Reagent card and kit for endotoxin detection and detection method and application thereof

Abstract

The invention provides a reagent card and a kit for endotoxin detection as well as a detection method and application thereof, and relates to the field of endotoxin detection. The recombinant C factor is applied to fluorescence chromatography for endotoxin detection, the prepared kit is accurate and stable in detection result, the detection limit is remarkably improved, and the production process is also simplified.

Description

Technical Field

[0001] The present invention belongs to the technical field of endotoxin detection, and specifically relates to a reagent card, a kit and a detection method and application thereof for endotoxin detection. Background Art

[0002] Endotoxin is a toxin released after the cell wall of Gram-negative bacteria is lysed. Because it can cause fever in the human body, it is also called "pyrogen". Its component is lipopolysaccharide. Lipopolysaccharide is composed of O-specific polysaccharide chains, core polysaccharides and lipid A, and its toxic active center is lipid A. Studies have shown that endotoxins embedded in the cell wall are not only released when the bacteria are lysed and die, but also continuously released to the surrounding during cell growth and division, and the released endotoxins will adhere to particulate matter and dust in the air. However, it is very difficult to remove endotoxins, because its chemical nature is lipopolysaccharide rather than protein, so general high temperature cannot destroy its biological toxicity. Even at 100°C, it still maintains most of its activity. It will only lose its toxicity at 160°C for 4 hours or higher. Therefore, only some high-temperature resistant instruments can use high-temperature methods to remove endotoxins. In addition, unlike exotoxins, which lose their toxicity after being treated with dilute formaldehyde, endotoxins also have a certain stability in the face of strong acids, strong bases and strong oxidants. The human body is extremely sensitive to endotoxins. Once trace amounts (1-5ng / kg body weight) of endotoxins enter the human blood, they can cause endotoxemia, shock, multiple organ failure, and even death. Therefore, biological products, medical devices, etc. must undergo strict endotoxin detection experiments before they are allowed to be used. At the same time, the development of highly sensitive endotoxin detection methods is one of the keys to the biopharmaceutical industry.

[0003] In the early 20th century, scientists S. Florence and others first proposed a method for endotoxin detection, called the rabbit pyrogen method. The specific operation is to inject the sample to be tested into the rabbit intravenously and observe whether the rabbit's body temperature rises to determine whether the sample contains endotoxin. The principle is that endotoxin acts on the central nervous system of mammals, causing the body temperature to rise. The detection sensitivity can reach 0.5EU / mL. This method was certified by the US FDA as a standard method for detecting endotoxin in the 1940s. However, this method has many disadvantages, such as unstable results due to individual differences, complex operation, and low sensitivity.

[0004] In 1956, Bang et al. conducted a landmark study on horseshoe crab hemolymph coagulation, pointing out that hemolymph coagulation of the American horseshoe crab L. Polyphemus was caused by injection of Gram-negative bacteria of the genus Vibrio (Bang F B. Abacterial disease of limulus polyphemus [J]. Bulletin of the Johns Hopkins Hospital, 1956.). The coagulation process of horseshoe crab hemolymph is explained as a series of cascade reactions caused by LPS-specific activation of zymogen ProC: horseshoe crab hemolymph contains zymogen ProC, zymogen ProB, clotting enzyme ProCE and coagulogen. First, zymogen ProC is activated by LPS and converted into active α-chelatase C, which activates zymogen ProB into chelatase B, which in turn activates clotting enzyme ProCE to become coagulase, causing coagulogen to be converted into coagulin gel through its head-to-tail polymerization; when these cascade reactions begin, the hemolymph flowing out of the wound will be blocked, and the invading Gram-negative bacteria will be fixed by the coagulin gel and killed by the secreted antimicrobial peptides.

[0005] The detection of endotoxin using horseshoe crab reagent is divided into gel method and photometric method. The gel method is to qualitatively or semi-quantitatively detect endotoxin by whether the cascade reaction finally forms a gel. The photometric method is further divided into turbidity method and colorimetric method. The turbidity method detects the endotoxin content based on the turbidity of the solution after the formation of gel; the colorimetric method is the reaction of the coagulase after the cascade reaction with the chromogenic substrate, and the endotoxin content is detected by measuring the absorbance A545nm. The gel method does not require professional instruments and is simple and convenient to operate. The linear detection range of the photometric method is wider, and the detection is sensitive and accurate. The disadvantage is that it will be interfered by (1-3)-β-D-glucan, because it can replace factor C to participate in the cascade reaction and activate the zymogen B to chelate B, resulting in false positive results.

[0006] Horseshoe crabs were first used as fertilizers, feeds, and food. Later, when horseshoe crab reagents were found to be useful for endotoxin testing, people began to use the puncture method as the standard for blood collection and preparation of horseshoe crab reagents. After blood collection, the horseshoe crabs will be released. It is estimated that the mortality rate after release is as high as 20%. In addition, the survival rate of artificial breeding is low and the cycle is long. In addition, with the deterioration of the environment and the wanton predation by humans, the number of horseshoe crabs has dropped sharply in recent years. In the 1970s, the number of horseshoe crabs in Chinese waters dropped by 80% to 90%. Drawing blood from live horseshoe crabs to prepare horseshoe crab reagents is greatly restricted. In addition, Japan listed horseshoe crabs on the endangered species list as early as the 1990s. In 2021, my country also announced that Chinese horseshoe crabs and round-tailed horseshoe crabs would be upgraded to national second-class protected animals. Horseshoe crabs are strictly controlled, and medical device companies are advised to replace other endotoxin detection methods as soon as possible and develop new endotoxin detection technologies.

[0007] Factor C is a serine protease. In 1991, Muta et al. cloned the cDNA of the full-length Factor C and confirmed that Factor C is composed of 1019 amino acids, the first 25 amino acids are its signal peptide, and the mature peptide is 994 amino acids. It is a double-chain protein with a heavy chain (R26-S690) and a light chain (S691-I1019), with a size of 109kDa. However, the application of the recombinant Factor C method has been slow, and it was not until 2020 that the recombinant Factor C method was included in the Chinese Pharmacopoeia. Because the horseshoe crab Factor C has a complex structure, a large molecular weight, a double-chain structure similar to an antibody, and 6 potential glycosylation sites, it is difficult to express the full-length and biologically active Factor C. At the same time, it is very easy to introduce exogenous LPS during the expression process, and the horseshoe crab Factor C is extremely sensitive to endotoxins in the environment. During the expression and purification process, the endotoxins in the culture medium or solution will be enriched, which greatly reduces the activity of the expressed natural Factor C.

[0008] In recent years, scientists have developed a new generation of endotoxin detection kits that do not rely on horseshoe crab lymph - recombinant factor C (rFC) endotoxin detection kits, which use genetic recombination technology to express horseshoe crab factor C. After binding to endotoxin, it is converted from an inactive protease to a biologically active protease, which recognizes and catalyzes downstream fluorescent substrates to produce fluorescent signals. The intensity of the fluorescent signal is positively correlated with the endotoxin concentration, thereby quantitatively detecting endotoxins. Compared with the horseshoe crab reagent method, the recombinant C-factor method is an improved method of the current horseshoe crab reagent endotoxin detection method. It uses a fluorescent microplate reader and the recombinant C-factor method to detect bacterial endotoxins, which has the advantages of reliability, permanence, and environmental protection. The main manifestations are: ① This method does not have the interference of G-factor bypass and has high specificity. The recombinant technology eliminates the interference of β-1,3-glucan in the horseshoe crab reagent on the test results to prevent false positives in the test; ② The reagents used in this method do not contain B factor, procoagulant, prothrombin, etc. Therefore, samples containing inhibitory or enhancing effects on the above substances are suitable for the recombinant C-factor method; ③ It does not rely on animal-derived ingredients, providing higher supply security, which is in line with the "3R" principle of experimental animal science. ④ The product is produced using recombinant technology and has good batch consistency; ⑤ Endpoint fluorescence determination is comparable to other traditional quantitative horseshoe crab reagent methods. Pei Yusheng et al. validated the recombinant factor C method for detecting bacterial endotoxins and compared it with the dynamic colorimetric method. They found that the recombinant factor C method has better specificity. It does not react with β-1,3-glucan and has a sensitivity range of 0.005-5EU / mL. It has good specificity, accuracy and precision and can meet the detection needs of related products (Pei Yusheng, Cai Tong, Chen Chen, et al. Validation of the recombinant factor C detection method for bacterial endotoxins [J]. Chinese Journal of Biologicals, 2020, 33(1):4.).

[0009] Endotoxin detection methods based on biochemical sensors such as fluorescent probes and bio-enzymes utilize the physical and chemical signal changes generated by the interaction between the analyte and the biological sensitive layer, which can be converted into electrochemical, optical, thermal, piezoelectric and other response signals through a signal converter, and the correlation between these signals and the analyte is analyzed, so as to achieve the purpose of analyzing and detecting the analyte. A large number of studies have shown that biosensors have the advantages of good selectivity, high sensitivity, fast analysis speed, low cost, and the ability to perform online continuous monitoring in complex systems. In particular, they are highly automated, miniaturized and integrated, and have been booming in the field of research and technology development in recent years. The huge advantages of biosensors have also shown good application prospects in endotoxin research. At present, a large number of biosensors have been used for endotoxin detection. However, the design and construction of the sensing layer of the biosensor is time-consuming, easily affected by background light, has poor anti-interference ability, has non-specific binding, and the life of the biorecognition element is not long.

[0010] Prior art CN114636816A provides a fluorescent microsphere probe for detecting endotoxins, and its preparation method and application. The preparation method of the fluorescent microsphere probe for detecting endotoxins includes the following steps: obtaining a recombinant protein horseshoe crab factor C protein; activating the carboxylated fluorescent microspheres to obtain activated fluorescent microspheres; coupling the activated fluorescent microspheres with the recombinant protein horseshoe crab factor C protein to obtain coupled microspheres; blocking the unreacted groups on the coupled microspheres with amino PEG and ethanolamine to obtain a fluorescent microsphere probe for detecting endotoxins, and then detecting endotoxins by fluorescence chromatography. This method requires coupling the recombinant factor C protein to the microspheres, which is cumbersome and has poor stability.

[0011] Prior art CN117586995A discloses a novel recombinant factor C and its preparation method and application. The invention transforms the existing horseshoe crab coagulation factor C, maintains the original sequence (functional domain) of the light chain region unchanged, and replaces the Sushi1 and Sushi2 regions in the heavy chain with the Sushi4 region in the light chain. Without changing the overall structure and functional domain of Factor C, the free energy of the protein itself is reduced, making the protein itself more stable and improving the ability to bind to substrates. In addition, the present invention adds a signal sequence to the N-terminus of the protein sequence to allow the target protein to be expressed extracellularly, which is more conducive to subsequent detection and purification, and finally designs a modified protein rFC-MBJ. This method performs endotoxin detection on a 96-well plate, and has poor portability and stability.

[0012] With the deepening of research, many immunological endotoxin detection methods have been continuously introduced, such as CN117310184A: A method for detecting endotoxins, which invents a method for qualitatively capturing HRG protein with endotoxins by antigen-antibody binding, and then separating endotoxins from proteins to detect endotoxins; CN110095600A: A test strip and kit for bacterial endotoxins, which prepares endotoxin antibodies and uses ELISA to detect endotoxins. The above methods all have their own theoretical advantages, but without exception, they have not been recognized by the "Chinese Pharmacopoeia", and their methods and operational feasibility need to be further verified. Summary of the invention

[0013] In view of the problems existing in the prior art, the present invention provides an endotoxin detection kit and its supporting reagent card and detection method. The kit is based on the fluorescence chromatography method of recombinant factor C, which can quickly detect endotoxins, meets the requirements of the pharmacopoeia, and can improve the detection efficiency and accuracy.

[0014] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0015] In one aspect, the present invention provides a reagent card for endotoxin detection, the structural schematic diagram of the reagent card is as follows Figure 1 As shown, the preparation method comprises the following steps:

[0016] S1. preparing a fluorescent colorimetric substrate solution: dissolving a fluorescent colorimetric substrate in a protein coating buffer to obtain a substrate protein solution; dissolving lipopolysaccharide in the substrate protein solution to obtain a substrate protein solution containing lipopolysaccharide;

[0017] S2, coating T line and C line: streaking the nitrocellulose membrane with substrate protein solution to obtain T line; streaking the same nitrocellulose membrane with substrate protein solution containing lipopolysaccharide to obtain C line; then drying the nitrocellulose membrane to obtain coated nitrocellulose membrane;

[0018] S3, assembly: sticking the coated nitrocellulose membrane, the water absorbent pad and the sample pad on the polyvinyl chloride plate in sequence, and pressing the water absorbent pad and the sample pad on both ends of the coated nitrocellulose membrane respectively to obtain a reagent plate;

[0019] S4, cutting: Cut the reagent plate into thin strips and install them in the card shell to obtain a reagent card.

[0020] Preferably, the fluorescent color developing substrate in step S1 is a protease fluorescent substrate coupled to a macromolecule.

[0021] Macromolecules refer to biological substances with a relative molecular mass of more than 5000, such as proteins, nucleic acids, polysaccharides, etc. Coupling the protease fluorescent substrate with the macromolecule through chemical bonds can increase the molecular weight of the protease fluorescent substrate and enhance its anchoring ability, so that the protease fluorescent substrate is firmly attached to the nitrocellulose membrane to prevent it from falling off.

[0022] Preferably, the macromolecule comprises bovine serum albumin, casein or ovalbumin.

[0023] Specifically, the macromolecule is bovine serum albumin (BSA).

[0024] Protease fluorescent substrates are important tools for monitoring protease activity. They produce fluorescence when reacting with proteases, allowing the activity of proteases to be assessed by changes in fluorescence intensity.

[0025] Preferably, the protease fluorescent substrate includes a serine protease fluorescent substrate such as chymotrypsin, elastase, or a chymotrypsin fluorescent substrate.

[0026] Preferably, the protease fluorescent substrate is a chymotrypsin fluorescent substrate.

[0027] Specifically, the specific structure of the chymosin fluorescent substrate is Nt-Boc-Val-Pro-Arg-AMC, wherein Nt-BOC is the abbreviation of the protective group N-tert-butyloxycarbonyl, Val-Pro-Arg is a tripeptide, and AMC is a fluorescent substrate; in addition, BSA protein is additionally coupled to the original peptide chain of the protease fluorescent substrate (obtaining Nt-Boc-Val-Pro-Arg-AMC-BSA), which can improve its tightness with the nitrocellulose membrane coating, and after the peptide chain is cut by the protease, the fluorescent group AMC will not be released.

[0028] Preferably, the components of the protein coating buffer in step S1 include surfactant, bovine serum albumin, sodium chloride, sugar reagent or phospholipid substance.

[0029] Preferably, the phospholipids include cephalin, phosphatidylcholine, serine phospholipids or phosphatidylinositol. Adding phospholipids to the protein coating buffer can better adsorb lipopolysaccharide.

[0030] Specifically, the phospholipid substance is cephalin.

[0031] Preferably, in step S2, 0.25-0.75 mg / mL substrate protein solution is used to draw T line, and the usage amount is 1 μL / cm; on the same nitrocellulose membrane, substrate protein solution containing 1 EU / mL lipopolysaccharide is used to draw C line, and the usage amount is 1 μL / cm.

[0032] In another aspect, the present invention provides a kit for endotoxin detection, the kit comprising a recombinant Factor C protein lyophilized powder, a diluent and the above-mentioned reagent card.

[0033] Preferably, the components of the recombinant Factor C protein lyophilized powder include recombinant Factor C protein, BSA and lyophilization excipients.

[0034] Preferably, the method for preparing the recombinant Factor C protein lyophilized powder comprises the following steps:

[0035] S1. Dissolve the lyophilized excipient in sterile water to obtain solution A;

[0036] S2, adding recombinant factor C protein and BSA to solution A, stirring to dissolve, to obtain solution B;

[0037] S3, freeze-drying solution B to obtain freeze-dried powder.

[0038] Preferably, the method for preparing the recombinant Factor C protein comprises the following steps: constructing a plasmid containing the Factor C gene sequence, transfecting the plasmid into cells (such as insect SF9, mammalian CHO, mammalian HEK293 and other cells), expressing and purifying to obtain the recombinant Factor C protein.

[0039] Preferably, the lyophilized excipients include mannitol, polyvinyl pyrrolidone, lactose, sucrose, raffinose, PEG-3000 or PEG-8000.

[0040] Preferably, the diluent comprises a buffer, a surfactant and a divalent cationic compound.

[0041] Preferably, the buffer comprises Tris buffer, PBS buffer, Tricine buffer or Hepes buffer.

[0042] Specifically, the buffer is Hepes buffer.

[0043] Preferably, the surfactant includes Tween-20, Tween-80, Triton-X100, SDS or sodium lauroyl sarcosinate.

[0044] Preferably, the divalent cationic compound includes magnesium chloride, magnesium sulfate, calcium chloride or zinc sulfate.

[0045] In another aspect, the present invention provides a method for detecting endotoxin, wherein the method uses the above-mentioned kit for detection, and comprises the following steps:

[0046] The recombinant factor C protein lyophilized powder was dissolved in a diluent, and different concentrations of endotoxin standard substances were added and mixed, and then dropped onto the sample pad of the reagent card, and the fluorescence intensity of the T-line area and the C-line area was measured. According to the changes in the fluorescence intensity of the T-line area caused by different concentrations of standard substances, a concentration standard curve was obtained;

[0047] Dissolve the recombinant Factor C protein lyophilized powder in diluent, add the sample to be tested and mix well, then drop it onto the sample pad of the reagent card, measure the fluorescence intensity of the T-line area and the C-line area, and calculate the endotoxin concentration in the sample to be tested based on the change in fluorescence intensity of the T-line area caused by the sample to be tested and the concentration standard curve.

[0048] Preferably, the steps of the detection method are as follows: adding the recombinant factor C protein lyophilized powder to the diluent and mixing it to a final concentration of 20 μg / mL, and then mixing it with endotoxin standards of different concentrations or samples to be tested at a volume ratio of 1:1 to obtain a mixed solution; placing the reagent card on a clean and flat table, and taking 100 μL of the mixed solution and dropping it on the sample pad of the reagent card; using a fluorescent immunoassay analyzer to scan the T line area and the C line area on the reagent card at 5 minutes and 20 minutes, respectively, to obtain the fluorescence signal RFU, and calculating the T line area fluorescence signal growth value ΔRFU corresponding to different samples (ΔRFU=end point RFU-starting point RFU); preparing an endotoxin concentration standard curve according to the T line area fluorescence signal growth value ΔRFU corresponding to the endotoxin standard of different concentrations; and calculating the endotoxin concentration value in the sample to be tested by combining the T line area fluorescence signal growth value of the sample to be tested with the standard curve.

[0049] In another aspect, the present invention provides a use of the above reagent card and kit in endotoxin detection.

[0050] Preferably, the application is an application other than disease diagnosis or treatment.

[0051] Preferably, the above reagent cards and kits are only used for the detection of endotoxins in the industrial market and are not used in clinical practice, so there is no problem of complexity in sample pre-processing.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The present invention performs endotoxin detection based on the principle of the recombinant factor C method and provides a new idea for kit design. On the one hand, the protease fluorescent substrate coupled to the macromolecule is coated on the reagent card, and on the other hand, the recombinant factor C protein is stored in the form of dry powder and dissolved and diluted before use. Finally, by optimizing the relevant buffer system, the obtained kit detection results are accurate and stable, the detection limit is significantly improved, and the production process is also simplified. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1Schematic diagram of the structure of the endotoxin detection reagent card.

[0055] Figure 2 Endotoxin concentration standard curve.

[0056] Figure 3 Schematic diagram of the principle of detecting endotoxin using recombinant C factor combined with fluorescent substrate (C in the horizontal axis represents different concentrations of endotoxin standards).

[0057] Figure 4 Recombinant factor C protein lyophilized powder prepared with different lyophilization excipients. DETAILED DESCRIPTION

[0058] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning Experiment Guide (Fourth Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0059] Example 1 Preparation of a reagent card for endotoxin detection

[0060] Tween-20, BSA (Solarbio, product number 9048-46-8), sodium chloride, trehalose and cephalin were dissolved in sterile deionized water to make the mass fraction of each component 0.01%, and stirred evenly to obtain a protein coating buffer; BSA-coupled chymosin fluorescent substrate (structure Nt-Boc-Val-Pro-Arg-AMC-BSA, synthesized by Shanghai Kepeptide Biotechnology Co., Ltd.) was added to the protein coating buffer to make the final concentration of the fluorescent substrate 0.5 mg / mL to obtain a fluorescent substrate solution.

[0061] Take a square nitrocellulose membrane, draw a T line on the nitrocellulose membrane using a 0.5 mg / mL fluorescent substrate solution in an amount of 1 μL / cm; draw a C line on the same nitrocellulose membrane using a 0.5 mg / mL fluorescent substrate solution containing 1 EU / mL lipopolysaccharide in an amount of 1 μL / cm; after the marking, place the nitrocellulose membrane in an oven (Shanghai Yiheng Scientific Instrument Co., Ltd., model DHP-9126) at a temperature of 37°C and a humidity of <30% for 24 hours to obtain a coated nitrocellulose membrane.

[0062] The coated nitrocellulose membrane was attached to the middle of the polyvinyl chloride plate; the absorbent pad was pressed on one end of the coated nitrocellulose membrane, with an overlapping area of ​​1-2 mm; the sample pad was pressed on the other end of the nitrocellulose membrane, with an overlapping area of ​​1-2 mm, to obtain a reagent plate; the reagent plate was cut into thin strips with a width of 4±0.4 mm (see the schematic diagram Figure 1 ), install the card shell; put the card shell and desiccant into an aluminum foil bag, label it and pack it into a box to get the finished reagent card.

[0063] Example 2: Preparation of a kit for endotoxin detection

[0064] The kit consists of a recombinant Factor C protein lyophilized powder, a diluent, and the reagent card in Example 1, wherein the preparation methods of the recombinant Factor C protein lyophilized powder and the diluent are as follows:

[0065] 1) Preparation of recombinant factor C protein lyophilized powder

[0066] The factor C gene of Tachypleus tridentatus was obtained from NCBI (GenBank: AAL75577.1), and then a signal peptide gene was added to the N-terminus of the factor C gene, and a His tag was added to the C-terminus. The expression vector HD293F was double-digested with EcoRI and HindIII and ligated with the recombinant factor C gene to construct a plasmid (sequence shown in SEQ ID NO.1), which was transfected into mammalian cells and expressed and purified to obtain the recombinant factor C protein (sequence shown in SEQ ID NO.2).

[0067] Lactose and PEG-8000 were dissolved as freeze-drying excipients in sterile deionized water to make the lactose mass fraction 3% and the PEG-8000 mass fraction 0.3%; recombinant factor C protein and BSA were then added to make the recombinant factor C protein concentration 20 μg / mL and the BSA mass fraction 2%, and stirred to dissolve; freeze-dried using a freeze dryer (Dongfulong, model LY0-0.5) according to the procedure in Table 1 to obtain recombinant factor C protein freeze-dried powder.

[0068] Table 1 Freeze-drying parameters

[0069]

[0070] 2) Preparation of dilution solution

[0071] Tween-20 and 0.01% sodium lauroyl sarcosinate were added to 0.2M Hepes buffer at pH 7.2, so that the mass fractions of Tween-20 and sodium lauroyl sarcosinate were both 0.01%; magnesium sulfate and calcium chloride were then added so that the final concentrations of magnesium sulfate and calcium chloride were both 0.1M to obtain a buffer solution.

[0072] The recombinant Factor C protein lyophilized powder and the buffer solution were placed in suitable containers respectively, and matched with the reagent card in Example 1 to obtain a kit for endotoxin detection.

[0073] Example 3: A method for detecting endotoxin

[0074] Take one 80EU endotoxin working standard (China Food and Drug Inspection Institute, catalog number 150601-202494), add 0.8mL inspection water to dissolve, and obtain a standard solution with a concentration of 100EU / mL, and then dilute it to 50, 5, 0.5, 0.05, and 0.005EU / mL of endotoxin standard.

[0075] The recombinant factor C protein lyophilized powder in Example 2 was added to the diluent and mixed to a final concentration of 20 μg / mL, and then mixed with endotoxin standards or test samples of different concentrations at a volume ratio of 1:1 to obtain different sample mixtures; the reagent card in Example 1 was placed on a clean and flat table, and 100 μL of the mixture was aspirated and dripped onto the sample pad of the reagent card; the T line area and C line area on the reagent card were scanned using a fluorescent immunoassay analyzer (excitation wavelength 380 nm, emission wavelength 440 nm) at 5 minutes and 20 minutes respectively to obtain the fluorescence signal RFU, and the T line fluorescence signal growth value ΔRFU corresponding to different samples was calculated (ΔRFU=end point RFU-start point RFU); according to the fluorescence signal growth value ΔRFU corresponding to the endotoxin standard of different concentrations (see Table 2), an endotoxin concentration standard curve was prepared (lgΔRFU=a×logC+b, see Figure 2 ); The fluorescence signal growth value of the sample to be tested is combined with the standard curve to calculate the endotoxin concentration value in the sample to be tested. The detection principle diagram is shown in Figure 3 shown.

[0076] Table 2 Fluorescence growth values ​​generated by different concentrations of endotoxin standards

[0077] Endotoxin standard concentration (EU / mL) ΔRFU 50 186096 5 64104 0.5 24729 0.05 8592 0.005 3338

[0078] Example 4: Optimizing freeze-dried excipient components

[0079] According to the method in Example 2, the lyophilized excipients were prepared according to the component design in Table 3 (the percentage before the component indicates its mass fraction in the lyophilized excipient solution), and then the recombinant Factor C protein lyophilized powder was prepared. Figure 4 As shown in the figure, from left to right are recombinant Factor C protein lyophilized powders numbered 1-10. The water content and solubility of these lyophilized powders were tested according to the conventional methods in the art. The results are shown in Table 3, indicating that the lyophilization excipient composed of 3% lactose and 0.3% PEG-8000 makes the recombinant Factor C protein lyophilized powder have the lowest water content and the best solubility.

[0080] Table 3 Water content and solubility of recombinant factor C protein lyophilized powder prepared with different lyophilized excipients

[0081] serial number Freeze-dried excipient components Water content (%) Solubility (+ represents the degree of solubility) 1 3% Raffinose + 0.3% PEG-8000 / + 2 3% Mannitol 7.62 ++ 3 3% Lactose 6.85 ++ 4 3% sucrose + 0.3% PEG-8000 4.67 ++ 5 3% lactose + 0.3% PEG-8000 2.76 +++ 6 1% polyvinyl pyrrolidone + 3% cottonseed sugar 8.62 + 7 1% polyvinylpyrrolidone + 3% mannitol 5.06 ++ 8 1% Polyvinylpyrrolidone + 0.3% PEG-3000 / + 9 1% Polyvinylpyrrolidone + 0.3% PEG-8000 / + 10 3% lactose + 0.3% PEG-3000 3.24 ++

[0082] Note: “ / ” indicates that the freeze-dried powder has no obvious solid structure and cannot be taken out for water content determination.

[0083] Example 5: Optimizing protein coating buffer formulation

[0084] According to the method in Example 1, according to the formula design of Table 4, different surfactants, BSA, sodium chloride, different sugar reagents and different phospholipid substances are mixed and dissolved, so that the mass fraction of each component is 0.01%, and a protein coating buffer is obtained, and then a reagent card is prepared. According to the detection method of Example 3, endotoxin-negative samples and standard products containing 1EU / mL endotoxin are detected respectively. In addition, the evaluation criteria of the kit are as follows: 1) the lower the C-line fluorescence value of the negative sample, the better, and the T-line fluorescence value>30000; 2) the C-line fluorescence value range of the standard product is 30000-34000, and the T-line fluorescence value>30000. The test results are shown in Table 4 (where the upper value is the C-line fluorescence value, and the lower value is the T-line fluorescence value), indicating that the optimal protein coating buffer formula is composed of Tween-20, BSA, sodium chloride, trehalose and phospholipids (mass fraction is 0.01%).

[0085] Table 4 Test results of the kit prepared with different protein coating buffers

[0086]

[0087] Example 6: Optimizing the concentration of fluorescent chromogenic substrate protein

[0088] According to the method in Example 1, the concentrations of the fluorescent substrate protein were prepared to 0.25, 0.5 and 0.75 mg / mL, respectively, and reagent cards were prepared respectively; according to the detection method of Example 3, the endotoxin-negative sample and the standard containing 1 EU / mL endotoxin were detected, respectively. The results are shown in Table 5, indicating that the fluorescence values ​​corresponding to the fluorescent substrate concentrations of 0.75 mg / mL and 0.5 mg / mL were very different, and increasing the concentration could not increase the fluorescence intensity, indicating that the optimal fluorescent substrate solution concentration is 0.5 mg / mL.

[0089] Table 5 Test results of the kit prepared with different fluorescent chromogenic substrate protein concentrations

[0090]

[0091]

[0092] Example 7: Optimization of diluent components

[0093] According to the method in Example 2 and the formula design in Table 6, different surfactants and different divalent cationic compounds were mixed and added to 0.2M Hepes buffer at pH = 7.2 to prepare different dilutions (the percentage before the component indicates its mass fraction in the dilution, and the concentration value before the component indicates its concentration in the dilution); according to the detection method in Example 3, the endotoxin-negative samples and the standard containing 1EU / mL endotoxin were tested respectively. At the same time, according to the conventional applicable methods in the field, the water absorption rate of the reagent card prepared in Example 1 for different buffers was determined, and the solubility of the recombinant C factor protein lyophilized powder prepared in Example 2 in different buffers was determined. The measurement results are shown in Table 6, and the data in the table are from top to bottom: 1) lyophilized powder solubility (+ represents the degree of solubility), 2) negative sample C line fluorescence value, 3)

[0094] 1EU / mL standard C line fluorescence value, 4) reagent card water absorption speed (s), the results showed that the optimal dilution formula was 0.01% Tween-20, 0.01% sodium lauroyl sarcosinate, 0.1M magnesium sulfate, 0.01M calcium chloride. This formula has good solubility, fast flow rate, low background and high sensitivity.

[0095] Table 6 Test results of the kit prepared with different dilutions

[0096]

[0097]

[0098] Example 8: Kit performance test

[0099] Unless otherwise specified, the method of Example 3 was used in this example to detect the endotoxin concentration of the sample.

[0100] 1) Determination of the detection limit of the kit:

[0101] Endotoxin working standard gradient solutions of 100, 50, 5, 0.5, 0.05, 0.005, 0.001, and 0 EU / mL were prepared for detection. Each gradient was tested 3 times and the average value was calculated. The results are shown in Table 7, indicating that the endotoxin detection limit of this kit is 50-0.005 EU / mL.

[0102] Table 7 Detection results of different endotoxin standard concentrations

[0103] Endotoxin standard concentration (EU / mL) ΔRFU average 100 206200±1500 50 187143±1500 5 65297±500 0.5 24022±350 0.05 8245±150 0.005 3299±125 0.001 1806±75 0 1615±50

[0104] 2) Sample reliability test:

[0105] Three endotoxin samples (sodium hyaluronate, cell culture fluid, pipette tip leaching fluid) with high, low, and negative levels were prepared and tested using the horseshoe crab gel method (using Dana Bio, kit No. NJF01060), the colorimetric method (using Dana Bio, kit No. XSF0596K-2), the recombinant factor C method (using Shanghai Ruinuo Bio, kit No. RAF-01) and the method of Example 3 to verify the reliability of the samples. The results show that the sample detection results of the kit are reliable, as shown in Table 8.

[0106] Table 8 Comparison of test results of different detection methods

[0107]

[0108] 3) Repeatability and accuracy:

[0109] Endotoxin standard solutions of three concentrations (2, 0.2, 0.02 EU / mL) were prepared for testing, with 8 replicates for each concentration. The CV and relative deviation of the test results were calculated. The results are shown in Table 9. The measured CV and relative deviation were both less than 10%, indicating that the kit has good repeatability and accuracy.

[0110] Table 9 Repeatability and accuracy evaluation

[0111] Endotoxin standard concentration (EU / mL) 2 0.2 0.02 Repeat 1 1.91 0.206 0.021 Repeat 2 1.96 0.214 0.022 Repeat 3 1.92 0.195 0.021 Repeat 4 1.91 0.189 0.018 Repeat 5 1.94 0.201 0.019 Repeat 6 1.89 0.201 0.02 Repeat 7 1.96 0.188 0.021 Repeat 8 1.86 0.181 0.022 CV 1.8％ 5.5％ 6.9％ Relative deviation 4.1％ 1.6％ 2.5％

[0112] 4) Stability:

[0113] Endotoxin standard solutions of three concentrations (2, 0.2, 0.02 EU / mL) were prepared, and the reagent card was placed in a 37°C incubator. The repeatability, accuracy and calibration curve of the reagent card were tested on the 1st, 3rd, 5th, 7th, 11th, 13th, 15th, 17th and 19th days, respectively. The results are shown in Table 10, indicating that the reagent card was stable within 15 days of thermal acceleration.

[0114] Table 10 Stability evaluation

[0115]

[0116] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A reagent card for endotoxin detection, characterized in that: The preparation method of the reagent card comprises the following steps: S1. preparing a fluorescent colorimetric substrate solution: dissolving a fluorescent colorimetric substrate in a protein coating buffer to obtain a substrate protein solution; dissolving lipopolysaccharide in the substrate protein solution to obtain a substrate protein solution containing lipopolysaccharide; S2, coating T line and C line: streaking the nitrocellulose membrane with substrate protein solution to obtain T line; streaking the same nitrocellulose membrane with substrate protein solution containing lipopolysaccharide to obtain C line; then drying the nitrocellulose membrane to obtain coated nitrocellulose membrane; S3, assembly: sticking the coated nitrocellulose membrane, the water absorbent pad and the sample pad on the polyvinyl chloride plate in sequence, and pressing the water absorbent pad and the sample pad on both ends of the coated nitrocellulose membrane respectively to obtain a reagent plate; S4, cutting: Cut the reagent plate into thin strips and install them in the card shell to obtain a reagent card.

2. The reagent card according to claim 1, characterized in that: The fluorescent color development substrate in step S1 is a protease fluorescent substrate coupled to a macromolecule.

3. The reagent card according to claim 2, characterized in that: The macromolecule is bovine serum albumin, and the protease fluorescent substrate is chymosin fluorescent substrate.

4. The reagent card according to claim 1, characterized in that: The components of the protein coating buffer in step S1 include surfactant, bovine serum albumin, sodium chloride, sugar reagent or phospholipid substance.

5. A kit for endotoxin detection, characterized in that: The kit comprises a recombinant Factor C protein lyophilized powder, a diluent and the reagent card according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The components of the recombinant factor C protein lyophilized powder include recombinant factor C protein, BSA and lyophilized excipients.

7. The kit according to claim 6, characterized in that The freeze-drying auxiliary materials include mannitol, polyvinyl pyrrolidone, lactose, sucrose, raffinose, PEG-3000 or PEG-8000.

8. The kit according to claim 5, characterized in that The components of the diluent include a buffer, a surfactant and a divalent cationic compound.

9. A method for detecting endotoxin, characterized in that: The detection method adopts the kit according to any one of claims 5 to 8 for detection, comprising the following steps: The recombinant factor C protein lyophilized powder was dissolved in a diluent, and different concentrations of endotoxin standard substances were added and mixed, and then dropped onto the sample pad of the reagent card, and the fluorescence intensity of the T-line area and the C-line area was measured. According to the changes in the fluorescence intensity of the T-line area caused by different concentrations of standard substances, a concentration standard curve was obtained; Dissolve the recombinant Factor C protein lyophilized powder in diluent, add the sample to be tested and mix well, then drop it onto the sample pad of the reagent card, measure the fluorescence intensity of the T-line area and the C-line area, and calculate the endotoxin concentration in the sample to be tested based on the change in fluorescence intensity of the T-line area caused by the sample to be tested and the concentration standard curve.

10. Use of the reagent card according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 8 in endotoxin detection, characterized in that: The application is not an application for disease diagnosis or treatment.

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