Method for quantitatively determining activity of blood coagulation factor XIII based on fluorescence

Abstract

The invention discloses a method for quantitatively determining the activity of a blood coagulation factor XIII based on fluorescence, which comprises the following steps: (1) treating a plasma sample with bentonite to obtain a plasma sample; (2) adding a plasma sample into the reaction system, and then incubating to obtain a to-be-detected sample; the reaction system comprises a buffer solution, calcium chloride, polyethylene glycol, glycine ethyl ester, himedinium bromide and a fluorescent substrate; (3) establishing a standard curve; and (4) FXIII activity detection: detecting the to-be-detected sample by using the fluorescence tester to obtain the fluorescence intensity of the to-be-detected sample, and calculating the concentration of the to-be-detected sample according to the standard curve. By optimizing the pretreatment process of the plasma sample, the influence of other proteins in the plasma on blood coagulation factor detection is remarkably reduced, and the measurement accuracy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of coagulation factor detection, and specifically relates to a method for determining the activity of coagulation factor XIII based on fluorescence quantitative determination. Background Art

[0002] Coagulation factors are a group of proteins that play a key role in the blood coagulation process. They participate in a complex cascade reaction to ensure that stable thrombi can be formed quickly when blood vessels are damaged, prevent bleeding, and promote wound healing. Among them, coagulation factor XIII (FXIII) is the last factor in the coagulation cascade reaction, which is mainly responsible for cross-linking soluble fibrin monomers into insoluble stable fibrin polymers, thereby enhancing the mechanical strength and stability of thrombi.

[0003] Since FXIII plays an integral role in the coagulation process, the activity test of FXIII is very important for the diagnosis and treatment of coagulation disorders. The current methods for quantitatively measuring FXIII activity include ammonia release assay, isopeptidase activity assay, and amine incorporation assay. Among them, the ammonia release method is a method for evaluating FXIII activity by measuring the amount of ammonia produced in the cross-linking reaction mediated by FXIII. In this process, FXIII catalyzes the formation of ε-lysyl-γ-glutamine bonds in specific substrates, and ammonia is released when these bonds are formed. By using appropriate buffers and pH indicators, the release of ammonia can be detected and the activity of FXIII can be calculated. The advantage of this method is that it is simple and rapid, but it may not be as sensitive as other methods and may be interfered by other ammonia sources in the sample. The isopeptidase activity assay is based on the use of a specific substrate for FXIII, which can undergo structural changes under the action of FXIII. Generally, such substrates are designed to contain a fluorophore and a quencher, which are separated by a peptide sequence that can be recognized and cross-linked by FXIII. When FXIII catalyzes the cross-linking of this polypeptide chain, the distance between the fluorophore and the quencher group increases, resulting in an increase in fluorescence intensity. By measuring the change in fluorescence, the activity of FXIII can be indirectly determined. The amine incorporation assay is a method that quantifies the activity of FXIII by exploiting the ability of FXIII to incorporate specifically labeled amine compounds (such as radioactive or fluorescently labeled amines) into fibrin or polypeptide substrates. In this process, FXIII catalyzes the formation of a covalent bond between the amine and a specific amino acid residue (usually lysine) on the substrate. By measuring the amount of labeled amine incorporated into the substrate, the activity level of FXIII can be inferred. This method is also highly sensitive and can provide accurate results, but it may involve the use of radioactive materials, which require special handling and safety measures.

[0004] The isopeptidase activity assay (i.e., the fluorescent substrate method for the determination of coagulation factor XIII) has the advantages of high sensitivity and good specificity, but still has the following disadvantages:

[0005] (1) Even under optimized conditions, the fluorescent substrate may nonspecifically bind to other proteins and impurities in the sample, resulting in increased background fluorescence. This background signal will reduce the signal-to-noise ratio and affect the accuracy of the results.

[0006] (2) Although the fluorescent substrate is designed to specifically recognize FXIIIa, in actual applications, there may be other enzymes (such as other transglutaminases) that are able to cleave the same substrate, resulting in cross-reactions, which will affect the accurate measurement of FXIIIa activity. Summary of the invention

[0007] The purpose of the present invention is to provide a method for determining the activity of coagulation factor XIII based on fluorescence quantification, so as to solve the problem that the above-mentioned fluorescent substrate cross-reacts in practical applications and other proteins in the sample affect the determination process, resulting in poor measurement accuracy.

[0008] To achieve the above object, the present invention provides a method for determining the activity of coagulation factor XIII based on fluorescence quantification, comprising the following steps:

[0009] (1) Sample pretreatment

[0010] The plasma sample is treated with bentonite to obtain a plasma test sample;

[0011] (2) Preparation of test samples

[0012] Adding the plasma sample into the reaction system and then incubating to obtain a sample to be tested;

[0013] The reaction system includes a buffer solution, calcium chloride, polyethylene glycol, glycine ethyl ester, hexamethylenetetramine bromide and a fluorescent substrate;

[0014] (3) Establishment of standard curve

[0015] Prepare a series of standard FXIII solutions of different concentrations, prepare standard samples of different concentrations according to step (2), detect the prepared standard samples on a fluorescence meter, and draw a standard curve with the concentration percentage of the standard FXIII solution as the horizontal axis and the slope of the fluorescence intensity-time curve during the incubation period as the vertical axis;

[0016] (4) FXIII activity detection

[0017] The sample to be tested is detected by a fluorescence meter to obtain the fluorescence intensity of the sample to be tested, and the concentration of the sample to be tested is calculated according to the standard curve.

[0018] Preferably, in step (1), bentonite is added to the plasma sample, the volume mass ratio of the plasma sample to bentonite is 1 mL: (20-50) mg, and a vortex oscillator is used to gently mix so that the bentonite is evenly distributed in the plasma sample. The mixing time is 10-30 s. After mixing, the mixture is allowed to stand at room temperature for 10-15 min, and then centrifuged at 1000-3000 r / min for 10-15 min. After centrifugation, the supernatant is taken as the plasma sample.

[0019] Preferably, in step (2), the concentration of the buffer solution in the reaction system is 50-100 mM, the concentration of calcium chloride is 8-12 mM, the mass fraction of polyethylene glycol is 0.05-0.15%, the concentration of glycine ethyl ester is 3-8 mM, the concentration of hexamethylenetetramine is 3-8 μg / mL, and the concentration of the fluorescent substrate is 30-80 μM.

[0020] Preferably, the buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, Hepes buffer solution, N,N-dihydroxyethylglycine, and tris(hydroxymethyl)aminomethane.

[0021] Preferably, in step (2), the volume ratio of the plasma sample to the reaction system is 1:(10-20).

[0022] Preferably, in step (2), the incubation temperature is 35-38° C., and the incubation time is 0.01-2 h.

[0023] Preferably, in step (2), the fluorescent substrate comprises a fluorescent group and a fluorescent quenching group, wherein the fluorescent group is 2'-aminobenzanilide and the fluorescent quenching group is 2,4-dinitrophenylhydrazine.

[0024] Preferably, in step (2), the sequence of the fluorescent substrate is:

[0025] (2-Abz)-Asn-Glu(CAD-DNP)-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys.

[0026] Preferably, in step (3), the excitation wavelength of the fluorescence meter is 313 nm.

[0027] Preferably, in step (3), the incubation time is 5 to 40 min, and a linear regression analysis is performed using the fluorescence intensity (y-axis)-time (x-axis) within this time period, each concentration percentage corresponds to a slope, and finally a standard curve is constructed using the slope (y-axis)-concentration percentage (x-axis).

[0028] Therefore, the present invention adopts a method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis using the above structure, which has the following beneficial effects:

[0029] (1) The present invention can effectively reduce the influence of high concentration of protein and other components in plasma on the determination of FXIII activity by diluting the plasma sample with a buffer solution at a ratio of (5-8):1. This precise dilution ratio ensures that the concentration of FXIII in the sample is within the appropriate detection range, reduces nonspecific interference, and improves the sensitivity and accuracy of the detection.

[0030] (2) The present invention adds an appropriate amount of bentonite (20-50 mg bentonite per 1 mL of plasma sample) to the diluted plasma sample, and through vortex oscillation and centrifugation, can effectively remove fibrinogen and other macromolecular proteins. This not only reduces the interference of these components on the fluorescent signal, but also avoids their non-specific binding with other components in the FXIII reaction system, thereby improving the specificity and reliability of the detection.

[0031] (3) The volume ratio of the plasma sample to the reaction system is set to 1:(10-20) in the present invention. This ratio ensures sufficient FXIII concentration for detection without affecting the reaction due to excessive plasma components. The appropriate volume ratio helps maintain the stability of the reaction system and reduce the interference of other components in the plasma, thereby improving the accuracy and sensitivity of the detection.

[0032] (4) The fluorescent substrate of the present invention contains a fluorescent group (such as 2'-aminobenzanilide) and a fluorescence quenching group (such as 2,4-dinitrophenylhydrazine). This design allows the fluorescent group to separate from the quenching group under the catalysis of FXIII, resulting in a significant change in the fluorescence signal. This fluorescence enhancement mechanism not only improves the sensitivity of the detection, but also effectively reduces the interference of background fluorescence, thereby enhancing the specificity and accuracy of the detection.

[0033] The technical solution of the present invention is further described in detail below through embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The standard curve measured in Example 1;

[0035] Figure 2 1 is the fluorescence intensity-time curve at various concentration percentages in Example 1. DETAILED DESCRIPTION

[0036] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the present invention is not limited to this embodiment.

[0037] Example 1

[0038] A method for determining the activity of coagulation factor XIII based on fluorescence quantification comprises the following steps:

[0039] (1) Sample pretreatment

[0040] Bentonite was added to the plasma sample, the volume mass ratio of the plasma sample to the bentonite was 1 mL:30 mg, and the sample was gently mixed using a vortex oscillator to evenly distribute the bentonite in the plasma sample. The mixing time was 20 s, and the sample was allowed to stand at room temperature for 15 min after mixing to allow the bentonite to have enough time to adsorb fibrinogen and other macromolecular proteins, and then centrifuged at 2000 r / min for 12 min. Fibrinogen and other macromolecular proteins can be efficiently removed by vortex oscillation and centrifugation. After centrifugation, the supernatant was taken as the plasma sample;

[0041] (2) Preparation of test samples

[0042] The plasma sample was added into the reaction system, the volume ratio of the plasma sample to the reaction system was 1:20, and then incubated at 37°C for 1.5h to obtain the sample to be tested;

[0043] The reaction system includes a buffer solution, calcium chloride, polyethylene glycol, glycine ethyl ester, hexamethylenetetramine bromide and a fluorescent substrate, the buffer solution is N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes buffer solution), N,N-dihydroxyethylglycine and tris(hydroxymethylaminomethane), the concentration of N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid in the reaction system is 50 mM, the concentration of N,N-dihydroxyethylglycine is 20 mM, the concentration of tris(hydroxymethylaminomethane) is 10 mM, the concentration of calcium chloride is 10 mM, the mass fraction of polyethylene glycol is 0.1%, the concentration of glycine ethyl ester is 5 mM, the concentration of hexamethylenetetramine bromide is 5 μg / mL, and the concentration of the fluorescent substrate is 50 μM;

[0044] The fluorescent substrate includes a fluorescent group and a fluorescent quenching group, wherein the fluorescent group is 2'-aminobenzanilide, and the fluorescent quenching group is 2,4-dinitrophenylhydrazine; the sequence of the fluorescent substrate is (2-Abz)-Asn-Glu(CAD-DNP)-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys.

[0045] The chemical formula of the fluorescent substrate is:

[0046]

[0047] (3) Establishment of standard curve

[0048] A series of standard FXIII solutions of different concentrations were prepared. Specifically, 1 IU / mL of FXIII solution was recorded as 100%, and serial dilution was performed to obtain 50%, 25%, 12.5%, 6.25%, and 3.125% FXIII solutions. Standard samples of different concentrations were prepared according to step (2). The prepared standard samples were detected on a fluorescence meter. The excitation wavelength of the fluorescence meter was 313 nm, and the emission wavelength of the measurement was 418 nm. The fluorescence intensity-time curves of each FXIII solution concentration within 5 to 30 min of incubation were then measured. The slopes of the fluorescence intensity-time curves at each concentration percentage are shown in the attached figure. Figure 2 Finally, a standard curve was drawn with the slope as the ordinate and the concentration percentage of the standard FXIII solution as the abscissa; the standard curve is shown in the attached Figure 1 .

[0049] (4) FXIII activity detection

[0050] The test sample is detected by a fluorescence meter to obtain the fluorescence intensity of the test sample within 5 to 30 minutes, and a linear regression analysis is performed using the fluorescence intensity (y-axis)-time (x-axis) to obtain the slope. Finally, the concentration of the test sample is calculated based on the standard curve.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for determining the activity of coagulation factor XIII based on fluorescence quantification, characterized in that: The following steps are involved: (1) Sample pretreatment The plasma sample is treated with bentonite to obtain a plasma test sample; (2) Preparation of test samples Adding the plasma sample into the reaction system and then incubating to obtain a sample to be tested; The reaction system includes a buffer solution, calcium chloride, polyethylene glycol, glycine ethyl ester, hexamethylenetetramine bromide and a fluorescent substrate; (3) Establishment of standard curve Prepare a series of standard FXIII solutions of different concentrations, prepare standard samples of different concentrations according to step (2), detect the prepared standard samples on a fluorescence meter, and draw a standard curve with the concentration percentage of the standard FXIII solution as the horizontal axis and the slope of the fluorescence intensity-time curve during the incubation period as the vertical axis; (4) FXIII activity detection The sample to be tested is detected by a fluorescence meter to obtain the fluorescence intensity of the sample to be tested, and the concentration of the sample to be tested is calculated according to the standard curve.

2. A method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1, characterized in that: In step (1), bentonite is added to the plasma sample, the volume mass ratio of the plasma sample to bentonite is 1 mL: (20-50) mg, and a vortex oscillator is used to gently mix so that the bentonite is evenly distributed in the plasma sample. The mixing time is 10-30 seconds. After mixing, the mixture is allowed to stand at room temperature for 10-15 minutes, and then centrifuged at 1000-3000 r / min for 10-15 minutes. After centrifugation, the supernatant is taken as the plasma sample.

3. The method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1, characterized in that: In step (2), the concentration of the buffer solution in the reaction system is 50-100 mM, the concentration of calcium chloride is 8-12 mM, the mass fraction of polyethylene glycol is 0.05-0.15%, the concentration of glycine ethyl ester is 3-8 mM, the concentration of hexamethylenetetramine is 3-8 μg / mL, and the concentration of the fluorescent substrate is 30-80 μM.

4. A method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1 or 2, characterized in that: The buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, Hepes buffer solution, N,N-dihydroxyethylglycine, and tris(hydroxymethyl)aminomethane.

5. The method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1, characterized in that: In step (2), the volume ratio of the plasma sample to the reaction system is 1:(10-20).

6. The method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1, characterized in that: In step (2), the incubation temperature is 35-38° C., and the incubation time is 0.01-2 h.

7. A method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1, characterized in that: In step (2), the fluorescent substrate comprises a fluorescent group and a fluorescent quenching group, wherein the fluorescent group is 2'-aminobenzanilide and the fluorescent quenching group is 2,4-dinitrophenylhydrazine.

8. A method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 7, characterized in that: In step (2), the sequence of the fluorescent substrate is: (2-Abz)-Asn-Glu(CAD-DNP)-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys.

9. The method for determining the activity of coagulation factor XIII based on fluorescence quantitative analysis according to claim 1, characterized in that: In step (3), the excitation wavelength of the fluorescence meter is 313 nm.

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