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

Abstract

The application provides a reagent card and kit for endotoxin detection, a detection method and application thereof, and relates to the field of endotoxin detection. The application applies recombinant C factor to fluorescence chromatography for endotoxin detection, and the prepared kit has accurate and stable detection results, a significantly improved detection limit, and a simplified production process.

Description

Technical Field

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

[0002] Endotoxins are toxins released by Gram-negative bacteria upon cell wall lysis. Because they cause fever upon entry into the human body, they are also called "pyrogens." They are composed of lipopolysaccharides (LPS). LPS consists of O-specific polysaccharide chains, a core polysaccharide, and lipid A, with lipid A being the active toxic site. Research has shown that endotoxins embedded in the cell wall are not only released upon bacterial lysis and death but are also continuously released into the surrounding environment during cell growth and division. Released endotoxins can also attach to airborne particles and dust. However, the removal of endotoxins is challenging. Because they are chemically lipopolysaccharides, not proteins, conventional high temperatures cannot destroy their biological toxicity. Even at 100°C, they retain most of their activity. Their toxicity is lost only at 160°C for 4 hours or higher. Therefore, only certain heat-resistant instruments can be used for high-temperature removal of endotoxins. Furthermore, unlike exotoxins, which lose their toxicity upon treatment with dilute formaldehyde, endotoxins are relatively stable in the presence of strong acids, bases, and oxidants. The human body is extremely sensitive to endotoxins. Even trace amounts (1-5ng / kg body weight) of endotoxins entering the human blood can cause endotoxemia, shock, multiple organ failure, and even death. Therefore, biological products and medical devices must undergo rigorous endotoxin testing before they are allowed to be used. At the same time, developing highly sensitive endotoxin detection methods is one of the keys to the biopharmaceutical industry.

[0003] In the early 20th century, scientists such as S. Florence first proposed a method for endotoxin detection, known as the rabbit pyrogen method. This method involves injecting a test sample intravenously into a rabbit and observing whether the rabbit's body temperature rises to determine whether the sample contains endotoxins. The principle is that endotoxins act on the mammalian central nervous system, causing a rise in body temperature. The detection sensitivity can reach 0.5 EU / mL, and this method was certified by the US FDA as a standard method for endotoxin detection in the 1940s. However, this method has several drawbacks, including unstable results due to individual variability, complex operation, and low sensitivity.

[0004] In 1956, Bang et al. made a landmark study on horseshoe crab hemolymph coagulation, pointing out that hemolymph coagulation of American horseshoe crab L. Polyphemus was caused by injection of Gram-negative bacteria of the genus Vibrio (Bang F BA bacterial disease of limulus polyphemus[J]. Bulletin of the Johns Hopkins Hospital, 1956.). The horseshoe crab hemolymph coagulation process 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 into coagulase, leading to the conversion of coagulogen into coagulin through head-to-tail polymerization. When these cascade reactions begin, hemolymph outflow from the wound is blocked, and invading Gram-negative bacteria are immobilized by the coagulin gel and killed by secreted antimicrobial peptides.

[0005] Endotoxin detection using Limulus amebocyte lysate reagent can be divided into gel method and photometric method. The gel method is a qualitative or semi-quantitative detection of endotoxins based on whether a gel is formed at the end of the cascade reaction. The photometric method is further divided into turbidimetric method and colorimetric method. The turbidimetric method detects the endotoxin content based on the turbidity of the solution after gel formation; the colorimetric method is a reaction between the coagulase after the cascade reaction and the chromogenic substrate, and the endotoxin content is detected by measuring the absorbance A545nm. The gel method does not require specialized instruments and is simple and convenient to operate. The photometric method has a wider linear detection range and is sensitive and accurate. The disadvantage is that it will be interfered with by (1-3)-β-D-glucan, because it can replace factor C in the cascade reaction and activate zymogen B to chelatase B, resulting in false positive results.

[0006] Horseshoe crabs were originally used as fertilizer, feed, and food. Later, after the discovery of limulus amebocyte lysate (LA-lysate) reagents for endotoxin testing, the standard method for preparing limulus amebocyte lysate reagents was adopted. After blood collection, the horseshoe crabs are released into the wild. An estimated 20% mortality rate is associated with captive breeding, with low survival rates and long breeding cycles. Furthermore, coupled with environmental degradation and rampant human predation, the horseshoe crab population has plummeted in recent years. Medical device companies are advised to urgently transition to alternative endotoxin testing methods and develop novel endotoxin detection technologies.

[0007] Factor C is a serine protease. In 1991, Muta et al. cloned the full-length Factor C cDNA and confirmed that Factor C consists of 1019 amino acids, with the first 25 amino acids serving as a signal peptide and the mature peptide of 994 amino acids. It is a two-chain protein with one heavy chain (R26-S690) and one light chain (S691-I1019), weighing 109 kDa. However, the application of recombinant Factor C has been slow, and it was not included in the Chinese Pharmacopoeia until 2020. Because horseshoe crab Factor C has a complex structure, a large molecular weight, a double-chain structure similar to an antibody, and six potential glycosylation sites, it is difficult to express full-length, biologically active Factor C. Furthermore, the expression process is highly susceptible to the introduction of exogenous LPS. Horseshoe crab Factor C is also extremely sensitive to endotoxins in the environment, and endotoxins in the culture medium or solution are enriched during expression and purification, significantly reducing the activity of the expressed native Factor C.

[0008] In recent years, scientists have developed a new generation of endotoxin detection kits that do not rely on horseshoe crab lymphocytes—the recombinant Factor C (rFC) endotoxin detection kit. This kit uses genetically recombinantly expressed horseshoe crab Factor C. Upon binding to endotoxins, the inactive protease is converted into a biologically active protease, which recognizes and catalyzes downstream fluorescent substrates to produce a fluorescent signal. The intensity of the fluorescent signal is positively correlated with the endotoxin concentration, enabling quantitative endotoxin detection. Compared with the horseshoe crab reagent method, the recombinant factor C method is an improved method of the current horseshoe crab reagent endotoxin detection method. It uses a fluorescent microplate reader and the recombinant factor C method to detect bacterial endotoxins. It has the advantages of reliability, permanence, and environmental protection. The main manifestations are: ① This method does not have the interference of factor G 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, preventing false positives in the test; ② The reagents used in this method do not contain factor B, procoagulant, coagulogen, etc. Therefore, samples containing inhibitory or enhancing effects on the above substances are suitable for the recombinant factor C method; ③ It does not rely on animal-derived ingredients, provides higher supply security, and complies 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 limulus amebocyte lysate reagent methods. Pei Yusheng et al. conducted a methodological validation of 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 Biological Products, 2020, 33(1):4.).

[0009] Endotoxin detection methods based on biochemical sensors such as fluorescent probes and enzymes utilize the physicochemical signal changes generated by the interaction between the analyte and a biosensitive layer. These signals are then converted by a signal converter into electrochemical, optical, thermal, or piezoelectric response signals. The correlation between these signals and the analyte is analyzed, thereby achieving the purpose of analyzing and detecting the analyte. Numerous studies have demonstrated that biosensors offer advantages such as good selectivity, high sensitivity, fast analysis speed, low cost, and the ability to perform online and continuous monitoring in complex systems. In particular, their high degree of automation, miniaturization, and integration have led to a booming research and technological development in recent years. These significant advantages have also led to their promising application in endotoxin research, and a large number of biosensors have been used for endotoxin detection. However, the design and construction of biosensor sensing layers is time-consuming, they are susceptible to background light, have poor anti-interference capabilities, are subject to nonspecific binding, and have a short lifespan for the biorecognition element.

[0010] Prior art CN114636816A provides a fluorescent microsphere probe for detecting endotoxins, as well as its preparation method and application. The preparation method for the fluorescent microsphere probe for detecting endotoxins includes the following steps: obtaining a recombinant limulus factor C protein; activating carboxylated fluorescent microspheres to obtain activated fluorescent microspheres; coupling the activated fluorescent microspheres with the recombinant limulus factor C protein to obtain coupled microspheres; blocking 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, its preparation method, and application. This invention modifies the existing horseshoe crab coagulation Factor C, maintaining the original sequence (functional domain) of the light chain region and replacing the Sushi1 and Sushi2 regions of the heavy chain with the Sushi4 region of the light chain. Without altering the overall structure and functional domains of Factor C, the free energy of the protein itself is reduced, making it more stable and improving its substrate binding ability. Furthermore, the invention adds a signal sequence to the N-terminus of the protein sequence, enabling extracellular expression of the target protein, facilitating subsequent detection and purification. The resulting modified protein, rFC-MBJ, is designed. This method performs endotoxin detection on a 96-well plate, which suffers from 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 containing endotoxins through antigen-antibody binding, and then separating the endotoxins from the protein for endotoxin detection; CN110095600A: A test strip and kit for bacterial endotoxins, which prepares endotoxin antibodies and uses ELISA for endotoxin detection. The above methods all have their own theoretical advantages, but without exception, none of them has been recognized by the "Chinese Pharmacopoeia", and their methods and operational feasibility need further verification. Summary of the Invention

[0013] In response to 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 and can rapidly detect endotoxins, which not only meets the requirements of the pharmacopoeia but also improves the detection efficiency and accuracy.

[0014] To achieve the above object, 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 diagram of the reagent card is as follows Figure 1 As shown, the preparation method comprises the following steps:

[0016] S1. Preparing a fluorescent chromogenic substrate solution: dissolving a fluorescent chromogenic 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: streak the nitrocellulose membrane with a substrate protein solution to obtain a T-line; streak the same nitrocellulose membrane with a substrate protein solution containing lipopolysaccharide to obtain a C-line; then dry the nitrocellulose membrane to obtain a coated nitrocellulose membrane;

[0018] S3. Assembly: Paste the coated nitrocellulose membrane, absorbent pad, and sample pad on the polyvinyl chloride plate in sequence, and press the absorbent pad and sample pad on both ends of the coated nitrocellulose membrane to obtain a reagent plate;

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

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

[0021] Macromolecules refer to biological substances with a relative molecular mass of more than 5000, such as proteins, nucleic acids, polysaccharides, etc. By chemically coupling the protease fluorescent substrate to the macromolecule, the molecular weight of the protease fluorescent substrate can be increased, while its anchoring ability is enhanced, making the protease fluorescent substrate firmly attached to the nitrocellulose membrane and preventing 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] Fluorescent protease substrates are important tools for monitoring protease activity. They produce fluorescence when reacting with proteases, allowing the activity of the protease 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 dissolution solution is used to draw T lines, and the amount used is 1 μL / cm; on the same nitrocellulose membrane, 1 EU / mL lipopolysaccharide substrate protein dissolution solution is used to draw C lines, and the amount used is 1 μL / cm.

[0032] In another aspect, the present invention provides a kit for endotoxin detection, 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. Add recombinant Factor C protein and BSA to Solution A and stir 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 cells, etc.), and expressing and purifying the recombinant Factor C protein.

[0039] Preferably, the lyophilization 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 endotoxins, wherein the method uses the above-mentioned kit for detection, comprising the following steps:

[0046] Dissolve the recombinant Factor C protein lyophilized powder in diluent, add different concentrations of endotoxin standards and mix thoroughly, then drip onto the sample pad of the reagent card. Measure the fluorescence intensity of the T-line area and the C-line area. Obtain a concentration standard curve based on the changes in fluorescence intensity of the T-line area caused by different concentrations of the standard.

[0047] Dissolve the recombinant Factor C protein lyophilized powder in diluent, add the test sample and mix thoroughly, then drip onto the sample pad of the reagent card. Measure the fluorescence intensity of the T-line area and the C-line area. Calculate the endotoxin concentration in the test sample based on the change in fluorescence intensity of the T-line area caused by the test sample and the concentration standard curve.

[0048] Preferably, the detection method comprises the following steps: adding lyophilized recombinant Factor C protein powder to a diluent and mixing to a final concentration of 20 μg / mL; then mixing the mixture with endotoxin standards or test samples of varying concentrations at a volume ratio of 1:1 to obtain a mixed solution; placing a reagent card on a clean, flat surface, and dripping 100 μL of the mixed solution onto the sample pad of the reagent card; scanning the T-line area and C-line area on the reagent card using a fluorescence immunoassay at 5 and 20 minutes, respectively, to obtain fluorescence signals (RFUs); calculating the fluorescence signal increase values ​​(ΔRFU) of the T-line area corresponding to different samples (ΔRFU = end point RFU - start point RFU); creating an endotoxin concentration standard curve based on the fluorescence signal increase values ​​(ΔRFU) of the T-line area corresponding to different concentrations of the endotoxin standard; and calculating the endotoxin concentration in the test sample using the fluorescence signal increase values ​​of the T-line area of ​​the test sample combined 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 uses the recombinant Factor C method to detect endotoxins and provides a new approach to kit design. On the one hand, a protease fluorescent substrate coupled to a macromolecule is coated onto a reagent card. On the other hand, the recombinant Factor C protein is stored in dry powder form and dissolved and diluted before use. Finally, by optimizing the relevant buffer system, the resulting kit provides accurate and stable test results, significantly improves the detection limit, and simplifies the production process. 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 endotoxin detection using recombinant Factor C combined with a fluorescent substrate (C on the horizontal axis represents different endotoxin standard concentrations).

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

[0058] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the examples. If the specific conditions are not specified in the examples, the experiments shall be carried out under conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, many specific details are given in the specific embodiments below. The specific embodiments described here are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present invention. Such structures and technologies are described in many publications, such as "Molecular Cloning Laboratory Manual (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] Dissolve Tween-20, BSA (Solarbio, Cat. No. 9048-46-8), sodium chloride, trehalose, and cephalin in sterile deionized water to a mass fraction of 0.01% for each component and stir evenly to obtain a protein coating buffer. Add BSA-conjugated chymotrypsin fluorescent substrate (structure: Nt-Boc-Val-Pro-Arg-AMC-BSA, synthesized by Shanghai Kepeptide Biotechnology Co., Ltd.) to the protein coating buffer to a final concentration of 0.5 mg / mL to obtain a fluorescent substrate solution.

[0061] Take a square nitrocellulose membrane and draw a T line on the nitrocellulose membrane using a 0.5 mg / mL fluorescent substrate solution at a volume of 1 μL / cm. On the same nitrocellulose membrane, draw a C line using a 0.5 mg / mL fluorescent substrate solution containing 1 EU / mL lipopolysaccharide at a volume of 1 μL / cm. After the marking is completed, the nitrocellulose membrane is placed 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 placed in the middle of the polyvinyl chloride plate; the absorbent pad was pressed on one end of the coated nitrocellulose membrane with an overlap area of ​​1-2 mm; the sample pad was pressed on the other end of the nitrocellulose membrane with an overlap area of ​​1-2 mm to obtain the 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 the aluminum foil bag, label it and pack it into the 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 described in Example 1. 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 Chinese horseshoe crab ( Tachypleus tridentatus ) was cloned from a Factor C gene (GenBank: AAL75577.1). A signal peptide gene was then 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). This plasmid was transfected into mammalian cells, and the recombinant Factor C protein was expressed and purified to obtain the recombinant Factor C protein (sequence shown in SEQ ID NO. 2).

[0067] Lactose and PEG-8000, used as lyophilization excipients, were dissolved in sterile deionized water to a lactose concentration of 3% and a PEG-8000 concentration of 0.3%. Recombinant Factor C protein and BSA were then added to a concentration of 20 μg / mL and a BSA concentration of 2%, and the mixture was stirred to dissolve. Lyophilization was performed using a freeze dryer (Dongfulong, Model LY0-0.5) according to the procedure in Table 1 to obtain lyophilized recombinant Factor C protein powder.

[0068] Table 1 Freeze-drying parameters

[0069]

[0070] 2) Prepare dilution solution

[0071] Tween-20 and 0.01% by mass of sodium lauroyl sarcosinate were added to 0.2 M 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 0.1 M to obtain a buffer solution.

[0072] Recombinant Factor C protein lyophilized powder and buffer were placed in appropriate containers, respectively, and used with the reagent card described 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. 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. The mixture was 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, flat surface, and 100 μL of the mixture was 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 (excitation wavelength 380 nm, emission wavelength 440 nm) at 5 and 20 minutes, respectively, to obtain the fluorescence signal RFU. The T-line fluorescence signal increase ΔRFU corresponding to different samples was calculated (ΔRFU = end point RFU - start point RFU). Based on the fluorescence signal increase ΔRFU corresponding to different concentrations of endotoxin standards (see Table 2), an endotoxin concentration standard curve was prepared (log ΔRFU = a × log C + b, see Table 2). 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 FIG. Figure 3 shown.

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

[0077]

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

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

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

[0081]

[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] Following the method in Example 1 and the formulation in Table 4, different surfactants, BSA, sodium chloride, different carbohydrate reagents, and different phospholipids were mixed and dissolved to a mass fraction of 0.01% for each component to obtain a protein coating buffer, which was then used to prepare a reagent card. Endotoxin-negative samples and standards containing 1 EU / mL of endotoxin were tested according to the detection method in Example 3. Furthermore, the evaluation criteria for the kit were as follows: 1) the lower the C-line fluorescence value of the negative sample, the better, with a T-line fluorescence value >30,000; 2) the C-line fluorescence value of the standard was in the range of 30,000-34,000 and a T-line fluorescence value >30,000. The test results are shown in Table 4 (where the upper values ​​represent the C-line fluorescence value and the lower values ​​represent the T-line fluorescence value), indicating that the optimal protein coating buffer formulation consists of Tween-20, BSA, sodium chloride, trehalose, and cephalin (all at a mass fraction of 0.01%).

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

[0086]

[0087] Example 6: Optimizing the concentration of fluorescent 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 for each. According to the detection method of Example 3, endotoxin-negative samples and standards containing 1 EU / mL endotoxin were tested, respectively. The results are shown in Table 5, which shows that the difference in fluorescence values ​​corresponding to the fluorescent substrate concentrations of 0.75 mg / mL and 0.5 mg / mL is very small, and increasing the concentration no longer increases the fluorescence intensity, indicating that the optimal fluorescent substrate solution concentration is 0.5 mg / mL.

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

[0090]

[0091] Example 7: Optimization of diluent components

[0092] Following the method in Example 2 and the formulations in Table 6, different surfactants and different divalent cationic compounds were mixed and added to 0.2 M Hepes buffer, pH 7.2, to prepare different dilutions (the percentage before the component indicates its mass fraction in the dilution, and the concentration before the component indicates its concentration in the dilution). Endotoxin-negative samples and standards containing 1 EU / mL of endotoxin were tested according to the detection method in Example 3. Simultaneously, the water absorption rate of the reagent card prepared in Example 1 in different buffers was measured, and the solubility of the recombinant Factor C protein lyophilized powder prepared in Example 2 in different buffers was measured according to conventional methods in the art. The test results are shown in Table 6. The data in the table are, from top to bottom: 1) lyophilized powder solubility (+ indicates easy solubility), 2) C-line fluorescence value of negative samples, 3) C-line fluorescence value of 1 EU / mL standard, and 4) water absorption rate of the reagent card (s). The results show that the optimal diluent formula is 0.01% Tween-20, 0.01% sodium lauroyl sarcosinate, 0.1 M magnesium sulfate, and 0.01 M calcium chloride. This formula has good solubility, fast flow rate, low background, and high sensitivity.

[0093] Table 6 Test results of the kit prepared with different dilutions

[0094]

[0095] Example 8: Kit Performance Testing

[0096] Unless otherwise specified, the method of Example 3 was used in all examples to detect the endotoxin concentration of the samples.

[0097] 1) Determination of the detection limit of the kit:

[0098] 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 repeated 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.

[0099] Table 7 Detection results of different endotoxin standard concentrations

[0100]

[0101] 2) Sample reliability test:

[0102] Three endotoxin samples (sodium hyaluronate, cell culture fluid, and pipette tip extract) with high, low, and negative endotoxin levels were prepared and tested using the Limulus Amebocyte Lysate (TAL) gel assay (using the kit NJF01060 from Dana Biosciences), the colorimetric assay (using the kit XSF0596K-2 from Dana Biosciences), the recombinant Factor C assay (using the kit RAF-01 from Shanghai Ruinuo Biosciences), and the method described in Example 3 to verify the reliability of the samples. The results demonstrated that the kits' sample detection results were reliable (see Table 8).

[0103] Table 8 Comparison of test results of different detection methods

[0104]

[0105] 3) Repeatability and accuracy:

[0106] Endotoxin standard solutions at three concentrations (2, 0.2, and 0.02 EU / mL) were prepared for testing, with eight 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.

[0107] Table 9 Repeatability and accuracy evaluation

[0108]

[0109] 4) Stability:

[0110] Endotoxin standard solutions of three concentrations (2, 0.2, and 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 days 1, 3, 5, 7, 11, 13, 15, 17, and 19, respectively. The results are shown in Table 10, indicating that the reagent card was stable within 15 days of thermal acceleration.

[0111] Table 10 Stability evaluation

[0112]

[0113] 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 kit for endotoxin detection, characterized in that: The kit includes recombinant Factor C protein lyophilized powder, diluent and reagent card; The components of the recombinant Factor C protein lyophilized powder include recombinant Factor C protein, BSA and lyophilized excipients; The lyophilized excipient consists of 3% lactose and 0.3% PEG-8000; The diluent consists of 0.01% Tween-20, 0.01% sodium lauroyl sarcosinate, 0.1M magnesium sulfate and 0.01M calcium chloride; The preparation method of the reagent card comprises the following steps: S1. Preparing a fluorescent chromogenic substrate solution: dissolving a fluorescent chromogenic 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: streak the nitrocellulose membrane with a substrate protein solution to obtain a T-line; streak the same nitrocellulose membrane with a substrate protein solution containing lipopolysaccharide to obtain a C-line; then dry the nitrocellulose membrane to obtain a coated nitrocellulose membrane; S3. Assembly: Paste the coated nitrocellulose membrane, absorbent pad, and sample pad on the polyvinyl chloride plate in sequence, and press the absorbent pad and sample pad on both ends of the coated nitrocellulose membrane to obtain a reagent plate; S4, cutting: cutting the reagent plate into thin strips and placing them in a card housing to obtain a reagent card; In step S1, the fluorescent color development substrate is a chymotrypsin fluorescent substrate coupled with BSA; The concentration of the fluorescent chromogenic substrate dissolved in the protein coating buffer is 0.5 mg / mL; The protein coating buffer in step S1 is composed of 0.01% Tween-20, 0.01% BSA, 0.01% sodium chloride, 0.01% trehalose and 0.01% cephalin.

2. A method for detecting endotoxin, characterized in that: The detection method is performed using the kit according to claim 1, comprising the following steps: Dissolve the recombinant Factor C protein lyophilized powder in diluent, add different concentrations of endotoxin standards and mix thoroughly, then drip onto the sample pad of the reagent card. Measure the fluorescence intensity of the T-line area and the C-line area. Obtain a concentration standard curve based on the changes in fluorescence intensity of the T-line area caused by different concentrations of the standard. Dissolve the recombinant Factor C protein lyophilized powder in diluent, add the test sample and mix thoroughly, then drip onto the sample pad of the reagent card. Measure the fluorescence intensity of the T-line area and the C-line area. Calculate the endotoxin concentration in the test sample based on the change in fluorescence intensity of the T-line area caused by the test sample and the concentration standard curve.

3. Use of the kit according to claim 1 in endotoxin detection, characterized in that: The application is not an application for disease diagnosis or treatment.

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