Method for detecting pyrogen in biological sample and detection kit

Abstract

The present invention relates to a method and a detection kit for detecting pyrogens in a biological sample, in which immune cells, preferably mononuclear cell line THP-1 or THP-1 macrophages, are contacted with a sample possibly containing pyrogens, thereby causing the expression of an immunomodulatory mediator, in which, after lysis of the immune cells, nucleic acids of the immune cells are extracted, and the nucleic acids of the immune cells are separated from the sample. And the expression of the immunoregulation mediator is detected through PCR (Polymerase Chain Reaction). At least one of three different cell factors / chemotactic factors (preferably IL-1beta, TNF alpha or CXCL8 / IL-8) is detected in parallel. The method can be used for detecting endotoxin and non-endotoxin pyrogen pollution in a medicine sample.

Description

Technical Field

[0001] The present invention generally relates to an improved monocyte activation test based on nucleic acid amplification technology (NAT-MAT). The present invention achieves rapid and reliable detection of pyrogens in pharmaceutical products by using polymerase chain reaction (PCR), quantitative real-time PCR (qPCR) and digital PCR (dPCR) techniques. The present invention includes the following processes: treating immunocompetent cells with the pharmaceutical product to be tested, extracting nucleic acids, and detecting cytokines by PCR technology. The present invention also includes the development and use of detection kits for the detection of endotoxins and non-endotoxin pyrogenic contaminants in pharmaceutical samples. The present invention is shown in the claims. Background Art

[0002] One of the key steps in the production of pharmaceutical products is safety testing. Medicinal products and devices for parenteral administration must be tested for the presence of fever-causing contaminants, i.e., pyrogens. Usually, pyrogens are of biological origin, but can also be non-biological. Pyrogens can trigger a systemic response after entering the circulatory system of humans or other mammals. This systemic response is called an inflammatory response or simply inflammation, and is mediated by a variety of different immune system cells. Inflammation is an important defense mechanism to protect the organism from serious infections caused by pathogens and to fight pathogenic invaders, or to start the healing process in the case of injuries. The immune defense system includes two main mechanisms. One mechanism is the translocation of immune cells (i.e., white blood cells, such as B and T lymphocytes or monocytes / macrophages) to the site of infection through the blood and lymphatic system. The other mechanism is an increase in the whole body temperature that causes fever. Fever causes the environment of the pathogen to become overheated, making it unfavorable for the pathogen. The invader itself is responsible for the temperature increase because its own compounds cause the fever response. As mentioned initially, these compounds are called pyrogens or pyrogenic compounds. People divide them into exogenous and endogenous pyrogens. Exogenous pyrogens are components of microbial entities such as bacteria, viruses or fungi. Typically, they are present on the surface of microorganisms, i.e., endotoxin lipopolysaccharide (LPS) on Gram-negative bacteria, lipoteichoic acid (LTA) on Gram-positive bacteria, or other components in viruses or fungi. Exogenous pyrogens trigger the release of endogenous pyrogens from infiltrating immune cells into the site of infection or injury. They are released by leukocytes, malignancies or tissue trauma. Endogenous pyrogens are proinflammatory mediators such as cytokines and chemokines, i.e., for example, interleukin (IL)-1, IL-6, tumor necrosis factor (TNFα) or CXCL8 / IL-8.

[0003] In addition to acting as pyrogens, the released pro-inflammatory mediators attract other immune cells to translocate to the site of infection and release further cytokines and chemokines. Complex regulatory mechanisms ensure that only enough pro-inflammatory cytokines and chemokines are present to fight the invader, as too many pro-inflammatory mediators can cause local damage to the organism, but can cause systemic damage to the organism when the invader enters the circulation and thereby provokes an immune response. Similarly, dysregulation of the immune response can lead to local or systemic malignant inflammation. Local and systemic malignant inflammation can lead to life-threatening septic shock. Factors that promote the development of sepsis depend on the type and amount of exogenous or endogenous pyrogens, as well as the sensitivity and susceptibility of the individual to malignant inflammation. Exogenous pyrogens can also be of abiotic origin. such as metal compounds in elastomers or rubber abrasion which may find its way into pharmaceutical products.

[0004] In order to prevent systemic inflammatory reactions that could ultimately lead to malignant inflammation and systemic shock or sepsis, the safety of medicinal products administered parenterally, intravenously, intramuscularly or subcutaneously, must be closely monitored for the presence of pyrogens. The European Pharmacopoeia describes two animal-based tests; the rabbit pyrogen test (RPT) and the limulus amoebocyte lysate (LAL), also known as the bacterial endotoxin test (BET). Currently, they are commonly used to test pharmaceutical parenteral products.

[0005] European Pharmacopoeia (EP 2.6.8) and United States Pharmacopoeia (USP <151> ) is described in the Rabbit Pyrogen Test. This is an in vivo test that detects an increase in body temperature in a statistically significant number of rabbits. A sterile solution of the test drug is injected intravenously and the mean body temperature of the test animals is calculated. The RPT is sensitive to a wide range of pyrogens, including endotoxins and non-endotoxin compounds, but the sensitivity is low. It is in the range of nanograms of endotoxin / ml compared to picograms of endotoxin / ml in other pyrogen tests such as LAL. Moreover, only an approximate correlation between the presence of pyrogens and febrile reactions can be drawn between different rabbit species, with differences of up to 10,000 times. Moreover, the use of the RTP has become more stringent since EU Directive 2010 / 63 (“for the protection of experimental animals”) and a 5-year re-approval has been refused since 2018. Therefore, differences between rabbit species, insensitivity, insufficient quantitative results, ethical reasons and especially the timely limited approval of this test make the RPT obsolete in the near future, at least in Europe.

[0006] An alternative to RTP is LAL. It is listed in the European Pharmacopoeia (EP 2.6.14) and the United States Pharmacopoeia (USP <85> ). This is an in vitro test in which an amoeba lysate is taken from horseshoe crabs (Limulus polyphemus). The red blood cells in the lysate react with bacterial endotoxins in medical products that need to be tested by clotting. The clotting can then be measured. Horseshoe crabs are found on the East Coast of the United States and their population is strictly monitored and managed. In order to remove the amoeba lysate from the animals, they need to be captured and transported to the blood collection site. After the blood is drawn, they are transported back and released into the environment. It is estimated that 5% to 15% of these animals will not survive, depending on who estimates it. This is an important issue because horseshoe crabs are listed as endangered. In addition, three other Asian horseshoe crab species are already listed as endangered for reasons other than LAL. Due to the increase in LAL testing being performed worldwide, the availability of animals is decreasing. This could jeopardize drug safety and spark a global discussion. LAL has other limitations, as it can only detect endotoxin LPS of Gram-negative bacteria, but not pyrogens of non-endotoxin origin (Moltz, 1993; Tilders et al., 1994; Zeisberger & Roth, 1993). Therefore, LAL does not fully reflect the potential effectiveness of non-endotoxin contamination in human drugs. Therefore, other tests are usually used in addition to RFP.

[0007] Both animal-based test systems have strong limitations in terms of availability, sensitivity, specificity and ethical reasons. Other non-animal tests are also needed. The requirements for these tests include high sensitivity, specificity for a variety of pyrogens and unlimited availability. A non-animal alternative that meets all these requirements is the monocyte activation test. It is described in the European Pharmacopoeia (EP 2.6.30). As early as the 1990s, models that exploit the ability of immune cells to react to pyrogens were described as potential alternative candidates to RPT and LAL (A et al., 1999; Eperon et al., 1997; Hartung & Wendel, 1995). Several different methods have been employed and described, using peripheral blood mononuclear cells (PBMC) from humans (Hartung and Wendel, 1995), monocytic cell lines such as MonoMac 6 (MM6) (Ziegler-Heitbroc et al., 1988) or THP-1 (Auwerx, 1991; Tsuchiya et al., 1980) and the mouse macrophage cell line RAW264.7. THP-1 cells can be cultured and used as monocytic cells, i.e., suspension cells, and can be differentiated into THP-1 derived macrophages, which are metabolically inactive but more sensitive to the detection of pyrogens by increasing cluster of differentiation (CD)-14. Readouts include the cytokines IL-1β, IL-6 or TNF as described in the European Pharmacopoeia (EP 2.6.30), and the non-pyrogenic metabolites neopterin or nitrite (Hartung et al., 2001).

[0008] Six different alternative pyrogen detection methods based on leukocyte lines or human whole blood have been summarized and described by the European Centre for Validation of Alternative Methods (ECVAM).

[0009] Pyrogenic tests based on leukocyte lines include tests using MM6 and THP-1 cells (Eperon & Jungi, 1996; Peterbauer et al., 2000; TAKTAKTAK et al., 1991; Werner Felmayer et al., 1995).

[0010] The MM6-based test uses a commercially available ELISA kit to detect IL-6 or TNF in the supernatant. The sensitivity of the Endotoxin Standard Biological Reference Preparation Batch 2 is 0.125 IU / ml, while the sensitivity of LAL is 0.03 IU / ml. The detection limit of LPS is 10 pg / ml. The presence of immunoglobulin G can inhibit this test. The MM6 test can be used in conjunction with LAL to test some bacterial vaccines (TAKTAKTAK et al., 1991).

[0011] Using THP-1 cells, it was demonstrated that the detection of gram-negative pyrogens correlates with the detection limit of LAL, but the detection of gram-positive pyrogens is superior to LAL. The detection of pyrogens is determined by measuring TNF or neopterin (Peterbauer et al., 2000). The detection limit is 1-10 pg / ml endotoxin.

[0012] Developed a pyrogen test based on fresh human whole blood to measure the production and secretion of proinflammatory cytokines IL-1, IL-6, TNF or prostaglandin E2 (PG2) by ELISA. These tests are related to the amount of pyrogen present in the test sample (Fennrich et al., 1999; Hartung & Wendel, 1996; Pool et al., 1998). Two tests were established to detect the endpoints of IL-1β or IL-6 by ELISA. As described in "Novel Pyrogen Tests Based on the Human Fever Reaction, The Report and Recommendations of ECVAM Workshop 43" (Hartung et al., 2001), the detection limit of the single plate test is <50pg / ml LPS / ml or 3pg / ml LPS, and the detection limit of the double plate test is 0.06IU / ml, i.e. 6pg / ml LPS.

[0013] Although several alternative methods comparable to LAL have been described, they are not yet widely and routinely used. Furthermore, the ban on rabbit pyrogen testing and the threat of extinction of horseshoe crabs (Limulus fasciatus) require the development of reliable and widely applicable pyrogen detection methods to avoid compromising drug safety. Summary of the invention

[0014] The present invention discloses an advanced in vitro pyrogen test based on a monocyte THP-1 cell line model of the monocyte / macrophage system. The model is designed to detect endotoxin and non-endotoxin pyrogens in medicinal products according to the requirements of monocyte activation tests, rather than detecting secreted proteins by ELISA as described in the European Pharmacopoeia (EP 2.6.30), measuring the amplification of messenger RNA (mRNA) by polymerase chain reaction (PCR) to detect the expression of pro-inflammatory cytokines, whether using the well-established quantitative real-time PCR (qPCR) or the newly established digital PCR (dPCR).

[0015] For the first time, a method has been developed from culturing monocytes / macrophages to detecting cytokines by PCR. The system works via monocytes or macrophages. The first step is to culture the cells, allowing them to differentiate into macrophages or remain as monocytes. The cells are plated on a 96-well scale on cell culture plates and combined with the drug product to be tested at three different dilutions in four replicates. After a certain incubation time, the next step is to lyse the cells and extract the nucleic acids. The extracts are amplified by qPCR or dPCR.

[0016] The present invention includes a primer / probe system for the simultaneous detection of at least three cytokines / chemokines IL-1β, IL-6 and IL-8. In addition, a fourth cytokine TNF or an internal extraction control can be added, which is a synthetic RNA.

[0017] The present invention is used to detect endotoxin pyrogens and non-endotoxin pyrogens.

[0018] The present invention provides a kit comprising a test system, which includes a cell system, a primer / probe system for detecting the cytokine / chemokine and a housekeeping gene to ensure data accuracy, two lyophilized master mixes containing or lacking reverse transcriptase for performing dPCR, and an analysis program that facilitates the analysis of data obtained by dPCR.

[0019] Other objects and features of the present invention will be described hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A The graph shown is the IL-1β baseline gene expression level measured by digital PCR in THP-1 monocytes at four different cell densities (150,000 cells / 100μl / 96-well plate, 200,000 cells / 100μl / 96-well plate, 300,000 cells / 100μl / 96-well plate, 400,000 cells / 100μl / 96-well plate) in the monocyte activation test, in untreated control cells, demonstrating the ability of the system to detect IL-1β gene expression.

[0021] Figure 1B The graph shown is the baseline gene expression level of TNFα measured by digital PCR in THP-1 monocytes at four different cell densities (150,000 cells / 100 μl / 96-well plate, 200,000 cells / 100 μl / 96-well plate, 300,000 cells / 100 μl / 96-well plate, 400,000 cells / 100 μl / 96-well plate) in untreated control cells in the monocyte activation test, demonstrating the ability of the system to detect IL-6 gene expression.

[0022] Figure 1C The graph shown is the baseline gene expression level of IL-8 measured by digital PCR in THP-1 monocytes at four different cell densities (150,000 cells / 100 μl / 96-well plate, 200,000 cells / 100 μl / 96-well plate, 300,000 cells / 100 μl / 96-well plate, 400,000 cells / 100 μl / 96-well plate) in the monocyte activation test, in untreated control cells, demonstrating the ability of the system to detect IL-8 gene expression.

[0023] Figure 1D The graph shown is the baseline gene expression level of IL-6 measured by digital PCR in THP-1 monocytes at four different cell densities (150,000 cells / 100 μl / 96-well plate, 200,000 cells / 100 μl / 96-well plate, 300,000 cells / 100 μl / 96-well plate, 400,000 cells / 100 μl / 96-well plate) in the monocyte activation test in untreated control cells, demonstrating the ability of the system to detect TNFα gene expression.

[0024] Figure 2A The graph shown is the baseline gene expression of IL-1β when different volumes of extract (0.5μl in a 12μl dPCR reaction, 1μl in a 12μl dPCR reaction, 2μl in a 12μl dPCR reaction, 3μl in a 12μl dPCR reaction, 4μl in a 12μl dPCR reaction, and 5μl in a 12μl dPCR reaction) were amplified using digital PCR, showing that gene expression increased linearly with increasing volume.

[0025] Figure 2B The graph shown is baseline gene expression of TNFα when different volumes of extract (0.5 μl in a 12 μl dPCR reaction, 1 μl in a 12 μl dPCR reaction, 2 μl in a 12 μl dPCR reaction, 3 μl in a 12 μl dPCR reaction, 4 μl in a 12 μl dPCR reaction, and 5 μl in a 12 μl dPCR reaction) were amplified using digital PCR, showing a linear increase in gene expression with increasing volume.

[0026] Figure 2C The graph shown is the baseline gene expression of IL-8 when different volumes of extract (0.5 μl in a 12 μl dPCR reaction, 1 μl in a 12 μl dPCR reaction, 2 μl in a 12 μl dPCR reaction, 3 μl in a 12 μl dPCR reaction, 4 μl in a 12 μl dPCR reaction, and 5 μl in a 12 μl dPCR reaction) were amplified using digital PCR, showing that gene expression increased linearly with increasing volume.

[0027] Figure 2D The graph shown is the baseline gene expression of IL-6 when different volumes of extract (0.5 μl in a 12 μl dPCR reaction, 1 μl in a 12 μl dPCR reaction, 2 μl in a 12 μl dPCR reaction, 3 μl in a 12 μl dPCR reaction, 4 μl in a 12 μl dPCR reaction, and 5 μl in a 12 μl dPCR reaction) were amplified using digital PCR, showing that gene expression increased linearly with increasing volume.

[0028] Figure 3A The graphs shown show IL-1β, TNFα and IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is directed to E. coli endotoxin lipopolysaccharide (LPS) (0, 0.005 EU / ml and 0.009 EU / ml).

[0029] Figure 3B The graph shown shows IL-1β gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression in response to E. coli lipopolysaccharide (LPS) (0, 0.005 EU / m l and 0.009EU / m l).

[0030] Figure 3C The graph shown shows TNFα gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression in response to E. coli lipopolysaccharide (LPS) (0, 0.005 EU / m l and 0.009EU / m l).

[0031] Figure 3D The graph shown shows IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression in response to E. coli lipopolysaccharide (LPS) (0, 0.005 EU / m l and 0.009EU / m l).

[0032] Figure 4A The graph shown shows IL-1β, TNFα and IL-8 gene expression as responses in a monocyte activation assay using THP-1 derived macrophages when amplified with digital PCR. The housekeeping gene TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is directed to lipoteichoic acid (0, 0.05 μg / ml and 0.05 μg / ml) of Staphylococcus aureus (a Gram-positive bacterium).

[0033] Figure 4B The graph shown shows IL-1β gene expression in response to a monocyte activation assay performed with THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is directed to lipoteichoic acid (0, 0.05 μg / ml and 0.05 μg / ml) of Staphylococcus aureus, a Gram-positive bacterium.

[0034] Figure 4C The graph shown shows TNFα gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is directed to lipoteichoic acid (00.05 μg / ml and 0.05 μg / ml) of Staphylococcus aureus, a Gram-positive bacterium.

[0035] Figure 4D The graph shown shows IL-8 gene expression as a response in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is directed to lipoteichoic acid (0, 0.05 μg / ml and 0.05 μg / ml) of Staphylococcus aureus, a Gram-positive bacterium.

[0036] Figure 5A The graph shown shows IL-1β, TNFα and IL-8 gene expression as responses in a monocyte activation assay using THP-1 derived macrophages when amplified with digital PCR. The housekeeping gene TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is directed to zymosan, which is a component of the fungus (0, 0.1 μg / ml and 7.5 μg / ml).

[0037] Figure 5BThe graph shown shows IL-1β gene expression as a response in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is directed to zymosan, a component of the fungus (0, 0.1 μg / ml and 7.5 μg / ml).

[0038] Figure 5C The graph shown shows TNFα gene expression as a response in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is directed to zymosan, a component of the fungus (0, 0.1 μg / ml and 7.5 μg / ml).

[0039] Figure 5D The graph shown shows IL-8 gene expression as a response in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is directed to zymosan, which is a component of the fungus (0, 0.1 μg / ml and 7.5 μg / ml).

[0040] Fig. 6A The graph shown shows IL-1β, TNFα and IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is Staphylococcus aureus peptidoglycan (0, 0.1μg / ml and 0.5μg / ml)

[0041] Figure 6B The graph shown shows IL-1β gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is peptidoglycan from Staphylococcus aureus (0, 0.1 μg / ml and 0.5 μg / ml)

[0042] Figure 6C The graph shown shows TNFα gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is peptidoglycan from Staphylococcus aureus (0, 0.1 μg / ml and 0.5 μg / ml)

[0043] Fig.6D The graph shown shows IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is peptidoglycan from Staphylococcus aureus (0, 0.1 μg / ml and 0.5 μg / ml)

[0044] Fig. 7A The graphs shown show IL-1β, TNFα and IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified by digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is peptidoglycan from Streptococcus spec. (0, 0.1 μg / ml and 0.5 μg / ml)

[0045] Figure 7B The graph shown shows IL-1β gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is Streptococcus peptidoglycan (0, 0.1 μg / ml and 0.5 μg / ml)

[0046] Figure 7C The graph shown shows TNFα gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is Streptococcus peptidoglycan (0, 0.1 μg / ml and 0.5 μg / ml)

[0047] Fig.7D The graph shown shows IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is Streptococcus peptidoglycan (0, 0.1 μg / ml and 0.5 μg / ml)

[0048] Fig. 8A The graph shown shows IL-1β, TNFα and IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is lipopolysaccharide (0, 0.004EU / ml, 0.005EU / ml and 0.01EU / ml)

[0049] Figure 8B The graph shown shows IL-1β, TNFα and IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. TATA binding protein (TBP) ensures the accuracy of the data. Cytokine gene expression is peptidoglycan of Staphylococcus aureus (0, 0.1 μg / ml and 0.5 μg / ml, 1 μg / ml).

[0050] Figure 8C The graph shown shows IL-1β, TNFα and IL-8 gene expression in response to a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. TATA binding protein (TBP) ensures data accuracy. Cytokine gene expression is streptococcal peptidoglycan (0, 0.1 μg / ml and 0.5 μg / ml)

[0051] Fig.8D The graphs shown show IL-1β, TNFα and IL-8 gene expression as responses in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine expression is zymosan (0, 0.1 μg / ml and 0.5 μg / ml).

[0052] Fig.8D The graphs shown show IL-1β, TNFα and IL-8 gene expression as responses in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine expression is zymosan (0, 0.1 μg / ml and 0.5 μg / ml).

[0053] Fig. 8E The graphs shown show IL-1β, TNFα and IL-8 gene expression as responses in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine expression is flagellin (0, 0.1 μg / ml and 0.5 μg / ml, 1 μg / ml).

[0054] Fig.8FThe graphs shown show IL-1β, TNFα and IL-8 gene expression as responses in a monocyte activation assay using THP-1 derived macrophages when amplified using digital PCR. The housekeeping gene TATA binding protein (TBP) ensures data accuracy. Cytokine expression is lipoteichoic acid (0, 0.01 μg / ml, 0.02 μg / ml, 0.05 μg and 0.075 μg / ml).

[0055] Fig. 9 The graph shows the difference in the number of cells per well when amplified using digital PCR (500,000 cells / 100 μl / 96-well plate, 1.5x10 6 Gene expression of IL-1β and IL-8 in response to a monocyte activation assay performed with THP-1 derived macrophages containing 10 cells / 100 μl / 96-well plate. Cytokine gene expression was directed to non-endotoxin pyrogenic lipoteichoic acid (LTA) at a concentration series of 0.05 μl / ml LTA, 0.1 μl / ml LTA, 0.25 μl / ml LTA, and 0.5 μl / ml LTA, as well as a negative control of 0 μg / ml LTA.

[0056] Fig. 10A The scatter plot shown depicts the relative fluorescence intensity of digital PCR amplification, representing the gene expression of a synthetic internal extraction control (IK_RNA) extracted at different concentrations (1 million copies per 12 μl PCR reaction, 0.5 million copies per 12 μl PCR reaction, 50,000 copies per 12 μl PCR reaction, and 25,000 copies per 12 μl PCR reaction).

[0057] Fig. 10B The graph shows the dPCR amplification and calculated from Fig. 8A Shown are scatter plots of quantification of gene expression of a synthetic internal extraction control (IK_RNA) extracted at different concentrations (1 million copies per 12 μl PCR reaction, 0.5 million copies per 12 μl PCR reaction, 50,000 copies per 12 μl PCR reaction, and 25,000 copies per 12 μl PCR reaction).

[0058] Fig.11A The graph depicts the baseline linear increase in housekeeping gene TATA binding protein (TBP) gene expression when digitally PCR amplified at different cell densities (30,000 cells / 96 wells / 100 μl, 40,000 cells / 96 wells / 100 μl, 50,000 cells / 96 wells / 100 μl). Gene expression represents the baseline of untreated control THP-1 monocytes.

[0059] Fig. 11BThe graph shown depicts the gene expression of the housekeeping gene TBP amplified by digital PCR when treated with three concentrations (0 μg / ml LTA, 0.05 μg / ml LTA, 1 μg / ml LTA) of non-endotoxin LTA in a monocyte activation assay using THP-1 monocytes, representing the stability of TBP during the treatment process.

[0060] Fig. 11C The graph shown shows the baseline gene expression of the housekeeping gene TBP when different volumes of extract were amplified by digital PCR (0.5 μl in 12 μl dPCR reaction, 1 μl in 12 μl dPCR reaction, 2 μl in 12 μl dPCR reaction, 3 μl in 12 μl dPCR reaction, 4 μl in 12 μl dPCR reaction, and 5 μl in 12 μl dPCR reaction), indicating that gene expression increased linearly with increasing volume.

[0061] Fig. 12A The scatter plots shown depict relative fluorescence intensities of digital PCR amplifications representing gene expression of the housekeeping gene TBP and the cytokines / chemokines IL-1β, TNFα, and IL-8 as a response of THP-1 derived macrophages to the non-endotoxin pyrogen LTA. A non-template PCR control (NTC) was included.

[0062] Fig. 12B The scatter plots shown depict relative fluorescence intensities of digital PCR amplifications representing gene expression of the housekeeping gene TBP and the cytokines / chemokines IL-1β, IL-6, TNFα, and IL-8 as a response of THP-1 derived macrophages to the non-endotoxin pyrogen LTA. A non-template PCR control (NTC) was included.

[0063] Fig. 12C The scatter plots shown depict relative fluorescence intensities of digital PCR amplifications representing gene expression of the housekeeping gene TBP and the cytokines / chemokines IL-1β, TNFα, and IL-8 as a response of THP-1 derived macrophages to the non-endotoxin pyrogen LTA, as well as an internal extraction control IK_RNA. A non-template PCR control (NTC) was included.

[0064] Fig.13A The graphs shown depict amplification curves of quantitative real-time PCR (qPCR) amplified gene expression of the cytokines / chemokines IL-1β, IL-6 and IL-8 and the housekeeping gene TBP as a response to the non-endotoxin pyrogen LTA in a monocyte expansion assay using THP-1 monocytes.

[0065] Fig. 13BThe graphs shown show the relative gene expression of the cytokines / chemokines IL-1β, IL-6, and IL-8 relative to the housekeeping gene TBP, as shown in Fig. 9 The Ct values ​​of the curve shown in A are related.

[0066] Fig.14A The scatter plot shown depicts the relative fluorescence intensity of digital PCR amplifications showing gene expression of five synthetic gene fragments of cytokines / chemokines IL-1β, IL-6, TNFα, and IL-8, with the housekeeping gene TBP serving as a positive PCR control in five replicate experiments (quintuplex).

[0067] Fig. 14B The graph depicts quantification of digital PCR amplification showing gene expression of five synthetic gene fragments of the cytokines / chemokines IL-1β, IL-6, TNFα, and IL-8, with the housekeeping gene TBP serving as a positive PCR control in five replicate experiments.

[0068] Fig.15A The scatter plots shown depict relative fluorescence intensities of digital PCR amplifications showing gene expression of five synthetic gene fragments of cytokines / chemokines IL-1β, IL-6, and IL-8, the housekeeping gene TBP, and the internal control IK_DNA (equivalent to the internal extraction control IK_RNA) as positive PCR controls in five replicate experiments.

[0069] Fig. 15B The graph shown depicts quantification of digital PCR amplification showing gene expression of five synthetic gene fragments of the cytokines / chemokines IL-1β, IL-6 and IL-8, the housekeeping gene TBP and the internal control IK_DNA (equivalent to the internal extraction control IK_RNA) as positive PCR controls in five replicate experiments. DETAILED DESCRIPTION

[0070] The present invention is based on the responsiveness of human immune cells to external stimuli (such as pyrogens), and expresses a variety of pro-inflammatory immunomodulators including TNFα, IL-1β, IL-6 and IL-8. The subsequent release of these expressed pro-inflammatory cytokines and chemokines will cause an immunomodulatory response of the organism's immune system, thereby leading to an inflammatory response. The present invention aims to measure the gene expression of cytokines / chemokines TNFα, IL-1β, IL-6 and IL-8 in response to external pyrogens. To this end, macrophages are incubated with medicinal samples and pyrogen standards for several hours, followed by isolation of mRNA, and gene expression is measured by polymerase chain reaction. Two polymerase chain reaction (PCR) methods can be applied-quantitative real-time PCR or digital PCR.

[0071] The monocyte activation assay presented herein was developed to detect endotoxins, i.e., gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa, Chlamydia trachomatis, Yersinia pestis), and non-endotoxin pyrogens, such as gram-positive bacteria (e.g., Staphylococcus aureus, Streptococcus, Bacillus, Clostridium) or fungi (e.g., Saccharomyces cerevisiae). Due to the ability of monocytes / macrophages to respond to a large number of pyrogens, the present invention can be used to detect a variety of different biological and non-biological agents. 1. Pyrogen test kit

[0072] The present invention can be used to detect endotoxin and non-endotoxin contamination in pharmaceutical products such as parenteral medical products, dialysate, vaccines, intravenous solutions and any other pharmaceutical or non-pharmaceutical liquids that are not taken orally or come into contact with body fluids. A. Endogenous immune mediators of inflammatory response

[0073] In theory, any immunomodulatory mediator expressed by the monocyte / macrophage system in response to pyrogens can be used to detect pyrogen contamination, including IL-1β, TNFα, or IL-6 or IL-8 ( et al., 2009). Chapter 2.6.30 “Monocyte activation test” of the European Pharmacopoeia (07 / 2017:20630) recommends the use of cytokines IL-1β, TNFα or IL-6 as assays in the monocyte activation test (MAT). These cytokines are known to be involved in the pathogenesis of fever and septic shock (E va n s et al., 2015). Although the role of the chemokine IL-8 in the development of fever and sepsis is less studied, it is produced in large quantities by the monocyte / macrophage system (Duque and Descoteaux, 2014), and macrophages are the first cells in the immune response to express and secrete IL-8 to attract other immune cells such as neutrophils to the site of infection (Duque and Descoteaux, 2014). Neutrophils are the main mediators involved in the rapid innate host defense against microbial pathogens and the first defenders to produce reactive oxygen species (ROS) ( 1984). Since ROS can activate the nuclear factor-B pathway, which is an important transcription factor for cytokines and chemokines, the link between increased ROS production and the progression of inflammatory diseases has become another factor in the inflammatory response (N aik and Dixit, 2011). This makes IL-8 an important detector of inflammatory response.

[0074] The present invention is based on the above-mentioned Chapter 2.6.30 of the European Pharmacopoeia (07 / 2017:20630). Therein, it is recommended to detect at least one of the cytokines IL-1β, TNFα or IL-6. The present invention relies on the parallel measurement of at least three cytokines / chemokines (IL-1βTNFαCXCL8 / IL-8) by dPCR or qPCR, the housekeeping gene TATA binding protein (TBP) to control data accuracy, and the additional opportunity to add a fourth cytokine IL-6 or an internal extraction control RNA (IK_RNA), which can be added before the treated monocytes / macrophages are extracted. Although the immunoreactive and secreted IL-6 protein appears to be a key cytokine in the proinflammatory response, unlike TNFαmRNA, the IL-6mRNA in macrophages binds to YB-1 and is easily secreted into the extracellular space, thereby reducing the IL-6mRNA in macrophages (Kang et al., 2014). In another immune cell type, namely dendritic cells, the situation is different. Here, YB-1 acts as an IL-6mRNA stabilizer. Therefore, the present invention provides a test substance for detecting IL-6 gene expression, optionally depending on the cell type, which is used for detecting pyrogen contamination in pharmaceutical products. B. Primer / probe systems for detecting endogenous immune mediators of inflammatory responses

[0075] After determining the endogenous immune regulatory mediator of inflammatory response, primer / probe system must be prepared for use in the present invention. Primer / probe system is used to detect gene expression, i.e., detect the mRNA level of cytokine IL-1β, IL-6, TNFα, chemokine CXCL-8 / IL-8, housekeeping gene TBP and synthetic mRNA IK_RNA. Primer / probe system has specificity to corresponding mRNA, and genomic DNA that may contaminate sample is not detected. Primer / probe system should be amplified simultaneously by dPCR or qPCR in one reaction.

[0076] The four-primer / probe system is an essential component of the master mix containing analytical reagents and reverse transcriptase for the QIAcuity-dPCR system (Qiagen) provided by QIAGEN. A fifth optional primer / probe set can be added, i.e., a primer / probe system for detecting IL-6 or a primer / probe system for detecting synthetic RNA (IK_RNA). The master mix can also be used for any qPCR system that allows simultaneous detection of at least four mRNAs (e.g., CFX96 from BioRad). TM real-time systems). C. Monocyte / macrophage system

[0077] The first line of nucleic acid amplification test monocyte activation test (NAT-MAT) is the immune competent cell system. The present invention relies on the monocyte / macrophage system combined with the sample to be tested. Basically, all cells that can express Toll-like receptors (TLR) or are made to express TLR can be used for the application of the present invention. TLR is a mediator of pyrogenic inflammatory response. They are preferably from the same source as the intended use object of the pharmaceutical product, that is, human monocytes / macrophages for pharmaceuticals and cells such as cats, dogs, mice for veterinary drugs. The cells are seeded in the wells of a 96-well cell culture plate.

[0078] One embodiment of the present invention is the monocytic cell line THP-1. They need to be present at a cell density of at least 30,000 cells per 96 wells, but can also work at a minimum of 40,000 cells per 96 wells, 50,000 cells per 96 wells, 60,000 cells per 96 wells, 70,000 cells per well, 80,000 cells per well, 90,000 cells per well, or 100,000 cells per well. Other monocytic cell lines can also be used.

[0079] Another embodiment of the present invention is THP-1 derived macrophages. THP-1 derived macrophages are more capable of responding to pyrogens than monocytes. The first step is to trigger monocytes to differentiate into macrophages. The present invention provides macrophages and inoculates them in 96-well cell culture plates. They need to be present at a cell density of at least 30,000 cells per 96 wells, but can also work at a density of at least 40,000 cells per 96 wells, 50,000 cells per 96 wells, 60,000 cells per 96 wells, 70,000 cells per well, 80,000 cells per well, 90,000 cells per well, or 100,000 cells per well. THP-1 derived macrophages require at least 24 hours to attach to the surface of a 96-well cell culture plate. 2. Experimental system

[0080] Pyrogenicity assays were performed in three different vessels. A. Inoculation and Treatment of Monocytes / Macrophages with Samples and Pyrogen Standards

[0081] In a first container (96-well cell culture plate), THP-1 monocytes or THP-1 derived macrophages are seeded and incubated with the sample and a pyrogen standard concentration series for different times. The volume of added sample is at least 50 μl and not more than 150 μl. Ideally, the incubation time is 2 to 4 hours. The incubation of cells with the sample is performed in the same container as the one in which the cells were seeded. B. Extraction to obtain mRNA for detection of endogenous immune mediators of inflammatory response

[0082] After the incubation time, three volumes of lysis buffer were added to the 96-well cell culture plate. Afterwards, the assay container is placed in an automated extraction system (e.g., KingFisher Flex from ThermoFisher Scientific), and a mixture of total DNA and total RNA as eluate or a total RNA mixture as eluate is obtained, depending on the extraction system used (e.g., the modified AniPath kit from IST Innuscreen). C. Quantitative polymerase chain reaction for detection of endogenous immune mediators of inflammatory responses

[0083] Both digital PCR and real-time PCR are methods that can quantitatively determine the amount of nucleic acids. Both methods are sensitive and accurate. The difference between the two lies in the readout. The newly emerging digital PCR is similar to traditional PCR, and endpoint PCR is different from quantitative real-time PCR, which monitors the progress of the reaction. Digital PCR does not require a standard curve for nucleic acid quantification. For the quantification of nucleic acids amplified by real-time PCR, a standard curve or reference, i.e., a housekeeping gene, is required to enable relative quantification. Digital PCR provides absolute quantitative determination of nucleic acids and does not require a standard curve.

[0084] The eluate is subjected to dPCR or qPCR in a reaction mixture containing PCR buffer, reverse transcriptase for reverse transcription of mRNA and a primer / probe system for simultaneous detection of IL-1β, IL-8, TNFα and the housekeeping gene TBP, with the option of adding additional primer / probe systems to detect synthetic RNA (IK_RNA) or IL-6 added to the cells before extraction.

[0085] According to the requirements of Chapter 2.6.30 "Monocyte Activation Test" of the European Pharmacopoeia (07 / 2017:20630), a concentration series of pyrogen standards were added to the cell system together with the samples and amplified. 3. Detection kit

[0086] The above embodiments can be incorporated into diagnostic kits for detecting pyrogenic contamination in pharmaceutical products or for performing pyrogenic tests. The kits can detect endotoxin pyrogens of Gram-negative bacteria and non-endotoxin pyrogens of Gram-positive bacteria and their components (such as lipoteichoic acid, peptidoglycan, flagellin) and fungi or their components (such as zymosan).

[0087] The kit can be used to detect endotoxin and non-endotoxin contamination in pharmaceutical products such as parenteral medical products, dialysate, vaccines, intravenous solutions and any other pharmaceutical or non-pharmaceutical fluids that are not taken orally or come into contact with body fluids. 4. Interpretation of Cytokine / Chemokine Gene Expression Data

[0088] The interpretation and analysis of gene expression data obtained by dPCR were performed according to the requirements of Chapter 2.6.30 “Monocyte activation assay” of the European Pharmacopoeia (07 / 2017:20630). A. Quantitative testing using the standard curve method

[0089] The quantitative test uses the pyrogen standard curve recommended by the European Pharmacopoeia (07 / 2017:20630) Chapter 2.6.30 "Monocyte Activation Test". This standard curve is generated by incubating monocytes / macrophages with an endotoxin USP reference standard. It allows quantification of cytokine production in response to monocyte / macrophage activation compared to a known endotoxin standard. Any other available pyrogen can be used to generate the standard curve. By using standard best fit data analysis software, a standard curve can be generated with any data point that significantly varies in concentration of the standard endotoxin or any other known pyrogen. The European Pharmacopoeia (07 / 2017:20630) Chapter 2.6.30 "Monocyte Activation Test" recommends five data points for the generation of the standard curve, ranging from R0 (no treatment; negative control) to R1-R4. The R1 data point represents the 0.5-x limit of detection of the known pyrogen used in the standard curve, R2 represents the 1-x limit of detection of the known pyrogen used in the standard curve, R3 represents the 2-x limit of detection of the known pyrogen used in the standard curve, and R4 represents the 4-x limit of detection of the known pyrogen used in the standard curve. Once the standard curve is generated, the equivalent endotoxin or endotoxin-free concentrations of the samples to be tested can be interpolated from the standard curve. By using USP reference standards, it is helpful to normalize the data obtained to USP / FDA defined Endotoxin Units (EU / mL), which are the same as the International Units (IU) defined by the World Health Organization and are the industry standard units for indicating pyrogen contamination. definition

[0090] The term "endotoxin" as used herein refers to the endotoxin lipopolysaccharide (LPS).

[0091] As used herein, the term "non-endotoxin pyrogens" refers to all other pyrogens except the endotoxin lipopolysaccharide (LPS).

[0092] The term "assay system" as used herein refers to any cell culture plate containing monocytes / macrophages, including test samples, standards, and primer / probe systems for detecting cytokine gene expression, housekeeping gene expression, and internal extraction control IK_RNA. Materials and methods Cell culture of THP-1 monocytes.

[0093] THP-1 monocytes (CLS Cell Lines Service GmbH) The cells were cultured in RPMI1640 medium containing glutamine and sodium bicarbonate (Capricorn Scientific) supplemented with sodium pyruvate (Sigma-Aldrich), HEPES (Gibco), and glucose at 37°C and 5% CO. 2 Cells were cultured at densities ranging from 10,000 to 50,000 cells per mL in the presence of 3.5 g / L glucose (Capricorn Technologies), heat-inactivated fetal bovine serum (Sigma-Aldrich), and beta-mercaptoethanol (PAN Biotech). Preparation of THP-1-derived macrophages.

[0094] THP-1 monocytes were cultured in RPMI1640 medium containing glutamine and sodium bicarbonate (Sigma-Aldrich) and supplemented with sodium pyruvate (Sigma-Aldrich), HEPES (Gibco) and glucose (Capricorn Technologies), heat-inactivated fetal bovine serum (Sigma-Aldrich) and β-mercaptoethanol (PAN Biotech). 15,000,000 THP-1 monocyte suspension cells were counted and treated with 10 to 20 nM Phorbol-12-myristic acid-13-acetate (PMA) (Sigma-Aldrich) for 4 to 8 days in an incubator at 37°C, 5% CO 2 In a T175 cell culture flask (175 cm 2 ) Differentiate THP-1 monocytes into THP-1-derived macrophages. Cell seeding of THP-1 derived macrophages.

[0095] THP-1 derived macrophages were seeded in 96-well plates in quadruplicate, with each well containing 50,000 cells in RPMI1640 medium (cell density ranged from 50,000 to 150,000 cells per well) containing glutamine and sodium bicarbonate (Sigma-Aldrich) supplemented with sodium pyruvate (Sigma-Aldrich), HEPES (Gibco) and glucose (Capricorn Technologies), heat-inactivated fetal bovine serum (Sigma-Aldrich) and β-mercaptoethanol (PAN Biotech). Standard treatment of cells was performed in the same medium in which the samples to be tested were dissolved, usually a 100 μl volume of physiological sodium chloride solution for one to four hours. Samples were diluted according to the requirements of the European Pharmacopoeia (07 / 2017:20630), Chapter 2.6.30 "Monocyte activation test". The samples to be tested will be applied to the cells at undiluted, 2-fold and 4-fold dilutions, as well as the same dilutions spiked with a mid-dose standard. mRNA was extracted from THP-1 derived macrophages after treatment with standard pyrogens and test samples.

[0096] After the incubation time, mRNA is extracted to measure the gene expression of cytokines and housekeeping genes. For this reason, the lysis buffer containing guanidine thiocyanate of equal volume (100 μ l) is added to the cells in the cell culture plate. This similar cell is separated from the cell culture plate surface. The lysed cells are transferred to deep well plates (KFFLX plate group, #845-KF-1296010, IST Innuscreen company) to automatically extract in KingFisher Flex system (Thermo Fisher Scientific). Nucleic acid (DNA and RNA) is captured by magnetic beads (#31-00641IST Innuscreen company), washed and eluted into elution plates in RNase-free water (#314-00851, IST Innuscreen company). Digital polymerase chain reaction (qPCR).

[0097] The extract was amplified with digital PCR (QIAcuity, QIAGEN). To this end, we used a one-step dPCR kit to combine the reverse transcription of messenger RNA (mRNA) with complementary DNA (cDNA). The reaction mixture was transferred to an 8.5k 96-well nanoplate (#250011, QIAGEN) for amplification, and the reaction mixture contained a probe master mix, reverse transcriptase, a gene-specific primer / probe mixture (containing specific primers and probes for cytokines / chemokines IL-1β, TNFα, IL-8, and TBP), and 6 μl of RNA extract (total volume of 12 μl). With the help of a pipetting robot QIAgility (QIAGEN), the reaction mixture was directly pipetted from the elution plate containing the extract onto the nanoplate. Amplification was performed with the help of digital PCR in a thermal cycler QIAcuity (QIAGEN). The results are shown in the number of copies per microliter calculated by the integrated analysis software QIAcuity Suite. Interpretation of cytokine production data.

[0098] The medicinal product is tested in different dilutions and the results are quantitatively analyzed according to the requirements of Chapter 2.6.30 “Monocyte activation test” of the European Pharmacopoeia (07 / 2017:20630). A. Standard curve method - qualitative method.

[0099] First, the validation of the standard curve consisting of R0 to R4 must be confirmed according to the following requirements: i) The regression of the response of the log10 dose should be statistically significant (p<0.01). ii) The regression of the response of the log10 dose must not deviate significantly from linearity (p>0.05). In the first step, the appropriate dilutions of the samples for analysis must be determined, where the samples are added in undiluted, two-fold and four-fold dilutions. A 96-well cell culture plate can test two different samples simultaneously in one assay; three dilutions are set for each sample (undiluted, two-fold and four-fold dilutions), and for each sample, the equivalents in each replicate must meet the following criteria: i) The average of the four replicates must be calculated, and the recovery of endotoxin is calculated - that is, by subtracting the average concentration of the unspiked sample from the average concentration of the sample dilutions to which the spike (spike) R3 was added; this value should be in the range of 50-200%. ii) Dilutions that do not meet the spike recovery criteria will not be considered in further analysis; if all dilutions do not meet these criteria, the assay cannot be analyzed and different dilutions must be reconsidered. iii) To be considered negative, the test sample must meet the following requirements: After dilution and concentration correction, the endotoxin equivalent measured in the unspiked sample must be less than the contaminant limit concentration (CLC) specified for the test sample. iv) The result of the additional negative control added to the PCR reaction to check the purity of the reagents used needs to be 0 copies per microliter for the assay to be valid for analysis.

[0100] The method according to the present invention comprises at least the following embodiments: 1. A method for detecting endotoxin or non-endotoxin pyrogen in a sample, comprising the following steps: Seed cryopreserved monocytes / macrophages at an appropriate cell density on a 96-well cell culture plate. Combine the sample with the monocyte / macrophage system, which is able to respond to the sample by producing certain pro-inflammatory cytokines. After an incubation time of 2 to 4 hours, add lysis buffer to the cells and transfer the cell culture plate containing monocytes / macrophages to an automated extraction system that utilizes magnetic beads. Nucleic acids can bind to the magnetic beads and elute after washing and purification from cell debris. Transfer the eluate containing nucleic acids DNA / RNA to an automated pipetting system for combining PCR reagents, in addition to being able to In addition to an enzyme for amplifying DNA, the PCR reagent also includes a buffer in which the enzyme can act, a primer / probe system, and an elution solution, wherein the primer / probe system contains primers / probes for three cytokines / chemokines IL-1β, TNFα, IL-8 and primers / probes for a housekeeping gene TBP; and transferred to an 8.5k96-well nanoplate, the nanoplate is transferred to a dPCR thermal cycler, and the gene expression of certain proinflammatory cytokines is subsequently analyzed, wherein an increased level of at least one of the three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicates the presence of pyrogens in the sample. 2. A method for detecting endotoxin or non-endotoxin pyrogen in a sample, comprising the following steps: seeding cryopreserved monocytes / macrophages at an appropriate cell density on a 96-well cell culture plate, allowing the sample to be combined with a monocyte / macrophage system, which is capable of responding to the sample by producing certain pro-inflammatory cytokines / chemokines; adding a lysis buffer to the cells after an incubation time of 2 to 3 hours, and transferring the cell culture plate containing the monocytes / macrophages to an automatic extraction system using magnetic beads, to which nucleic acids can be bound and eluted after washing and purification from cell debris; transferring the eluate containing nucleic acid DNA / RNA to an automatic pipetting system for combining PCR reagents, which, in addition to an enzyme capable of amplifying DNA, also include a buffer in which the enzyme can act, a primer / probe system, and an eluate, wherein the primer / probe system includes primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6 and a primer / probe for a housekeeping gene TBP; and transferring to an 8.5k The nanoplate was transferred to a dPCR thermal cycler on a 96-well nanoplate and subsequently analyzed for gene expression of certain pro-inflammatory cytokines, among which an elevated level of at least one of the four fever-related cytokines / chemokines (IL-1β, TNFα, IL-8, IL-6) indicated the presence of pyrogens in the sample. 3. A method for detecting endotoxin or non-endotoxin pyrogen in a sample, comprising the following steps: Cryopreserved monocytes / macrophages are plated at an appropriate cell density on a 96-well cell culture plate. The sample is combined with a monocyte / macrophage system that is capable of responding to the sample by producing certain proinflammatory cytokines / chemokines. An internal extraction control, which is a synthetic RNA molecule, is added to each well of the 96-well plate containing monocytes / macrophages. After 2 to 3 hours of incubation, a lysis buffer is added to the cells, and the cell culture plate containing monocytes / macrophages is transferred to an automated extraction system. The eluate is transferred to an automated pipetting system to combine PCR reagents, which, in addition to an enzyme capable of amplifying DNA, also include a buffer in which the enzyme can function, a primer / probe system, and an eluate, wherein the primer / probe system includes primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6 and a primer / probe for a housekeeping gene TBP; and transferred to an 8.5k The nanoplate was transferred to a dPCR thermal cycler on a 96-well nanoplate and subsequently analyzed for gene expression of certain pro-inflammatory cytokines, among which elevated levels of at least one of the three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicated the presence of pyrogens in the sample. 4. A method for detecting endotoxin or non-endotoxin pyrogen in a pharmaceutical product for parenteral administration, comprising the following steps: Seed cryopreserved monocytes / macrophages at an appropriate cell density on a 96-well cell culture plate. Bind the pharmaceutical product to the monocyte / macrophage system, which is able to respond to the sample by producing certain pro-inflammatory cytokines / chemokines. After an incubation time of 2 to 3 hours, add lysis buffer to the cells and transfer the cell culture plate containing monocytes / macrophages to an automated extraction system utilizing magnetic beads. Nucleic acids can bind to the magnetic beads and elute after washing and purification from cell debris. Transfer the eluate containing nucleic acid DNA / RNA to an automated pipetting system for binding to PCR reagents, except In addition to an enzyme capable of amplifying DNA, the PCR reagent also includes a buffer in which the enzyme can act, a primer / probe system, and an elution fluid, wherein the primer / probe system contains primers / probes for three cytokines / chemokines IL-1β, TNFα, IL-8 and primers / probes for a housekeeping gene TBP; and transferred to an 8.5k96-well nanoplate, the nanoplate is transferred to a dPCR thermal cycler, and the gene expression of certain proinflammatory cytokines is subsequently analyzed, wherein an increased level of at least one of the three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicates the presence of pyrogens in the sample. 5. A method for detecting endotoxin or non-endotoxin pyrogen in a pharmaceutical product for parenteral administration, comprising the following steps: Seed cryopreserved monocytes / macrophages at an appropriate cell density on a 96-well cell culture plate. Combine the sample with the monocyte / macrophage system, which is able to respond to the sample by producing certain pro-inflammatory cytokines / chemokines. After an incubation time of 2 to 3 hours, add lysis buffer to the cells and transfer the cell culture plate containing monocytes / macrophages to an automated extraction system that utilizes magnetic beads. Nucleic acids can bind to the magnetic beads and elute after washing and purification from cell debris. Transfer the eluate containing nucleic acids DNA / RNA to an automated pipetting system for combining with PCR reagents, which, in addition to being able to amplify D In addition to the enzyme of NA, the PCR reagent also includes a buffer in which the enzyme can act, a primer / probe system, and an eluent, wherein the primer / probe system contains primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6 and primers / probes for the housekeeping gene TBP; and transferred to an 8.5k96-well nanoplate, the nanoplate is transferred to a dPCR thermal cycler, and the gene expression of certain proinflammatory cytokines is subsequently analyzed, wherein an increased level of at least one of the four fever-related cytokines / chemokines (IL-1β, TNFα, IL-8, IL-6) indicates the presence of pyrogens in the sample. 6. A method for detecting endotoxin or non-endotoxin pyrogens in a pharmaceutical product for parenteral administration, comprising the following steps: Cryopreserved monocytes / macrophages are seeded at an appropriate cell density on a 96-well cell culture plate. The sample is combined with a monocyte / macrophage system that is capable of responding to the sample by producing certain proinflammatory cytokines / chemokines. An internal extraction control, which is a synthetic RNA molecule, is added to each well of the 96-well plate containing monocytes / macrophages. After incubation for 2 to 3 hours, a lysis buffer is added to the cells, and the cell culture plate containing monocytes / macrophages is transferred to an automated extraction system. The eluate is transferred to an automated pipetting system to combine PCR reagents, which, in addition to an enzyme capable of amplifying DNA, also include a buffer in which the enzyme can act, a primer / probe system, and an eluate, wherein the primer / probe system includes primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6 and a primer / probe for a housekeeping gene TBP; and transferred to an 8.5k The nanoplate was transferred to a dPCR thermal cycler on a 96-well nanoplate and subsequently analyzed for gene expression of certain pro-inflammatory cytokines, among which elevated levels of at least one of the three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicated the presence of pyrogens in the sample.

[0101] In vitro pyrogen testing is performed as follows: Embodiment 1: (a) Combine the monocyte / macrophage system and the test sample in a cell culture plate (b) incubating the monocyte / macrophage system and the sample so that when the sample contains a pyrogen, the monocyte / macrophage system responds by expressing pro-inflammatory cytokines (c) Transferring the contents of the combined monocyte / macrophage system and sample to an automated extraction system containing magnetic beads, to which the nucleic acids bind and are eluted in a subsequent step (d) transferring the eluate to an 8.5k 96-well nanoplate along with a PCR reagent containing all reagents required to amplify the gene of interest, the PCR reagent including a primer / probe system capable of amplifying at least one of the fever-associated proinflammatory cytokines / chemokines IL-1β, TNFα, IL-8 and the housekeeping gene TBP (e) detecting the presence of cytokines / chemokines in the amplicon, wherein a level of one of the detected cytokines / chemokines above the baseline indicates the presence of a pyrogen. Embodiment 2: (a) Combine the monocyte / macrophage system and the test sample in a cell culture plate (b) incubating the monocyte / macrophage system and the sample so that when the sample contains a pyrogen, the monocyte / macrophage system responds by expressing pro-inflammatory cytokines / chemokines (c) Transferring the contents of the combined monocyte / macrophage system and sample to an automated extraction system containing magnetic beads, to which the nucleic acids bind and are eluted in a subsequent step (d) transferring the eluate to an 8.5k 96-well nanoplate along with a PCR reagent containing all reagents required to amplify the gene of interest, wherein the PCR reagent includes a primer / probe system capable of amplifying at least one of the fever-related proinflammatory cytokines IL-1β, TNFα, IL-8, IL-6 and the housekeeping gene TBP (e) detecting the presence of cytokines in the amplified product, wherein an increase in one of the detected cytokines indicates the presence of a pyrogen. Embodiment 3: (a) Combine the monocyte / macrophage system and the test sample in a cell culture plate (b) incubating the monocyte / macrophage system and the sample so that when the sample contains a pyrogen, the monocyte / macrophage system responds by expressing pro-inflammatory cytokines (c) Transferring the contents of the combined monocyte / macrophage system and sample to an automated extraction system containing magnetic beads, to which the nucleic acids bind and are eluted in a subsequent step (d) transferring the eluate to an 8.5k 96-well nanoplate along with a PCR reagent containing all reagents required to amplify the gene of interest, the PCR-reagent including a primer / probe system capable of amplifying at least one of the fever-associated proinflammatory cytokines IL-1β, TNFα, IL-8, an internal extraction control, and a housekeeping gene TBP (e) detecting the presence of cytokines in the amplified product, wherein an increase in one of the detected cytokines indicates the presence of a pyrogen.

[0102] The samples in Examples 1 to 3 are drugs, nutrients or pharmaceutical products.

[0103] In vitro pyrogen testing can detect two proinflammatory cytokines IL-1β and TNFα and the chemokine CXCL8 / IL-8.

[0104] In vitro pyrogen testing can also be based on three proinflammatory cytokines IL-1β, TNFα, IL-16 and the chemokine CXCL8 / IL-8.

[0105] Three pro-inflammatory cytokines IL-1β, TNFα, and IL-8 can also be detected.

[0106] In vitro pyrogen testing was quality controlled using the housekeeping gene TBP or an internal extraction control.

[0107] In one embodiment of the in vitro pyrogen test, the monocyte / macrophage system consists of human THP-1 cells, which is a monocytic cell line isolated from the peripheral blood of a patient with acute monocytic leukemia.

[0108] In the in vitro pyrogen test, the monocyte / macrophage system contains a sufficient number of cells to provide at least 50,000 cells per well of the test system. The test kit for in vitro pyrogen testing includes a microtiter Plates, dPCR nanoplates, cryopreserved monocyte / macrophage system, PCR reagent mix, and standard samples. References A, P., ER, W., & G, W.-F. (1999). “Further development of cell culture models for the detection of bacterial pyrogens.” ALTEX, 16(1), 3–8. https: / / pubmed.ncbi.nlm.nih.gov / 11148756 / Auwerx, J. (1991). The human leukemia cell line, THP-1: a multifaceted model of monocyte-macrophage differentiation. Experientia, 47(1), 22–31. https: / / doi.org / 10.1007 / BF02041244 Duque, GA, & Descoteaux, A. (2014). Macrophage cytokines: involvement in immunity and infectious diseases. Frontiers in Immunology, 5, 1–12. Eperon, S., de Groote, D., Werner-Felmayer, G., & Jungi, TW (1997). 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Claims

1. A method for detecting pyrogens in biological samples, wherein immune cells are contacted with a sample that may contain pyrogens, thereby causing expression of immunomodulatory mediators, It is characterized in that After the immune cells are lysed, the nucleic acid of the immune cells is extracted, and the expression of immune regulatory mediators is detected by PCR.

2. The method according to claim 1, It is characterized in that The immune cells are cells that express Toll-like receptors (TLRs) or can be induced to express TLRs.

3. The method according to claim 1 or 2, It is characterized in that Cultured monocytes / macrophages are used as immune cells, preferably the monocytic cell line THP-1 or THP-1 macrophages.

4. The method according to any one of claims 1 to 3, It is characterized in that The immunomodulatory mediator is a proinflammatory mediator, preferably a cytokine or a chemokine, in particular IL-1β, TNFα or CXCL8 / IL-8.

5. The method according to any one of claims 1 to 4, It is characterized in that Expression is detected by measuring messenger RNA (mRNA) amplification using quantitative real-time PCR (qPCR) or digital PCR (dPCR).

6. The method according to any one of claims 1 to 5, It is characterized in that At least one of three different cytokines / chemokines is tested in parallel, preferably IL-1β, TNFα or CXCL8 / IL-8.

7. The method according to any one of claims 1 to 6, It is characterized in that A housekeeping gene is added to control data accuracy, preferably the housekeeping gene TATA binding protein (TBP).

8. The method according to any one of claims 1 to 7, It is characterized in that A fourth cytokine IL-6 or internally extracted control RNA (IK_RNA) was additionally added.

9. The method according to any one of claims 3 to 7, The following steps are involved: a) Seeding cryopreserved monocytes / macrophages in cell culture plates preferably with 96 wells b) combining the sample with the monocyte / macrophage system, which is capable of responding to the sample by producing certain pro-inflammatory cytokines c) Add lysis buffer and transfer the cell culture plate containing monocytes / macrophages to the automated extraction system so that the released nucleic acids bind to the solid phase. d) Washing and purification of nucleic acids bound to the solid phase e) DNA amplification of nucleic acids and PCR to detect gene expression of immune regulatory mediators.

10. The method according to claim 9, wherein primers / probes for three cytokines (IL-1β, TNFα, IL-8) or four cytokines (IL-1β, TNFα, IL-8, IL-6) and primers / probes for the housekeeping gene TBP are added to the DNA amplification step of the nucleic acid.

11. The method according to claim 9 or 10, in, An elevated level of at least one of the fever-associated cytokines (IL-1β, TNFα, IL-8, or IL-6) indicates the presence of a pyrogen in the sample.

12. A detection kit for implementing the method according to any one of claims 1 to 11, include: a) Cultured monocytes / macrophages b) Cell culture plates, preferably with 96 wells c) at least one lysis buffer and a buffer for binding the nucleic acids released by lysis d) Solid phase, preferably magnetic beads e) PCR reagents for DNA amplification, a primer / probe system comprising primers / probes for at least three cytokines / chemokines.

13. The kit as claimed in claim 12, further comprising include: f) housekeeping gene, preferably housekeeping gene TATA binding protein (TBP) g) The fourth cytokine IL-6 or internally extracted control RNA (IK_RNA) h) Primers / probes targeting the housekeeping gene TBP i) Primers / probes directed against a fourth cytokine.

14. Use of the method of any one of claims 1 to 11 or the test kit of claim 12 or 13 to detect endotoxin or non-endotoxin contamination in: a) a pharmaceutical product, preferably a parenteral medicinal product, a dialysis solution, a vaccine or an intravenous solution; or b) Non-oral or non-medicinal liquids that do not come into contact with body fluids c) Pharmaceutical products d) Nutritional supplements.

15. Use of the method according to any one of claims 1 to 11 or the test kit according to claim 12 or 13 in detecting IL-6 gene expression.

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