A screening method for recombinant factor b activity and sensitivity for endotoxin detection

Abstract

The application discloses a screening method for activity and sensitivity of recombinant factor B for endotoxin detection. The method comprises the following steps: adding a series of endotoxin standard solution with different concentrations in an endotoxin-free 96-well plate, and adding endotoxin-free water as a control; adding an activity detection reagent, wherein the activity detection reagent comprises a buffer, a mixed solution of recombinant factor C and a to-be-detected recombinant factor B, and a fluorescent substrate; reading a zero-point fluorescence value, and then placing the 96-well plate in a 37 DEG C incubator for incubation; reading the fluorescence value again, calculating a fluorescence intensity change value Delta RFU, and determining the result. The method realizes independent and direct evaluation of the activity and sensitivity of the recombinant factor B through a 'two-factor' (only containing the recombinant factor C and the recombinant factor B) detection system, and completely gets rid of the dependence on downstream coagulase activity. The method can help screening of the recombinant factor B with high activity and sensitivity, and the recombinant factor B can be used in a recombinant limulus reagent for endotoxin detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. More specifically, it relates to a method for screening the activity and sensitivity of recombinant factor B for endotoxin detection. Background Technology

[0002] Bacterial endotoxins are among the most important pyrogens. When their concentration in the blood exceeds the body's clearance threshold, they trigger a cascade of inflammatory factors, leading to fever, endotoxemia, and even septic shock. Therefore, pharmacopoeias in various countries list endotoxin testing as a key quality control indicator for pharmaceuticals, medical devices, and biological products.

[0003] Traditional endotoxin detection techniques rely on horseshoe crab (LMS) reagents, extracted from the blood of horseshoe crabs. Hemocyanin in horseshoe crab blood specifically recognizes endotoxins, activating a cascade reaction of factor C → factor B → coagulogen, ultimately converting the coagulogen into an insoluble gel. With the rapid development of the biopharmaceutical industry, traditional horseshoe crab reagents face two major challenges: first, the sustainability pressure on horseshoe crab resources—only four species of horseshoe crabs are available for blood collection globally, and their numbers are dwindling; horseshoe crab resources are currently strictly controlled in many countries; second, the batch-to-batch activity variations of natural reagents lead to detection variability.

[0004] To address these issues, researchers have begun developing non-animal-derived detection reagents using gene recombination technology. Currently, the mainstream approaches fall into two categories: single-factor recombinant reagents (containing only recombinant factor C) and three-factor recombinant reagents (containing recombinant factor C, factor B, and coagulase). Single-factor systems, lacking a cascade amplification effect, rely on fluorescently labeled substrates for detection; while three-factor systems, by mimicking natural cascade reactions, can utilize chromogenic substrates (such as p-nitroaniline-labeled horseshoe crab tripeptide) for conventional ELISA reader detection, ensuring accuracy and sensitivity. Their sensitivity can reach 0.005 EU / ml, and they also offer advantages in terms of ease of operation and cost control.

[0005] Factor B is a crucial link in the three-factor recombinant reagent cascade reaction. Activated by the activated Factor C, it further activates the downstream coagulase, leading to a stepwise increase in the endotoxin detection signal through this cascade reaction. The activity of Factor B is one of the key factors determining the sensitivity of the entire reaction system. Currently, existing methods for detecting recombinant Factor B activity do not rely on independent detection but rather on functional verification through its role in the cascade reaction. However, this method is limited by the activity of the subsequent coagulase, resulting in low screening efficiency. Therefore, identifying and screening highly active recombinant Factor B without relying on coagulase, thereby ensuring convenient quality control during the recombinant Factor B process development and maintaining sensitivity and accuracy in endotoxin detection, is a key issue that requires attention.

[0006] Therefore, there is an urgent need in this field for a method that can directly, accurately, and efficiently detect the activity and sensitivity of recombinant factor B, independent of coagulase, for the optimization of the production process and quality control of recombinant factor B, and is of great strategic significance for accelerating the technological iteration of the industry and ensuring the safety of medical products. Summary of the Invention

[0007] To overcome existing problems, the present invention aims to provide a screening method for the activity and sensitivity of recombinant factor B for endotoxin detection. This method, through a "two-factor" detection system (containing only recombinant factor C and recombinant factor B), achieves independent and direct evaluation of the activity and sensitivity of recombinant factor B, completely eliminating dependence on downstream coagulogen activity. This method can help screen for recombinant factor B with high activity and sensitivity for use in recombinant horseshoe crab reagents for endotoxin detection.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] This invention provides a method for screening the activity and sensitivity of recombinant factor B for endotoxin detection, comprising the following steps:

[0010] A series of endotoxin standard solutions of different concentrations were added to an endotoxin-free 96-well plate, along with endotoxin-free water as a control. The lowest concentration of the series of endotoxin standard solutions was 0.005 EU / ml.

[0011] Add an activity detection reagent, which includes a buffer solution, a mixture of recombinant factor C and the recombinant factor B to be tested, and a fluorescent substrate;

[0012] Read the zero-point fluorescence value, and then incubate the 96-well plate in a 37°C incubator;

[0013] Read the fluorescence value again and calculate the fluorescence intensity change value ΔRFU;

[0014] Result determination:

[0015] When each ΔRFU is a positive number that increases sequentially, it is determined that the recombinant factor B to be tested meets the activity requirements for endotoxin detection.

[0016] A standard curve is plotted with the logarithm of ΔRFU on the ordinate and the logarithm of the endotoxin standard concentration on the abscissa. When the correlation coefficient R of the standard curve is... 2 If the value is ≥0.98, the recombinant factor B to be tested is determined to meet the sensitivity requirements for endotoxin detection.

[0017] In this invention, a series of endotoxin working standard solutions need to be prepared. The lowest recommended concentration is 0.005 EU / ml, which ensures that the recombinant factor B screened by the method of this invention can meet the industry's requirements for endotoxin detection. Other concentrations can be selected according to experimental needs.

[0018] Furthermore, the buffer solution comprises the following components: 50-200 mM HEPES buffer at pH 7.4, 50-200 mM magnesium sulfate, 0.1-0.5% (w / v) gelatin, 0.5-2.5% (w / v) mannitol, and 0.1-0.5% (w / v) CHAPS (3-[(3-cholanamide supplement) dimethylacetyl inner salt]-1-propanesulfonic acid inner salt).

[0019] Furthermore, the buffer solution comprises the following components: 100 mM HEPES buffer at pH 7.4, 100 mM magnesium sulfate, 0.2% (w / v) gelatin, 1.5% (w / v) mannitol, and 0.2% (w / v) CHAPS.

[0020] Furthermore, in the mixture of recombinant factor C and recombinant factor B to be tested, the content ratio of recombinant factor C to recombinant factor B to be tested is (1-3):1.

[0021] Furthermore, in the mixture of recombinant factor C and recombinant factor B to be tested, the content ratio of recombinant factor C to recombinant factor B to be tested is 1:1.

[0022] Furthermore, the fluorescent substrate is selected from any one of Boc-Asp-Pro-Arg-MCA, Boc-Leu-Thr-Arg-MCA, and Boc-Val-Pro-Arg-MCA.

[0023] Furthermore, in the activity detection reagent, the final concentration of the fluorescent substrate is 50-500 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested is 10-100 μg / ml.

[0024] Furthermore, in the activity detection reagent, the final concentration of the fluorescent substrate is 100 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested is 100 μg / ml.

[0025] Furthermore, the fluorescence detection conditions are: excitation wavelength 360 nm, emission wavelength 460 nm.

[0026] Furthermore, incubate at 37°C for 30 minutes.

[0027] The beneficial effects of this invention are as follows:

[0028] The screening method of this invention does not rely on coagulase precursors and directly assesses factor B activity and sensitivity. Its detection results show high consistency with the validation results of traditional three-factor recombinant horseshoe crab reagents (see Experimental Example 3), demonstrating the effectiveness and reliability of the method, while also being simpler to operate. Furthermore, the screening method of this invention can be used in high-throughput screening reagent systems, is suitable for 96-well plate microplate reader detection, and has the advantages of simple operation, high sensitivity, good repeatability, and suitability for industrial production scenarios. Attached Figure Description

[0029] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0030] Figure 1 The results of screening different detection reagents for the sensitivity of recombinant factor B are shown.

[0031] Figure 2 The results show the screening results of the optimal detection reagent for different recombinant factor B sensitivities.

[0032] Figure 3 The results of verifying the sensitivity of different recombinant factor B using the recombinant horseshoe crab three-factor reagent method are shown. Detailed Implementation

[0033] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Prepare endotoxin standard solutions. Dissolve the endotoxin standard in a disposable pyrogen-free glass tube to 20 EU / ml, then perform serial dilutions (5, 0.5, 0.05, 0.005 EU / ml) to obtain endotoxin standard solutions of various concentrations. Add 100 μl of each concentration of endotoxin standard and endotoxin-free water (as a 0 EU / ml control) to an endotoxin-free 96-well plate. Perform 2-3 replicates for each reaction and preheat the plate at 37°C.

[0036] Prepare the activity assay reagent. Take an appropriate volume of sterile water for injection and add HEPES buffer (pH 7.4) to a final concentration of 200 mM, 50 mM magnesium sulfate solution, 0.5% (w / v) gelatin, 0.5% (w / v) mannitol, and 0.5% (w / v) CHAPS sequentially. After thorough dissolution and mixing, bring the volume to a final level and filter through a 0.1 μm filter membrane to obtain the buffer solution. Prepare the activity assay reagent by taking the recombinant factor C solution, the recombinant factor B solution to be tested, the fluorescent substrate solution, and the buffer solution. In the activity assay reagent, the final concentration of the fluorescent substrate (Boc-Asp-Pro-Arg-MCA) is 500 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested (the ratio of recombinant factor C to the recombinant factor B to be tested is 3:1) is 10 μg / ml.

[0037] After adding 100 μl of activity detection reagent to preheated endotoxin standard or endotoxin-free water, immediately read the zero-point fluorescence value. The fluorescence detection conditions are excitation wavelength 360 nm and emission wavelength 460 nm. Then, place the 96-well plate in a 37°C incubator for 30 min, and read the values ​​again using a microplate reader.

[0038] First, calculate the RFU difference between 30 minutes and 0 hours for each concentration sample (5, 0.5, 0.05, 0.005 EU / ml). Then, subtract the difference of the 0 EU / ml control group from these differences. The result is the final change in fluorescence units (ΔRFU).

[0039] Example 2

[0040] Prepare the activity assay reagent. Take an appropriate volume of sterile water for injection and add sequentially the following: 50 mM HEPES buffer (pH 7.4), 200 mM magnesium sulfate solution, 0.1% (w / v) gelatin, 2.5% (w / v) mannitol, and 0.1% (w / v) CHAPS. After thorough dissolution and mixing, bring the volume to a final level and filter through a 0.1 μm filter membrane to obtain the buffer solution. Prepare the activity assay reagent by taking the recombinant factor C solution, the recombinant factor B solution to be tested, the fluorescent substrate solution, and the buffer solution. In the activity assay reagent, the final concentration of the fluorescent substrate (Boc-Leu-Thr-Arg-MCA) is 200 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested (the ratio of recombinant factor C to recombinant factor B to be tested is 2:1) is 30 μg / ml.

[0041] Other operations are the same as in Example 1.

[0042] Example 3

[0043] Prepare the activity assay reagent. Take an appropriate volume of sterile water for injection, and add sequentially the following: 100 mM MEPES buffer (pH 7.4), 100 mM magnesium sulfate solution, 0.2% (w / v) gelatin, 1.5% (w / v) mannitol, and 0.2% (w / v) CHAPS. After thorough dissolution and mixing, bring the volume to a final level and filter through a 0.1 μm filter membrane to obtain the buffer solution. Prepare the activity assay reagent by taking the recombinant factor C solution, the recombinant factor B solution to be tested, the fluorescent substrate solution, and the buffer solution. In the activity assay reagent, the final concentration of the fluorescent substrate (Boc-Val-Pro-Arg-MCA) is 100 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested (the ratio of recombinant factor C to recombinant factor B to be tested is 1:1) is 100 μg / ml.

[0044] Other operations are the same as in Example 1.

[0045] Comparative Example 1

[0046] Preparation of the contrast activity assay reagent. Take an appropriate volume of sterile water for injection, and add sequentially a final concentration of 20 mM Tris-HCl buffer (pH 8.0) and 150 mM sodium chloride solution (reference: Yamashita K, et al. A mutant equipped with a regenerated disulphide for the missing His loop of a serineprotease zymogen in the horseshoe crab coagulation cascade. J Biochem. 2021 Dec 4;170(4):489-500. doi: 10.1093 / jb / mvab064.). After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1 μm filter membrane to obtain the contrast buffer. Prepare the activity detection reagent by taking recombinant factor C solution, recombinant factor B solution to be tested, fluorescent substrate solution and buffer solution. The final concentration of fluorescent substrate (Boc-Leu-Thr-Arg-MCA) is 400 μM, and the final concentration of the mixture of recombinant factor C and recombinant factor B to be tested (the content ratio of recombinant factor C to recombinant factor B to be tested is 2:1) is 30 μg / ml.

[0047] Other operations are the same as in Example 1.

[0048] Comparative Example 2

[0049] Preparation of comparative activity detection reagents. The sample preparation method and detection system refer to patent CN105866080B "Recombinant Limulus Amebocyte Lysate (LAL) Tri-factor Reagent and Its Detection of Endotoxins", that is, the system adds recombinant LAL coagulase factor, and the activity of the recombinant LAL tri-factor reagent is identified to be tested for recombinant factor B.

[0050] Other operations are the same as in Example 1.

[0051] Experimental Example 1: Detection of the activity of recombinant factor B using different activity assay reagents

[0052] Endotoxin tests were performed on recombinant factor B, identified as active using the recombinant horseshoe crab tri-factor reagent, according to Examples 1-3 and Comparative Example 1. The endotoxin reactions of different activity detection reagents on the same batch of active recombinant factor B were compared, and the results are shown in Table 1. A standard curve was plotted with the logarithm of ΔRFU on the ordinate and the logarithm of the endotoxin standard concentration on the abscissa. Figure 1 .

[0053] Table 1. Results of recombinant factor B activity assay using different detection reagents

[0054]

[0055] The negative ΔRFU value in Comparative Example 1 indicates that it cannot be used to detect the activity of recombinant factor B at 0.005-5 EU / ml, and the correlation coefficient R of the standard curve corresponding to Comparative Example 1 is also negative. 2 A value <0.98 indicates that it cannot detect recombinant factor B at a sensitivity of 0.005-5 EU / ml, possibly due to high background noise or inability to effectively activate the reaction at low endotoxin concentrations. In contrast, the ΔRFU values ​​in Examples 1-3 are progressively increasing positive numbers, and the correlation coefficient R of the standard curve is... 2 ≥0.98, proving that the system and method of Examples 1-3 can be used to determine the activity and sensitivity of the recombinant factor B to be tested.

[0056] Experimental Example 2: Screening for high and low activity and sensitivity of recombinant factor B using optimal detection reagents

[0057] The activity screening of various recombinant factor B was performed using the activity detection reagents and screening methods described in Example 3. The results are shown in Table 2. A standard curve was plotted with the logarithm of ΔRFU as the ordinate and the logarithm of the endotoxin standard concentration as the abscissa. Figure 2 When each ΔRFU is a positive number that increases sequentially, the recombinant factor B to be tested is determined to meet the activity requirements for endotoxin detection; when the correlation coefficient R of the 0.005-5 EU / ml standard curve is... 2 If the value is ≥0.98, the recombinant factor B to be tested is determined to meet the sensitivity requirements for endotoxin detection.

[0058] Table 2 Results of screening recombinant factor B activity using optimal detection reagents

[0059]

[0060] From Table 2 and Figure 2 It can be seen that the ΔRFU of factors B#1 and B#4 are negative, and the correlation coefficient R of the corresponding standard curves is also negative. 2 The value <0.98 indicates that the activity and sensitivity of factors B #1 and #4 at endotoxin concentrations of 0.005-5 EU / ml do not meet the requirements. Meanwhile, the ΔRFU values ​​for factors B #2 and #3 are both positive numbers that increase sequentially, and the correlation coefficient R of the 0.005-5 EU / ml standard curve is low. 2 A value ≥0.98 indicates that the activity and sensitivity of factors B #2 and #3 both meet the requirements. Furthermore, factor B #3 has the highest reading, indicating that its activity is higher than that of #2. Based on these two comparisons, #3 exhibits the best performance and can be used for subsequent production of recombinant horseshoe crab reagents.

[0061] Experimental Example 3: Verification of the accuracy of recombinant factor B activity detection results using recombinant horseshoe crab three-factor reagent.

[0062] The recombinant horseshoe crab tri-factor reagent of Comparative Example 2 was used to verify the activity of recombinant factors B #1 and #3 screened in Experimental Example 2. The endotoxin concentrations and corresponding reaction times are shown in Table 3. A standard curve was plotted with the logarithm of ΔRFU as the ordinate and the logarithm of the endotoxin standard concentration as the abscissa. Figure 3 .

[0063] Table 3 Results of Factor B activity validation using recombinant horseshoe crab reagent

[0064]

[0065] Through Table 3 and Figure 3 It can be seen that the reaction time of Factor B #1 at 5 EU / ml is close to one hour, and the reaction time at 0.005-0.5 EU / ml is also greater than one hour. Within the 0.005-0.5 EU / ml endotoxin range, the reaction time required for this protein to reach the detection limit is longer than the maximum reaction time, indicating that its activity is very low. Furthermore, the correlation coefficient R of the standard curve is low. 2 The value <0.98 indicates that its sensitivity cannot meet the requirement of 0.005-5 EU / ml. Factor B #3, on the other hand, requires a very short reaction time, indicating high activity. Furthermore, according to the standard curve, within the 0.005-5 EU / ml range, the correlation coefficient R of the standard curve for #3 is [value missing]. 2 A value ≥0.98 indicates that its sensitivity also meets the requirements. This result is consistent with the result of Experiment Example 2, proving that the screening method of the present invention is effective. Furthermore, because it does not depend on recombinant coagulase, the detection results are not affected by coagulase activity, and the experimental operation is simpler.

[0066] 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 method for screening the activity and sensitivity of recombinant factor B for endotoxin detection, characterized in that, Includes the following steps: A series of endotoxin standard solutions of different concentrations were added to an endotoxin-free 96-well plate, along with endotoxin-free water as a control. The lowest concentration of the series of endotoxin standard solutions was 0.005 EU / ml. Add an activity detection reagent, which includes a buffer solution, a mixture of recombinant factor C and the recombinant factor B to be tested, and a fluorescent substrate; the buffer solution contains the following components: 50-200 mM HEPES buffer at pH 7.4, 50-200 mM magnesium sulfate, 0.1-0.5% w / v gelatin, 0.5-2.5% w / v mannitol, and 0.1-0.5% w / v CHAPS; Read the zero-point fluorescence value, and then incubate the 96-well plate in a 37°C incubator; Read the fluorescence value again and calculate the fluorescence intensity change value ΔRFU; Result determination: When each ΔRFU is a positive number that increases sequentially, it is determined that the recombinant factor B to be tested meets the activity requirements for endotoxin detection. A standard curve is plotted with the logarithm of ΔRFU on the ordinate and the logarithm of the endotoxin standard concentration on the abscissa. When the correlation coefficient R of the standard curve is... 2 If the value is ≥0.98, the recombinant factor B to be tested is determined to meet the sensitivity requirements for endotoxin detection.

2. The screening method according to claim 1, characterized in that, The buffer solution contains the following components: 100 mM HEPES buffer at pH 7.4, 100 mM magnesium sulfate, 0.2% w / v gelatin, 1.5% w / v mannitol, and 0.2% w / v CHAPS.

3. The screening method according to claim 1, characterized in that, In the mixture of recombinant factor C and recombinant factor B to be tested, the content ratio of recombinant factor C to recombinant factor B to be tested is (1-3):

1.

4. The screening method according to claim 1, characterized in that, In the mixture of recombinant factor C and recombinant factor B to be tested, the content ratio of recombinant factor C to recombinant factor B to be tested is 1:

1.

5. The screening method according to claim 1, characterized in that, The fluorescent substrate is selected from any one of Boc-Asp-Pro-Arg-MCA, Boc-Leu-Thr-Arg-MCA, and Boc-Val-Pro-Arg-MCA.

6. The screening method according to claim 1, characterized in that, In the activity detection reagent, the final concentration of the fluorescent substrate is 50-500 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested is 10-100 μg / ml.

7. The screening method according to claim 1, characterized in that, In the activity detection reagent, the final concentration of the fluorescent substrate is 100 μM, and the final concentration of the mixture of recombinant factor C and the recombinant factor B to be tested is 100 μg / ml.

8. The screening method according to claim 1, characterized in that, The fluorescence detection conditions were: excitation wavelength 360 nm and emission wavelength 460 nm.

9. The screening method according to claim 1, characterized in that, Incubate at 37°C for 30 minutes.

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