferably a buffer or a cell medium which are compatible with the cells, i.e. the cells are able to survive and able to maintain the ability to produce ROS if subjected to a pyrogen challenge in the buffer or cell medium in the time period of ROS production. The time period of ROS production is defined as the period of time from exposing the sample to the cells of the invention to the final measurement of the amount of reactive oxygen species (ROS) produced by said cells upon exposure to the sample.

If ROS production is measured by chemiluminescent methods, it is preferred to perform a measurement that is continuous over a period of time since release of ROS results in essentially immediate stimulation of light production through interaction with chemiluminescent reporter probe molecules.

If ROS production is measured by fluorescent methods, it is possible to perform "end-point measurements" since the fluorescent products of interaction between ROS and fluorescent reporter probe molecules are accumulated in the sample. In theory, it is thus possible to store the sample for weeks, preferably in a cool place, before measuring the fluorescent signal in the sample. For practical reasons it preferred to perform the measurement within a week, and most preferably within a few hours.

In one embodiment, the length of the time period of ROS production is any interval from 15 minutes to 1 day. Preferably, 15-360 minutes, such as e.g. 30- 300 minutes, such as e.g. 60-270 minutes, such as e.g. 90-240 minutes, such as e.g. 120-210 minutes, such as e.g. 150-200 minutes, such as e.g. 170-190 minutes. This means that the measurement of ROS production is started at time zero (0 minutes), when the sample is e.g. mixed with the cells, and continued 15- 360 minutes, such as e.g. 30-300 minutes, such as e.g. 60-270 minutes, such as e.g. 90-240 minutes, such as e.g. 120-210 minutes, such as e.g. 150-200 minutes, such as e.g. 170-190 minutes. In another embodiment, the length of the time period of ROS production is about 15 minutes, or about 30 minutes, or about 60 minutes, or about 90 minutes, or about 120 minutes, or about 150 minutes, or about 180 minutes, or about 210 minutes, or about 240 minutes, or about 270 minutes, or about 300 minutes, or about 360 minutes, i.e. the measurement of ROS production is started at time zero (0 minutes), when the sample is e.g. mixed with the cells, and continued about 15 minutes, or about 30 minutes, or about 60 minutes, or about 90 minutes, or about 120 minutes, or about 150 minutes, or about 180 minutes, or about 210 minutes, or about 240 minutes, or about 270 minutes, or about 300 minutes, or about 360 minutes.

Examples of buffers or cell media which are most likely compatible with the cells of the invention are the standard cell culturing media such as: RPMI 1640 Media, Dulbecco's Modified Eagle Media, Iscove's Modified Dulbecco's Medium, and Leibovitz's L-15 Media and probably several of more specially prepared media. Furthermore, cells can for a limited time period (less than 4-6 hours) remain suspended in buffered saline solution such as HBSS, PBS, TRIS, or similar. Composition of assay buffers and media can influence the assay and variations hereof are obvious to a person skilled within the art.

When the sample is a gaseous sample, the pyrogens can be collected on a e.g. membrane filter. To desorb the pyrogens the filter is shaken with pyrogen-free cell media or buffer such as e.g. HBSS and the cell media or buffer is then tested by the method of the invention e.g. in the HL-60 assay. If the sample is a solid it can be dissolved or suspended in cell media or buffer such as e.g. HBSS and the cell media or buffer is tested by the method of the invention e.g. in the HL-60 assay.

In a preferred embodiment of the invention, the sample is a liquid or a solid sample and is exposed to cells originating from a HL-60 cell line. In a more preferred embodiment, the sample is a liquid or a solid sample and is exposed for 15-360 minutes to cells originating from a HL-60 cell line, such as e.g. 30-300 minutes, or such as e.g 60-270 minutes, or such as e.g. 90-240 minutes, or such as e.g. 120-210 minutes, or such as e.g. 150-200 minutes. The measurement of ROS production of the cells originating from a HL-60 cell line is started at time zero (0 minutes), when the sample is e.g. mixed with the cells, and continued for 15-360 minutes, such as e.g. 30-300 minutes, or such as e.g 60-270 minutes, or such as e.g. 90-240 minutes, or such as e.g. 120-210 minutes, or such as e.g. 150-200 minutes.

Cells of the invention

The cells of the invention are in the broadest aspect characterized by that they are able to produce reactive oxygen species (ROS) when challenged with a pyrogen.

The term "reactive oxygen species (ROS)" as used herein include oxygen ions, free radicals and peroxides both inorganic and organic, such as but not limited to superoxide (O₂ ^") and hydrogen peroxide (H₂O₂). ROS are generally very small molecules and are highly reactive due to the presence of unpaired valence shell electrons. ROS form as a natural by-product of the normal metabolism of oxygen. ROS are produced and released by various cells such as but not limited to polymorphonuclear leukocytes and other professional phagocytes during an inflammatory response.

The ROS are produced during the interaction of metabolism with oxygen by the NADPH-oxidase. The enzyme complex is formed by two membrane-bound components and at least three cytosolic components which in the non-activated cell are mainly intertwined in an autoinhibitory conformation. Upon activation of the cell by e.g. a microbial substance such as a pyrogen the cytosolic components are phosphorylated thus altering the conformation and associates with the membrane bound components. This will assemble the NADPH-oxidase and start a two electron reduction of molecular oxygen and thereby produce the superoxide anion (O₂") which can be either spontaneously or enzymatic converted into other reactive oxygen species as e.g. H₂O₂ or HOCI. The oxidative burst of the phagocyte can be delivered to both the interior of the cell (to phagolysosomes) or to the exterior environment. Preferred cells according to the present invention are "myeloid-like cells". A "myeloid like cell" or a "cell derived from a myeloid like cell" is any cell originating from the myeloid compartment with an NADPH oxidase system capable of generating ROS. It is believed that the requirements for such cell lies in the combination of presence of appropriate surface receptors in combination with a functional NADPH oxidase system capable of generating ROS. Myeloid-like cells include myeloid progenitor cells, neutrophils, eosinophils, basophils, mast cell precursors, mast cells, monocytees, macrophages, as well as cell lines derived from such cells. Myeloid cells also comprise any such corresponding cell from a different animal, preferably a mammal.

In one embodiment of the present invention, the cells are derived from a pluripotent stem cell. A pluripotent stem cell is a cell that can differentiate into most cell types, i.e. into any of the three primary tissue types: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). The endoderm is the innermost layer of the embryo. The mesoderm is in between, and the ectoderm is outermost. Pluripotent stem cells can eventually specialize in any bodily tissue.

In another embodiment of the invention, the cells are polymorphonuclear (PMN) leukocytes, and/or "PMN-like cells" expressing e.g. one or more PMN-specific surface-receptors. In the present invention the terms "PMN cells" and "PMN-like cells" are used interchangeably. Cells derived from a myeloid like cell according to the present invention may in particular be a cell with the capability of being differentiated into a PMN-like cell, such as e.g. a HL-60 cell or a NB-4 cell. In order to function efficiently, this differentiation process usually requires stimulation by differentiation agents.

Polymorphonuclear leukocytes (PMN leukocytes) are granulocytes including neutrophil, eosinophil and basophil granulocytes, which are a category of leukocytes characterised by the presence of granules in their cytoplasm. The term "polymorphonuclear" refers to the varying shapes of the nucleus, which is usually lobed into three segments. PMN leukocytes or granulocytes are characterized by the capability to produce and release reactive oxygen species (ROS) upon exposure to pyrogens. In a preferred embodiment, the cells are polymorphonuclear leukocyte-like cells. Polymorphonuclear leukocyte-like cells are cells that resemble granulocytes functionally in that they are able to produce and release reactive oxygen species (ROS) upon stimulation with a substance such as e.g. a pyrogen or an inflammatory substance. Polymorphonuclear leukocyte-like cells can be cells either derived from the human body or a cell line.

In another preferred embodiment, the cells are derived from a cell line. In a more preferred embodiment the cell line is selected from the group consisting of NB-4, THP-I, KG-I, K562, KCL22, PLB-985, U937, Mono Mac 6, X-CDG, PL-21, ML-I, ML-3, MHH-225, AML-193, HL-60 and variants thereof. The cell lines are either the wild type or a variant of mutated/transfected cell types. In the most preferred embodiment, the cells are derived from a cell line selected from the group consisting of NB-4 and HL-60 and variants thereof. The NB-4 or the HL-60 cell line is either the wild type or a variant of mutated/transfected cell types.

In a specific preferred embodiment, the cells are derived from the HL-60 cell line or variants thereof. The term "HL-60 cell line" as used herein refers to a human promyelocytic leukaemia cell line (ATCC, CCL-240). The HL-60 cell line was established in 1977 from a patient with acute myeloid leukaemia. The cells largely resemble promyelocytes but can be induced to differentiate terminally in vitro. Some reagents cause HL-60 cells to differentiate to granulocyte-like cells, others to monocyte/macrophage-like cells. The cells exhibit phagocytic activity and responsiveness to chemotactic stimuli.

In a more preferred embodiment of the present invention the cell line is the HL-60 cell line, the wild type obtained from ATCC (ATCC CCL-240), whose stock was obtained at passage 8 and is distributed at passage 21.

By using a cell line such as the HL-60 cell line, more reproducible results can be obtained compared with the use of primary isolated human cells or whole blood which always will be bias related since they will reflect the physiological state of the donor. Furthermore, the HL-60 cells represent a well-tested and investigated cell line easily differentiated to a granulocyte-like cell characterized by the capability of producing ROS.

Amount of cells The cells of the invention are either attached to a solid surface or suspended in the assay buffer of the method of the invention. The "assay buffer" is defined as a liquid comprising the components of the method of the invention. The assay buffer can be any buffer or cell media which are compatible with the cells of the invention, as outlined above. Examples of an assay buffer are provided hereinabove.

The components of the invention are defined as the sample, the cells and ingredients essential for the method of the invention. Ingredients essential for the invention are evident from the present description and examples.

The total volume of the components of the method of the invention and the assay buffer constitute the "assay volume".

In the broadest aspect of the present invention the minimum amount of cells or concentration of cells required to conduct the method of the present invention is the amount of cells or concentration of cells that show significant ROS production when stimulated with a pyrogen or an inflammatory substance.

In one preferred embodiment of the invention the cell concentration is about 10² cells/ml of the assay volume, or more preferably about 10³ cells/ml of the assay volume, or more preferably about 10⁴ cells/ml of the assay volume, or more preferably about 10⁵ cells/ml of the assay volume, or more preferably about 10⁶ cells/ml of the assay volume, or more preferably about 10⁷ cells/ml of the assay volume, or more preferably about 10⁸ cells/ml of the assay volume, or more preferably about 10⁹ cells/ml of the assay volume. In another embodiment, the cell concentration is 10²-10⁹ cells/ml of the assay volume, such as e.g. 10³-10⁸ cells/ml of the assay volume, or such as e.g. 10⁴-10⁷ cells/ml of the assay volume, or such as e.g 10⁵-10⁷ cells/ml of the assay volume, or such as e.g. 10⁶- 10⁷ cells/ml of the assay volume.

In a preferred embodiment, the cell concentration is about 10⁶ cells/ml of the assay volume, or the cell concentration is about 1.5xlO⁶ cells/ml of the assay volume, or the cell concentration is about 2xlO⁶ cells/ml of the assay volume, or the cell concentration is about 2.5xlO⁶ cells/ml of the assay volume, or the cell concentration is about 3xlO⁶ cells/ml of the assay volume, or the cell concentration is about 3.5xlO⁶ cells/ml of the assay volume, or the cell concentration is about 4xlO⁶ cells/ml of the assay volume, or the cell concentration is about 4.5xlO⁶ cells/ml of the assay volume, or the cell concentration is about 5xlO⁶ cells/ml of the assay volume. In a specific preferred embodiment of the invention, the cell concentration is about 2.5xlO⁶ cells/ml of the assay volume.

In another preferred embodiment, the cells are originating from a HL-60 cell line and the concentration of HL-60 cells is about 10⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 1.5xlO⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 2xlO⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 2.5xlO⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 3xlO⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 3.5xlO⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 4xlO⁶ cells/ml, or the concentration of HL-60 cells is about 4.5xlO⁶ cells/ml of the assay volume, or the concentration of HL-60 cells is about 5xlO⁶ cells/ml of the assay volume. In a more preferred embodiment, the cells are originating from a HL-60 cell line and the concentration of HL-60 cells is about 2.5xlO⁶ cells/ml of the assay volume.

In the experiment described in Example 1 and illustrated in Figure 1 the graphs depict the ROS production of HL-60 cells either stimulated or non-stimulated with the yeast cell wall component, zymosan. The amount of HL-60 cells is 2.500.000 cells/ml of the assay volume.

In general, the amount of cells seems to correlate directly with the ROS production of the cells, given that the higher number of cells the more pronounced oxidative burst. However, it is possible that another amount/concentration of cells (being either higher or lower) is preferable when detecting a pyrogen in concentrations close to the detection limit of the assay.

Differentiating agents

In order to increase the ability of the cells used in the method of the invention to produce and/or release ROS, the cells can be differentiated with one or more differentiating agents prior to exposing the sample to the cells. Thus, in one embodiment, the invention relates to a method further comprising differentiation of the cells with one or more differentiating agents capable of increasing the ability of the cells to produce and/or release ROS when stimulated/exposed to an inflammatory contaminant.

In the broadest sense differentiating agents are substances capable of increasing the ability of the cells of the invention to produce and/or release ROS upon stimulation with a pyrogen or an inflammatory substance. Preferably, differentiating agents result in morphological changes of the cells of the invention such as an altered receptor expression and/or an increase of certain proteins such as for instance components of the NADPH-oxidase.

In order to achieve differentiation of the cells of the invention, the cells are incubated in the presence of one or more differentiating agents for a given period of time, defined as the differentiating period, described below.

In a preferred embodiment of the present invention, the one or more differentiating agents are selected from the group consisting of:

- Retinoids, defined as a collectively notation for both natural forms and synthetic analogues of vitamin A, such as, but not limited to all trans retinoic acid (ATRA) or 9-cis retinoic acid,

- polar solvents, such as but not limited to Dimethylformamide (DMF) or Dimethylsulphoxide (DMSO),

- Dibutyryl cyclic AMP,

- Actinomycin D, Hypoxanthine, Antithymocyte globulin, Tunicamycin, 6- thioguanine, L-ethionine,

- or a mixture thereof.

All differentiating agents can be used either alone or in combination.

The term "ATRA" as used herein refers to all-trans retinoic acid which is a form of vitamin A. It is known that ATRA is capable of diffentiating HL-60 cells to cells that functionally and morphologically resemble mature granulocytes.

In one embodiment of the present invention, the cells are differentiated with one or more differentiating agents capable of increasing the ability of the cells to produce ROS. Preferably, the differentiating agents are selected from the group consisting of all trans retinoic acid (ATRA), 9-cis retinoic acid, dimethylformamide (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP, Actinomycin D, hypoxanthine, antithymocyte globulin, tunicamycin, 6-thioguanidine, L-ethionine and combinations thereof. More preferably, the differentiating agents are selected from the group consisting of all trans retinoic acid (ATRA), 9-cis retinoic acid, dimethylformamide (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP and combinations thereof.

In another preferred embodiment of the present invention, differentiating agents of the invention include one or more substances such as but not limited to, vitamin D3, granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), IFN-γ, TNF-α, thalidomide and its metabolites, magnolol, honokiol, caffeic acid, auranofin. The one or more substances can be used either alone or in combination with the differentiating agents: all trans retinoic acid (ATRA), 9-cis retinoic acid, dimethylformamide (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP, Actinomycin D, hypoxanthine, antithymocyte globulin, tunicamycin, 6-thioguanidine or L- ethionine.

In a specific preferred embodiment of the present invention the differentiating agent is all trans retinoic acid (ATRA).

To differentiate the cells of the invention, the one or more differentiating agents are added to the buffer or cell media, wherein the cells are maintained and/or grown, for a given period of time, defined as the differentiating period, defined below.

In one embodiment, the concentration of differentiating agent used to differentiate the cells of the invention is 10 nM-100 μM in the buffer or cell media, wherein the cells are maintained and/or grown, such as e.g. 20 nM-90 μM, or such as e.g. 30 nM-80 μM, or such as e.g. 40 nM-70 μM, or such as e.g. 50 nM- 60 μM, or such as e.g. 60 nM-50 μM, or such as e.g. 70 nM-40 μM, or such as e.g. 80 nM-30 μM, or such as e.g. 90 nM-20 μM, or such as e.g. 100 nM-10 μM, or such as e.g. 500 nM-5 μM.

In another embodiment, the concentration of differentiating agent used to differentiate the cells of the invention is about 10 nM in the buffer or cell media, wherein the cells are maintained and/or grown, or about 20 nM, or about 30 nM, or about 40 nM, or about 50 nM, or about 60 nM, or about 70 nM, or about 80 nM, or about 90 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or about 400 nM, or about 450 nM, or 5 about 500 nM, about 550 nm, or about 600 nM, or about 650 nM, or about 700 nM, or about 750 nM, or about 800 nM, or about 850 nM, or about 900 nM, or about 950 nM, or about 1 μM, or about 2 μM, or about 3 μM, or about 4 μM, or about 5 μM, or about 6 μM, or about 7 μM, or about 8 μM, or about 9 μM, or about 10 μM, or about 20 μM, or about 30 μM, or about 40 μM, or about 50 μM, or about 10 60 μM, or about 70 μM, or about 80 μM, or about 90 μM, or about 100 μM. In a preferred embodiment, the concentration of the differentiating agent is about 1 μM.

In a more preferred embodiment, the differentiating agent is all trans retinoic acid (ATRA) which is used in a concentration of 10 nM-100 μM in the buffer or cell 15 media, wherein the cells are maintained and/or grown, such as e.g. 20 nM-90 μM, or such as e.g. 30 nM-80 μM, or such as e.g. 40 nM-70 μM, or such as e.g. 50 nM-60 μM, or such as e.g. 60 nM-50 μM, or such as e.g. 70 nM-40 μM, or such as e.g. 80 nM-30 μM, or such as e.g. 90 nM-20 μM, or such as e.g. 100 nM-10 μM, or such as e.g. 500 nM-5 μM.

20 In a most preferred embodiment, the differentiating agent is all trans retinoic acid (ATRA) which is used in a concentration of about 10 nM in the buffer or cell media, wherein the cells are maintained and/or grown, or about 20 nM, or about 30 nM, or about 40 nM, or about 50 nM, or about 60 nM, or about 70 nM, or about 80 nM, or about 90 nM, or about 100 nM, or about 150 nM, or about 200 nM, or

25 about 250 nM, or about 300 nM, or about 350 nM, or about 400 nM, or about 450 nM, or about 500 nM, about 550 nm, or about 600 nM, or about 650 nM, or about 700 nM, or about 750 nM, or about 800 nM, or about 850 nM, or about 900 nM, or about 950 nM, or about 1 μM, or about 2 μM, or about 3 μM, or about 4 μM, or about 5 μM, or about 6 μM, or about 7 μM, or about 8 μM, or about 9 μM, or about

30 10 μM, or about 20 μM, or about 30 μM, or about 40 μM, or about 50 μM, or about 60 μM, or about 70 μM, or about 80 μM, or about 90 μM, or about 100 μM. In a preferred embodiment, the concentration of ATRA is about 1 μM. Furthermore, environmental factors such as pH of the buffer used in the method of the invention and multiple other chemical inducers can facilitate the differentiation of cells, however modifications and variations of environmental factors are obvious to a person skilled within the art.

The capability of HL-60 cells to produce a robust oxidative burst (production and release of ROS) upon pyrogen challenge is greatly improved when the HL-60 cells are differentiated into granulocyte-like cells by one or more differentiating agents. Thus, in one embodiment of the invention, the cells are originating from a HL-60 cell line and are differentiated by one or more differentiating agents, as described above. Preferably, the cells are originating from a HL-60 cell line and differentiated with a differentiating agent selected from the group consisting of all trans retinoic acid (ATRA), 9-cis retinoic acid, 13-cis retinoic acid, 9,13-di-cis retinoic acid, dimethylformamide (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP. More preferably, the cells are originating from a HL-60 cell line and differentiated with all trans retinoic acid (ATRA).

Differentiation period

As described above, in order to achieve differentiation of the cells used in the method of the invention, the cells are incubated in the presence of one or more differentiating agents for a given period of time, defined as the differentiating period.

In the broadest sense, the minimum differentiation period is the time that yields cells with a ROS production greater than non-differentiated cells when stimulated with a pyrogen.

In a preferred embodiment of the present invention the differentiation period is the amount of time that provides the cells used in the method of the invention most proficient to produce ROS when stimulated. If the cells used in the method of the invention require differentiation to achieve optimal ROS production when stimulated, the differentiation period is of great importance. The explanation most likely is that the amount of receptors recognizing various microbial cell wall components and/or NADPH oxidase components are up regulated during the differentiation period. The optimal amount of time the cells should be differentiated (and perhaps co-differentiated) is highly relevant for the invention. In one embodiment, the cells are differentiated with one or more differentiating agents for a period of 2-12 days before exposing the sample to the cells. Preferably, the cells are differentiated with one or more differentiating agents for a period of 4-10 days before exposing the sample to the cells. More preferably, the cells are differentiated with one or more differentiating agents for a period of 6-8 days before exposing the sample to the cells. Most preferably the cells are differentiated with one or more differentiating agents for 7 days before exposing the sample to the cells.

In a preferred embodiment, the cells are differentiated with ATRA for a period of 2-12 days before exposing the sample to the cells. Preferably, the cells are differentiated with ATRA for a period of 4-10 days before exposing the sample to the cells. More preferably, the cells are differentiated with ATRA for a period of 6- 8 days before exposing the sample to the cells. Most preferably the cells are differentiated with ATRA for 7 days before exposing the sample to the cells.

It has been shown that ATRA-differentiation of the cell lines HL-60 and NB-4 is optimal after 3-4 days (Fleck et al., 2003) and that viability generally decreases after 3-4 days of culturing (Collins et al. 1990). However, the present inventors have shown that differentiation of HL-60 cells with ATRA for more than 4 days such as up to 10 days under optimal assay conditions results in a higher ROS production and/or ROS release when stimulated with a pyrogen or inflammatory substance, cf. Example 2.

Figure 2 illustrates ROS production from HL-60 cells differentiated with ATRA for 4-8 days. The top graph in Figure 2 depicts the ROS production from zymosan- stimulated HL-60 cells differentiated with 1 μM ATRA for 4, 5, 6, 7, and 8 days respectively, and the lower graph in Figure 2 depicts the ROS production from non-stimulated HL-60 cells differentiated with 1 μM ATRA for 4, 5, 6, 7, and 8 days, respectively.

Thus, in a preferred embodiment of the invention, the cells are originating from a HL-60 cell line and are differentiated with ATRA for a period of 4-10 days before exposing the sample to the cells. More preferably, the cells are originating from a HL-60 cell line and are differentiated with ATRA for a period of 6-8 days before exposing the sample to the cells. Most preferably, the cells are originating from a HL-60 cell line and are differentiated with ATRA for 7 days of the sample to the cells. However, the HL-60 cells can also be differentiated in a shorter period of time such as e.g. 2 days or 3 days.

Priming agents

In addition to differentiation of the cells as described above leading to an 5 increased ROS production and/or ROS release, priming of the cells can also lead to an increased ROS productions upon stimulation with a pyrogen or inflammatory substance.

In the broadest sense, priming of the cells of the invention is pre-treatment of cells with a substance in order to achieve increased responses to activating 10 stimuli. In the literature, priming is believed to render the cells more responsive to subsequent stimulation due to one or several events leading to:

• Increased kinase activity and/or

• Increases of intracellular calcium and/or

• Partial phosphorylation of NADPH oxidase components and/or

15 • Alterations of Phospholipid-dependent signal transduction

Thus, in another aspect the invention relates to a method further comprising pre- treating the cells with one or more priming agents before exposing the sample to the cells.

The term "priming agent" as used herein refers to a substance capable of 20 increasing the ability of the cells of the invention to produce and/or release ROS upon stimulation with a pyrogen or an inflammatory substance by rendering the cells more responsive to stimulation due to one or more cellular events leading to one or more cellular changes as e.g. outlined above. Preferably a priming agent is a substance which upon incubation with the cells of the invention for a period of 25 from >0-24 hours is capable of increasing the ability of the cells to produce and/or release ROS upon stimulation with a pyrogen or an inflammatory substance. The time period of incubation with the priming agent is >0-24 hours such as e.g. about 1 minute, or about 2 minutes, or about 3 minutes, or about 4 minutes, or about 5 minutes, or about 10 minutes, or about 15 minutes, or about 30 20 minutes, or about 30 minutes, or about 40 minutes, or about 50 minutes, or about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 5 hours, or about 6 hours, or about 7 hours, or about 8 hours, or about 9 hours, or about 10 hours, or about 11 hours, or about 12 hours, or about 13 hours, or about 14 hours, or about 15 hours, or about 16 hours, or about 17 hours, or about 18 hours, or about 19 hours, or about 20 hours, or about 21 hours, or about 22 hours, or about 23 hours, or about 24 hours. The length of the incubation period is depended on the priming agent used.

Priming can be achieved with a widely diverse group of substances comprising of, but not limited to cytokines as defined below and as for instance, but not limited to, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-IO, IL-Il, IL-12, IL-13, IL-15, TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ, GM-CSF, G-CSF, M-CSF, chemokines as defined below and as for instance, but not limited to IL-8, NAP-2, plasma proteins as defined below or peptides, as for instance, but not limited to PAF, IgG, HGF, "nerve growth factor", FAS, CD40, Substance P, complement factors as defined below and as for instance, but not limited to, C5a, microbial substances as for instance, but not limited to LPS, fMLP etc.

The term "Cytokine" as used herein refers to a low molecular weight protein that is produced by a wide variety of haemopoietic and non-haemopoietic cell types, and which is critical to the function of both innate and adaptive immune responses. Cytokines play a critical role in the development and function of the immune system, as well as in a variety of immunological, inflammatory and infectious diseases. Cytokines have been variously named as lymphokines, interleukins and chemokines, based on their presumed function, and their cell of secretion or target of action. The term interleukin was initially used by researchers for those cytokines whose presumed targets are principally leukocytes. The term chemokine referred to a specific class of cytokines that mediated chemoattraction (chemotaxis) between cells. The latter term alone has been retained (see below); interleukins are now used largely for designation of newer cytokine molecules discovered every day, and have little significance attached to their presumed function. Of note, IL-8 (interleukin-8) is the only chemokine originally named an interleukin. Cytokines have now been classified into four different types based on structural homology, which has been partly able to separate cytokines that do not demonstrate a considerable degree of redundancy.

1) Four α-helix bundle family, the three dimensional structures of whose members have four bundles of α-helices. This family in turn is divided into three sub-families, the IL-2 subfamily, the interferon (IFN) subfamily and the IL-IO subfamily. The first of these three subfamilies is the largest, and contains several non-immunological cytokines including erythropoietin(EPO) and thrombopoietin (THPO). Alternatively four helix bundle cytokines can be grouped into long chain and short chain cytokines.

2) IL-I family, which primarily includes IL-I, IL-16, TGFβ, IL-18 and IL-25.

3) IL-17 family, which consists of related molecules and are listed alphabetically IL-17A, IL-17B, IL-17C, IL17D, IL-17E and IL-17F. IL-17 is a proinflammatory cytokine secreted by activated T cells.

4) Chemokines.

A more clinically and experimentally useful classification divides immunological cytokines into those that promote the proliferation and functioning of helper T- cells type 1 (example, IL-2, INF-γ etc.) and helper T-cells type 2 (IL-4, IL-IO, IL- 13, TGF-β etc.), respectively. It is remarkable that the cytokines that belong to one of these sub-sets tend to inhibit the effects of their counterparts - a tendency under intensive study for their possible role in the pathogenesis of autoimmune disorders. Specific examples of cytokines are GM-CSF, IFN-α, IFN-β, IFN-γ, IFN-λ, TNF-α, IL-6, IL-lβ, IL-8 and IL-18.

Thus, in one embodiment of the invention, the one or more priming agents are cytokines, as defined above. In a preferred embodiment, the one or more cytokines are selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-9, IL-IO, IL-Il, IL-12, IL-13, IL-15, TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ, HGF, NGF, IGF, TGF, GM-CSF, G-CSF and M-CSF.

In a second embodiment of the invention, the one or more priming agents are chemokines selected from the group consisting of IL-8 and NAP-2. In a third embodiment of the invention, the one or more priming agents are plasma proteins or peptides selected from the group consisting of PAF, IgG, FAS, CD40 and Substance P.

In a further embodiment of the invention, the one or more priming agents are complement factors, such as but not limited to C5a.

In yet a further embodiment of the invention, the one or more priming agents are microbial substances. In a preferred embodiment, the microbial substances are selected from the group consisting of LPS and fMLP.

The primers can be sub-divided into to groups: "primers" and "dedicated primers" the term primers when used herein refers to substances that will both prime and activate the cells, and the term "dedicated primers" when used herein refers to substances that will prime without activation of the cells. In a preferred embodiment of the present invention a dedicated primer is used to prime the cells. The dedicated primers are substances such as, but not limited to TNF-α, GM-CSF, G-CSF and HGF.

In Example 3 and in Figure 3 the ROS production (quantified as luminol enhanced chemiluminescence) of GM-CSF primed and non-primed cells are shown. As seen the priming of the cells almost doubles the peak height of the ROS response with only a small increase of the response of the non-zymosan stimulated control cells (data not shown). Thus, in a specific preferred embodiment of the invention the one or more priming agents are GM-CSF and/or G-CSF used either alone or in combination.

The concentration of the chosen primers and the optimal priming time can vary from primer to primer, however most primers seem effective in sub-nano to nano- molar or micro-molar concentrations, and with priming times varying from a few minutes to 24 hours.

Preservation of cells of the invention

Laboratory cell preservation and storage of cells and cell lines are well-known in the art. Freezing the cells and thawing the cells prior to use is not uncommon, but the viability and functionality of the cells after this process is generally severely affected . The present inventors have found that it is possible to preserve the cells of the invention after the cells have been differentiated, e.g. by cryopreservation or lyophilisation, and retain the functional activity of the cells after reconstitution of the cells prior to applying the cells in the method of the invention.

The cells of the invention can be preserved after differentiation of the cells according to methods known in the art, e.g. cryopreservation, lyophilization etc. Prior to exposing the sample to the cells, the cells are then reconstituted (thawed) and prepared for application in the method of the invention.

Preservation by freezing/Cryopreservation The prior art teaches that ROS production is severely damaged in PMN cells that have been subject to freezing (see e.g. Timm et al., page 253, column 1, lines 26- 30 as well as Yamashita (Cryobiology 17, 112-119, 1980 , table 6)). The present inventors have found that it is possible to preserve the cells of the invention by cryopreservation after differentiation of the cells. The cryopreservation methods employed by the inventors are not deleterious to the cells, and permits reconstitution of the cryopreserved cells to form cells which possess from 10- 100% of the level of functional activity compared to identical cells, which have not been cryopreserved. In some cases the level of functional activity of the reconstituted cryopreserved cells can be even higher compared to the level of functional activity of identical cells, which have not been cryopreserved. According to the present invention, the cells may be preserved at any time during the differentiation period, or even before initiating differentiation. However, in connection with kits according to the present invention, the cells are preferably ready to use and do not require further differentiation. The inventors have furthermore discovered that freezing of cells at -190 ⁰C instead of -80 ⁰C increases storage time without negatively affecting the sensitivity of the cells (results not shown).

The term "identical cells" as used herein refers to cells that are treated exactly the same way as the cryopreserved cells, i.e. with respect to differentiation, priming, maintenance of the cells etc. The only difference between the cryopreserved cells and the "identical cells" is that the cryopreserved cells have been exposed to cryopreservation. The cells are cryopreserved by applying methods known in the art e.g. as described in Zerbe et al., J. Vet. Med A 50, 179-184 (2003); Schindler el al. J Immunol Methods. 2004 Nov;294(l-2):89-100; Malawista S. E. et al. J. Clin. Invest. VoI 83, February 1989, 728-732;Takahashi et al. Journal of Immunology, 5 vol 134, no.6, junw 1985; Malawista S. E. et al. Cell motility and the cytoskeleton 63:254-257 (2006); Rowley, J.Hematotherapy 1, 233 (1992); Trickett et al., J.AIDS and Hum. Retro., 17, 129 (1998); Areman et al., Transfusion, 28, 151 (1988) Bone marrow and stem cell precessing: A manual of current techniques, edited by Areman et al. F.A. Davis Company (1992); US 5,759,764, WO 10 97/35472, WO 2006/052835, US 7,112,576

Zerbe et al. (2003) describes cryopreservation of polymorphonuclear neutrophil granulocytes (PMN). The PMN cells were suspended in a cryoprotective solution (equine plasma with 5% (v/v) dimethylsulphoxide (DMSO)) and frozen in liquid nitrogen. A temperature gradient with low cooling velocity (l°C/min between 4 15 and -70⁰C) resulted in highest numbers of viable cells after thawing. Zerbe et al. (2993) is hereby incorporated by reference.

Schindler et al (2004) describes a cryoprotective solution containing 10 %v/v DMSO. The cryoprotective solution containing the cells were transferred into pre- cooled cryotubes and put into the rack of a programmable freezer with a TP type

20 nitrogen container (Nicoll Plus PC, Air Liquide, Marne-la-Valle'e Cedex 3, France), precooled to 4°C. A temperature probe was inserted into an extra aliquot containing the same volume of cell suspension to follow the freezing process. The freezing program was started 5 min. after closing the freezer. The cell suspension was cooled down to -5°C at a rate of l°C/min. In order to compensate for the

25 latent heat of fusion generated by the cell suspension when changing from the liquid to the solid state. The temperature Tx in the freezing chamber was set to - 30⁰C. The crystallization temperature was -12°C. When this temperature was reached, the cell suspension was cooled down to -40⁰C at a rate of 2°C/min. The cell suspension was given 120 seconds to stabilize before being cooled down to -

30 120⁰C at a rate of 10°C/min. After freezing, the tubes were removed from the freezer and transferred immediately into the vapour phase of liquid nitrogen (nitrogen tank, Air Liquide, Kryotechnik, Dusseldorf, Germany). Schindler et al (2004) is hereby incorporated by reference. In US 7,112,576, hereby incorporated by reference, a cryopreservation method is described wherein the crypreservation medium comprising arabinogalactan or a functional equivalent thereof. Preferably, the cryopreservation medium does not comprise DMSO. In addition to arabinogalactan, a biological or a functional equivalent threof, the cryopreservation medium preferably further comprises a cryoprotective agent that penetrates the cell membrane, e.g. glycerol or propylene glycol. The medium may also comprise a cryoprotective agent other than arabinogalactan or a biological or a functional equivalent thereof which does not penetrate the cell membrane.

As described above, the cells of the invention can be preserved after differentiation of the cells. Thus, in the broadest aspect of the invention, the invention relates to a method, wherein the cells have been preserved after differentiation of the cells. In one embodiment, the invention relates to a method, wherein the cells have been cryopreserved after differentiation of the cells. The cells of the invention can be cryopreserved by cryopreservation methods known in the art, as outlined above and by the method described by the inventors in example 9.

Prior to exposing the sample to the cells, the cells are reconstituted from cryopreservation. The cells are reconstituted by methods known in the art, see e.g. the references listed above, and by the method outlined by the inventors in example 9. The cells can be rapidly thawed by addition of e.g. preheated cell media and centrifuged, the cell can be washed one or more times in preheated assay buffer or other buffers known in the art, e.g. HBSS buffer, before resuspension of the cells in assay buffer. The cells can then be applied in the method of the invention.

The inventors have surprisingly found that the cells of the invention retain the level of functional activity after reconstitution from cryopreservation (e.g. example 9 and figures 13-15). Thus, in one embodiment, the invention relates to a method, wherein the cells after reconstitution from cryopreservation possess a level of functional activity which is 10-100% of the level of functional activity of identical cells, which have not been cryopreserved, such as e.g. 10-90%, or such as e.g. 10-80%, or such as e.g. 10-70%, or such as e.g. 10-60%, or such as e.g. 10-50%, or such as e.g. 10-40%, or such as e.g. 10-30%, or such as e.g. 10- 20% of the level of functional activity of identical cells, which have not been cryopreserved, or such as about 10% of the level of functional activity of identical cells, which have not been cryopreserved, or such as e.g. about 20%, or such as e.g. about 30%, or such as e.g. about 40%, or such as e.g. about 50%, or such 5 as e.g. about 60%, or such as e.g. about 70%, or such as e.g. about 80%, or such as e.g. about 90%, or such as e.g. about 100% of the level of functional activity of identical cells, which have not been cryopreserved.

In some cases the level of functional activity of the reconstituted cryopreserved cells can be even higher compared to the level of functional activity of identical

10 cells, which have not been cryopreserved. Therefore in a second embodiment, the invention relates to a method, wherein the cells after reconstitution from cryopreservation possess a level of functional activity which is higher than the level of functional activity of identical cells, which have not been cryopreserved, such as e.g. 100-1000% of the level of functional activity of identical cells, which

15 have not been cryopreserved, or such as e.g. 100-500%, or such as e.g. 100- 400%, or such as e.g. 100-300%, or such as e.g. 100-200% of the level of functional activity of identical cells, which have not been cryopreserved, or such as e.g. about 200%, or such as e.g. about 300%, or such as e.g. about 400%, or such as e.g. about 500%, or such as e.g. about 600%, or such as e.g. about

20 700%, or such as e.g. about 800%, or such as e.g. about 900%, or such as e.g. about 1000% of the level of functional activity of identical cells, which have not been cryopreserved.

As also outlined above, the term "identical cells" as used herein refers to cells that are treated exactly the same way as the cryopreserved cells, i.e. with respect to 25 differentiation, priming, maintenance of the cells etc. The only difference between the cryopreserved cells and the "identical cells" is that the cryopreserved cells have been exposed to cryopreservation.

The term "functional activity" as used herein refers to general cellular functions, e.g. the ability to generate reactive oxygen species (ROS) upon stimulation with a 30 pyrogen. The ROS production can be measured by methods known in the art or by the method of the invention as outlined in the description and the examples.

In one embodiment, the invention relates to a method, wherein the cells immediately after thawing are able to produce ROS upon stimulation with a pyrogen. In another embodiment, the invention relates to a method, wherein said cells are able to produce ROS in an amount of at least 10% of the amount of ROS produced by identical cells which have not been cryopreserved or lyophilised, such as e.g. at least 20 %, or such as e.g. at least 30%, or such as e.g. at least 40%, or such as e.g. at least 50%, or such as e.g. at least 60%, or such as e.g. at least 70%, or such as e.g. at least 80%, or such as e.g. at least 90%, or such as e.g. at least 100%.

In a further embodiment, the invention relates to a method, wherein the sample is exposed to the cells immediately after thawing of the cells.

The term "immediate after" as used herein refers to 1 min to 5 hours after thawing of the cells, such as e.g. 1 min to 4 hours, or such as e.g. 1 min to 3 hours, or such as e.g. 1 min to 2 hours, or such as e.g. 1 min to 60 min, or such as e.g. 1 min to 45 min, or such as e.g. 1 min to 30 min, or such as e.g. 1 min to 20 min, or such as e.g. 1 min to 15 min, or such as e.g. 1 min to 10 min, or such as e.g. 1 min to 5 min after thawing of the cells. About 30 minutes seems to work particularly well.

In another aspect, the invention relates to cryopreserved differentiated polymorphonuclear leukocyte-like cells, wherein said cells are differentiated prior to cryopreservation, and wherein said cells immediately after thawing are able to produce ROS upon stimulation with a pyrogen. The cells can be cryopreserved by methods known in the art as outlined above.

In one embodiment, the invention relates to cryopreserved differentiated polymorphonuclear leukocyte-like cells, wherein said cells are able to produce ROS in an amount of at least 10% of the amount of ROS produced by identical cells which have not been cryopreserved, such as e.g. at least 20 %, or such as e.g. at least 30%, or such as e.g. at least 40%, or such as e.g. at least 50%, or such as e.g. at least 60%, or such as e.g. at least 70%, or such as e.g. at least 80%, or such as e.g. at least 90%, or such as e.g. at least 100% of the amount of ROS produced by identical cells which have not been cryopreserved. The amount of ROS produced can be measured by the method of the invention as defined in the claims, the description and the examples. In another embodiment, the cells are originating from a cell line selected from the group consisting of NB-4, THP-I, KG-I, K562, KCL22, PLB-985, U937, Mono Mac 6, X-CDG, PL-21, ML-I, ML-3, MHH-225, AML-193, HL-60 and variants thereof. In a preferred embodiment, the cells are originating from a HL-60 cell line or variants thereof. The cells can be differentiated with one or more differentiating agents selected from the group consisting of all trans retinoic acid (ATRA), 9-cis retinoic acid, dimethylformamide 5 (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP and combinations thereof. In a preferred embodiment, the differentiating agent is all-trans retinoic acid (ATRA). In another preferred embodiment, the cells are differentiated with one or more differentiating agents for a period of 2-12 days.

In a preferred embodiment, the invention relates to cryopreserved differentiated 10 HL-60 cells, wherein said cells are able to produce ROS in an amount of at least 10% of the amount of ROS produced by identical HL-60 cells which have not been cryopreserved, such as e.g. at least 20 %, or such as e.g. at least 30%, or such as e.g. at least 40%, or such as e.g. at least 50%, or such as e.g. at least 60%, or such as e.g. at least 70%, or such as e.g. at least 80%, or such as e.g. at least 15 90%, or such as e.g. at least 100% of the amount of ROS produced by identical HL-60 cells which have not been cryopreserved. In a more preferred embodiment, the HL-60 cells are differentiated with ATRA for a period of 2-12 days.

In yet a preferred embodiment, the invention relates to cryopreserved differentiated HL-60 cells, wherein said cells are able to produce ROS in a higher 20 amount compared to the amount of ROS produced by identical HL-60 cells which have not been cryopreserved, such as e.g. about 100 %, or such as e.g. about

200%, or such as e.g. about 300%, or such as e.g. about 400%, or such as e.g. about 500%, or such as about 600%, or such as e.g. about 700%, or such as e.g. about 800%, or such as e.g. about 900%, or such as e.g. about 1000% of the 25 amount of ROS produced by identical HL-60 cells which have not been cryopreserved. In a more preferred embodiment, the HL-60 cells are differentiated with ATRA for a period of 2-12 days.

Lyophilisation

The cells of the invention can be lyophilised after differentiation. The cells are 30 lyophilised by applying methods known in the art e.g as described in US

5,045,446, US 5,648,206, US 6,960,464 and Han Y. et al. Cryobiology 51 (2005) 152-164, which are not deleterious to the structure and the functional activity of the cells, and which permits the reconstitution of the lyophilised cells to form cells which are identical or almost identical to the natural cells with respect to the functional activity of the cells.

One way to lyophilise the cells of the invention is described in US 5,045,446 and US 5,648,206, which are hereby incorporated by reference. In this method, the lyophilisation process comprises immersing a plurality of cells in an essentially isotonic aqueous solution containing a carbohydrate, and which preferably includes an amphipathic polymer, freezing the solution, and drying the solution to yield freeze-dried cells which, when reconstituted, produce a significant percentage of intact and viable cells. For further description of the lyophilisation method see US 5,045,446 and US 5,648,206.

As described in US 6,960,464, hereby incorporated by reference, a cryoprotectant may be added directly to the cells in suspension. Preferably, the cells are collected and then resuspended in a cryoprotectant. The concentration of the cryoprotectant will vary depending on the cell type, buffers used, the type of cryoprotectant and other factors. Optimal conditions can be determined by one skilled in the art without undue experimentation. Cryoprotectants provide protection of the cells during the freezing process by depressing the freezing point, minimizing the effect of solution changes external to the cell, penetrating the cell to protect against solute concentration effects, and/or shifting the optimum colling rate to lower values (F. P. Simione, Journal of Parenteral Science & Technology, 46(6):226-232 (1992)). Cryoprotectants that can be used in the present invention include, but are not limited to carbohydrates and carbohydrate derivatives such as trehalose, sucrose, lactose, maltose, mannitol, galactose, ribose, fructose, xylose, mannose, dextrose, glucose, and sorbitol, and polymers such as polyethyleneamine, polyvinylpyrrolidone (PVP), ficoll tec. Other cryoprotectants which can be used in accordance with the invention, such as acacia gum, albumin, gelatine, and sugar alcohols, will be readily recognised by one skilled in the art.

After the cells have been mixed with the cryoprotectant, the cell suspension may be aliquoted into containers to be used for lyophilisation and storage, such as chilled cryovials, e.g. NUNC tubes (Gibco BRL, Gaithersburg, Md., Cat. No 366656), or glass vials (Wheaton, Millville, NJ.). Prior to lyophilisation the cellsare frozen at about -20⁰C to about -180⁰C, preferably at about -180°C, or preferably about -80⁰C.

Methods of freezing a sample to a temperature from about -80⁰C to about -180⁰C are well-known in the art. These include overnight storage (about 16 hrs) of the vials which contain the cells in a -80⁰C freezer, or immersion of the vials which contain the cells in dry ice, or in a low temperature bath, such as dry ice ethanol, or in a bath containing liquid nitrogen. Other such systems are disclosed in The chemist's companion; A handbook of practical data, techniques, and references, Gordon, AJ. et al., eds, John Wiley and Sons, NY (1972).

The cells are then lyophilised by techniques which are well-known in the art. Lyophilisation is a process by which ice and/or moisture is removed from frozen cells by sublimation under vacuum at low, subzero temperatures (e.g. -40⁰C to - 50⁰C). Any residual moisture associated with the "dried" preparation is then removed by gradually raising the temperature, resulting in evaporation. Thus, according to the invention, lyophilisation comprises subjecting frozen cells to a vacuum under conditions sufficient to substantially remove moisture and/or ice from said cells (also referred to herein as substantially dried cells). The substantially dried cells may then be stored at various temperatures (room temperature to about -180°C, preferably about 4°C to about -80°C, more preferably about -20°C to about -80°C, and most preferably about -20°C.

One such process for lyophilizing cells comprises the steps of

(a) loading a container containing frozen cells into a lyophiliser, the lyophiliser having a temperature of about -40⁰C to about -50⁰C,

(b) subjecting the cells to a vacuum, and

(c) substantially drying the cells.

Preferably, the vacuum is less than about 100 μm Hg, and the cells are dried by:

(i) holding the temperature of the chamber at about -45°C for about 2 hours, and (ii) increasing the temperature of the chamber from about -45°C to about 10⁰C at the rate of about 0.1°C/hr to 1.0°C/hr (preferably, 0.5°C/hr to 0.8°C/hr or preferably 0.6°C/hr to 0.8°C/hr).

The cell container may then be sealed and stored for extended time at various temperatures.

As outlined above, the cells of the invention can be lyophilised after differentiation of the cells. Thus, in one aspect of the invention, the invention relates to a method, wherein the cells have been preserved by lyophilization. In one embodiment, the invention relates to ajnethod, wherein the cells after reconstitution from lyophilisation possess a level of functional activity which is 10- 100% of the level of functional activity of identical cells, which have not been lyophilised.

Thus, in one embodiment, the invention relates to a method, wherein the cells after reconstitution from lyophilisation possess a level of functional activity which is 10-100% of the level of functional activity of identical cells, which have not been lyophilised, such as e.g. 10-90%, or such as e.g. 10-80%, or such as e.g. 10-70%, or such as e.g. 10-60%, or such as e.g. 10-50%, or such as e.g. 10- 40%, or such as e.g. 10-30%, or such as e.g. 10-20% of the level of functional activity of identical cells, which have not been lyophilised, or such as about 10% of the level of functional activity of identical cells, which have not been lyophilised, or such as e.g. about 20%, or such as e.g. about 30%, or such as e.g. about 40%, or such as e.g. about 50%, or such as e.g. about 60%, or such as e.g. about 70%, or such as e.g. about 80%, or such as e.g. about 90%, or such as e.g. about 100% of the level of functional activity of identical cells, which have not been lyophilised.

In some cases the level of functional activity of the reconstituted lyophilised cells can be even higher compared to the level of functional activity of identical cells, which have not been lyophilised. Therefore in a second embodiment, the invention relates to a method, wherein the cells after reconstitution from lyophilisation possess a level of functional activity which is higher than the level of functional activity of identical cells, which have not been lyophilised, such as e.g. 100- 1000% of the level of functional activity of identical cells, which have not been lyophilized, or such as e.g. 100-500%, or such as e.g. 100-400%, or such as e.g. 100-300%, or such as e.g. 100-200% of the level of functional activity of identical cells, which have not been lyophilised, or such as e.g. about 200%, or such as e.g. about 300%, or such as e.g. about 400%, or such as e.g. about 500%, or such as e.g. about 600%, or such as e.g. about 700%, or such as e.g. about 800%, or such as e.g. about 900%, or such as e.g. about 1000% of the level of functional activity of identical cells, which have not been lyophilised.

In another embodiment, the invention relates to a method , wherein said cells immediately after thawing from lyophilisation are able to produce ROS upon stimulation with a pyrogen.

In a further embodiment, the invention relates to a method, wherein said cells are able to produce ROS in an amount of at least 10% of the amount of ROS produced by identical cells which have not been lyophilised, such as e.g. at least 20%, or such as e.g. at least 30%, or such as e.g. at least 40%, or such as e.g. at least 50%, or such as e.g. at least 60%, or such as e.g. at least 70%, or such as e.g. at least 80%, or such as e.g. at least 90%, or such as e.g. at least 100% of the amount of ROS produced by identical cells which have not been lyophilised, or about 10%, or about 20%, or about 30% or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100% of the amount of ROS produced by identical cells which have not been lyophilised.

In yet a further embodiment, the invention relates to a method, wherein the sample is exposed to the cells immediately after thawing/reconstitution of the cells.

In another aspect, the invention relates to lyophilized polymorphonuclear leukocyte-like cells, wherein said cells are differentiated prior to lyophilization, and wherein said cells immediately after thawing are able to produce ROS upon stimulation with a pyrogen. The cells can be lyophilised by methods known in the art as outlined above.

In one embodiment, the invention relates to lyophilised differentiated polymorphonuclear leukocyte-like cells, wherein said cells are able to produce ROS in an amount of at least 10% of the amount of ROS produced by identical cells which have not been lyophilised, such as e.g. at least 20 %, or such as e.g. at least 30%, or such as e.g. at least 40%, or such as e.g. at least 50%, or such as e.g. at least 60%, or such as e.g. at least 70%, or such as e.g. at least 80%, or such as e.g. at least 90%, or such as e.g. at least 100% of the amount of ROS produced by identical cells which have not been lyophilised. The amount of ROS produced can be measured by the method of the invention as defined in the claims, the description and the examples. In another embodiment, the cells are originating from a cell line selected from the group consisting of NB-4, THP-I, KG- 1, K562, KCL22, PLB-985, U937, Mono Mac 6, X-CDG, PL-21, ML-I, ML-3, MHH- 225, AML-193, HL-60 and variants thereof. In a preferred embodiment, the cells are originating from a HL-60 cell line or variants thereof. The cells can be differentiated with one or more differentiating agents selected from the group consisting of all trans retinoic acid (ATRA), 9-cis retinoic acid, dimethylformamide (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP and combinations thereof. In a preferred embodiment, the differentiating agent is all-trans retinoic acid (ATRA). In another preferred embodiment, the cells are differentiated with one or more differentiating agents for a period of 2-12 days.

In a preferred embodiment, the invention relates to lyophilised differentiated HL- 60 cells, wherein said cells are able to produce ROS in an amount of at least 10% of the amount of ROS produced by identical HL-60 cells which have not been lyophilised, such as e.g. at least 20 %, or such as e.g. at least 30%, or such as e.g. at least 40%, or such as e.g. at least 50%, or such as e.g. at least 60%, or such as e.g. at least 70%, or such as e.g. at least 80%, or such as e.g. at least 90%, or such as e.g. at least 100% of the amount of ROS produced by identical HL-60 cells which have not been lyophilised. In a more preferred embodiment, the HL-60 cells are differentiated with ATRA for a period of 2-12 days.

Inflammatory contaminants/ 'pyrogens

The method of the invention is able to detect the presence of one or more pyrogens in a sample.

The terms "pyrogen" or "inflammatory substance/contaminants" as used herein are interchangeable terms and refer to certain chemical or biological compounds capable of producing an inflammatory response. When humans or other mammals are exposed to an inflammatory compound, an either local or systemic inflammatory response can occur. A local response to inflammatory compounds is characterized by an activation of leukocytes, normally leading to a redness and swelling of surrounding tissue due to local vasodilation. The systemic response occurs when inflammatory compounds are brought into contact with the circulatory system. Even low concentrations of inflammatory compounds can result in septic shock characterized by loss of blood pressure, edema and high fever. "Inflammatory substances" or "pyrogens" include compounds capable of evoking a systemic inflammatory response characterized by fever. Microorganisms and substances originating from microorganisms are well-known fever-producing substances. Examples of fever-producing substances are but are not limited to a microorganism or related substance including a Gram-positive bacteria, a Gram- negative bacteria, a cell envelope constituent from Gram-positive bacteria, a cell envelope constituent from a Gram-negative bacteria, a fungi, fungal hyphae or spores, a yeast cell, a cell envelope constituent from a yeast cell, an endospore or a virus.

In one embodiment, the invention relates to a method, wherein the pyrogen is selected from the group consisting of a Gram-positive bacteria, a Gram-negative bacteria, a cell envelope constituent from Gram-positive bacteria, a cell envelope constituent from a Gram-negative bacteria, a fungi, a yeast cell and a cell envelope constituent from a yeast cell, a fungal spore and a virus.

The term "Gram-positive bacteria" as used herein refers to bacteria that are stained dark blue or violet by Gram staining, in contrast to Gram-negative bacteria, which cannot retain the stain, instead taking up the counterstain and appearing red or pink. The stain is caused by a high amount of peptidoglycan in the cell envelope, which typically, but not always lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria.

Gram-positive bacteria include but are not limited to Bacillus ssp., Listeria ssp., Staphylococcus ssp., Streptococcus ssp., Enterococcus ssp., and Clostridium ssp.. Gram-positive bacteria also include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms. The Deinococcus-Thermus bacteria also have Gram-positive stains, although they are structurally similar to Gram-negative bacteria. Specific examples of Gram-positive bacteria include B. subtilis, S. aureus, S. epidermidis, Streptococcus, Pneumococcus. In one embodiment, the invention relates to a method wherein the Gram-positive bacteria is selected from the group consisting of Staphylococcus ssp., Enterococcus ssp., Streptococcus ssp., Listeria ssp. and Bacillus ssp. In a preferred embodiment, the Gram-positive bacteria is selected from the group consisting of B. subtilis and S. aureus.

The term "Gram-negative bacteria" as used herein refers to bacteria that do not retain crystal violet dye in the Gram staining protocol. Gram-positive bacteria will retain the dark blue dye after an alcohol wash, whereas Gram-negative do not. In a Gram stain test, a counterstain is added after the crystal violet, which colors all Gram-negative bacteria a red or pink color. Many species of Gram-negative bacteria are pathogenic, meaning they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram- negative cell envelopes, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer.

Gram-negative bacteria include but are not limited to Klebsiella ssp., Shigella ssp., Escherichia ssp., Salmonella ssp., Pseudomonas ssp., Moraxella ssp., Helicobacter ssp., Stenotrophomonas ssp., Bdellovibrio ssp., acetic acid bacteria, Legionella ssp.. Gram-negative bacteria also include the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria. Gram-negative cocci include Neisseria gonorrhea, Neisseria meningitidis and Moraxella catarrhalis. Gram-negative bacilli include a multitude of species Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis and Salmonella typhi.

In one embodiment of the invention the Gram-negative bacteria is selected from the group consisting of Salmonella ssp., Esherichia ssp., Shigella ssp., Pseudomonas ssp. and Klebsiella ssp. In a preferred embodiment, the Gram- negative bacteria are selected from the group consisting of S. typhimurium and E. coli.

The term "Cell envelope constituent" as used herein refers to a constituent or component of the cell envelope of a microorganims, such as Gram-positive or Gram-negative bacteria or viruses. The cell envelope is the outer portion of a microorganism or virus and is defined as the cell membrane and cell wall plus the outer membrane. The term "envelope" therefore relates to the entire exterior of the microorganisms or virus. Examples of cell envelope constituents are peptidoglycan, lipopolysaccharride (LPS) or endotoxin, lipid A, lipoteichoic acid (LTA).

The term "LPS" as used herein refers to lipopolysaccharide (LPS) which is a large molecule that contains both lipid and polysaccharide. It is a major constituent of the cell envelope of Gram-negative bacteria and protects them from host immune defenses. LPS comprises three parts: polysaccharide (O) side chains; core polysaccharides; and lipid A. Lipid A contains unusual fatty acids (e.g. hydroxy- myristic acid) and is inserted into the outer membrane while the rest of the LPS projects from the surface. Core polysaccharide contains unusual sugars (e.g. KDO, keto-deoxyoctulonate and heptulose). It contains two glucosamine sugar derivatives each containing three fatty acids with phosphate or pyrophosphate attached. The core polysaccharide is attached to lipid A, which is also in part responsible for the toxicity of Gram-negative bacteria.

The polysaccharide sidechain is referred to as the O-antigen of the bacteria. O side chain (O antigen) is also a polysaccharide chain that extends from the core polysaccharide. The composition of the O side chain varies between different Gram-negative bacterial strains. O side chains are easily recognized by the antibodies of the host, however, the nature of the chain can easily be modified by Gram-negative bacteria to avoid detection. LPS also increases the negative charge of the cell envelope and helps stabilize the overall membrane structure.

In one embodiment of the invention, the cell envelope constituent from a Gram- negative bacteria is lipopolysaccharride (LPS).

The term "LTA" as used herein refers to Lipoteichoic acid which is a surface- associated adhesion molecule from Gram-positive bacteria and regulator of autolytic wall enzymes (muramidases). It is released from the bacterial cells mainly after bacteriolysis induced by lysozyme, cationic peptides from leukocytes, or beta-lactam antibiotics. It binds to target cells either non-specifically, to membrane phospholipids, or specifically, to Toll-like receptors. LTA bound to targets can interact with circulating antibodies and activate the complement cascade to induce a passive immune kill phenomenon. It also triggers the release from neutrophils and macrophages of reactive oxygen and nitrogen species, acid hydrolases, highly cationic proteinases, bactericidal cationic peptides, growth factors, and cytotoxic cytokines, which may act in synergy to amplify cell damage. Thus, LTA shares with endotoxin (lipopolysaccharide, LPS) many of its pathogenetic properties. In animal studies, LTA has induced arthritis, nephritis, uveitis, encephalomyelitis, meningeal inflammation, and periodontal lesions, and also triggered cascades resulting in septic shock and multiorgan failure. Binding of LTA to targets can be inhibited by antibodies, phospholipids, and specific antibodies to CD14 and Toll, and in vitro its release can be inhibited by non- bacteriolytic antibiotics and by polysulphates such as heparin, which probably interfere with the activation of autolysis. From all this evidence, LTA can be considered a virulence factor that has an important role in infections and in postinfectious sequelae caused by Gram-positive bacteria. In one embodiment of the invention, the cell envelope constituent from a Gram-positive bacteria is lipoteichoic acid (LTA).

The term "fungi" as used herein refers to nonphototrophic eucaryotic microorganisms that contain rigid cell walls and produce spores.

In one embodiment of the invention the fungi is selected from the group consisting of Candida ssp., Aspergillus ssp., Histoplasma ssp., Coccidioides ssp. and Cryptococcus ssp. In a preferred embodiment, the fungus is Candida albicans.

The term "fungal spore" as used herein refers to ascospores, basidiospores, zygospore and oospores produced by fungi.

In a preferred embodiment of the invention the fungal spore is Aspergillus niger spores.

The term "virus" as used herein refers to a microorganism that contains either DNA or RNA as the genetic element and replicates in cells but is characterized by having an extracellular state. Examples of vira are hepatitis A virus, herpes simplex-I virus, herpes simplex-II virus, hepatitis B, hepatitis C virus, influenza A, influenza B, influenza C virus, human immune deficiency-I virus and human immune deficiency-II virus.

Examples of yeast are Candida albicans and Saccharomyces cerevisiae.

Positive detection of inflammatory contaminants/ pyrogens As outlined above, the invention relates to a method capable of detecting a broad range of pyrogens in a sample. The method of the invention is highly sensitive compared to existing pyrogen tests.

In one embodiment, the invention relates to a method characterized in that the method is capable of detecting the Gram-positive bacteria at a concentration of >

10⁴ bac/ml.

In a second embodiment, the invention relates to a method characterized in that the method is capable of detecting the Gram-negative bacteria at a concentration of > 10³ bac/ml.

In a third embodiment, the invention relates to a method characterized in that the method is capable of detecting the cell envelope constituent from a Gram- negative bacteria at a concentration of > 5 pg/ml.

In another embodiment, the invention relates to a method characterized in that the method is capable of detecting the cell envelope constituent from a Gram- positive bacteria at a concentration of > 10 ng/ml.

In yet another embodiment, the invention relates to a method characterized in that the method is capable of detecting the fungal spores at a concentration of >

10⁵ spores/ml.

In a further embodiment, the invention relates to a method characterized in that the method is capable of detecting the yeast at a concentration of > 10⁴ yeast cells/ml.

In the examples of the present application, embodiments of the invention are described wherein positive detection limits as outlined above, are obtained.

P re-treatment of the sample to be tested

In order to optimize the interaction between the one or more pyrogens potentially present in the sample to be tested and the cells of the invention, the sample can be exposed to various pre-treatments before exposing the sample to the cells, since pretreatment has turned out to yield an increased sensitivity for some applications. In order to disrupt the envelope of a microorganism potentially present in the sample to be tested, the sample can be pre-treated with one or more envelope disruptors before exposing the sample to the cells. The disruption of the envelope can for instance be the shedding of LPS from a Gram-negative bacteria further exposing the highly active lipid A portion of the LPS due to a treatment with a envelope disruptor. This will make it possible for the cells of the present invention to be exposed to a higher free concentration of pyrogen thus resulting in an increased production of ROS towards a given microbial challenge thus decreasing the detection limit and/or the positive detection of a pyrogen of the method of the invention. This is exemplified in figure 5.

Thus, in one embodiment, the invention relates to a method further comprising pre-treating the sample with an envelope disruptor before exposing the sample to the cells. The term "envelope disruptor" as used herein refers to chemical or physical agents that disrupt the outer membrane and/or entire envelope of microorganisms.

In one embodiment of the invention, physical envelope disruption is conducted by ultrasonication. In a preferred embodiment the sample is subjected to from 5-35 seconds sonication and 5-35 seconds pause cycles of ultrasonic treatment over a period of 1-20 minutes using an ultrasonicator. In a more preferred embodiment, the sample is subjected to 15 seconds sonication and 15 seconds pause cycles of ultrasonic treatment over a period of 5 minutes using an ultrasonicator with an effective power of 130 W.

Preferably, the envelope disruptor is selected from the group consisting of sodium dodecyl sulfate (SDS), Triton XlOO, chloroform and chelating agent as ethylenediaminetetraacetic acid (EDTA). In a preferred embodiment, the envelope disruptor is EDTA.

Yet another approach for optimizing the detection of the one or more pyrogens potentially present in the sample to be tested and the cells of the invention, is ultra-filtrating the sample before exposing the sample to the cells. When using the HL-60 assay as described in Example 8, there is a risk of falsification of the test results by interfering substances in the product. Low molecular weight substances can be separated from most samples by ultrafiltration. The ultrafilter can be e.g a Teflon filter or a cellulose such as e.g. a cellulose triacetate filter with e.g. a cut off of e.g. 20 000 Da. By ultrafiltration the low molecular weight compounds can be flushed out and pyrogenic, high molecular weight substances is retained and concentrated. To desorb the pyrogens the filter is shaken with pyrogen-free cell media or buffer such as e.g. HBSS and the homogenized pyrogens is then tested in the HL-60 assay, cf. Example 5. Thus, in another embodiment, the invention relates to a method further comprising ultra-filtrating the sample before exposing the sample to the cells.

Various components of the immune system are described in the following :

Yet another approach for optimizing the interaction between the one or more pyrogens potentially present in the sample to be tested and the cells of the invention is opsonising the sample. Thus, in one embodiment, the invention relates to a method further comprising opsonising the sample before exposing the sample to the cells. The term "opsonization" as used herein refers to the process whereby one or more opsonins binds to the surface of the one or more pyrogens (or antigens) thereby mediate the binding of the pyrogens to the cells of the invention.

The term "opsonin" as used herein refers to any molecule that acts by binding to the surface of the pyrogens (or antigens) and thereby mediating the binding of the pyrogens and the cells of the invention. During the process of opsonization, pyrogens associate with one or more opsonins such as but not limited to an antibody and/or complement molecules. The cells of the invention, such as cells derived from a pluripotent stem cell, e.g. polymorphonuclear leukocytes or polymorphonuclear leukocyte-like cells, or HL-60 cells express receptors that bind opsonins. By coating the one or more pyrogens potentially present in the sample by opsonins, recognition of the pyrogens by the cells of the invention is greatly enhanced. Examples of opsonins include but are but not limited to antibodies and complement factors, as defined below. Opsonins can be either purified from human or animal blood or generated as recombinant proteins, as single soluble molecules, dimers, trimers or polymers. In a preferred embodiment of the present invention the opsonin is a single soluble purified opsonin from human, guinea pig or rabbit blood.

The term "antibody" as used herein refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term antibody is used to mean whole antibodies and binding fragments thereof. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin isotypes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 KDa) and one "heavy" chain (about 50-70 KDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region. The Fc portion of the antibody molecule corresponds largely to the constant region of the immunoglobulin heavy chain, and is responsible for the antibody's effector function (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N. Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.

Antibodies also include single-armed composite monoclonal antibodies, single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, as well as diabodies, tribodies, and tetrabodies (Pack et al. (1995) J MoI Biol 246:28; Biotechnol 11 : 1271; and Biochemistry 31 : 1579). The antibodies are, e.g., polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, fragments produced by a Fab expression library, or the like.

The term "complement factor" as used herein refers to At least one of the proteins of the complement system which consists of more than 35 soluble and cell-bound proteins, 12 of which are directly involved in the complement pathways involved in the destructions of pathogens. The proteins account for 5% of the serum globulin fraction. Most of these proteins circulate as zymogens, which are inactive until proteolytic cleavage. The complement proteins are synthesized mainly by hepatocytes; however, significant amounts are also produced by monocytes, macrophages, and epithelial cells in the gastrointestinal and genitourinary tracts. The complement cascade can be activated on the surface of a pathogen through one or more of the three pathways; classical, lectin and alternative pathway. Examples of complements factors and proteins related to the complement system are C5a, C3a and C4a which mediates inflammation and C5b, C6, C7, C8, C9 which bind to the membrane of the foreign pathogen. Furthermore, but not limited to proteins like Factor B and D and properdin is involved in the alternative pathway of complement activation. Complement factors further comprise isolated or recombinant complement component Cl, C3, C4, C5 or one or several of their respective proteolytic cleavage products. Furthermore other plasma proteins such as: MBL, C-reactive protein, sCD14 and/or LBP can influence opsonization. In a specific embodiment of the invention, the complements factors are C3b, C4b and metabolic products hereof, and the plasma protein mannose-binding-lectin (MBL).

In another aspect, the invention relates to a method for detection of one or more pyrogens in a sample comprising the steps of:

(a) optionally, treating the sample with a envelope disruptor

(b) optionally, ultrafiltrating the sample

(c) optionally, opsonising the sample

(d) optionally, adding one or more antibodies to the sample, said antibodies being specific to one or more specific inflammatory contaminants, optionally along with immune components and a ROS reporter probe,

(e) exposing the sample to cells characterized by producing reactive oxygen species (ROS) if exposed to a pyrogen, optionally said cells are pre-treated with one or more priming agents and/or one or more differentiating agents before exposure to the sample,

(f) measuring the amount of reactive oxygen species (ROS) produced by said cells, and

(g) determining the presence of said one or more pyrogens in the sample by evaluation of the data obtained in step (f).

Plasma

It is possible to optimize the ROS production of the cells of the invention upon pyrogen stimulation, by adding mammalian blood plasma to the assay buffer. Most likely, the presence of plasma results in an increase in the ROS production because the plasma leads to opsonization of one or more pyrogens potentially present in the sample and thereby binding of the pyrogens to the cells of the invention is enhanced.

It has been observed that the presence of plasma in the assay buffer reduces the ROS background signal, and thereby improves pyrogen detection limits since pyrogen responses close to the detection limit can be blocked out by the elevated background of a non plasma supplemented sample, furthermore plasma addition reduces onset time of the ROS response, cf. Example 4 and Figure 4.

In the broadest aspect of the present invention the plasma is of mammal origin. In one embodiment of the present invention the plasma is from a mammal such as, but not limited to human, rabbit, guinea pig, cow, horse. In a preferred embodiment of the present invention the plasma is human plasma.

In one embodiment of the present invention 0.01%-25% plasma is added to the assay buffer. In another preferred embodiment of the present invention 0.1%- 20% plasma is added to the assay buffer. In another preferred embodiment of the present invention between 0.5%-15% plasma is added to the assay buffer. In another preferred embodiment of the present invention l%-10% plasma is added to the assay buffer. In another preferred embodiment of the present invention 2%-5% plasma is added to the assay buffer. In a specific preferred embodiment of the present invention about 2.5% plasma is added to the assay buffer.

In one embodiment, the invention relates to a method, wherein one or more anticoagulants are added to whole blood to obtain the plasma used in the method of the invention. The choice of anticoagulants for the plasma also influences the method of the invention since several anticoagulants interact with the activation of the complement system, which is speculated to play a key role in the method of the invention. Anticoagulants that have the least influence on the complement system are sodium-heparin or recombinant hirudin (lepirudin). Thus, in a second embodiment of the invention the one or more anticoagulants are sodium-heparin and/or lepirudin.

It should be understood that the use of plasma includes use of isolated lyophilised plasma proteins in any concentrations and combinations, this relates to both natural sources and to recombinant inclusive engineered, truncated or fused proteins, alone or in combination. A non-limiting example of an isolated plasma protein could be human serum albumin. According to the present invention it is demonstrated that the assay tends to be more sensitive upon addition of lyophilized/freeze dried plasma components rather than e.g. use of plasma components that have been preserved by regular freezing procedures, even though use of both plasma types alone or in any combination is a part of the present invention. In particular, the present inventors have discovered that the use of lyophilized plasma results in better cell responds towards zymosan that with plasma frozen at -80 ⁰C. The response to LPS stimulation seems to be equivalent, or slightly higher with the use of lyophilized plasma, instead of frozen plasma (data not shown). Furthermore, the inventors have discovered that in case frozen plasma (-80 ⁰C) is used in the current methods, 2.5% appears to be the most optimal concentration to use. When using lyophilized plasma however, it seems that higher concentrations could benefit the assay in that you get improved responses without elevated backgrounds (data not shown).

Incubation of the sample with plasma and/or plasma proteins Prior to exposing the sample to the cells, the sample can be incubated with plasma, as defined above, and/or one or more isolated and/or recombinant plasma proteins such as, but not limited to, complement proteins (as defined above) or acute phase reactants. The incubation with plasma and/or one or more plasma proteins can result in an increased ROS production of the cells of the invention upon pyrogen-stimulation.

The term "plasma protein" as used herein refers to protein present in blood plasma, such as albumins, globulins, fibrinogens and hemoglobins. Examples of plasma proteins are ovalbumin, serum albumin, such as e.g. human serum albumin or bovine serum albumin, alphal-globulins, alpha2-globulins, beta- globulins, gamma-globulins or antibodies as defined above, complement factors as defined above; fibrin, fibrinogen and hemoglobin.

The incubation period of the sample with plasma and/or one or more plasma proteins can vary accordingly to the type of inflammatory contaminant potentially present in the sample and to the plasma protein of choice. In a preferred embodiment of the present invention, the incubation period of the plasma and/or one or more plasma proteins with the cells of the invention is 1-120 minutes, such as 2-90 minutes, or such as 3-60 minutes, or such as 5-30 minutes.

The incubation can be performed, but is not limited to be, with plasma, prepared accordingly to the above mentioned respect to choice of anti coagulants; or one or several of the here mentioned plasma proteins: Isolated or recombinant complement components Cl, C3, C 4 or C5 either in active or inactive form, or one or several of their respective proteolytic cleavage products or by MBL, C-reactive protein, sCD14 or LBP. It should be understood that the use of plasma as opsonin includes use of isolated plasma proteins in any concentrations and combinations, this relates to both natural sources and to recombinant inclusive engineered, truncated or fused proteins, alone or in combination. By means of ultrafiltration, the inventors have discovered that the opsonising activity in plasma primarily resides in the high molecular fraction (> 30.000 Dalton) (data not shown).

ROS detection (step (H) of the method of the invention) As outlined above, in step i) of the method of the invention the sample is exposed to the cells of the invention, i.e. the sample is mixed with the cells of the invention. Upon mixture with the cells, the potential one or more pyrogens in the sample will stimulate the cells to produce and release ROS into the reaction- mixture. The measurement of the amount of ROS produced by the cells (step ii of the method of the invention) can in general be performed by any assay capable of detecting ROS in a given sample and/or reaction-mixture.

Several assays can be applied in order to detect the ROS produced and released by the cells of the invention. Most likely, such an assay will comprise of a compound or substrate, capable of binding or reacting with the produced ROS in order to produce a response. Such a response could be, but is not limited to, a chemiluminometric, fluorescent or colormetric/chromogenic response. Thus, in one embodiment of the invention, the amount of reactive oxygen species (ROS) is determined by fluorometry or chemiluminescence.

Fluorometry utilizes the conversion of a non-fluorescent probe to a fluorescent product (fluorophore) upon reaction with ROS. When adding the non-fluorescent probe to the assay buffer and subsequently activating the cells by an inflammatory contaminant, the cells will produce an oxidative burst releasing ROS to the interior and exterior of the cell. The produced ROS will then react with the non-fluorescent probe converting it to a fluorophore. The fluorophore has the ability of absorbing light, converting the fluorophore to a higher energy state also called an excited state, this higher energy state can not be sustained for long resulting in an emission of light energy at a lower energy, thus longer wavelength than the absorbed light. The intensity of the emitted light subsequent to an excitation (measured by a fluorometer or FACS) will therefore provide a measurement of how much non-fluorescent probe has been converted to the fluorescent product, and thereby how much ROS has been produced by the cells.

The terms "fluorescent probe" or "fluorophore" are used interchangeably herein and refers to a compound that upon a chemical reaction with reactive oxygen species (ROS) is capable of emitting light when exitated. Non-limiting examples of fluorescent probes are hydroethidin, dihydrorhodamine such as 1,2,3 dihydrorhodamine, ADHP (lO-acetyl-3, 7 dihydroxyphenoxazine), T₁T- dichlorofluorescein diacetate (DCFH-DA) such as H₂DCFDA or carboxy-H₂DCFDA (5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate) and other commercially produced dyes such as but not limited to MitoSOX Red, a specific superoxide indicator. The fluorescent light emitted by the fluorescent probe can be measured by a method such as, but not limited to: FACS analysis or in a 5 fluorometer.

In one embodiment, the invention relates to a method wherein the amount of reactive oxygen species (ROS) produced by the cells of the invention upon exposure to the sample to be tested is measured by fluorometry. In a preferred embodiment, the invention relates to a method, wherein the amount of reactive

10 oxygen species (ROS) is measured by fluorometry by use of a fluorescent probe selected from the group consisting of hydroethidin, 1,2,3 dihydrorhodamine, ADHP (lO-Acetyl-3, 7-dihydroxyphenoxazine), H₂DCFDA (2',7'- dichlorodihydrofluorescein diacetate), carboxy-H₂DCFDA (5-(and 6-)carboxy-2',7'- dichlorodihydrofluorescein diacetate) and other commercial fluorescent probes,

15 such as but not limited to MitoSOX Red. In a specific preferred embodiment of the invention, the fluorescent probe is 1,2,3-dihydrorhodaminee, and the detection is conducted using a fluorometer.

In one embodiment of the invention, the concentration of the fluorescent probe is from 0.01-1000 μM in the assay buffer, such as e.g. from 0.01-500 μM in the

20 assay buffer, or such as e.g. from 0.01-100 μM in the assay buffer, or such as e.g. from 0.1-100 μM in the assay buffer, or such as e.g. from 0.5-50 μM in the assay buffer, or such as e.g. from 1-50 μM in the assay buffer, or such as e.g. from 1-40 μM in the assay buffer, or such as e.g. from 1-30 μM in the assay buffer, or such as e.g. from 1-20 μM in the assay buffer, or such as e.g. from 1-

25 10 μM in the assay buffer. In a specific preferred embodiment of the invention the concentration of the fluorescent probe is from 1-10 μM in the assay buffer.

In another embodiment of the invention, the concentration of the fluorescent probe is about 0.01 μM in the assay buffer, or about 0.1 μM in the assay buffer, or about 1 μM, or about 2 μM, or about 3 μM, or about 4 μM, or about 5 μM, or about 30 6 μM, or about 7 μM, or about 8 μM, or about 9 μM, or about 10 μM, or about 20 μM, or about 30 μM, or about 40 μM, or about 50 μM, or about 60 μM, or about 70 μM, or about 80 μM, or about 90 μM, or about 100 μM, or about 200 μM, or about 300 μM, or about 400 μM, or about 500 μM, or about 600 μM, or about 700 μM, or about 800 μM, or about 900 μM, or about 1000 μM. In a specific preferred embodiment of the invention the concentration of the fluorescent probe is about 6 μM in the assay buffer. The concentration of the fluorescent probe required depends on the individual probe used.

Chemiluminescence utilizes the reaction between a chemiluminometric probe and ROS to generate light. Normally, chemiluminescence involves the production of an excited species which goes on to release visible light (by emitting photons) in order to revert to its ground state energy. When adding the chemiluminometric probe to the assay buffer and subsequently activating the cells by inflammatory contaminants the cells will produce an oxidative burst releasing ROS to the interior and exterior of the cell. The produced ROS will then react with the chemiluminometric probe converting it to an exited species. The exited state of the chemiluminometric probe can not be substained for long and will, on return to the ground state release light. The intensity of the emitted light (measured by a luminometer) will therefore provide a measurement of how much chemiluminometric probe has been converted, and therefore how much ROS has been produced by the cell.

In another embodiment, the invention relates to a method, wherein the amount of reactive oxygen species (ROS) produced by the cells are measured by chemiluminescence. In a preferred embodiment, the invention relates to a method, wherein the amount of reactive oxygen species (ROS) produced by the cells are measured by chemiluminescence by use of a chemiluminescent probe.

The term "chemiluminescent probe" as used herein refers to a compound which is capable of emitting light upon a chemical reaction, such as upon reaction with reactive oxygen species, for example superoxide or hydrogen peroxide. Non- limiting examples of chemiluminescent probes are luminol, isoluminol, lucigenin or pholasin. Thus, in one embodiment of the invention, the amount of reactive oxygen species (ROS) produced by the cells are measured by chemiluminescence by use of a chemiluminescent probe selected from the group consisting of luminol, isoluminol, lucigenin and pholasin. In a specific preferred embodiment of the invention the chemiluminomescent probe is luminol.

In one embodiment of the invention, the concentration of the chemiluminescent probe is from 1-10.000 μM in the assay buffer, such as e.g. from 10-1000 μM in the assay buffer, or such as e.g. from 100-500 μM in the assay buffer, or such as e.g. from 150-350 μM in the assay buffer, or such as e.g. from 200-350 μM in the assay buffer. In a specific preferred embodiment of the invention the concentration of the chemiluminescent probe is from 250-320 μM in the assay buffer.

In another embodiment of the invention, the concentration of the chemiluminescent probe is about 1 μM in the assay buffer, about 10 μM, or about 100 μM, or about 200 μM, or about 300 μM, or about 400 μM, or about 500 μM, or about 600 μM, or about 700 μM, or about 800 μM, or about 900 μM, or about 1000 μM in the assay buffer. In a specific preferred embodiment of the invention the concentration of the chemiluminescent probe is about 283 μM in the assay buffer.

Variations of probes and concentration can be relevant for the invention, and can be modified or altered by a professional skilled within the art.

When applying chemiluminescence for measuring the amount of ROS produced by the cells of the invention, the chemiluminescent probe can be added to the mixture of the sample to be tested and the cells of the invention in step i) of the method of the present invention, i.e. the chemiluminescent probe can be added when the sample is exposed to the cells of the invention. The amount of reactive oxygen species (ROS) is determined by measuring the intensity of the emitted light from the chemiluminescent probe. The emitted light is measured by e.g. a luminometer.

When applying fluorometry for measuring the amount of ROS produced by the cells of the invention, the fluorescent probe can be added to the mixture of the sample to be tested and the cells of the invention in step i) of the method of the present invention, i.e. the fluorescent probe can be added when the sample is exposed to the cells of the invention. The amount of reactive oxygen species (ROS) is determined by measuring the intensity of the emitted light from the fluorescent probe by e.g. a fluorometer.

In one embodiment, the invention relates to a method, wherein the amount of reactive oxygen species (ROS) is measured continuously for 15-360 minutes after exposing the sample to the cells, such as e.g. for 30-360 minutes after exposing the sample to the cells, such as e.g. for 60-300 minutes after exposing the sample to the cells, such as e.g. for 90-240 minutes after exposing the sample to the cells, such as e.g. for 120-240 minutes after exposing the sample to the cells, such as e.g. for 150-210 minutes after exposing the sample to the cells, such as e.g. for 160-200 minutes after exposing the sample to the cells. This means that the measurement of the amount of ROS produced is started at time zero (when the sample is exposed to the cells of the invention, e.g. when the sample is mixed with the cells) and continued for 15-360 minutes, such as e.g. for 30-360 minutes, or such as e.g. for 60-300 minutes, or such as e.g. for 90-240 minutes, or such as e.g. for 120-240 minutes, or such as e.g. for 150-210 minutes, or such as e.g. for 160-200 minutes.

The amount of reactive oxygen species (ROS) can by measured continuously as outlined by e.g. chemiluminescence.

In another embodiment, the invention relates to a method, wherein the amount of reactive oxygen species (ROS) is measured continuously for a time period selected from the group consisting of about 120 minutes after exposing the sample to the cells, about 140 minutes after exposing the sample to the cells, about 160 minutes after exposing the sample to the cells and about 180 minutes after exposing the sample to the cells. This means that the measurement of the amount of ROS is started at time zero (when the sample is exposed to the cells of the invention, e.g. when the sample is mixed with the cells) and continued for about 120 minutes after exposing the sample to the cells, or about 140 minutes after exposing the sample to the cells, or about 160 minutes after exposing the sample to the cells or about 180 minutes after exposing the sample to the cells. In a preferred embodiment, the amount of reactive oxygen species (ROS) is measured continuously for a time period of about 180 minutes after exposing the sample to the cells. The term "continuously" as used herein is defined as a measurement every 1 second - 5 minutes, such as e.g. every 30 seconds - 4 minutes, such as e.g. 1 minute - 3 minutes, or a measurement about every 1 second, or about every 5 seconds, or about every 10 seconds, or about every 30 seconds, or about every 1 minute, or about every 2 minutes, or about every 3 minutes, or about every 4 minutes, or about every 5 minutes.

In one embodiment, the invention relates to a method, wherein the amount of reactive oxygen species (ROS) is measured by a single measurement 30-360 minutes after exposing the sample to the cells, such as e.g. 60-300 minutes after exposing the sample to the cells, such as e.g. 90-240 minutes after exposing the sample to the cells, such as e.g. 120-240 minutes after exposing the sample to the cells, such as e.g. 150-210 minutes after exposing the sample to the cells, such as e.g. 160-200 minutes after exposing the sample to the cells. In a preferred embodiment, the ROS is measured by a single measurement as outlined above by e.g. preferably by fluorometry.

In another embodiment, the invention relates to a method, wherein the amount of reactive oxygen species (ROS) is measured by a single measurement about 120 minutes after exposing the sample to the cells, or about 140 minutes after exposing the sample to the cells, about 160 minutes after exposing the sample to the cells or about 180 minutes after exposing the sample to the cells. The amount of reactive oxygen species (ROS) can be measured by e.g. chemiluminescence or fluorometry. In a preferred embodiment, the amount of reactive oxygen species (ROS) is measured by a single measurement about 180 minutes after exposing the sample to the cells. In a preferred embodiment, the ROS is measured by a single measurement as outlined above by e.g. fluorometry.

Data evaluation (step (Hi) of the invention)

The ROS production from the cells of the invention is measured in step (iii) of the invention as a function of time. Data obtained during the measuring period can be evaluated in respect to several parameters. The preferred and easiest quantifiable are: Area Under Curve, (AL)C) in the entire measuring period, peak height or onset time. However several others can be used. Data obtained can subsequently be analysed by a program as for instance Excel, GraphPad Prism, SigmaPlot and/or SigmaStat. Data should always be compared with the results obtained using a non-stimulated reference of equal treatment and volume.

From the response of the serial diluted "standard solution" of a known pyrogen, a standard curve is prepared. The response of a given test solution is then compared to the standard curve, in terms of e.g. peak height or AL)C, and the response can be quantified. The ROS-stimulatory activity of the test solution can then be converted into equivalents of the known pyrogen. Thus the response from a given test sample can be quantified in to "total immunogenic units" (TIU) equivalent to a given concentration of known pyrogen e.g. LPS or zymosan. It is important to know that this response does not necessarily refer to an in-vivo inflammatory effect of the test sample, however exists solely as a quantifiable term.

Qualitative determination

The method of the invention provides some knowledge as to which type of inflammatory contaminant that has been detected, based on the onset time of the ROS response. The onset time of the ROS response is concentration dependent (the higher concentration of pyrogen, the earlier onset time), however in relevant concentrations the differences are pronounced. Below some examples of the differences of fast activators (yeasts and yeast cell wall fragments (zymosan)), fast to intermediate activators (Gram-positive bacteria and LTA) and slow activators (Gram-negative bacteria and LPS) is shown. The response patterns to different inflammatory contaminants are likewise visualized in some of the figures.

Also the kinetic of the response can help determine the type of pyrogen, though no finally established pattern has been determined, some responses are bi- or triphasic while others are monophasic.

In order to optimize the quantitative detection of pyrogens potentially present in the sample, or to optimize qualitative determination of the pyrogens, the sample can be incubated (opsonized) with antibodies (as defined above) directed to one or more specific pyrogens. Opsonization with antibodies can be performed either alone or in combination with the hereinabove described plasma and/or plasma proteins. Opsonization for optimizing the quantitative pyrogen detection has been described above, cf. the section on "pre-treatment of the sample to be tested".

However, one or more antibodies directed to one or more specific pyrogens can also be added to the sample before exposing the sample to the cells in order to determine the type of the one or more pyrogens potentially present in the sample, i.e. qualitative determination of the pyrogens present in the sample. Thus, in one embodiment, the invention relates to a method further comprising adding one or more antibodies directed to one or more specific pyrogens to the sample before exposing the sample to the cells. Relevant antibodies are as defined above. The specific antibodies are preferable monoclonal, but can also (if they can be free from pyrogen contamination from the antigen the animal was initially immunised with) be polyclonal, chimeric and any combination of such antibodies. The antibodies must be able to activate human cells and are therefore preferably, but not limited to, of rabbit origin, since these antibodies in studies has proven to be compatible with the human Fc receptor. The antibody may be an IgM, IgG, IgA or another immunoglobulin or immunoglobulin fragment that can form an immune complex with the antigen and activate the aforementioned cells to an oxidant production.

The qualitative determination of the one or more pyrogens potentially present in the sample can be determined by the use of specific antibodies, as described above. Incubation of the sample with specific antibodies directed against specific conserved parts of various pyrogens will result in a more rapid (and most likely, more robust) response to the sample. Thus, a sample that has been tested positive for pyrogenic contamination by the method of the invention can be subsequently incubated with several different specific antibodies (but only one antibody per test) recognizing typically and specific parts of different types of pyrogens (e.g. antibody directed against e.g. LPS reveal Gram-negative bacterial contamination, an antibody directed against e.g. LTA reveal Gram-positive bacterial contamination, and an antibody directed against e.g. 1,3 β-glucan reveal fungal contamination etc.). Furthermore, when the method of the invention has detected e.g. a Gram-negative pyrogen another subset of antibodies could determine the precise origin of contamination using specific antibodies towards either the distinct genus (Salmonella, Escherichia or Campylobacter) or even species (e.g. Salmonella typhi, Salmonella typhimurium or Salmonella enteritidis. The principle is exemplified in Figure 7.

Use

The invention also relates to use of the method of the invention, wherein the sample is from a product for human or animal use or a raw material for the production of these. In a preferred embodiment, the product is a pharmaceutical composition, ingredient for pharmaceutical composition, infusion liquids such as e.g. a peritoneal dialysing fluid, a cosmetic product, a nutrient product such as but not limited to parenteral nutrition and materials for medical use. In another preferred embodiment, the sample is an environmental sample selected from the group consisting of air, soil or water.

Kit

In order to perform a test for presence of inflammatory contaminants a kit comprising the different components can be provided according to the present invention. When using the kit, the cells of the invention, mentioned hereinabove may either be provided as primary cells or a cell line needing culturing, sub- culturing and perhaps differentiation or the cells can be provided as cryo- preserved or lyophilised cells, ready to use within few minutes or hours, such as e.g. 1 min to 5 hours. Pyrogen-free reference solutions and suitable standards as well as cell media and buffers can also be provided enabling the buyer to conduct the entire assay within 4-6 hours if cell material has been prepared. Furthermore, the kit can provide additional reagents such as: differentiating agents, priming agents, ROS reactive chemiluminogenic and/or fluorescent probes, membrane disruptors, opsonins, plasma or isolated components hereof, specific antibodies, ultra- and/or membrane filters and various materials such as plates for conducting the assay, and other pyrogen free plastic components.

Cells of the kit can be of any origin mentioned hereinabove. If differentiation of the cells is necessary in the method of the invention, the cells provided with the kit can either be terminally differentiated and e.g. be cryopreserved or lyophilised or the cells can be non-differentiated. In the latter case the kit comprises one or more differentiating agents.

Thus, in one embodiment the kit of the invention comprises cryopreserved or lyophilised differentiated HL-60 cells. The cells are preserved by deep freezing in a suitable medium and are to be thawed before use.

In another embodiment, the invention relates to a kit further comprising one or more differentiating agents as defined above. In yet another embodiment, the invention relates to a kit further comprising one or more priming agents as defined above. In a further embodiment, the invention relates to a kit further comprising one or more envelope disruptors as defined above. In yet a further embodiment, the invention relates to a kit further comprising of an opsonin e.g. plasma or plasma components as described herein above. In yet a further embodiment, the invention relates to a kit further comprising one or more specific antibodies directed to one or more specific pyrogens, as defined above. The kit may also comprise an ultrafilter e.g a teflon filter or a cellulose filter such as e.g. a cellulose triacetate filter with a cut off of e.g. 20 000 Da. or a membrane filter such as a Teflon filter with a pore size of e.g. 0,2 μm. The kit may also comprise a plate suitable for conducting the assay. In one embodiment, the invention relates to a kit, wherein the cells are originating from a HL-60 cell line or variants thereof. In another embodiment, the invention relates to a kit, wherein the cells are differentiated with one or more differentiating agents capable of increasing the ability of the cells to produce ROS. The differentiating agents can be selected from the group consisting of all trans retinoic acid (ATRA), 9-cis retinoic acid, dimethylformamide (DMF), Dimethylsulphoxide (DMSO), dibutyryl cyclic AMP and combinations thereof. In a preferred embodiment, the differentiating agent is all-trans retinoic acid (ATRA). In another preferred embodiment, the cells are differentiated with one or more differentiating agents for a period of 2-12 days, such as e.g. 4-8 days, or such as e.g. 6-8 days, or such as e.g. 7 days.

In a further embodiment, the invention relates to a kit, wherein the cells are preserved after differentiation of the cells. The differentiated cells can be preserved by cryopreservation or lyophilisation, as outlined in details above.

In a preferred embodiment, the invention relates a kit for performing the method of the invention comprising :

(i) cryopreserved differentiated HL-60 cells, and

(ii) a chemiluminescent ROS reporter probe, and/or

(iii) a fluorescent ROS reporter probe,

wherein the HL-60 cells are differentiated according to the invention, and wherein the differentiated HL-60 cells are cryopreserved by methods known in the art.

In one embodiment, the differentiated HL-60 cells are cryopreserved in a cryopreservation solution comprising about 10% DMSO and 20% foetal calf serum. In another embodiment, the differentiated HL-60 cells are differentiated with ATRA for a period of 2-12 day, such as e.g. 6-8 days, or such as 7 days.

In another preferred embodiment, the invention relates a kit for performing the method of the invention comprising :

(iv) lyophilised differentiated HL-60 cells, and

(v) a chemiluminescent ROS reporter probe, and/or (vi) a fluorescent ROS reporter probe,

wherein the HL-60 cells are differentiated according to the invention, and wherein the differentiated HL-60 cells are lyophilised by methods known in the art. In another embodiment, the lyophilised differentiated HL-60 cells are differentiated with ATRA for a period of 2-12 day, such as e.g. 6-8 days, or such as 7 days.

In vitro assay by Timm et al.

Timm et al. 2006 describes an in vitro assay for detection of microorganisms and related substances utilizing HL-60 cells for chemiluminescence. The assay is based upon the production of reactive oxygen species (ROS) by differentiated HL-60 cells measured by luminol-enhanced chemiluminescence. The assay is capable of measuring the presence of a wide variety of microorganisms including yeast cells, however endospores are not mentioned. Several detection limits are described, e.g. LPS (100 pg/ml) and Gram-negative bacteria, S.typhimurium (10⁶ bac/ml). The article Timm et al. (2006) Eur.J.Pharm.Sci. 27.2-3:252-58 is hereby incorporated by reference.

In Timm et al. 2006 a new pyrogen assay using the human leukemia cell line HL- 60 is described. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to Salmonella typhimurium but displays good sensitivity towards Bacillus subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts Candida albicans and Saccharomyces cerevisiae is considerably better than obtained in other in-vitro cell systems. Overall these results indicate that the HL- 60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine. In Timm et al 2006 the possibility of establishing a fast and sensitive assay, capable of detecting microorganisms and related substances, is examined. The assay is based upon differentiated HL-60 cells production of ROS measured by luminol enhanced chemiluminescence. Apart from examining the sensitivity to bacteria and bacterial cell wall components, the sensitivity of the assay towards yeasts is examined since these have proven complicated to detect in other in-vitro pyrogen cell assays.

Materials and methods

Reagents

Candida albicans ATCC 10231, Salmonella typhimurium ATCC 14028 and Bacillus subtilis ATCC 6633 were purchased from OXOID, GB. Saccharomyces cerevisiae ATCC 9763 was kindly provided by Abeline Christensen (Novo Nordisk, Denmark). LPS prepared from Escherichia coli 055 :B5 (Bio-Whittaker, Walkersville, Maryland, USA) and LTA isolated from Bacillus subtilis (Sigma Chemical CO) were used. Both LPS and LTA were reconstituted in Hanks Balanced Salt Solution (HBSS) with CaCI₂ and MgCI₂ (GIBCO™ Invitrogen, Carlsbad, California, USA). The same batch of HBSS with CaCI₂ and MgCI₂ was used throughout the experiments and will subsequently only be denoted HBSS. Human plasma was obtained by centrifugation (1000 x g, 20 min) of whole blood collected from healthy volunteers and stored at -20 ⁰C until use.

Maintenance of cell line and differentiation procedure

The human promyelocytic leukaemia cell line HL-60 (ATCC, CCL-240) had undergone 10 passages after acquisition from ATCC. The cells were used up to 10 weeks after thawing and underwent passage twice a week. The cells were maintained in growth medium: RPMI 1640 (cat. no. : 01-106-1A, Biological industries, kibbutz beit haemek, Israel), supplemented with 10 % heat inactivated fetal bovine serum (cat. no. : 04-001-lA, Biological industries, kibbutz beit haemek, Isreal), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine (Sigma-Aldrich, St. Louis, USA). The cells were incubated in humidified atmosphere containing 5 % CO2 and 95 % air at 37 ⁰C. The cells were seeded at Ix IO⁵ cells/ml and kept under 10⁶ cells/ml throughout their use. To introduce differentiation along the granulocytic pathway, cells were seeded at 3x lO⁵ cells/ml in growth medium supplemented with 1 μM all-trans retinoic acid (ATRA) (Fluka, Fluka and Riedel-de Haen, Sigma-Aldrich St. Louis, USA). The cells were allowed to differentiate for 6-7 days without any replacement of growth medium. The 6-7 days differentiation period was determined as optimal for ROS production upon pyrogen challenge (data not shown).

Preparation of microorganisms

C. albicans was grown at 37 ⁰C overnight in RPMI 1640, centrifuged (2000 x g, 10 min) and subsequently washed twice with HBSS. The yeasts were resuspended in HBSS and the suspension was standardized to 10⁷ yeasts/ml using a Bϋrker-Tϋrk chamber.

S. cerevisiae was grown at 37 ⁰C overnight in Sabouraud Dextrose agar, centrifuged (2000 x g, 10 min) and subsequently washed twice with HBSS. The yeasts were resuspended in HBSS and the suspension was standardized to 10⁷ yeasts/ml using a Bϋrker-Tϋrk chamber. Both suspensions were prepared immediately before the experiments were conducted, and were exposed to L)V irradiation for one minute to inactivate the yeasts.

S. typhimurium and B. subtilis were incubated at 37 ⁰C in RPMI 1640 and harvested after 24 h. The bacteria were isolated by centrifugation (2000 x g, 10 min) and washed twice with HBSS. The bacteria were resuspended at 10⁸ bacteria/ml in HBSS and exposed to L)V irradiation for one minute to kill the bacteria. No cfu were detected after spreading and incubation of 0.1 ml of the irradiated suspension on TGY plates. The bacteria were standardized by spectrophotometry (OD ₄₅₀) to 10⁷ bacteria/ml. B. subtilis: OD₄₅₀ = 1 equals IxIO⁸ bacteria/ml; dry weight = 2.4xlO^"12 g/bacteria. S. typhimurium: OD₄₅₀ = 1 equals 8xlO⁸ bacteria/ml; dry weight = 2.3xlO^"13 g/bacteria.

Preparation of differentiated HL 60 cells

The differentiated HL-60 cell culture was centrifuged (125 x g, 10 min) and the pellet washed once in preheated (37 ⁰C) HBSS. The cells were resuspended in preheated HBSS and standardized to 10⁷ cells/ml using a Bϋrker-Tϋrk chamber.

Chemiluminescence measurements Chemiluminescence was measured with a 96 well ORION II Microplate luminometer (Berthold Detection Systems, Pforzheim, Germany) at 37 ⁰C using a polystyrene LumiNunc™ 96 Well Plate, (Nunc, Roskilde, Denmark). Each well contained a final volume of 200 μl. Prior to measurements a reaction mixture was prepared in each well containing a final concentration of 5xlO⁵ differentiated HL- 60 cells/well, 283 μM luminol (Across Organics, New Jersey, USA) and 2.5 % human plasma. The reaction mixture was supplemented with HBSS to a volume of 100 μl and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of 100 μl test solution. The microplate was then read for 1 s/well with a measure/delay- repeat cycle of 99. Due to software configurations, this cycle enables the plate to be read approximately once a minute for 104 minutes. The inter-well and inter- day variations were determined by activation with 100 μg/ml zymosan, a substance that for many years has served as a model for microbial activation of the innate immune system. The average inter-well CV % was determined to be 3.66 % (n=6, average CV % of 5 experiments each examining variations for 6 wells) and the inter-day CV % was determined to be 12.4 % (n = 5, average AL)C for 6 wells each day).

Statistical analysis

Statistical analysis was carried out using Wilcoxon signed rank test. Unless otherwise stated the results are displayed as median with error bars displaying the 25th and 75th percentiles. p<0.05 is considered significant and indicated with " * " when obtained.

Results

The assay was conducted in accordance to materials and methods using six different kinds of pyrogens. After addition of all-trans retinoic acid (ATRA) differentiated HL-60 cells to the 96 well micro plate containing plasma and luminol the plate is incubated at 37 ⁰C in order to temperature equilibrate for 15 min. Afterwards the plate is placed in the thermostat-regulated luminometer (37 ⁰C) and the preheated (37 ⁰C) test solution or control solution (HBSS) added. Cells "stimulated" with control solution (HBSS) will subsequently be denoted "non- stimulated cells". Closed triangles in fig. 9. a. displays the light emission evoked by addition of a test solution containing Candida albicans (10⁶ yeasts/ml), in comparison open circles shows the light emission from non-stimulated cells. As displayed, the ROS production and subsequent light emission due to the reaction with luminol, is rapid and obtains maximal RLL) value after 15 min. This however is not always the case, and other stimulatory substances can show a prolonged time to obtain the maximal RLL) value and display different kinetics. Fig. 9.b. displays the response from LTA stimulation that also generates a rapid response but displays an almost bi-phasic response with peaks at both 15 and 60 min. Stimulation with LPS displays a late response with no significant ROS production until 15-30 min after addition of test solution (data not shown). Because various pyrogens display different kinetics, the preferred parameter for quantification of chemiluminescent time profile measurements is chosen to be the area under the curve (AL)C) for the entire measuring period.

Stimulation of differentiated HL-60 cells with S. typhimurium and B. subtilis

As an example of prokaryotic challenge of differentiated HL-60 cells, the gram- negative S. typhimurium and the gram-positive B. subtilis were used as test solutions in concentrations of 10⁴ - 10⁶ bacteria/ml. Fig. 10. a. and 10. b. show the results of stimulation of differentiated HL-60 cells with S. typhimurium and B. subtilis, respectively. As seen in fig. 10. a. only stimulation with 10⁶ bacteria/ml of S. typhimurium resulted in activation of the differentiated HL-60 cells to produce a significant chemiluminometric response compared to non-stimulated cells (n=8, p<0.05). Challenge with B. subtilis (fig. 10. b.) results in a concentration dependent increase in the chemiluminometric response in the tested concentrations 10⁴ - 10⁶ bacteria/ml. All responses were significantly different from the ROS release of non-stimulated cells (n=8, p<0.05).

Stimulation of differentiated HL-60 cells with S. cerevisiae and C. albicans

As an example of eukaryotic challenge, the differentiated HL-60 cells were exposed to S. cerevisiae and C. albicans. Both yeasts were used in concentrations of 10⁴ - 10⁶ yeasts/ml. Fig. 11. a. and 11. b. depict the results of S. cerevisiae and C. albicans challenge. A concentration dependent increase in the chemiluminometric response was observed in the range 10⁴ - 10⁶ yeasts/ml and all concentrations showed a ROS release significantly different from that of non- stimulated cells (n=8, p<0.05).

Stimulation of differentiated HL-60 cells with LPS and LTA Fig 12. a. and 12. b. depict the challenge of differentiated HL-60 cells with cell wall components. LPS and LTA were used as test solutions in concentrations of 10² - 10⁴ pg/ml and 10⁴ - 10⁶ pg/ml, respectively. At all concentrations the response was significantly different from the ROS generation from non-stimulated cells, and the response displayed concentration dependence in the examined interval.

Discussion

As shown, the HL-60 chemiluminescence assay is capable of detecting the gram- positive bacteria B. subtilis in a concentration dependent manner in the interval 10⁴-10⁶ bacteria/ml. This provides a detection of 24 ng/ml B. subtilis, which is in proximity of the Mono Mac 6 detection limit of 5 ng/ml (Moesby et al., 2003) and better than the detection limit of the rabbit pyrogen test shown to be 120 μg/kg (Himanen et al. 1993). If the maximum administered volume to the pyrogen rabbit is used (10 ml/kg, Ph. Eur. 5th Ed.) this result in a detection limit of 12 μg/ml. S. typhimurium only induced a significant response at 10⁶ bacteria/ml. The Mono Mac 6 assay however provides a much lower detection limit, calculated to l.δxlO² bacteria/ml (Moesby et al. 1999). The reason for the difference between the responses to the two bacteria has not been further elucidated. The possibility that a general difference in the detection limits of gram-positive and gram- negative bacteria exists, is currently being examined.

Eukaryotic cells such as yeasts and moulds have previously shown difficult to detect in cell assays. The Mono Mac 6 assay fails to detect 10⁶ C.

(Moesby et al., 1999). The HL-60 chemiluminescence assay however detects 10⁴- 10⁶ yeasts/ml of both C. albicans and S. cerevisiae in a concentration dependent manner. Furthermore, the responses to the yeasts display higher AL)C values than any of the other test substances in the respective concentrations. These results are promising, since fungi represent a frequent contaminant of raw materials and furthermore because tests for pyrogens in recombinant drugs derived from yeast expression systems also have to be considered (Hartung et al. (2001) The Report and Recommendation of ECVAM workshop 43).

The bacterial components LPS and LTA also led to significant activation of the differentiated HL-60 cells. LPS could be detected in a concentration of 100 pg/ml, which is lower than the detection limit obtained in other studies using the rabbit pyrogen test (Hansen and Christensen 1990). LTA is pyrogenic in rabbits in a dose of 3 μg/kg (Moesby et al., 2003) with the maximum administered volume of 10 ml/kg rabbit this equals a detection limit of 300 ng/ml. As shown, the HL-60 cell assay detects 10 ng/ml LTA. This provides an increased sensitivity compared to the rabbit pyrogen test, and sensitivity similar to the Mono Mac 6 assay (Moesby et al., 2003). Others have shown that commercially produced LTA preparations can be contaminated with LPS and therefore give false positive response to LTA. However the kinetics of the LTA response in the HL-60 cell assay (peaks at 15 and 60 min) differ from the one observed with LPS stimulation (no significant increase ROS production until after 15-30 min) therefore it is probably not a LPS contamination that facilitates the peak response at 15 min, however elaborating studies must be preformed to render the results conclusive.

The sensitivity of the differentiated HL-60 cells towards LPS however leaves somewhat of a conundrum. The assay seems highly sensitive towards LPS from E. coli but is almost insensitive towards LPS from S. typhimurium. The E. coli contains about 3.4 % LPS so if the same percentile is used for S. typhimurium the failure to detect 10⁵ S. typhimurium/vr\\ seems strange since there should be approximately 800 pg LPS/ml present. However, the possibility that the bacterial cell wall components could "hide" the highly active lipid A part of LPS, or that LPS from S. typhimurium is less reactive than the used E. coli LPS cannot be excluded. Prior work has shown that ultrasonication can increase the sensitivity of the Mono Mac 6 assay towards some pyrogens (Moesby et al. 2000). It is therefore speculated if this procedure also can improve the sensitivity of the HL-60 cell assay.

A previous study has shown that the HL-60 cells only express low levels of CD14 in the membrane (Trayner et al. 1998) and since CD14 is generally recognized as the main receptor for LPS this also rise a question of how the recognition takes place. It seems likely that plasma proteins participate in the recognition of pyrogens probably via complement opsonization and other plasma proteins such as soluble CD14 (sCD14), lipopolysaccharide binding protein (LBP) etc. as shown for the recognition of LPS. Studies on PMN's show that opsonization is important and occasionally essential for e.g. zymosan recognition and it therefore seems likely that opsonization also facilitate recognition of pyrogens in the HL-60 cell assay. However it seems that the recognition of different pyrogens occur through different pathways since the response to some pyrogens seems bi-phasic and some mono-phasic. This could also help to explain the variations in the time before significant ROS release, possible indicating either rapid or slow pyrogen recognition. The HL-60 cell interaction with the pyrogen also seems relevant since it has not been further elucidated whether the activation and subsequent ROS production happens dependently or independently of phagocytosis.

Concluding remarks

This study shows that the HL-60 cell assay is capable of detecting various pyrogenic substances. The assay is fast and can provide results in less than 3 hours, preferably less than 2 hours. The ATRA differentiated HL-60 cells respond to the pyrogenic challenge with a respiratory burst generating a concentration dependent production of ROS, which can be measured using luminol enhanced chemiluminescence. The results show that the assay responds well to both prokaryotic and eukaryotic challenges as well as cell wall fragments from prokaryotes. The HL-60 cell assay therefore provides an assay that can be used to detect both yeasts and bacterial cell wall fragments. The response to LPS and LTA show that the assay has reduced sensitivity towards LPS compared to the Mono Mac 6 assay, but similar sensitivity towards LTA. Furthermore, the HL-60 assay displays better sensitivity than the rabbit pyrogen test to all the tested organisms and cell wall fragments with the exception of S. typhimurium. The lack of sensitivity towards the gram-negative bacteria S. typhimurium is a limitation of the assay, and the test is therefore in its current design, not suitable as a standalone test for pyrogens. However the good sensitivity towards the yeasts and LTA indicates that a combination of in-vitro human cell line assays possible could provide a detection diversity precluding the need for pyrogen testing on laboratory animals. However, validation, establishment of detection limits and using studies addressing the likely matrix influence of pharmaceutical preparations is necessary for the HL-60 cell assay. However, since the assay is fast and easy to conduct, it is relevant to examine whether or not the assay can be used as an "in-process" pyrogen control for pharmaceutical preparations. It also seems relevant to examine the use of priming agents in order to further improve the sensitivity of the HL-60 cell assay. The following examples of certain preferred embodiments of the invention are meant to be merely illustrative of non-limiting methods for carrying out the present invention.

EXAMPLES

Example 1

ROS production of ATRA differentiated HL-60 cells stimulated with zymosan in a luminol enhanced chemiluminometric assay. The ROS production as a function of time is illustrated in figure 1.

The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 7 days in an initial flask concentration of 3x lO⁵ cells/ml without any replacement of growth medium in the differentiation period. In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to a centrifuge tube and centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and the cell suspension is adjusted to 10⁷ cells/ml. 50 μl of cell suspension is added to each well giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of 100 μl "test solution" (100 μg/ml zymosan) or reference solution (pyrogen free HBSS). The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

As seen the addition of test solution containing zymosan activates the cells to produce ROS Example 2

ROS production from HL-60 cells differentiated with ATRA for 4-8 days. The differences in ROS production is illustrated in figure 2.

The HL-60 cells used in this experiment have been differentiated in different flasks 5 by lμM ATRA for 4, 5, 6, 7 and 8 days respectively in an initial flask concentration of 3x lO⁵ cells/ml without any replacement of growth medium in the differentiation period. In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to a 96-WeII Plate. The volume is adjusted to

10 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to separate centrifuge tubes and centrifuged at 125xg for 10 min, the supernatants are discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10

15 min, the supernatants are discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and the cell suspensions are adjusted to 10⁷ cells/ml. On the 96-well plate a row of wells is selected to conduct the experiment with the 4 days differentiated cells, another for the 5 day differentiated cells and so on. From the separate centrifuge tube containing the

20 differentiated cells, 50 μl of cell suspension is added to the designated wells giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of 100 μl "test solution" (100 μg/ml zymosan) or reference solution (pyrogen free HBSS). The plate is then immediately placed in the luminometer for plate reading

25 following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

The top graph in Figure 2 depicts the ROS response of zymosan-stimulated HL-60 cells differentiated with 1 μM ATRA for 4, 5, 6, 7, and 8 days respectively, and the bottom graph in Figure 2 depicts the ROS response from non-stimulated HL-60 30 cells differentiated with 1 μM ATRA for 4, 5, 6, 7, and 8 days respectively. Example 3

ROS production (quantified as luminol enhanced chemiluminescence) of GM-CSF primed and non-primed cells. The difference in ROS production between primed and non-primed cells is illustrated in figure 3.

The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 7 days in an initial flask concentration of 3xlO⁵ cells/ml without any replacement of growth medium in the differentiation period. 4 hours before the assay is conducted the cells are divided in to two separate flasks. The one flask is supplemented with GM-CSF to obtain a flask concentration of 25 ng/ml. The flasks are then incubated for additional 4 hours. In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to separate centrifuge tubes and centrifuged at 125xg for 10 min, the supernatants are discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatants discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and the cell suspensions are adjusted to 10⁷ cells/ml. On the 96-well plate a row of wells is selected to conduct the experiment with the non-primed differentiated cells, another for the primed differentiated cells. From the separate centrifuge tube containing the non-primed and primed differentiated cells respectively, 50 μl of cell suspension is added to the designated wells giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of 100 μl "test solution" (100 μg/ml zymosan) or reference solution (pyrogen free HBSS). The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

As seen the peak height and AL)C of the response are enhanced when measured using primed cells. Example 4

The effect of plasma in the assay buffer reduces the ROS background signal, improves pyrogen detection limits, and reduces onset time of the ROS response. The difference in the ROS production between a plasma supplemented and non plasma supplemented assay is illustrated in figure 4.

The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 7 days in an initial flask concentration of 3x lO⁵ cells/ml without any replacement of growth medium in the differentiation period. In a clean environment (e.g. laminar air flow unit) a reaction mixture either comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) or comprising only of luminol (final concentration of 283 μM) are added to designated wells in a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to a centrifuge tube and centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and the cell suspension is adjusted to 10⁷ cells/ml. 50 μl of cell suspension is added to each well giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of 100 μl "test solution" (100 μg/ml zymosan) or reference solution (pyrogen free HBSS). The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

As seen the addition of plasma to the assay decreases onset time (top graph) and reduces background response (bottom graph).

Example 5

Pre treatment of a sample with EDTA can improve detection limits. The differences in ROS production to a sample of S. typhimurium either pre-treated with EDTA or not pre-treated with EDTA is illustrated in figure 5. The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 7 days in an initial flask concentration of 3xlO⁵ cells/ml without any replacement of growth medium in the differentiation period. In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to designated wells in a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to a centrifuge tube and centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in pre-warmed (37°C) HBSS. The cells are then counted and the cell suspension is adjusted to 10⁷ cells/ml. 50 μl of cell suspension is added to each well giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of etiher 100 μl reference solution (pyrogen free HBSS), 100 μl "test solution 1" (comprising of a sample of S. typhimurium 10⁴ bacteria/ml) or 100 μl "test solution 2" (comprising of a sample prepared by treating a suspension of S. typhimurium with a EDTA/tris solution, final S. typhimurium concentration of 10⁴ bacteria/ml) The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

As seen the EDTA pre-treatment allow detection of 10⁴ bacteria/ml (S. typhimurium) while a similar setup without pre-treatment can not detect the same concentration.

Example 6

This example shows the time frame necessary for the present invention. The differences in ROS production to a sample of S. typhimurium and a non- stimulated sample is illustrated in figure 6. Top graph illustrate ROS production from 0-105 min. Bottom graph illustrate ROS production from 0-180 min.

The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 7 days in an initial flask concentration of 3x lO⁵ cells/ml without any replacement of growth medium in the differentiation period. In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to designated wells in a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to a centrifuge tube and centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and the cell suspension is adjusted to 10⁷ cells/ml. 50 μl of cell suspension is added to each well giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of etiher 100 μl reference solution (pyrogen free HBSS) or 100 μl "test solution 1" (S. typhimurium 10⁵ bacteria/ml) The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

As seen detection of S. thyphimurium inflammatory contaminant can at low concentrations be initiated very late. The example shows that before the 105 min, no differences in the ROS production of the non-stimulated and the stimulated cells can be detected. However beyond this time a slow increase of ROS production commences leading to a positive detection at the 180 min.

Inflammatory contaminants derived from gram negative bacteria may in many cases be difficult to detect when present in low concentrations. According to the present invention however, even low concentrations of S. typhimurium inflammatory contaminant can be detected after about 180 minutes.

Example 7

The method of the present invention can be used to detect an environmental contaminate. The differences in ROS production between a non-pyrogenic sample and an air sample from an outhouse is illustrated in figure 8.

A sample from an outhouse is acquired by filtering approximately 500 I air through a membrane filter. Contaminants on the filter is then desorbed in pyrogen free HBSS by vigorous shaking. The HBSS containing the possible contaminant from the filter is then designated "test solution 1". The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 7 days in an initial flask concentration of 3x lO⁵ cells/ml without any replacement of growth medium in the differentiation period. In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to designated wells in a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The ATRA differentiated HL-60 cells are transferred to a centrifuge tube and centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and the cell suspension is adjusted to 10⁷ cells/ml. 50 μl of cell suspension is added to each well giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of either 100 μl reference solution of equal volume and treatment (pyrogen free HBSS) or 100 μl "test solution 1" (possible contaminant containing HBSS from desorbed filter) The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 180 min.

As seen a reaction occurs after the 105 min indicating presence of one or more inflammatory contaminants in the air sample from the outhouse. Due to the late on-set of the response the contamination was speculated to most likely be largely endotoxin. This assumption was later confirmed by a positive LAL test.

Example 8

HL-60 assay

Definitions:

Supplemented RPMI=growth medium:

RPMI 1640 supplemented with 10% heat inactivated foetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2mM glutamine (must be sterile and pyrogen free) In the assay a white, flat- bottomed, sterile, pyrogen free, polystyrene 96-WeII Plate is used. However utilization of a round- or V-bottomed well-plate with fewer of more wells, in any colour, transparency, or material is within the scope of the present invention.

All cell work; culturing, sub culturing, differentiation and assay procedures are carried out under strictly sterile and pyrogen free conditions. All laboratory utensils must be pyrogen free and all cell flasks must be sterile, non pyrogenic filter-capped.

Start up of the cell line

The Cryopreserved human promyelocytic leukaemia cell line HL-60 (ATCC, CCL- 240) is quickly thawed, under strictly sterile and pyrogen free conditions, by addition of 10 ml of pre warmed (37°C) supplemented RPMI, the cell suspension is then centrifuged at 125xg for 10 min and the supernatant discarded, the pellet is resuspended in 5 ml supplemented RPMI, and placed in a sterile, non pyrogenic filter-capped, 50 ml cell flask. The cells are placed in a humidified atmosphere containing 5% CO₂ and 95% air at 37°C. After the first day additional 5 ml of supplemented RPMI is added. When the cells are at a concentration between 5x lO⁵ -10⁶ cells/ml, they are sub cultured.

Sub culturing of cells

The subculturing of the stable cell line takes place twice a week. To sub culture, the cell suspension is centrifuged at 125xg for 10 min, the supernatant discarded and the cells resuspended in an appropriate amount of supplemented (pre warmed) RPMI. The cells are counted in a Bϋrger-Tϋrk chamber and seeded for continuously differentiation and/or reseeded for sub culturing where the cells are seeded at Ix IO⁵ cells/ml. The cells are maintained in growth medium and incubated in humidified atmosphere containing 5% CO2 and 95% air at 37°C.

Continuous differentiation.

In order to continuously provide differentiated cells, a flask is set up at a cell concentration of 4x lO⁵ cells/ml, every day half the volume of the flask is drawn for differentiation and the flask is reconstituted with the same amount of fresh supplemented (pre warmed) RPMI as was drawn. This procedure is maintained for one week after which the procedure is terminated by differentiation of the entire flask content, in order to reduce accumulation of by-products.

To introduce differentiation along the granulocytic pathway, the cell suspension from the above mentioned flask is centrifuged at 125xg for 10 min, the supernatant discarded and the cells resuspended in an appropriate amount of supplemented (pre warmed) RPMI. The cells are counted using a Bϋrger-Tϋrk chamber and seeded in a new flask in a concentration of 3x lO⁵ cells/ml in (pre warmed) growth medium supplemented with 1 μM ATRA (all-trans retinoic acid) (stock solution of ATRA is 1 mM in DMSO). The cells are allowed to differentiate for 7 days without any replacement of growth medium.

Assay procedure

In a clean environment (e.g. laminar air flow unit) a sterile, pyrogen free white polystyrene 96-WeII Plate is unwrapped, and to each well to which test or control solution is subsequently added, plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells.

Cells that have undergone differentiation for 7 days are transferred to a centrifuge tube and centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently washed with appropriate quantities of pre warmed (37°C) HBSS (we use 5 ml of HBSS per 10-15 ml cell suspension). The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in an appropriate volume of pre warmed (37°C) HBSS (we use proximally 500 μl/5ml washed cell suspension) the cells are then counted using a Bϋrger-Tϋrk chamber, and the cell suspension is adjusted to 10⁷ cells/ml.

(attention: cells will sediment if not used immediately! Therefore gentle mixing of the cell suspension can be necessary in order to ensure a homogenous cell suspension) 50 μl of cell suspension is added to each well giving a final concentration of 5x lO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of 100 μl test, standard or reference solution.

Preparation of reference/standard/test solution Reference solution is a non-pyrogenic sample as for instance, pyrogen free and sterile HBSS.

Standard solution is prepared by serial dilution of a know pyrogen e.g. LPS in HBSS.

The test/standard solution is preferably diluted/reconstituted/dissolved in pre warmed HBSS (37°C), but must otherwise equilibrated at 37 ⁰C for 15-30 min.

To optimize detection, the test solution can be treated with, one or more types of, detergent, organic solvent, enzymes or other type of membrane/envelope disruptor such as, but not limited to, EDTA, chloroform, SDS or Triton-X prior to addition.

To optimize detection, test solution can be ultrasonicated prior to addition.

To remove any cell toxic detergents, interfering substances or matrix components from the test sample, the test can be ultra filtrated in an ultra filter with a low molecular weight cut of, (eg. 1.000-20.000 kD), and the detained test debris can then be reconstituted in appropriate quantities of pre warmed HBSS.

To improve "sterility/pyrogenecity assurance level" the test sample can be concentrate by means of ultra filtration in an ultra filter with a low molecular weight cut of, (eg. 5.000-20.000 kD). The detained test debris can then be reconstituted in appropriate quantities of pre warmed HBSS.

To optimize detection, test substance can be incubated (opsonized) in the presence of plasma, or isolated/recombinant plasma proteins as, but not limited to, complement proteins or acute phase reactants.

To optimize detection, or to induce a qualitative marker in the test, the test substance can be incubated (opsonized) in the presence specific antibodies.

When the test or control solution has been prepared, and the cells have temperature equilibrated for the designated time, the test/control solution is added to the 96 well micro plate and the plate is immediately placed in the luminometer for plate reading.

Preferably all tests are run as duplicates or triplicates. Plate reading

The micro plate is transferred to a pre warmed temperature controlled (37°C) luminometer (we use an Orion II, Berthold detectionsystems), and the assay is started immediately. The software settings for the luminometer should allow the plate to be read for example, but not limited to, 1 s/well with a measure/delay cycle allowing each well to be read every 2nd minute. The repeat cycle should be no less than 90 times, allowing the plate to be read for a proximally 3 hours or more.

Data analysis

Data obtained during the measuring period (preferably 3 hours or more) can be evaluated in respect to several parameters. The preferred and easiest quantifiable are: Area Under Curve, (AL)C) in the entire measuring period, peak height or onset time. However several others can be used.

Data should always be compared with the results obtained using a non-stimulated reference of equal treatment and volume.

From the response of the serial diluted "standard solution" of a known pyrogen, a standard curve is prepared. The response of a given test solution is then compared to the standard curve, in terms of e.g. peak height or AL)C, and the response can be quantified. The ROS-stimulatory activity of the test solution can then be converted into equivalents of the know pyrogen. Thus the response from a given test sample can be quantified in to "total immunogenic units" (TIU) equivalent to a given concentration of known pyrogen e.g. LPS or zymosan. It is important to know that this response do not necessarily refer to an in-vivo immunogenic effect of the test sample, however exists solely as a quantifiable term.

Test for matϊx interference in a new test substance

In order to test if there is an attenuation of the response due to matrix components in the test solution such as for instance excess EDTA or azide, two parallel tests are conducted. The first sample comprises of a known concentration of a pyrogenic compound added to a well in the microtiter plate. To another well the test sample is added and then spiked with an equal concentration of the aforementioned known pyrogen. If the matrix components interfere with the assay, the response of the spiked test sample will be of lesser magnitude than the directly stimulated. In such conditions ultra filtration of the test solution can be necessary.

Alternatively the test substance can be ultrafiltrated, and two parallel tests conducted. The first sample comprise of a known concentration of a pyrogenic compound added to a well in the microtiter plate. To another well the eluent of the ultrafiltrated test sample is added and then spiked with an equal concentration of the aforementioned known pyrogen. If the matrix of the test sample gives rise to either an attenuation or amplification of the response, this will be revealed by a significant difference in the results obtained from the two experiments.

Below are shown experimental data of positive detections by the method of the present invention. Some results are mean of several experiments others of a single experiment. Data are compared with the response of a reference sample (comprising of pyrogen free HBSS) of equal volume and treatment.

* All positive detections by the method of the present invention relates to the concentration of the pyrogenic sample, and not the concentration in the well. The well concentration of the pyrogen is only half the one listed since all samples are mixed 1 : 1 with cell containing assay mixture.

Example 9

ROS production of ATRA differentiated HL-60 cells which have been 5 cryopreserved, reconstituted from cryopreservation and immediately after thawing been stimulated with zymosan and LPS respectively in a luminol enhanced chemiluminometric assay. The ROS production as a function of time is illustrated in figures 13-15.

The HL-60 cells used in this experiment have been differentiated by lμM ATRA for 10 7 days in an initial flask concentration of 3xlO⁵ cells/ml without any replacement of growth medium in the differentiation period.

After differentiation the cells are centrifuged 125xg 10 min. and reconstituted in supplemented RPMI in a concentration of approx. l-5x lθ⁶ cells/ml. A cryopreservation liquid comprising of: supplemented RPMI 1640, 10% DMSO and 15 20 % foetal calf serum is slowly added to the cell suspension (1-2 min) to a final ratio of 1 : 1 between cryopreservation liquid and cell suspension. The cells suspended in the cryo-protectant solution are transferred to cryo-tubes with a volume of 1,5 ml and are immediately placed in a -80⁰C freezer.

In this experiment the cells have then been kept frozen for 1-2 days.

20 In a clean environment (e.g. laminar air flow unit) a reaction mixture comprising of plasma (final concentration of 2,5 %) and luminol (final concentration of 283 μM) are added to a 96-WeII Plate. The volume is adjusted to 50 μl by addition of HBSS. The plate is placed in an incubator and allowed to equilibrate at 37 ⁰C for 15 min prior to addition of cells. The cryopreserved cells are rapidly thawed by

25 addition of preheated supplemented RPMI the cells are transferred to a centrifuge tube and are centrifuged 125xg 10 min the supernatant is discarded and the cells are gently washed with pre warmed (37°C) HBSS. The cells are then again centrifuged at 125xg for 10 min, the supernatant is discarded and the cells are gently resuspended in pre warmed (37°C) HBSS. The cells are then counted and

30 the cell suspension is adjusted to 10⁷ cells/ml. 50 μl of cell suspension is added to each well giving a final concentration of 5xlO⁵ differentiated HL-60 cells/well the plate is then again allowed to equilibrate at 37 ⁰C for 15 min prior to the addition of 100 μl "test solution" (100 μg/ml zymosan, 10 ng/ml LPS and 100 pg/ml LPS respectively) or reference solution (pyrogen free HBSS). The plate is then immediately placed in the luminometer for plate reading following a protocol allowing each well to be read for 1 sec every 2^nd min for 200 min.

As seen the addition of test solution containing zymosan or LPS activates the cells to produce ROS

Example 10

ROS production by differentiated NB-4 cells

NB-4 cells have been used instead of HL-60 cells in an assay according to the present invention and as disclosed in example 1. The results from various concentrations of LPS stimulation of these cells as well as the positive detection of zymosan are shown in figures 20-21. It appears from this figure that it is possible to make quantitative measurements of pyrogens in NB-4 cells.

Example 11

HL-60 ROS dose-response data for stimulation by known pyrogens A HL-60 cell dose response testing of various doses of LPS, LTA, and peptidoglycans, respectively is shown in figures 16-18. The methods according to Example 1 were employed. It appears from these figures that it is possible to obtain a qualitative and quantitative estimate of some pyrogens in HL-60 cells using chemiluminescent measurements.

Example 12

Use of antibodies to amplify of the response to an inflammatory contamination and obtain qualitative pyrogen determination.

If the product prior to testing in the HL-60 assay is incubated with plasma and antibody directed against the contamination e.g. LTA the response is amplified. Use of antibodies to different classes of contamination e.g. LTA, LPS and peptidoglycans can furthermore reveal the origin of the contamination. The use of specific antibodies is illustrated in figure 19 that shows the response of ATRA differentiated HL-60 cells (5xlO⁵ cells/well) supplemented with 283 μM luminol and 2.5% plasma to HBSS (control), LTA (lipoteichoic acid from S. aureus) lng/mL, LTA (lipoteichoic acid from S. aureus) lng/mL+ plasma(5%*)+antibody raised against LTA from S. aureus (1 :50) preincubated 1 hr. at 37°C, and HBSS+plasma (5%*)+ antibody raised against LTA from S. aureus (1 :50) preincubated 1 hr. at 37°C (reference solution). "*" indicates that final plasma concentration in the well is 2.5%. Example 12 shows that use of antibodies make it possible to detect 1 ng/ml LTA which can not be detected without the use of antibodies. Furthermore the example illustrates that antibodies can be used as a qualitative determination of the antigen present since antibodies in them selves only to a small extend elicit a ROS release and therefore only the antigen bound by its specific antibody will elicit responses greater than the antigen it self.

Example 13

Use of the method of the present invention to find and quantify LPS in medicinal honey

Using the present invention the pyrogen content of two different active manuka honeys was examined. Manuka honey is a product intended for wound care and/or consumption. The response to the honey preparations thus obtained correlate with the LPS concentration determined by the LAL test conducted accordingly to the Ph. Eur. This has been substantiated by several tests that can isolate/indicate the present of LPS (Polymyxin test, ultrafiltration, heat treatment). This example therefore shows proof-of-principle, i.e. that it is possible to determine the presence and concentration of an LPS contamination present in honey.

Example 14

Use of the method of the present invention to achieve spike recovery in saline

By using both fresh and Cryopreserved cells in methods according to the present invention, a physiological saline solution been spiked with endotoxin as a positive control. The spike recovery was in both cases within Ph. Eur. demands, thereby indicating that method of the present invention can be used to test for LPS contamination in saline solutions.

Example 15 Use of the method of the present invention to achieve spike recovery in the pharmaceutical GC-globulin

In a pharmaceutical preparation containing GC-globulin, using spike recovery as a positive control, accordingly to Ph. Eur., indicates that the method of the present invention can be used to test pharmaceuticals for inflammatory contamination.

Example 16

Use of the method of the present invention to achieve spike recovery in the aluminium containing pharmaceutical DϊTe-Booster In a pharmaceutical preparation containing DiTe-Booster (an aluminium containing vaccine), the inventors have achieved spike recovery accordingly to Ph. Eur. indicating that the method of the present invention can be used to test aluminium containing pharmaceuticals. This example thus provides proof-of- concept that the present invention can be used for testing vaccine formulations for presence of inflammatory contamination, even if such formulations comprise e.g. aluminium ions.

References

Collins et al. (1990) MoI Cell Biol. 10(5):2154-63.

Fleck et al. (2003) In Vitro Cell Dev Biol Anim. 39(5-6):235-42

Fundamental Immunology, W. E. Paul, ed., Raven Press, N. Y. (1993)

Hansen and Christensen (1990) J. Clin. Pharm. Ther. 15(6), 425-433.

Himanen et al. (1993) J. Gen. Microbiol. 139, 2659-2665.

Moesby et al. (2003) Eur.J. Pharm. Sci. 19.4:245-51.

Moesby et al. (2000) Eur.J. Pharm. Sci 11.1 :51-57.

Moesby et al. (1999) Int. J. Pharm. 191, 141-149. Pack et al. (1995) J MoI Biol 246:28; Biotechnol 11:1271; and Biochemistry 31:1579

Timm etal. (2006) Eur.J.Pharm.Sci.27.2-3:252-58. Trayner et al. (1998) Leuk. Res.22(6), 537-547.

Claims

1. A method for detection of one or more inflammatory contaminants in a sample, said method comprising the steps of:

(i) exposing the sample to a cell derived from a myeloid-like cells in the presence of a reactive oxygen species (ROS) reporter probe,

(ii) measuring the amount of ROS produced by said cells, and

2. A method according to claim 1 for detection of one or more pyrogens in a sample comprising the steps of:

(i) exposing the sample to PMN like cells in the presence of one or more components of the immune system and a ROS probe,

(ii) measuring the amount of ROS produced by said cells, and

(iii) determining the presence of said one or more pyrogens in said sample by evaluation of the data obtained in step (ii).

3. The method according to claims 2, wherein the immune components comprise lyophilized plasma components.

4. The method according to any one of claims 1-3, wherein the cells are originating from a cell line selected from the group consisting of NB-4, THP-I, KG- 1, K562, KCL22, PLB-985, U937, Mono Mac 6, X-CDG, PL-21, ML-I, ML-3, MHH- 225, AML-193, HL-60 and variants thereof.

5. The method according to any one of claims 1-4, wherein the cells are differentiated with one or more differentiating agents for a period of 2-12 days before exposing the sample to the cells.

6. The method according to claim 5, wherein the cells have been differentiated for 7 days before exposing the sample to the cells.

7. The method according to any one of claims 5 or 6, wherein the cells have been preserved by a freezing process after differentiation of the cells.

8. The method according to claim 7, wherein the sample is exposed to the cells immediately after thawing of the cells.

5 9. The method according to any one of the preceding claims, wherein the cells are pre-treated with one or more priming agents before exposure of the sample to the cells.

10. The method according to any of the preceding claims, wherein the amount of ROS produced by the cells is measured by chemiluminescence.

10 11. The method according to claims 1-9, wherein the amount of ROS produced by the cells is measured by fluorescence.

12. The method according to claims 10, wherein the amount of reactive oxygen species (ROS) is measured continuously for 15 minutes to 1 day after exposing the sample to the cells.

15 13. The method according to claim 11, wherein the amount of ROS is measured 60 minutes to 7 days after exposing the sample to the cells.

14. The method according to any one of claims 1-13, wherein the sample is selected from the group consisting of: a pharmaceutical composition, an ingredient for a pharmaceutical composition, an infusion liquid, a biological

20 material, and a parenteral nutrition.

15. The method according to any one of claims 1-13, wherein the sample is selected from the group consisting of air, soil and water.

16. A kit for detecting presence of one or more inflammatory contaminants in a sample, wherein the kit comprises:

25 (i) cells derived from myeloid like cells,

(ii) one or more components of the immune system, and

(iii) at least one ROS reporter probe.

17. A kit according to claim 16 for detecting presence of one or more pyrogens in a sample, wherein the kit comprises:

(i) PMN like cells,

(ii) one or more components of the immune system, and

5 (iii) at least one ROS reporter probe.

18. The kit according to claim 17, wherein the immune system components comprise lyophilized plasma components.

19. The kit according to any one of claims 16-18, wherein the cells are differentiated with one or more differentiating agents for a period of 2-12 days.

10 20. The kit according to claim 19, wherein the cells are differentiated for a period of 7 days.

21. The kit according to any one of claims 16-20, wherein said cells are preserved by a freezing process.

22. The kit according to any one of claims 16-21, wherein said kit further 15 comprises at least one chemiluminescent probe.

23. The kit according to any one of claims 16-21, wherein said kit further comprises at least one fluorescent probe.

24. Use of myeloid-like cells that have been preserved by a freezing process for detecting presence of inflammatory contaminants in a sample.

20