A reaction buffer for improving the sensitivity of recombinant limulus reagent in detecting endotoxin, a kit containing the same and application thereof

Abstract

The application discloses a reaction buffer for improving the sensitivity of recombinant Limulus reagent in endotoxin detection, a kit containing the same and application. The reaction buffer comprises the following components: Tris-HCl buffer, magnesium ions, sodium chloride, sucrose, ethylene glycol diethyl ether diamine tetraacetic acid, a non-ionic surfactant, cyclodextrin or a derivative thereof. The application also discloses a preparation method of the reaction buffer and a kit containing the same. The sensitivity of recombinant Limulus reagent in endotoxin detection can be improved by one order of magnitude by using the reaction buffer or the kit, the minimum detection limit can reach 0.0005 EU / ml, the reaction buffer has strong anti-interference ability, the reaction time is short, and the detection efficiency is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a reaction buffer solution for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins, a kit containing the buffer solution, and its applications. Background Technology

[0002] Endotoxins are lipopolysaccharides (LPS) found in the outer membrane of Gram-negative bacterial cell walls. Even trace amounts entering the bloodstream can cause fever, while high doses exhibit strong toxicity, potentially leading to endotoxic shock and even death. Because Gram-negative bacteria are widespread in the environment, their endotoxins can be introduced during the production of pharmaceuticals or medical devices. Furthermore, due to their heat resistance and difficulty in inactivation, strict endotoxin control is essential for injectable drugs and medical devices. Endotoxin management is particularly critical in emerging therapeutic areas such as regenerative medicine, vaccines, antibody drugs, and nucleic acid drugs.

[0003] Currently, the mainstream method for endotoxin detection uses horseshoe crab reagents, which work by utilizing the reaction between clotting system components in horseshoe crab blood cell extracts and endotoxins. However, to protect horseshoe crab resources, ensure a stable supply of reagents, reduce batch-to-batch variability, and improve detection reproducibility, several manufacturers have begun developing alternative detection reagents based on recombinant proteins. These mainly fall into two categories: one is the "recombinant factor C reagent" (i.e., single-factor reagent) which uses recombinant factor C, and the other is the "recombinant horseshoe crab reagent" (i.e., three-factor reagent) which uses three recombinant proteins: factor C, factor B, and prothrombin.

[0004] Recombinant factor C reagent contains only factor C protein, has a single reaction pathway, lacks cascade amplification, and produces protease with low activity, requiring the use of a fluorescent substrate for detection. In contrast, recombinant horseshoe crab reagent (three-factor reagent) can achieve multi-stage enzymatic amplification, exhibits strong enzyme activity, and can catalyze colorimetric reactions, thus allowing detection using ordinary microplate readers. Studies have indicated that factor B helps improve the specificity of the reaction against endotoxins; therefore, the three-factor system is more similar to the natural horseshoe crab reagent in its detection mechanism.

[0005] While recombinant horseshoe crab reagents offer advantages in batch-to-batch consistency and raw material sustainability, their detection sensitivity still needs improvement. Currently, commercially available recombinant horseshoe crab reagents can quantify endotoxins as low as 0.005 EU / ml, but in real-world samples with numerous interfering substances, high-fold dilution is often required, affecting detection accuracy. Therefore, lowering the limit of detection (LOD) of the reagents helps expand the effective dilution range for complex samples.

[0006] Therefore, developing a reaction buffer that can significantly improve the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins has become a key problem that urgently needs to be solved in this field. Summary of the Invention

[0007] In view of the above technical requirements, the first objective of this invention is to provide a reaction buffer solution that can significantly improve the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins, thereby solving the problems of low sensitivity and poor anti-interference ability in existing detection technologies.

[0008] A second objective of this invention is to provide a method for preparing the above-mentioned reaction buffer solution.

[0009] A third object of the present invention is to provide a kit containing the above-described reaction buffer.

[0010] A fourth object of the present invention is to provide the application of the above-described reaction buffer or kit in the preparation of endotoxin detection products. In particular, these highly sensitive detection products can be used for pharmaceutical quality control and endotoxin detection in medical devices.

[0011] The fifth object of the present invention is to provide a method for detecting endotoxins.

[0012] To achieve the above objectives, the present invention adopts the following technical solution.

[0013] In a first aspect, the present invention provides a reaction buffer for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins. The reaction buffer, based on the final concentration of each component, comprises the following components: 20-100 mM Tris-HCl buffer with a pH of 6.8-8.0, 1-10 mM magnesium ions, 50-200 mM sodium chloride, 1-5% (w / v) sucrose, 0.1-1 mM ethylene glycol diethyl ether diaminetetraacetic acid, 0.01-0.1% (w / v) nonionic surfactant, and 0.1-1 mM cyclodextrin or a derivative thereof.

[0014] In this invention, magnesium ions act as a cofactor for the enzymatic reaction; sodium chloride acts as an ionic strength regulator; sucrose and ethylene glycol diethyl ether diaminetetraacetic acid (EGTA) act as stabilizers; and cyclodextrin or its derivatives act as sensitivity enhancers. The reaction buffer solution of this invention can significantly improve the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins, with a detection limit as low as 0.0005 EU / ml.

[0015] Furthermore, according to a specific embodiment of the present invention, the reaction buffer, based on the final concentration of each component, comprises the following components: 40-60 mM Tris-HCl buffer with a pH of 7.2-7.6, 3-6 mM magnesium ions, 80-120 mM sodium chloride, 2-4% (w / v) sucrose, 0.3-0.7 mM ethylene glycol diethyl ether diaminetetraacetic acid, 0.03-0.07% (w / v) nonionic surfactant, and 0.3-0.7 mM cyclodextrin or its derivatives.

[0016] Furthermore, the magnesium ions are provided by magnesium chloride, magnesium sulfate, or magnesium acetate.

[0017] Furthermore, the nonionic surfactant is a combination of polysorbate-80 and polysorbate-20, Triton X-100, or 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS). For example, polysorbate-80 and polysorbate-20 are used in a ratio of (1-10):1; polysorbate-80 and Triton X-100 are used in a ratio of (1-3):1; and polysorbate-80 and CHAPS are used in a ratio of (1-5):1.

[0018] Furthermore, the cyclodextrin or its derivative is selected from one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl-β-cyclodextrin.

[0019] Secondly, the present invention provides a method for preparing the above-mentioned reaction buffer solution, the method comprising: dissolving all components in water according to the formula concentration, mixing thoroughly, and filtering to obtain the solution; Preferably, a 0.1 μm filter membrane is used for filtration; the endotoxin level of the reaction buffer is controlled below 0.005 EU / ml.

[0020] Thirdly, the present invention provides a kit for detecting endotoxins using recombinant horseshoe crab reagent, the kit comprising the above-mentioned reaction buffer for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins.

[0021] Furthermore, the kit also includes a "recombinant horseshoe crab reagent" composed of recombinant factor C, recombinant factor B, and recombinant procoagulant, wherein the recombinant factor C, recombinant factor B, and recombinant procoagulant can be prepared using methods already disclosed in the prior art or purchased commercially.

[0022] Fourthly, the present invention provides the application of the above-mentioned reaction buffer or a kit containing the reaction buffer in the preparation of endotoxin detection products.

[0023] Fifthly, the present invention also provides a method for detecting endotoxins, the method using the above-mentioned reaction buffer or kit, specifically comprising the following steps: Prepare a series of endotoxin standard solutions of different concentrations; The recombinant horseshoe crab reagent was reconstituted with the reaction buffer solution to obtain a recombinant horseshoe crab reagent solution; The recombinant horseshoe crab reagent solution was mixed with endotoxin standard solutions of each concentration, incubated at 37°C for a period of time, the change of reaction signal was monitored, and the reaction time to reach the limit was recorded. A standard curve was established with the concentration of endotoxin standard solution as the x-axis and the reaction time as the y-axis. The sample to be tested is diluted to prepare the test sample. The recombinant horseshoe crab reagent solution is mixed with the test sample and incubated at 37°C for a period of time. The change of reaction signal is monitored and the reaction time to reach the limit is recorded. The endotoxin content of the test sample is calculated according to the established standard curve. The endotoxin content of the initial undiluted test sample is calculated according to the dilution factor.

[0024] Furthermore, if necessary, the sample to be tested can be pretreated according to existing methods before dilution.

[0025] The beneficial effects of this invention are as follows: Significantly improved sensitivity: Using the reaction buffer of the present invention, the sensitivity of the recombinant horseshoe crab reagent for detecting endotoxins can be improved by an order of magnitude (approximately 10 times), with the lowest detection limit reaching 0.0005 EU / ml.

[0026] Enhanced anti-interference capability: Thanks to the synergistic effect of multiple components in the buffer solution, it can still maintain high detection sensitivity in complex sample matrices (such as serum, protein solutions, etc.), solving the problem of traditional methods being susceptible to interference.

[0027] Improved reaction kinetics: The reaction time is shortened, and detection can be completed in half an hour, which improves the detection efficiency. At the same time, the linear correlation coefficient of the standard curve meets the requirement of R²≥0.98. Attached Figure Description

[0028] Figure 1 Standard curves for detecting endotoxin standards using different reaction buffers are shown. Detailed Implementation

[0029] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, all reagents and consumables used in the following embodiments were purchased from conventional biochemical reagent manufacturers, and all experimental methods used were conventional methods in the art.

[0031] Example 1 Prepare the reaction buffer according to the following components and concentrations: Tris-HCl buffer: 50 mM, pH 7.4; Magnesium acetate: 5 mM; NaCl: 100 mM; Sucrose: 3% (w / v); EGTA: 0.5 mM; Polysorbate-80: 0.06% (w / v); Polysorbate-20: 0.01% (w / v); Hydroxypropyl-β-cyclodextrin: 0.5 mM.

[0032] Dissolve the above components in endotoxin-free water, stir well, adjust the pH to 7.4, filter through a 0.1 μm filter membrane, dispense and store at 4°C for later use.

[0033] Example 2 Prepare the reaction buffer according to the following components and concentrations: Tris-HCl buffer: 40 mM, pH 7.6; Magnesium chloride: 5 mM; NaCl: 80 mM; Sucrose: 2% (w / v); EGTA: 0.3 mM; Polysorbate-80: 0.03% (w / v); Triton X-100: 0.03% (w / v); Hydroxypropyl-β-cyclodextrin: 0.3 mM.

[0034] Dissolve the above components in endotoxin-free water, stir well, adjust the pH to 7.6, filter through a 0.1 μm filter membrane, dispense and store at 4°C for later use.

[0035] Example 3 Prepare the reaction buffer according to the following components and concentrations: Tris-HCl buffer: 60 mM, pH 7.2; Magnesium sulfate: 5 mM; NaCl: 120 mM; Sucrose: 4% (w / v); EGTA: 0.7 mM; Polysorbate-80: 0.05% (w / v); CHAPS: 0.01% (w / v); Hydroxypropyl-β-cyclodextrin: 0.7 mM.

[0036] Dissolve the above components in endotoxin-free water, stir well, adjust the pH to 7.2, filter through a 0.1 μm filter membrane, dispense and store at 4°C for later use.

[0037] Comparative Example 1 The reaction buffer for the comparative example was prepared according to Chinese patent CN105866080B, with the specific components and concentrations as follows: Tris-HCl buffer: 100 mM, pH 6.8; Polysorbate-20: 0.1% (w / v).

[0038] Dissolve the above components in endotoxin-free water, stir well, filter through a 0.1 μm filter membrane, dispense into containers, and store at 4°C for later use.

[0039] Comparative Example 2 Prepare the reaction buffer according to the following components and concentrations: Tris-HCl buffer: 50 mM, pH 7.4; Magnesium acetate: 5 mM; NaCl: 100 mM; Sucrose: 3% (w / v); EGTA: 0.5 mM; Polysorbate-20: 0.06% (w / v); Dissolve the above components in endotoxin-free water, stir well, adjust the pH to 7.4, filter through a 0.1 μm filter membrane, dispense and store at 4°C for later use.

[0040] Experimental Example 1: Effect of different reaction buffers on the sensitivity of recombinant horseshoe crab reagent Recombinant horseshoe crab reagents (using proteins and substrates from patent CN105866080B) were prepared using the reaction buffers of the present invention (Examples 1, 2, and 3) and conventional buffers (Comparative Example 1 and Comparative Example 2), respectively, to detect endotoxin standards at a series of concentrations (0.0005, 0.005, 0.05, 0.5, 5 EU / ml).

[0041] The results showed that, using the reaction buffer of the present invention, the reaction systems of Examples 1, 2, and 3 were able to detect endotoxin at a concentration of 0.0005 EU / ml, with a correlation coefficient R of 1 / 2 for the standard curve. 2 The correlation coefficient of the standard curve was ≥0.98, while the endotoxin concentration of 0.0005 EU / ml could not be detected in the conventional buffer comparisons 1 and 2, only 0.005 EU / ml. 2 ≥0.96, compared to the control buffer, the detection sensitivity of the reaction buffer of this invention is improved by 10 times. Furthermore, the reaction time of the endotoxin working standard in the reaction buffer of this invention is less than 30 min, while the reaction time required for conventional buffers is approximately 2 h for Comparative Example 1 and approximately 1 h for Comparative Example 2. The reaction buffer of this invention significantly shortens the detection time required for the experiment. Results are shown in Table 1 and... Figure 1 .

[0042] Table 1. Experimental results showing the effect of different reaction buffers on the sensitivity of recombinant horseshoe crab reagent.

[0043] Experimental Example 2: Actual Sample Testing The reaction buffer of the present invention (Example 1) was used to detect endotoxins in actual drug samples (various injections, human serum albumin, vaccine preparations), and compared with the buffer of the comparative example, and compared with the traditional horseshoe crab reagent colorimetric method.

[0044] The results showed that the reaction buffer of the present invention enabled the recombinant horseshoe crab reagent to more accurately detect trace amounts of endotoxin in the sample, and the detection results were consistent with the standard method. It also exhibited good anti-interference ability, allowing for the determination of endotoxin content at low dilutions, particularly excelling in complex biological samples. In contrast, the comparative buffer used for recombinant horseshoe crab reagent detection of endotoxin had a high detection limit, failing to detect precise trace amounts of endotoxin, and showed weak detection capability for complex samples (see Table 2). Specifically, Example 1 and Comparative Example 1 showed significant differences in results for human serum albumin injection and pembrolizumab formulation. This is because the reaction buffer of the present invention has strong anti-interference ability, enabling accurate detection of endotoxin at low dilutions; while the comparative buffer has weak anti-interference ability, requiring high dilutions, and exhibits weak detection signal intensity, easily affected by background noise, resulting in data anomalies.

[0045] Table 2 Results of endotoxin content detection in samples using different buffer solutions

[0046] The reaction buffers prepared in other embodiments also have the same excellent properties as the reaction buffer in Example 1, and will not be listed here.

[0047] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A reaction buffer solution for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins, characterized in that, Based on the final concentration of each component, the reaction buffer consists of the following components: 40-60 mM Tris-HCl buffer with a pH of 7.2-7.6, 3-6 mM magnesium ions, 80-120 mM sodium chloride, 2-4% (w / v) sucrose, 0.3-0.7 mM ethylene glycol diethyl ether diaminetetraacetic acid, 0.03-0.07% (w / v) nonionic surfactant, and 0.3-0.7 mM cyclodextrin or its derivatives.

2. The reaction buffer solution according to claim 1, characterized in that, The magnesium ions are provided by magnesium chloride, magnesium sulfate, or magnesium acetate.

3. The reaction buffer solution according to claim 1, characterized in that, The nonionic surfactant is a combination of polysorbate-80 and polysorbate-20, Triton X-100 or 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt.

4. The reaction buffer solution according to claim 1, characterized in that, The cyclodextrin or its derivatives are selected from one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin.

5. The method for preparing the reaction buffer solution for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins according to any one of claims 1-4, characterized in that, The preparation method includes: dissolving all components in water according to the formula concentration, mixing thoroughly, and then filtering to obtain the final product.

6. The preparation method according to claim 5, characterized in that, Filtration was performed using a 0.1 μm filter membrane; the endotoxin level in the reaction buffer was controlled below 0.005 EU / ml.

7. A kit for detecting endotoxins using recombinant horseshoe crab reagent, characterized in that, The reaction buffer solution for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins, as described in any one of claims 1-4.

8. The reaction buffer solution for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins as described in any one of claims 1-4, or the kit for detecting endotoxins using recombinant horseshoe crab reagent as described in claim 7, in the preparation of endotoxin detection products.

9. A method for detecting endotoxins, characterized in that, The method uses the reaction buffer solution for improving the sensitivity of recombinant horseshoe crab reagent in detecting endotoxins as described in any one of claims 1-4 or the kit for detecting endotoxins using recombinant horseshoe crab reagent as described in claim 7, and includes the following steps: Prepare a series of endotoxin standard solutions of different concentrations; The recombinant horseshoe crab reagent was reconstituted with the reaction buffer solution to obtain a recombinant horseshoe crab reagent solution; The recombinant horseshoe crab reagent solution was mixed with endotoxin standard solutions of each concentration, incubated at 37°C for a period of time, the change of reaction signal was monitored, and the reaction time to reach the limit was recorded. A standard curve was established with the concentration of endotoxin standard solution as the x-axis and the reaction time as the y-axis. The sample to be tested is diluted to prepare the test sample. The recombinant horseshoe crab reagent solution is mixed with the test sample and incubated at 37°C for a period of time. The change of reaction signal is monitored and the reaction time to reach the limit is recorded. The endotoxin content of the test sample is calculated according to the established standard curve. The endotoxin content of the initial undiluted test sample is calculated according to the dilution factor.

10. The method according to claim 9, characterized in that, It also includes a pretreatment step of the sample to be tested before dilution.

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