A fluorescent microsphere probe for detecting endotoxin and a preparation method and application thereof

Abstract

The application provides a fluorescent microsphere probe for detecting endotoxin and a preparation method and application thereof. The preparation method of the fluorescent microsphere probe for detecting endotoxin comprises the following steps: obtaining recombinant protein factor C of limulus; activating carboxylated fluorescent microspheres to obtain activated fluorescent microspheres; coupling the activated fluorescent microspheres and the recombinant protein factor C of limulus to obtain coupled microspheres; and blocking unreacted groups on the coupled microspheres with aminated PEG and ethanolamine to obtain the fluorescent microsphere probe for detecting endotoxin. The application has the advantages of simple operation, short reaction time, good repeatability and high sensitivity, does not have special requirements for detection equipment, is convenient to carry, has short reaction time and is very suitable for the needs of various timely monitoring of endotoxin levels.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacterial endotoxin detection, in particular to a fluorescent microsphere probe for detecting endotoxin and a preparation method and application thereof. BACKGROUND

[0002] Endotoxin is the main component of the outer wall of the cell membrane of gram-negative bacteria (GNB), and plays an important role in the stability of bacteria. The bacterial endotoxin molecule is an amide lipid complex with a double sugar structure, and its molecular weight is generally between 2000 and 2300. Because of its small molecular weight, it can easily invade from the mesentery or tissue fluid and enter the circulatory system with the blood. Endotoxin is almost ubiquitous and is a highly toxic inflammatory and pyrogenic substance, and is the main pathogenic medium of endotoxemia and septic shock. Endotoxemia is a condition caused by the release of a large amount of bacterial endotoxin from bacteria in the blood or lesions, or by the input of a large amount of endotoxin-contaminated liquid. When the body is injured (including surgery), burned, infected, treated with radiotherapy, chemotherapy, and long-term traditional parenteral nutrition, the permeability of the intestinal mucosa increases, and the permeability of the intestinal mucosa to bacteria and endotoxin increases, allowing a large amount of bacteria and endotoxin to enter the blood.

[0003] Currently commonly used endotoxin detection methods include gel method, turbidity method and colorimetric method. Among them, the gel method is a qualitative or semi-quantitative detection method by observing whether a gel is formed as the end point of the reaction. This method is simple, economical and convenient, but has poor precision, and the formation of the gel is easily affected by many factors in the reaction, so it cannot be accurately quantified. The turbidity method is a method for measuring the content of endotoxin by measuring the turbidity change during the reaction of detecting limulus reagent and endotoxin. This method has a wide detection range and is simple to operate, and is widely used in clinical practice, but requires matching instruments and reagents. The colorimetric method is a method for measuring the content of endotoxin by measuring the color release of specific substrates produced by coagulase during the reaction of limulus reagent and endotoxin, but the required instruments and reagents are relatively expensive and the operation is relatively complex. The measurement range of the above-mentioned detection methods reaches 0.005-300.000 EU / mL, but is greatly affected by personal temporary operation and experimental environment, and is restricted by instruments, so the results differ greatly each time, affecting the precision of endotoxin detection.

[0004] In recent years, the colorimetric method has been further developed, and the fluorescent detection technology based on recombinant C factor has further improved the speed and sensitivity of endotoxin detection. However, the current endotoxin detection method is difficult to achieve fast and convenient on-site detection, which causes great difficulty for many applications that need to be monitored at any time. SUMMARY

[0005] The present application provides a high-sensitivity endotoxin fluorescent microsphere probe which can quickly and conveniently detect endotoxin.

[0006] To solve the above problems, the present application provides a preparation method of a fluorescent microsphere probe for detecting endotoxin, comprising the following steps:

[0007] obtaining recombinant protein factor C of limulus;

[0008] activating carboxylated fluorescent microspheres to obtain activated fluorescent microspheres;

[0009] carrying out coupling reaction on the activated fluorescent microspheres and the recombinant protein factor C of limulus to obtain coupled microspheres;

[0010] blocking unreacted groups on the coupled microspheres with aminated PEG and ethanolamine to obtain the fluorescent microsphere probe for detecting endotoxin.

[0011] Further, the recombinant protein factor C of limulus is prepared by the following method:

[0012] obtaining recombinant protein factor C gene, and amplifying the recombinant protein factor C gene;

[0013] carrying out double enzyme digestion and DNA enzyme ligation on the His-tagged vector pET-21a and the amplified recombinant protein factor C gene in sequence to obtain PET-21a-rFC1 plasmid;

[0014] introducing the PET-21a-rFC1 plasmid into E. coli for expression and purification to obtain the recombinant protein factor C of limulus.

[0015] Further, the recombinant protein factor C of limulus is prepared by the following method:

[0016] obtaining recombinant protein factor C gene, and amplifying the recombinant protein factor C gene;

[0017] carrying out double enzyme digestion and DNA enzyme ligation on the Fc-tagged vector pINFUSE-hIgG1-Fc2 and the amplified recombinant protein factor C gene in sequence to obtain pINFUSE-hIgG1-Fc2-rFC2 plasmid;

[0018] introducing the pINFUSE-hIgG1-Fc2-rFC2 plasmid into HEK293 / CHO cells for expression and purification to obtain the recombinant protein factor C of limulus.

[0019] Further, the adding amount of the recombinant protein factor C of limulus is 1-10 times of the molar amount of carboxyl on the activated fluorescent microspheres.

[0020] The second aspect of the present application provides a fluorescent microsphere probe for detecting endotoxin, which is prepared by the preparation method of any one of the first aspect.

[0021] The third aspect of the present application provides an endotoxin detection test paper, comprising the fluorescent microsphere probe for detecting endotoxin of the second aspect.

[0022] Further, it further comprises a sample pad, a fluorescent microsphere combination pad, a nitrocellulose membrane, a water absorption plate and a bottom plate, the nitrocellulose membrane is arranged in the middle of the bottom plate, the nitrocellulose membrane is provided with an endotoxin reaction detection line and an endotoxin quality control line, one end of the bottom plate is provided with the water absorption plate, the other end of the bottom plate is provided with the sample pad, the sample pad is arranged on the fluorescent microsphere combination pad, and the two ends of the nitrocellulose membrane are respectively connected with the water absorption plate and the fluorescent microsphere combination pad, and the fluorescent microsphere probe for detecting endotoxin is fixed on the fluorescent microsphere combination pad.

[0023] Further, the fluorescent microsphere combination pad comprises glass fibers, the glass fibers are sprayed with the fluorescent microsphere probe for detecting endotoxin, and the glass fibers and the fluorescent microsphere probe for detecting endotoxin are soaked with the same buffer.

[0024] Further, the endotoxin reaction detection line is fixed with a coupling complex, the coupling complex is formed by covalent coupling of endotoxin and a macromolecule; and the endotoxin quality control line is fixed with a His-tag antibody or an Fc-tag antibody.

[0025] The fourth aspect of the present application provides a detection method of endotoxin, which detects by using the endotoxin detection test paper of any one of the third aspect, and comprises the following steps:

[0026] Different concentrations of endotoxin standard samples are added to the sample pad, after chromatography, the fluorescence intensities of the endotoxin reaction detection line and the endotoxin quality control line are measured, and a standard curve is prepared according to the change of the fluorescence intensity of the endotoxin reaction detection line;

[0027] After the sample to be tested with endotoxin is diluted with a buffer, it is added to the sample pad, after chromatography, the fluorescence intensities of the endotoxin reaction detection line and the endotoxin quality control line are measured, and the content of endotoxin in the sample to be tested is calculated according to the standard curve.

[0028] The recombinant protein E. coli C factor protein is coupled to the fluorescent microspheres in the application, and the unreacted groups on the fluorescent microspheres are blocked by amino-PEG and ethanolamine to form a fluorescent microsphere detection probe, which can eliminate the interference of endotoxin in the environment on the sample detection result, and is beneficial to improve the accuracy and reliability of the detection result. In addition, the fluorescent microsphere detection probe is assembled to form an endotoxin detection test paper, the endotoxin in the sample to be detected is detected by chromatography, and the content of the endotoxin is calculated by comparison with a standard curve, thereby avoiding the subjectivity of manual interpretation. The endotoxin detection test paper has the advantages of simple operation, short reaction time, good repeatability and high sensitivity. No special requirements are required for the detection equipment. The endotoxin detection test paper is convenient to carry and has a short reaction time, and is very suitable for the needs of timely monitoring of endotoxin levels, such as outdoor conditions without fixed equipment conditions for environmental monitoring, or monitoring of endotoxin levels in nucleic acid protein purification at any time during the research and development process. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A structural schematic diagram of the endotoxin detection test paper provided by the embodiment of the application is shown in the figure.

[0030] Figure 2 A standard curve graph of endotoxin in the embodiment 3 of the application is shown in the figure.

[0031] Figure 3 A result graph of the endotoxin detection recovery rate in different samples provided by the embodiment 3 of the application is shown in the figure.

[0032] BRIEF DESCRIPTION OF DRAWINGS:

[0033] 1 - sample pad; 2 - fluorescent microsphere combination pad; 3 - nitrocellulose membrane; 4 - water absorption plate; 5 - bottom plate; 31 - endotoxin reaction detection line; 32 - endotoxin quality hole line. DETAILED DESCRIPTION

[0034] It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0035] In addition, the terms "comprising", "including", "containing", "having" have the meaning of non-limitation, that is, other steps and other components can be added without affecting the results. Unless otherwise specified, the materials, devices and reagents are commercially available.

[0036] In addition, although the steps in the preparation are described in the form of S10, S20, S30, etc. in the application, this description method is only for easy understanding, and the form of S10, S20, S30 does not represent the limitation of the sequence of the steps.

[0037] To solve the above problems, the embodiment of the present application provides a preparation method of a fluorescent microsphere probe for detecting endotoxin, comprising the following steps:

[0038] Step S10, obtaining recombinant protein factor C protein;

[0039] Step S20, activating the carboxylated fluorescent microspheres to obtain activated fluorescent microspheres;

[0040] Step S30, coupling the activated fluorescent microspheres and the recombinant protein factor C protein to obtain coupled microspheres;

[0041] Step S40, blocking the unreacted groups on the coupled microspheres with aminated PEG and ethanolamine to obtain the fluorescent microsphere probe for detecting endotoxin.

[0042] The preparation method of the fluorescent microsphere probe for detecting endotoxin provided by the embodiment of the present application couples the recombinant protein factor C protein to the fluorescent microspheres, and blocks the unreacted groups on the fluorescent microspheres with aminated PEG and ethanolamine to form the fluorescent microsphere detection probe, which can eliminate the interference of endotoxin in the environment on the sample detection result, and is beneficial to improve the accuracy and reliability of the detection result. In addition, the fluorescent microsphere probe for detecting endotoxin provided by the embodiment of the present application has the advantages of simple operation, short reaction time, good repeatability and high sensitivity, and can be directly observed by naked eyes, avoiding the subjectivity of manual interpretation.

[0043] In step S10, if the recombinant protein factor C (rFC for short) protein is expressed in a prokaryotic system, the following method can be used to prepare it:

[0044] Step S11, obtaining the recombinant protein factor C gene, and amplifying the recombinant protein factor C gene by using PCR technology;

[0045] Step S12, performing double digestion (Nde I and Xho I double digestion) on the carrier pET-21a with a His tag and the amplified recombinant protein factor C gene, and then connecting by using a DNA ligase to obtain a PET-21a-rFC1 plasmid;

[0046] Step S13, introducing the PET-21a-rFC1 plasmid into an E. coli shuffle competent cell for expression, so that the protein has a 6x His tag, and then purifying by using a nickel column to obtain the recombinant protein factor C protein, which is referred to as rFC1 protein below, and the rFC1 protein is stored at -80℃.

[0047] If the recombinant protein factor C protein is expressed in a eukaryotic system, the following method can be used to prepare it:

[0048] Step S11, obtaining the recombinant protein factor C gene, and using PCR technology to amplify the recombinant protein factor C gene;

[0049] Step S12, performing double enzyme digestion (Bgl II and EcoR I double enzyme digestion) on the carrier pINFUSE-hIgG1-Fc2 with Fc tag and the amplified recombinant protein factor C gene, and then connecting by using DNA ligase to obtain the pINFUSE-hIgG1-Fc2-rFC2 plasmid;

[0050] Step S13, introducing the pINFUSE-hIgG1-Fc2-rFC2 plasmid into HEK293 / CHO cells for expression, so that the protein has a 2x Fc tag, and then performing purification by using a protein A column to obtain the recombinant protein factor C protein, which is hereinafter referred to as rFC2 protein, and storing the rFC2 protein at-80℃.

[0051] The recombinant protein factor C gene can be purchased from the market, for example, from Nanjing Kings River Company.

[0052] In step S20, the carboxylated fluorescent microspheres are activated with an activator at room temperature for 15-30 min to obtain activated fluorescent microspheres, wherein the activator is EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NSH is greater than 2:5.

[0053] The amount of the activator added is 10-100 times the molar amount of the carboxyl groups in the carboxylated fluorescent microspheres, so that the carboxylated fluorescent microspheres can be fully activated.

[0054] The carboxylated fluorescent microspheres refer to latex microspheres with carboxyl groups and capable of emitting fluorescence. In this embodiment, the specific type of the carboxylated fluorescent microspheres is not further limited, and a person skilled in the art can select according to actual needs. In some optional embodiments, the carboxylated fluorescent microspheres are carboxylated europium-containing fluorescent microspheres commercialized by Thermos Scientific Company. The excitation light of the carboxylated europium-containing fluorescent microspheres is 300-360 nm, and the emission light is 600-630 nm.

[0055] The particle size of the carboxylated fluorescent microspheres is related to the efficiency and angle of transmission and scattering, which affects the optical density of the fluorescent microsphere aggregation and the sensitivity requirement of the final test. Therefore, the particle size of the carboxylated fluorescent microspheres is not further limited in this embodiment, and a person skilled in the art can select according to actual needs. In some optional embodiments, the particle size of the carboxylated fluorescent microspheres is 200 nm.

[0056] In step S30, the activated fluorescent microspheres are washed and mixed with borate buffer (pH 8.0) and recombinant protein factor C protein, so that the final concentration of the activated fluorescent microspheres is 0.1% w / v. After coupling reaction at room temperature for 2-3 hours, the coupled microspheres are obtained.

[0057] The amount of the recombinant protein factor C protein added is 1-10 times the molar amount of the carboxyl groups on the activated fluorescent microspheres.

[0058] The concentration of the borate buffer (pH 8.0) is 25-50 mmol / L.

[0059] In step S40, the coupled microspheres are mixed with aminated PEG and ethanolamine, so that the final concentration of the coupled microspheres is 0.1% w / v. After overnight blocking at 4°C, the unreacted groups on the coupled microspheres are blocked. After centrifugation to remove the supernatant and washing, the coupled microspheres are resuspended to a concentration of 1% w / v to obtain the fluorescent microsphere probe for detecting endotoxin, which is stored at 4°C.

[0060] The amount of the aminated PEG and ethanolamine added is 10-100 times the molar amount of the carboxyl groups on the coupled microspheres, so as to sufficiently block the excess carboxyl sites on the coupled microspheres.

[0061] The second aspect of the embodiments of the present application provides a fluorescent microsphere probe for detecting endotoxin, which is prepared by the preparation method of the first aspect.

[0062] The third aspect of the embodiments of the present application provides an endotoxin test paper, which comprises the fluorescent microsphere probe for detecting endotoxin of the second aspect.

[0063] In combination Figure 1 As shown, the endotoxin test paper comprises a sample pad 1, a fluorescent microsphere combination pad 2, a nitrocellulose membrane 3, a water absorption plate 4 and a base plate 5. The nitrocellulose membrane 3 is arranged in the middle of the base plate 5, and the nitrocellulose membrane 3 is provided with an endotoxin reaction detection line 31 and an endotoxin quality control line 32. The water absorption plate 4 is arranged at one end of the base plate 5, and the sample pad 1 is arranged at the other end of the base plate 5. The sample pad 1 is arranged on the fluorescent microsphere combination pad 2. The two ends of the nitrocellulose membrane 3 are respectively connected to the water absorption plate 4 and the fluorescent microsphere combination pad 2. The fluorescent microsphere probe for detecting endotoxin is fixed on the fluorescent microsphere combination pad 2.

[0064] The endotoxin is grafted onto a macromolecule to form a coupled complex, and the coupled complex is fixed on the nitrocellulose membrane 3 as the endotoxin reaction detection line 31.

[0065] The macromolecule refers to a substance with a molecular weight greater than 60 kDa. Grafting endotoxin to the macromolecule can increase the molecular weight of endotoxin, better anchor the endotoxin, and stably fix the endotoxin on the nitrocellulose membrane 3, so as to avoid the endotoxin from falling off. Specifically, the macromolecule can be bovine serum albumin (abbreviated as BSA), casein or ovalbumin OVA.

[0066] The His-tag antibody or the Fc-tag antibody is fixed on the endotoxin control line 32. Which one is fixed can be selected according to the recombinant protein Limulus C factor protein. If the recombinant protein Limulus C factor protein has a 6x His tag, the His-tag antibody is fixed on the endotoxin control line 32. If the recombinant protein Limulus C factor protein has a 2x Fc tag, the Fc-tag antibody is fixed on the endotoxin control line 32.

[0067] The sample pad 1 and the fluorescent microsphere combination pad 2 both include glass fibers, and the glass fibers need to be treated before being used for the sample pad 1 and the fluorescent microsphere combination pad 2 as follows:

[0068] The glass fibers are soaked in a 0.1 mol / L NaOH or dilute hydrochloric acid solution overnight to remove endotoxin in the glass fibers, and then washed with pyrogen-free water (such as sterile water for injection) and dried at 35°C.

[0069] The glass fibers are then soaked in a solution consistent with the buffer solution of the fluorescent microsphere probe for detecting endotoxin (for example, 10-50 mmol / L borate buffer (pH 8.0, 0.01-0.5% v / v Tween-20, 0.5-2% w / v BSA, 0.1-5% sucrose, 0.01-2% PVP (polyvinylpyrrolidone)), and then dried to obtain the treated glass fibers. In this way, the glass fibers are treated as described above, so that the fluorescent microspheres for detecting endotoxin are prevented from being blocked on the glass fibers.

[0070] The treated glass fibers can be directly used as the sample pad 1.

[0071] The treated glass fibers are fixed on a sputtering instrument, and after setting the parameters of the sputtering instrument, the fluorescent microsphere probe for detecting endotoxin is sprayed on the treated glass fibers, which are then dried at 35°C overnight to obtain the fluorescent microsphere combination pad 2. The parameters of the sputtering instrument can be set by the person skilled in the art according to the actual situation, and the embodiments of the present application do not make further limitations thereon.

[0072] It should be noted that the endotoxin detection test paper in the embodiments of the present application can have a blood filtration membrane added on the sample pad 1 as needed.

[0073] After the endotoxin detection test paper is cut and assembled, it can be assembled in a card shell or directly used, but if directly used, the use environment of the endotoxin detection test paper needs to be strictly controlled in a sterile environment, and when used, the sample pad 1 can be downwardly immersed in a sample liquid, and after chromatography for 15-30 min, the fluorescence intensities of the endotoxin reaction detection line 31 and the endotoxin quality control line 32 are measured, and the content of endotoxin in the sample liquid is calculated.

[0074] The endotoxin detection test paper provided by the embodiment of the present application couples the recombinant protein factor C protein to the fluorescent microspheres, and blocks the unreacted groups on the fluorescent microspheres with amino-PEG and ethanolamine to form a fluorescent microsphere detection probe, which can eliminate the interference of endotoxin in the environment on the sample detection result, and is beneficial to improve the accuracy and reliability of the detection result. In addition, the fluorescent microsphere detection probe is assembled to form an endotoxin detection test paper, the endotoxin in the sample to be detected is detected by chromatography, and the content of the endotoxin is calculated by comparison with a standard curve, which avoids the subjectivity of manual interpretation. The endotoxin detection test paper has the advantages of simple operation, short reaction time, good repeatability and high sensitivity. There is no special requirement for the detection equipment. The endotoxin detection test paper is convenient to carry and has a short reaction time, and is very suitable for various needs of timely monitoring of endotoxin levels, such as environmental monitoring under outdoor conditions without fixed equipment conditions, or timely monitoring of endotoxin levels in nucleic acid protein purification during the research and development process.

[0075] The fourth aspect of the embodiment of the present application provides a method for detecting endotoxin, which uses the endotoxin detection test paper of the third aspect for detection, and specifically includes the following steps:

[0076] Different concentrations of endotoxin standard samples are added to the sample pad 1, and after chromatography for 10-15 min, the fluorescence intensities of the endotoxin reaction detection line 31 and the endotoxin quality control line 32 are measured, and a standard curve is prepared according to the change of the fluorescence intensity of the endotoxin reaction detection line 31.

[0077] The sample to be detected with endotoxin is diluted with a buffer solution and added to the sample pad 1, and after chromatography for 10-15 min, the fluorescence intensities of the endotoxin reaction detection line 31 and the endotoxin quality control line 32 are measured, and the content of endotoxin in the sample to be detected is calculated according to the standard curve.

[0078] The method for detecting endotoxin provided in the embodiments of the present application couples the recombinant protein factor C protein of limulus to a fluorescent microsphere, and blocks the unreacted groups on the fluorescent microsphere with aminated PEG and ethanolamine to form a fluorescent microsphere detection probe, which can eliminate the interference of endotoxin in the environment on the detection result of the sample, and is beneficial to improve the accuracy and reliability of the detection result. In addition, the fluorescent microsphere detection probe is assembled to form an endotoxin detection test paper, the endotoxin in the sample to be detected is detected by chromatography, and the content of the endotoxin is calculated by comparison with a standard curve, which avoids the subjectivity of manual interpretation, and the endotoxin detection test paper has the advantages of simple operation, short reaction time, good repeatability and high sensitivity. The endotoxin detection test paper does not have special requirements for the detection equipment, is convenient to carry, has a short reaction time, and is very suitable for the needs of various timely monitoring of the endotoxin level, such as environmental monitoring under outdoor conditions without fixed equipment conditions, or timely monitoring of the endotoxin level in the process of nucleic acid protein purification.

[0079] In order to further illustrate the present application, the present application will be further illustrated by combining specific examples. The experimental methods used in the examples in the present application are all conventional methods unless otherwise specified; the materials, reagents, etc. used in the examples in the present application are all obtained by market purchase unless otherwise specified.

[0080] Example 1

[0081] The present embodiment provides a preparation method of a fluorescent microsphere probe for detecting endotoxin, comprising the following steps:

[0082] (1) Take 0.2 mg of carboxylated europium-containing fluorescent microspheres with a particle size of 200 nm (purchased from Thermos Scientific, excitation light at 330±30 nm, and emission light at 615±15 nm), and wash twice with 25-50 mmol / L MES buffer (pH 6.0); add a certain volume of MES buffer, calculate the carboxyl content, and add 10-100 times the molar amount of EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) (molar ratio >2:5), and oscillate at room temperature for 15-30 min;

[0083] (2) centrifugation to remove supernatant, then washed with coupling buffer (same as the storage buffer of recombinant protein rFC1 or rFC2) for 2-3 times, then mixed with a certain amount of 25-50 mmol / L borate buffer (pH 8.0) and a certain amount of solution of recombinant protein rFC1 or rFC2, the final volume of the mixed solution was 200 ul, so that the final concentration of the activated fluorescent microspheres was 0.1% w / v, then reacted for 2-3 h at room temperature, then centrifuged to remove supernatant, then washed with 25-50 mmol / L borate buffer (pH 8.0) for 2-3 times, to obtain the coupled microspheres, then added a certain amount of Tris buffer or borate buffer to make the concentration of the coupled microspheres about 0.1% w / v;

[0084] (3) took amino-PEG and ethanolamine, added to the coupled microspheres solution in step (2) to make the final concentration of the coupled microspheres about 0.1% w / v, then blocked overnight at 4℃, to block the unreacted groups on the coupled microspheres, then centrifuged to remove supernatant, then washed with Tris buffer or borate buffer, then resuspended to 1% w / v microspheres solution, to obtain the fluorescent microspheres probe for detecting endotoxin, then stored at 4℃.

[0085] Example 2

[0086] The present embodiment provides a method for preparing an endotoxin detection test paper, comprising the following steps:

[0087] (1) took endotoxin (referred to as LPS) and activated with EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) for 30 min, then added a certain proportion of bovine serum albumin (referred to as BSA) and reacted for 3 h, wherein the proportion of LPS and BSA was 0.75 mg / mL:4 mg / mL, then added PBS buffer in an ultrafiltration tube, then added ethanolamine or hydroxylamine, blocked overnight at 4℃ or blocked for 30 min at room temperature, to obtain BSA-LPS coupled complex, then suspended the BSA-LPS coupled complex in PBS buffer and stored at -80℃.

[0088] (2) soaked glass fibers in 0.1 mol / L NaOH solution overnight to remove endotoxin in the glass fibers, then washed with sterile water, then dried the glass fibers at 35℃, then soaked the glass fibers in 50 mmol / L borate buffer (pH 8.0, 0.05% v / v Tween-20, 2 w / v% sucrose, 0.5% PVP) for 1 h, then dried overnight at 35℃, to obtain treated glass fibers, which were directly used as sample pad;

[0089] (3) Take the partially treated glass fiber in step (2) and fix it on a gold spraying instrument. After setting the parameters of the gold spraying instrument, prepare the fluorescent microsphere probe for detecting endotoxin into a microsphere solution with a concentration of 0.01-0.05% w / v, and spray the solution on the treated glass fiber. Dry the glass fiber at 35°C overnight to obtain a fluorescent microsphere binding pad;

[0090] (4) Take a nitrocellulose membrane and fix it on a marking instrument. Prepare the BSA-LPS conjugate complex in step (1) into an endotoxin solution with a concentration of 0.5-10 EU / mL. Mark and fix the endotoxin solution on the endotoxin reaction detection line of the nitrocellulose membrane. Mark and fix His-tag antibody (for rFC1 adaptation) or Fc-tag antibody (for rFC2 adaptation) with a concentration of 0.25-2 mg / mL on the endotoxin quality control line of the nitrocellulose membrane. Dry the treated nitrocellulose membrane at 35°C overnight to obtain a treated nitrocellulose membrane.

[0091] (5) Place the treated nitrocellulose membrane in step (4) in the middle of a PVC base plate. Place a water absorption plate at one end of the PVC base plate and a sample pad at the other end. Place the sample pad on the fluorescent microsphere binding pad. Overlap and connect the two ends of the nitrocellulose membrane with the water absorption plate and the fluorescent microsphere binding pad, respectively. After assembly, cut the test strip into a strip with a width of 2.8-4 mm using a strip cutter. Assemble the test strip in a card shell to obtain an endotoxin test strip.

[0092] Example 3

[0093] The present embodiment provides a method for detecting endotoxin by an endotoxin test strip, which comprises the following steps:

[0094] (1) Dissolve the endotoxin lyophilized powder in a negative buffer solution (i.e., 10 mmol / L BB pH 8.0, 0.01-2% w / v PVP, 0.01-0.5% v / v Tween-20, 0.1-5% w / v sucrose, and 0.01-0.5% w / v BSA). Prepare the endotoxin lyophilized powder into an endotoxin solution with a concentration of 0.01, 0.05, 0.1, 0.5, 1, 2, and 4 EU / mL using the negative buffer solution as a negative control, i.e., the concentration of endotoxin is 0.

[0095] Take different concentrations of endotoxin solution, respectively. Add 50-200 ul of endotoxin solution to the sample pad of the chromatographic test strip using a pipette (add to the sample groove in the card shell), and perform chromatography for 10-20 min. Then, measure the fluorescence intensity at the T line and the C line (330 nm excitation and 610 nm reception). Calculate the standard curve according to the signal change. The fluorescence intensity results of different concentrations of endotoxin solution at the T line and the C line are shown in Table 1, and the standard curve is shown in Figure 2 ​

[0096] (2) Take the sample to be tested with endotoxin, and dilute the sample to be tested with the negative buffer solution in step (1) by 4, 8, 16, 32, and 64 times, respectively. After dilution, each sample is divided into two parts, one of which is added with 0.5 EU / mL of endotoxin standard as a control, and the other is recorded as F1. Each sample is measured in triplicate, and the average value is taken.

[0097] Use a pipette to take 50-200 uL of each sample to be tested, and drop 50-200 ul of the sample solution to be tested on the sample pad of the chromatographic test strip (drop in the sample groove if the card is used). After chromatography for 10-20 min, the fluorescence intensity at the T line and the C line is measured, and the endotoxin concentration is calculated according to the standard curve in Table 1. Figure 2

[0098] Table 1 Fluorescence intensity of endotoxin standard

[0099]

[0100]

[0101] Table 2 Concentration of endotoxin in the endotoxin sample to be tested

[0102]

[0103] From the calculation results in Table 2, it can be concluded that the sample to be tested with endotoxin contains about 2.1 EU / mL of endotoxin. The concentration of endotoxin in the sample to be tested with endotoxin can also be accurately calculated by adding 0.5 EU / mL as a control. This also shows that the endotoxin detection test paper provided in this embodiment improves the accuracy and reliability of the detection results, and does not require the use of expensive instruments, has short detection time, good repeatability, and high sensitivity.

[0104] In order to detect the detection effect of the endotoxin detection test paper, the same amount of endotoxin is added to various samples, and the recovery rate is calculated as the detection result value / endotoxin addition amount. It can be found that the recovery rates of different samples are quite different, Figure 3 Figure showing the endotoxin detection recovery rate in different samples, from Figure 3 It can be seen that the recovery rate in sterile water for injection is 0.98, indicating that the endotoxin detection test paper in this embodiment has obvious effect on the detection of endotoxin. Based on the characteristics of endotoxin, endotoxin naturally exists in plasma samples (rabbit plasma or human plasma), while serum and sterile water for injection perform well, indicating that the endotoxin detection test paper in this embodiment can be used for the detection of endotoxin in water and serum.

[0105] Example 4

[0106] ​The embodiment provides performance index test of the endotoxin detection test paper

[0107] (1) Sensitivity test

[0108] Six samples with different endotoxin contents, i.e., the concentrations of endotoxin are 0.001 EU / mL, 0.005 EU / mL, 0.01 EU / mL, 0.02 EU / mL, 0.05 EU / mL and 0.1 EU / mL, are measured for 10 times, and the average and SD (standard deviation) values are calculated to obtain the results shown in Table 3. As shown in Table 3, the sensitivity of the endotoxin detection test paper in the embodiment is 0.005 EU / mL.

[0109] Table 3

[0110]

[0111] (2) High-value linear determination

[0112] Six samples with different endotoxin contents, i.e., the concentrations of endotoxin are 0.05 EU / mL, 0.1 EU / mL, 0.5 EU / mL, 1 EU / mL, 2 EU / mL and 4 EU / mL, are measured for 3 times, and the results shown in Table 4 are obtained. As shown in Table 4, the highest detection range of the endotoxin detection test paper in the embodiment can reach 4 EU / mL, and the determination is based on r 2 ≥ 0.990.

[0113] Table 4

[0114]

[0115]

[0116] (3) Precision test

[0117] Two samples with different endotoxin contents are used to determine the intra-batch precision of the endotoxin detection test paper in the embodiment, 20 times of determination are performed on one sample by using three batches of endotoxin detection test paper, the inter-batch relative range of the endotoxin detection test paper in the embodiment is calculated, and the results shown in Table 5 and Table 6 are obtained. The results show that the intra-batch precision of the endotoxin detection test paper in the embodiment is 12.41% (see Table 5, the maximum error value), and the inter-batch relative range is 4.89% (see Table 6, the maximum error value).

[0118] Table 5

[0119]

[0120] Table 6

[0121]

[0122] (4) Stability test

[0123] Under the storage condition of 2-8℃, the same serum sample was determined at 0 day, 2 weeks, 1 month, 2 months, 4 months, 6 months, 8 months and 12 months, respectively, 6 times for each sample, and the average value was taken (see Table 7). The results showed that the measured values before 6 months had little difference compared with 0 day, indicating that the endotoxin detection test paper of the application could be stable for 6 months under the storage condition of 2-8℃.

[0124] Table 7

[0125]

[0126] From the above examples, it can be seen that the endotoxin detection test paper provided by the examples of the application has the advantages of high sensitivity, good specificity, good accuracy, good anti-interference property and low production cost.

[0127] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited to this. Various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the present disclosure, and these changes and modifications shall fall within the protection scope of the present disclosure.

Claims

1. An endotoxin test strip, characterized by, The detection endotoxin fluorescent microsphere probe comprises a sample pad, a fluorescent microsphere combination pad, a nitrocellulose membrane, a water absorption plate and a bottom plate, the nitrocellulose membrane is arranged in the middle of the bottom plate, the nitrocellulose membrane is provided with an endotoxin reaction detection line and an endotoxin quality control line, one end of the bottom plate is provided with the water absorption plate, the other end of the bottom plate is provided with the sample pad, the sample pad is arranged on the fluorescent microsphere combination pad, and the two ends of the nitrocellulose membrane are connected with the water absorption plate and the fluorescent microsphere combination pad respectively, and the detection endotoxin fluorescent microsphere probe is fixed on the fluorescent microsphere combination pad. The detection endotoxin fluorescent microsphere probe is prepared by the following method: The recombinant protein factor C of limulus is obtained by the following method: The carboxylated fluorescent microspheres are activated to obtain activated fluorescent microspheres; The activated fluorescent microspheres and the recombinant protein factor C of limulus are coupled in borate buffer to obtain coupled microspheres; The unreacted groups on the coupled microspheres are blocked by aminated PEG and ethanolamine to obtain the detection endotoxin fluorescent microsphere probe; The endotoxin reaction detection line is fixed with a coupled complex, and the coupled complex is formed by covalent coupling of endotoxin and macromolecules; and the endotoxin quality control line is fixed with a His-tag antibody or an Fc-tag antibody.

2. The endotoxin test strip according to claim 1, characterized in that The recombinant protein factor C of limulus is prepared by the following method: The recombinant protein factor C gene is obtained, and the recombinant protein factor C gene is amplified; The vector pET-21a with a His tag and the amplified recombinant protein factor C gene are sequentially subjected to double enzyme digestion and DNA enzyme ligation to obtain a PET-21a-rFC1 plasmid; The PET-21a-rFC1 plasmid is introduced into E. coli for expression and purification to obtain the recombinant protein factor C of limulus.

3. The endotoxin test strip according to claim 1, characterized in that, The recombinant protein factor C of limulus is prepared by the following method: The recombinant protein factor C gene is obtained, and the recombinant protein factor C gene is amplified; The vector pINFUSE-hIgG1-Fc2 with an Fc tag and the amplified recombinant protein factor C gene are sequentially subjected to double enzyme digestion and DNA enzyme ligation to obtain a pINFUSE-hIgG1-Fc2-rFC2 plasmid; The pINFUSE-hIgG1-Fc2-rFC2 plasmid is introduced into HEK293 / CHO cells for expression and purification to obtain the recombinant protein factor C of limulus.

4. The endotoxin test strip according to claim 1, characterized in that, The added amount of the recombinant protein factor C of limulus is 1-10 times of the molar amount of carboxyl groups on the activated fluorescent microspheres.

5. The endotoxin test strip according to claim 1, wherein The fluorescent microsphere combination pad comprises glass fibers, the glass fibers are sprayed with the detection endotoxin fluorescent microsphere probe, and the glass fibers and the detection endotoxin fluorescent microsphere probe are soaked in the same buffer.

6. A method for detecting endotoxin, characterized by, The endotoxin detection test paper is used for detection, and the detection comprises the following steps: The endotoxin standard of different concentrations is added dropwise to the sample pad, and after chromatography, the fluorescence intensity of the endotoxin reaction detection line and the endotoxin quality control line is determined, and a standard curve is prepared according to the change of the fluorescence intensity of the endotoxin reaction detection line; The sample to be tested with endotoxin is diluted with buffer and added dropwise to the sample pad, and after chromatography, the fluorescence intensity of the endotoxin reaction detection line and the endotoxin quality control line is determined, and the content of endotoxin in the sample to be tested is calculated according to the standard curve.

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