High-sensitivity in-vitro pyrogen detection method

Abstract

The invention provides a high-sensitivity in-vitro pyrogen detection method, which is characterized in that on the basis of an in-vitro pyrogen detection method (reporter gene method), a transgenic cell which expresses a Toll-like receptor and transfects an NF-kB reaction element and a luciferase gene is used for detecting a pyrogen, the pyrogen is combined with the Toll-like receptor on the surface of the cell to activate an NF-kB signal channel, and the NF-kB reaction element and the luciferase gene are converted into the NF-kB signal channel. Therefore, the expression of luciferase is activated.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically, it relates to a highly sensitive in vitro pyrogen detection method. Background Technology

[0002] Pyrogen-contaminated drugs or medical devices may cause fever or even septic shock in patients. Therefore, pyrogen detection is a crucial quality control item in safety testing. Traditional pyrogen detection methods include the rabbit assay and bacterial endotoxin detection methods (Limulus amebocyte lysate (LAL) assay). The rabbit assay is prone to false positives and has poor repeatability due to factors such as breed and experimental conditions. The endotoxin assay is limited in its ability to detect only endotoxins from Gram-negative bacteria. Furthermore, the monocyte activation test (MAT) is a hot research topic in pyrogen detection methods, offering advantages such as a wide detection range and quantitative analysis of pyrogens, but it has a long testing cycle and is cumbersome to operate.

[0003] Pyrogen detection is a crucial test for ensuring the safety of parenteral medications, especially intravenous medications. Bacterial lipopolysaccharide (LPS) is the core structure of pyrogens, which can trigger a strong immune inflammatory response in the human body, even leading to severe sepsis. Traditional methods for pyrogen detection include rabbit assays and endotoxin assays. While both are classic methods for pyrogen detection, they each have certain limitations.

[0004] Compared with traditional pyrogen testing methods, in vitro pyrogen detection methods (reporter gene assays) have become a research hotspot due to their advantages such as speed and ease of operation. The mononuclear cell activation reaction assay, designed based on the mechanism of pyrogen-induced fever in humans, has advantages such as not using animals, broad detection spectrum of pyrogens, and quantitative analysis of pyrogens. Although this method has been included in the European Pharmacopoeia (EP 10.0), the British Pharmacopoeia (BP 2021), and the Chinese Pharmacopoeia (ChP 2020), its detection sensitivity still needs to be improved. Summary of the Invention

[0005] The purpose of this invention is to provide a highly sensitive in vitro pyrogen detection method.

[0006] In a first aspect of the present invention, a highly sensitive in vitro pyrogen detection method is provided, the method comprising the steps of: S1: Provide transgenic cells that express Toll-like receptors and are exogenously transfected with NF-κB response elements and luciferase genes; pyrogens activate the NF-κB signaling pathway by binding to Toll-like receptors on the surface of the transgenic cells, thereby activating luciferase expression to indicate pyrogen levels; S2: Take the transgenic cells in the logarithmic growth phase, prepare a cell suspension, add cofactor and test sample solution to the cell suspension, incubate, and then add chromogenic solution; S3: Detect the chemiluminescence value (RLU value) of the cell suspension and calculate the pyrogen content in the sample solution based on the chemiluminescence value.

[0007] In another preferred embodiment, in step S1, the transgenic cells are selected from the group consisting of: human promyelocytic leukemia cells HL-60, mononuclear cell line MM6, human adenocarcinoma alveolar basal epithelial cells A549, and mouse mononuclear macrophage cell line RAW264.7.

[0008] In another preferred embodiment, the transgenic cells are exogenously transfected with the pNL3.2.NF-κB-RE plasmid.

[0009] In another preferred embodiment, the transgenic cells are monoclonal stable transgenic cell lines.

[0010] In another preferred embodiment, in step S2, the density of the cell suspension is approximately 1 × 10⁻⁶. 6 per ml.

[0011] In another preferred embodiment, in step S2, the final concentration of the cofactor is 10-1000 ng / ml, preferably 50-200 ng / ml, and more preferably 100 ng / ml.

[0012] In another preferred embodiment, in step S2, the incubation conditions are: 37°C, 5% carbon dioxide for 3-3.5 hours.

[0013] In another preferred embodiment, in step S2, after incubation, the mixture is equilibrated to room temperature before the colorimetric solution is added.

[0014] In another preferred embodiment, the method further includes the steps of: using pyrogen standard solutions with different concentration gradients as the sample solutions to be tested, performing a four-parameter logistic regression with the concentration of the pyrogen standard solutions as the abscissa and the average RLU value as the ordinate, and fitting a standard curve; and calculating the pyrogen content in the sample solutions to be tested based on the standard curve.

[0015] In another preferred embodiment, the concentrations of the pyrogen standard solutions at different concentration gradients are as follows: 1000.000 EU / ml, 250.000EU / ml, 62.500 EU / ml, 15.625 EU / ml, 3.906 EU / ml, 0.977 EU / ml, 0.244 EU / ml, 0.061EU / ml, 0.015 EU / ml, 0.004 EU / ml.

[0016] In another preferred embodiment, the standard curve is selected from the group consisting of: , ,and .

[0017] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the detection method of the present invention. Detailed Implementation

[0019] Through extensive and in-depth research, the inventors have developed a highly sensitive in vitro pyrogen detection method. This method, based on the in vitro pyrogen detection method (reporter gene method), uses transgenic cells expressing Toll-like receptors and transfected with NF-κB response elements and luciferase genes to detect pyrogens. Pyrogens activate the NF-κB signaling pathway by binding to Toll-like receptors on the cell surface, thereby activating luciferase expression. The invention unexpectedly discovered that adding a cofactor during the detection process can significantly improve detection sensitivity and anti-interference ability. Based on this, the present invention was completed.

[0020] In vitro pyrogen detection method (reporter gene method) The principle of this method is to exogenously transfect NF-κB response element and luciferase gene into cells expressing Toll-like receptors. Pyrogens activate the NF-κB signaling pathway by binding to Toll-like receptors on the cell surface, thereby activating luciferase expression to detect pyrogen content.

[0021] In the inventors’ previous research, it was found that Toll-like receptors (TLRs) 2, 4 and 6 are highly expressed on the surface of human promyelocytic leukemia (HL-60) cells. After pyrogens bind to TLR2, TLR4 and TLR6 on the surface of HL-60 cells, they stimulate the secretion of cytokines through the nuclear factor-κB (NF-κB) signaling pathway.

[0022] Based on the mechanism of pyrogen stimulation of HL-60 cells to secrete cytokines, plasmids carrying NF-κB and luciferase reporter genes were transfected into HL-60 cells to construct a stable HL-60-pNL3.2 cell line. It was found that after stimulation by pyrogens from different sources [such as LPS from Gram-negative bacteria, zymosan from yeast, and lipoteichoic acid (LTA) from Gram-positive bacteria], the amount of luciferase secreted by this cell line showed a dose-response relationship with the pyrogen concentration.

[0023] Based on this phenomenon, a pyrogen detection method using the HL-60 / NF-κB reporter gene was established, and the method showed good linearity, repeatability, recovery rate, and sensitivity. (See "Application of HL-60 / NF-κB reporter gene pyrogen detection method in the detection of pyrogens in monoclonal antibody drugs", China Pharmaceutical Industry Journal, 2022) This invention provides a highly sensitive in vitro pyrogen detection method, the method comprising the following steps: S1: Provide transgenic cells that express Toll-like receptors and are exogenously transfected with NF-κB response elements and luciferase genes; pyrogens activate the NF-κB signaling pathway by binding to Toll-like receptors on the surface of the transgenic cells, thereby activating luciferase expression to indicate pyrogen levels; S2: Take the transgenic cells in the logarithmic growth phase, prepare a cell suspension, add cofactor and test sample solution to the cell suspension, incubate, and then add chromogenic solution; S3: Detect the chemiluminescence value (RLU value) of the cell suspension and calculate the pyrogen content in the sample solution based on the chemiluminescence value.

[0024] In a preferred embodiment of the present invention, the cofactor is a protein comprising the following amino acid sequence: TTPEPCELDDEDFRCVCNFSEPQPDWSEAFQCVSAVEVEIHAGGLNLEPFLKRVDADADPRQYADTVKALRVRRLTVGAAQVPAQLLVGALRVLAYSRLKELTLEDLKITGTMPPLPLEATGLALSSLRLRNVSWATGRSWLAELQQWLKPGLKVLSIAQAHSPAFSCEQVRAFPALTSL DLSDNPGLGERGLMAALCPHKFPAIQNLALRNTGMETPTGVCAALAAAGVQPHSLDLSHNSLRATVNPSAPRCMWSSALNSLNLSFAGLEEQVPKGLPAKLRVLDLSCNRLNRAPQPDELPEVDNLTLDGNPFLVPGTALPHEGSMNSGVVPACARSTLSVGVSGTLVLLQGARGFA (SEQ ID NO.1) In a preferred embodiment, the amino acid sequence of the cofactor is shown in SEQ ID NO.1.

[0025] In a preferred embodiment, in step S1, the transgenic cells are selected from the group consisting of: human promyelocytic leukemia cells HL-60, mononuclear cell line MM6, human adenocarcinoma alveolar basal epithelial cells A549, and mouse mononuclear macrophage cell line RAW264.7.

[0026] In a preferred embodiment, the transgenic cells are exogenously transfected with the pNL3.2.NF-κB-RE plasmid.

[0027] In a preferred embodiment, the transgenic cells are monoclonal stable transgenic cell lines.

[0028] In a preferred embodiment, in step S1, the transgenic cells are HL-60-pNL3.2 stable transgenic cell lines, purchased from Shanghai Zhangjiang Biotechnology Co., Ltd.

[0029] In a preferred embodiment, in step S2, the density of the cell suspension is approximately 1 × 10⁻⁶. 6 per ml.

[0030] In a preferred embodiment, in step S2, the final concentration of the cofactor is 10-1000 ng / ml, preferably 50-200 ng / ml, and more preferably 100 ng / ml.

[0031] In a preferred embodiment, in step S2, the incubation conditions are: 37°C, 5% carbon dioxide for 3-3.5 hours.

[0032] In a preferred embodiment, in step S2, after incubation, the mixture is brought to room temperature before the colorimetric solution is added.

[0033] In a preferred embodiment, the method further includes the steps of: using pyrogen standard solutions with different concentration gradients as the sample solutions to be tested, performing a four-parameter logistic regression with the concentration of the pyrogen standard solutions as the abscissa and the average RLU value as the ordinate, and fitting a standard curve; and calculating the pyrogen content in the sample solutions to be tested based on the standard curve.

[0034] In a preferred embodiment, the concentrations of the pyrogen standard solutions at different concentration gradients are as follows: 1000.000 EU / ml, 250.000EU / ml, 62.500 EU / ml, 15.625 EU / ml, 3.906 EU / ml, 0.977 EU / ml, 0.244 EU / ml, 0.061EU / ml, 0.015 EU / ml, 0.004 EU / ml.

[0035] In another preferred embodiment, the standard curve is selected from the group consisting of: , ,and .

[0036] The beneficial effects of this invention include: 1) The addition of cofactor protein to the in vitro pyrogen detection (reporter gene method) method of the present invention can significantly improve the sensitivity of the method, which is at least 19 times higher than that of the prior art.

[0037] 2) The method of the present invention not only significantly reduces the detection limit of pyrogen detection, but also solves the interference problem in sample pyrogen detection, with strong anti-interference ability and stable detection results.

[0038] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.

[0039] Example 1 1. Technical Solution In HL60 human promyelocytic leukemia cells expressing Toll-like receptors, NF-κB response element and luciferase genes were exogenously transfected. With the help of cofactors, pyrogen LPS activated the NF-κB signaling pathway, thereby activating luciferase expression to detect pyrogen levels.

[0040] 2. Construction of cell lines HL60 cells in the logarithmic growth phase (purchased from the Chinese Academy of Sciences Type Culture Collection Committee) were seeded in culture dishes with IMDM medium containing 10% fetal bovine serum, and transfected one week later.

[0041] During transfection, cells were resuspended in IMDM and the cell density was adjusted to 7.0 × 10⁶ cells / mL. 6 Cells / ml: 700 μl of cell suspension was mixed with 200 ng of pNL3.2.NF-κB-RE plasmid (purchased from Promega, USA, sequence pNL 3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro]) in a 1.5 mL centrifuge tube. The complex was then added to a 0.4 cm... 2 Electroporation chambers were incubated in an ice bath for 5 minutes, followed by electroporation transfection with a pulse of 300 volts every 20 milliseconds.

[0042] Subsequently, 10 mL of IMDM containing 10% fetal bovine serum was added to the cell suspension. After transfection, the cell suspension was gently added to each well of a 24-well plate and incubated at 37°C with 5% CO2 for 48 hours. Then, 100 μg / mL hygromycin B was added. After antibiotic selection for 2 to 4 weeks, the cells were resuspended in IMDM containing 10% fetal bovine serum and 100 μg / mL hygromycin B and seeded into 96-well plates (100 μL per well, i.e., 1 cell per well). The cells were cultured for 2 to 3 weeks to select monoclonal cells, expand and prepare monoclonal cells, and obtain stable transfected cell lines.

[0043] 3. Testing Steps (1) Preparation of pyrogen standard solution Take one vial of LPS national standard, add 1 ml of sterile water for injection to reconstitute it completely, and obtain an LPS stock solution with a concentration of 9000 EU / ml.

[0044] Take 100 μl of LPS stock solution + 800 μl of test culture medium → standard solution S1 (1000 EU / ml) The standard solution S1 was diluted 9 times at a ratio of 4 to obtain solutions S2 to S10.

[0045] The concentrations of the S1 to S10 standard solutions are as follows: 1000.000 EU / ml, 250.000EU / ml, 62.500 EU / ml, 15.625 EU / ml, 3.906 EU / ml, 0.977 EU / ml, 0.244 EU / ml, 0.061EU / ml, 0.015 EU / ml, 0.004 EU / ml.

[0046] The specific dilution process is shown in the table below.

[0047] (2) Operating steps Cells in the logarithmic growth phase were collected, and the cell suspension was placed in a 15 ml centrifuge tube. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to prepare a suspension with a density of approximately 1 × 10⁻⁶ cells / mL. 6 Cell suspensions of 50 μl / well were prepared (a cell suspension with cofactor added to a final concentration of 100 ng / ml was prepared as the experimental group) and seeded into white opaque flat-bottomed 96-well plates.

[0048] Add 50 μl of S1-S10 standard series solutions to each well, with the negative control being a dilution solution. Incubate at 37°C and 5% CO2 for 3-3.5 hours. After incubation, remove the 96-well plate, equilibrate to room temperature, and add 100 μl of chromogenic solution (NANO® Lucifurase Assay purchased from Promega, USA) to each well. Shake at room temperature for 15 minutes and read the chemiluminescence value (RLU value) using a microplate reader.

[0049] The operating steps are as follows Figure 1 .

[0050] (3) Data processing Using the concentration of the pyrogen standard solution as the x-axis (logarithmic scale) and the average RLU value as the y-axis, a four-parameter logistic regression was performed to fit the standard curve. Three independent experiments were conducted, and the standard curves are shown in the table below.

[0051] (4) Analysis of experimental results The concentration value obtained by adding three times the standard deviation of the negative control signal value to the standard curve and then calculating it is the limit of detection (LOD).

[0052] The detection limit was calculated from the results of the three experiments and is shown in the table below.

[0053] Adding a cofactor at a final concentration of 100 ng / ml to the reaction system of the pyrogen detection reporter gene assay improved the method's limit of detection (LOD) to between 0.014 and 0.040, while without the cofactor, the method's LOD was between 0.765 and 1.602, resulting in a sensitivity improvement of at least 19 times.

[0054] Example 2 Application Case: Reporter Gene Therapy for Pyrogen Detection in Intravenous Immunoglobulin (IVIG) ① Calculation of Maximum Dilution Factor (MVD) When performing pyrogen detection, a method interference test must first be conducted to evaluate whether the drug interferes with the pyrogen detection method. Therefore, a method interference test was conducted to determine whether intravenous immunoglobulin (IVIG) would interfere with the in vitro pyrogen detection method (reporter gene method).

[0055] According to industry regulations, the first step is to calculate the maximum dilution factor (MVD), which refers to the maximum dilution factor that the test sample solution is allowed to reach during the test. The contaminant limit is then tested at a concentration not exceeding this dilution factor. The specific MVD value is calculated using the following formula: MVD = CLC × c / LOD; CLC stands for pyrogen contaminant limit. The pyrogen contaminant limit CLC for intravenous immunoglobulin is 1.0 EU / ml. When CLC is expressed in EU / ml, then c equals 1.0 ml / ml. Based on historical laboratory test results, LOD is set at 0.05 EU / ml.

[0056] Therefore, the maximum dilution factor (MVD) of intravenous immunoglobulin is 1.0 × 1 / 0.05 = 20 times.

[0057] ② Interference with the preparation of the test solution The intravenous immunoglobulin (IVIG) was diluted 5, 10, and 20 times. The preparation process was as follows: the test sample was diluted 2.5, 5, and 10 times with the detection culture medium. The diluted solutions were then mixed with an equal volume of 30 EU / ml LPS (30 EU / ml is the LPS concentration near the midpoint of the pyrogen standard curve) to prepare 5, 10, and 20 times interference test sample solutions. The interference test sample solutions contained an LPS concentration of 15 EU / ml.

[0058] Take 1000 μl of intravenous immunoglobulin solution + 1500 μl of test culture medium → 2500 μl (test solution T0, 2.5×). Take 500 μl of test solution T0 + 500 μl of detection culture medium → 1000 μl of test solution T1, 5× Take 500 μl of test solution T1 + 500 μl of detection culture medium → 1000 μl of test solution T2, 10× Take 500 μl of test solution T2 + 500 μl of detection culture medium → 1000 μl of test solution T3, 20× Add 30 EU / ml LPS to the above solution to obtain interfering test solutions R1~R3: Take 200 μl of the test solution T0 + 200 μl of the standard solution with a concentration of 30 EU / ml → 400 μl (Interference with test solution R1, 5×) Take 200 μl of the test solution T1 + 200 μl of the standard solution with a concentration of 30 EU / ml → 400 μl (Interference with test solution R2, 10×) Take 200 μl of the test solution T2 + 200 μl of the standard solution with a concentration of 30 EU / ml → 400 μl (Interference with test solution R3, 20×) ③ Interference test procedure According to the method in Example 1 (2), an interference experiment was conducted by adding a series of standard solutions to interfere with the test solutions R1~R3 ​​and T1~T3.

[0059] Substitute the chemiluminescence values ​​of interfering test solutions R1~R3 ​​and test solutions T1~T3 into the standard curve to calculate the LPS value of the interfering test solution (A) and the LPS value of the test solution (B).

[0060] The LPS recovery rate (R) under the experimental conditions was calculated using the formula below.

[0061] R = (AB) / C × 100% When the recovery rate is between 50% and 200% (as required by industry regulations), it is considered that the sample solution does not have any interfering effect under these test conditions.

[0062] If the recovery rate is not within the specified range, interference factors must be eliminated, and the interference test should be repeated to find the cause of the interference.

[0063] ④ Interference test results and analysis The standard curves obtained by processing the data according to Example 1 (3) are shown in the table below.

[0064] When a cofactor with a final concentration of 100 ng / ml is added to the reaction system of the pyrogen detection reporter gene assay, the recovery rate of the interference test is between 65% and 157%, which meets the industry requirement of a recovery rate of 50% to 200%.

[0065] Without the addition of cofactors, the recovery rate of the 20-fold dilution was 79%, which met the requirements. However, the recovery rates of the 5-fold and 10-fold dilutions failed to meet the method's recovery rate requirements, as detailed in the table below.

[0066] ⑤ Sample pyrogen determination results After confirming that intravenous immunoglobulin (IVIG) does not interfere with the in vitro pyrogen testing method (reporter gene method), the established method was used for pyrogen determination. The test solutions T1~T3 were substituted into the standard curve with added cofactors. The results are detailed in the table below. The results show that the established in vitro pyrogen testing method (reporter gene method) is suitable for the determination of pyrogens in IVIG.

[0067] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A highly sensitive in vitro pyrogen detection method, characterized in that, The method includes the following steps: S1: Provide transgenic cells that express Toll-like receptors and are exogenously transfected with NF-κB response elements and luciferase genes; pyrogens activate the NF-κB signaling pathway by binding to Toll-like receptors on the surface of the transgenic cells, thereby activating luciferase expression to indicate pyrogen levels; S2: Take the transgenic cells in the logarithmic growth phase, prepare a cell suspension, add cofactor and test sample solution to the cell suspension, incubate, and then add chromogenic solution; S3: Detect the chemiluminescence value (RLU value) of the cell suspension and calculate the pyrogen content in the sample solution based on the chemiluminescence value.

2. The method as described in claim 1, characterized in that, In step S1, the transgenic cells are selected from the following group: human promyelocytic leukemia cells HL-60, mononuclear cell line MM6, human adenocarcinoma alveolar basal epithelial cells A549, and mouse mononuclear macrophage cell line RAW264.

7.

3. The method as described in claim 1, characterized in that, The transgenic cells were exogenously transfected with the pNL3.2.NF-κB-RE plasmid.

4. The method as described in claim 1, characterized in that, The transgenic cells are monoclonal stable transgenic cell lines.

5. The method as described in claim 1, characterized in that, In step S2, the density of the cell suspension is approximately 1 × 10⁻⁶. 6 per ml.

6. The method as described in claim 1, characterized in that, In step S2, the final concentration of the cofactor is 10-1000 ng / ml, preferably 50-200 ng / ml, and more preferably 100 ng / ml.

7. The method as described in claim 1, characterized in that, In step S2, the incubation conditions are: 37°C, 5% carbon dioxide for 3-3.5 hours.

8. The method as described in claim 1, characterized in that, The method further includes the following steps: using pyrogen standard solutions with different concentration gradients as the sample solutions to be tested, performing a four-parameter logistic regression with the concentration of the pyrogen standard solutions as the abscissa and the average RLU value as the ordinate, and fitting a standard curve; calculating the pyrogen content in the sample solutions to be tested based on the standard curve.

9. The method as described in claim 8, characterized in that, The concentrations of pyrogen standard solutions at different concentration gradients are as follows: 1000.000 EU / ml, 250.000EU / ml, 62.500 EU / ml, 15.625 EU / ml, 3.906 EU / ml, 0.977EU / ml, 0.244 EU / ml, 0.061EU / ml, 0.015 EU / ml, 0.004 EU / ml.

10. The method as described in claim 9, characterized in that, The standard curves are selected from the following group: , ,and .

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