Kit as well as preparation method and application thereof

Abstract

The invention discloses a kit and a preparation method and application thereof.The kit is used for detecting the content of plasmin-alpha 2 plasmin inhibitor composites.The kit comprises a first reagent and a second reagent, the first reagent comprises a magnetic particle coated by a plasmin-alpha 2 plasmin inhibitor compound antibody and a magnetic particle buffer solution, and the second reagent comprises a plasmin-alpha 2 plasmin inhibitor compound antibody marked by a marker and an acridine diluent. The kit does not need to be diluted when a sample is tested, and compared with a commercially available Hisenmekang PIC kit, the kit has remarkable correlation; the preparation method can reduce the antibody dosage and simplify the test procedure, and has the advantages of simple operation, low cost and short result obtaining time.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection reagents, and in particular to a reagent kit and a preparation method and application thereof. Background Art

[0002] Plasmin antiplasmin complex (PIC) is a complex formed by the 1:1 binding of plasmin (Plm) and α2-plasmin inhibition (α2-PI). It is a biomarker that directly reflects the degree of activation of the fibrinolytic system. Once a thrombus is formed, the fibrinolytic system is immediately activated. Its concentration reflects the degree of plasmin activation, indicating hyperfibrinolytic activity and increased bleeding risk. It is the most useful marker that directly reflects the generation of plasmin.

[0003] Plasminogen (Plg) is the precursor zymogen of plasmin (Plm), which is a serine protease essential for the dissolution of fibrin (Fibrin, FIB). α2-PI is a single-chain glycoprotein, the main component of the fibrinolytic system and the main physiological inhibitor of Plm.

[0004] Under physiological conditions, in vivo Plg activators such as tissue-Plasminogen Activator (tPA) and urokinase-Plasminogen Activator (uPA) activate Plg into plasmin, which rapidly acts on FIB to degrade it, which is called the fibrinolytic process, while α2-PI in the body forms PIC with free plasmin in a 1:1 ratio to inactivate it. In the process of activating plasminogen to plasmin and plasmin action, there is a balance between the formation of plasmin-antiplasmin complex and the binding and action of plasmin on the surface of fibrin. After binding to fibrin, plasmin cannot bind to α2-PI for a short time. However, once the fibrin is completely dissolved, the plasmin released from the surface of fibrin immediately binds to a2-PI.

[0005] The interaction between plasmin and α2-PI is divided into two stages. In the first stage, the lysine residues in the terminal domain of the α2-PI molecule interact with the complementary lysine binding site of the plasmin molecule (Plm) kringle1 to form a non-covalent enzyme inhibitor complex Pm*α2-PI.

[0006] The first stage is a reversible second-order reaction that is completed in a few seconds with a reaction rate of 3.8×107m -1 ·s -1The second stage: The Arg364–Met365 (P1-P1') bond at the active center of the α2-PI molecule breaks, and then a covalent bond is formed between the broken Arg364 residue and the Ser741 residue at the active site of plasmin. This stage is a slower, irreversible first-order reaction with a reaction rate of about 4.0×10 -3 m -1 ·s -1 The above two stages form an extremely stable covalent complex Pm-α2-PI (PIC). The molecular weight of PIC is about 150KD. The half-life of plasmin is only a few seconds, the PIC index is easier to detect, and the plasma concentration is not affected by antiplatelet and anticoagulant drugs.

[0007] Currently, most of the test kits on the market rely on imports. Sysmex, a well-known in vitro diagnostic (IVD) manufacturer in Japan, was the first to establish a chemiluminescence method for quantitative determination of PIC. Its coating antibody is an anti-plasminogen monoclonal antibody, and the labeled antibody is an anti-α2 plasmin inhibitor antibody. The linear range of the Sysmex PIC test kit is 0.05-40ug / mL. When the concentration of the analyte reaches a certain value without dilution, the double antibody sandwich complex cannot be formed, that is, the high-dose hook effect, resulting in inaccurate measurements. The Sysmex PIC test kit needs to dilute the sample 40 times before testing. The test steps are cumbersome and increase the time to dilute the sample.

[0008] Currently, in the case of undiluted sample testing, a large amount of coated naked antibody / antibody-coated magnetic particles can be added in the first step of the reaction to capture excess PIC antigens, but the resulting kit cost will be relatively high. Summary of the invention

[0009] In order to solve one or more problems existing in the prior art, the present invention provides a kit. The technical solution adopted by the present invention to solve the above problems is: the kit is used to detect the content of plasmin-α2 plasmin inhibitor complex, and the kit includes: a first reagent and a second reagent, the first reagent includes: magnetic particles coated with plasmin-α2 plasmin inhibitor complex antibody and magnetic particle buffer, and the second reagent includes: plasmin-α2 plasmin inhibitor complex antibody labeled with a marker and acridine diluent.

[0010] In some embodiments, the magnetic particle buffer and the acridine diluent contain a buffer system, a stabilizer and a preservative.

[0011] In some embodiments, the magnetic particle buffer contains a plasmin inhibitor.

[0012] Furthermore, the plasmin inhibitor is lysine or a lysine analog, wherein the lysine analog includes: 6-aminocaproic acid, tranexamic acid, and aminobenzoic acid.

[0013] In some embodiments, the concentration of the plasmin inhibitor is 1-50 mM, wherein preferably the concentration of the plasmin inhibitor is 10 mM.

[0014] And the preparation method of the above kit, the plasmin-α2 plasmin inhibitor complex antibody is referred to as antibody for short; the first reagent is prepared, the antibody is coupled with tosyl magnetic beads, after the coupling is completed, the excess uncoupled antibody is removed, and then the uncoupled tosyl magnetic beads are blocked using a blocking night, after the blocking is completed, the magnetic beads are washed with a magnetic bead storage solution and redissolved into a mother liquor, and the mother liquor is diluted with a magnetic particle buffer to obtain the first reagent; The second reagent is prepared by labeling, incubating and purifying the antibody to obtain a labeled antibody solution, and diluting the antibody solution with an acridine diluent to obtain the second reagent.

[0015] Furthermore, it also includes: preparing a magnetic particle buffer, adding a plasmin inhibitor to the magnetic bead storage solution, and then using hydrochloric acid or sodium hydroxide to adjust the pH of the solution to 7.3-7.5.

[0016] And the application of the above-mentioned kit, adding the first reagent to the sample and performing the first incubation, performing magnetic separation and cleaning after the first incubation, then adding the second reagent and performing the second incubation, performing magnetic separation and cleaning after the second incubation, then adding pre-excitation solution and excitation solution and reading the luminescent signal value, comparing the standard curve according to the luminescent intensity and calculating the concentration of the plasmin-α2 plasmin inhibitor complex.

[0017] The technical effect achieved by the present invention is that the above-mentioned kit does not need to be diluted when testing the sample, and the correlation comparison between the above-mentioned kit and the commercially available Sysmex PIC kit shows significant correlation. The preparation method of the above-mentioned kit can reduce the amount of antibody used and simplify the testing procedure, and has the advantages of simple operation, low cost and short time to produce results. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a linear diagram of an embodiment of the present invention; Figure 2 is a linear graph of a comparative example of the present invention; Figure 3 This is a correlation diagram of the present invention. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned objects, features and advantages of the present invention more understandable, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from this description, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0020] The present invention discloses a kit for detecting the content of plasmin-α2 plasmin inhibitor complex (PIC), and the kit comprises: a first reagent and a second reagent, wherein the first reagent comprises: magnetic particles coated with plasmin-α2 plasmin inhibitor complex antibody and a magnetic particle buffer, and the second reagent comprises: plasmin-α2 plasmin inhibitor complex antibody labeled with a marker and an acridine diluent; The magnetic particle buffer and the acridine diluent contain a buffer system, a stabilizer and a preservative, the magnetic particle buffer contains at least one plasmin inhibitor, the plasmin inhibitor is lysine or a lysine analog, the lysine analog includes: 6-aminocaproic acid, tranexamic acid, aminotoluene. Furthermore, the concentration of the plasmin inhibitor is 1-50mM, preferably the concentration of the plasmin inhibitor is 10mM.

[0021] Specifically, the preparation method of the kit is as follows: the antibody of plasmin-α2 plasmin inhibitor complex is referred to as antibody for short; the first reagent is prepared, the antibody is coupled with tosyl magnetic beads, after the coupling is completed, the excess uncoupled antibody is removed, and then the uncoupled tosyl magnetic beads are blocked with blocking night, after the blocking is completed, the magnetic beads are washed with magnetic bead storage solution and redissolved into a mother liquor, and the mother liquor is diluted with a magnetic particle buffer to obtain the first reagent; Preparation of the second reagent, labeling, incubating and purifying the antibody to obtain a labeled antibody solution, and diluting the antibody solution with an acridine diluent to obtain the second reagent; To prepare the magnetic particle buffer, add plasmin inhibitor to the magnetic bead storage solution, and then use hydrochloric acid or sodium hydroxide to adjust the pH of the solution to 7.3-7.5.

[0022] The embodiment of the preparation method is: 1. Preparation of coated antibody: the coated antibody is the self-produced antibody 1 (item number: YM0227), antibody 1 is coupled with tosyl magnetic beads (manufacturer is JSR or Thermo), the coupling reaction buffer is borate buffer (CB), pH 9.6, and the coupling catalyst is a 3M ammonium sulfate solution; According to the antibody-magnetic bead ratio (15ug antibody / mg magnetic beads-35ug antibody / mg magnetic beads), magnetic bead reaction volume (10-20mg / ml), reaction buffer (2 / 3 coupling reaction buffer, 1 / 3 coupling catalyst), react at 37℃ for 16-24h. After coupling, wash the magnetic beads 3 times with PBST at a concentration of 10mg / mL to remove excess uncoupled antibodies. Add 1% casein, 2% BSA in PBS, pH7.4 blocking solution to block the uncoupled magnetic beads for 22-26h. After blocking, wash the magnetic beads 3 times with magnetic bead storage solution (50mM TBS, 0.5% BSA, 0.1% Tween 20, 0.05% Proclin300, pH7.3-7.5) at a concentration of 10mg / mL, and re-dissolve to 10mg / mL mother solution for use.

[0023] 1.1. Preparation of the first reagent: Preparation of the control magnetic bead diluent: magnetic bead storage solution (50mM TBS, 0.5% BSA, 0.1% Tween 20, 0.05% Proclin 300, pH 7.3-7.5; Example Preparation of magnetic bead diluent: Add one or more plasmin inhibitors to the magnetic bead storage solution. The plasmin inhibitor is lysine or a lysine analog, wherein the lysine analog includes: 6-aminocaproic acid, tranexamic acid, aminobenzoic acid, etc., preferably 6-aminocaproic acid. The concentration of the plasmin inhibitor is 1-50mM, preferably 10mM. Then adjust the pH to 7.3-7.5 with hydrochloric acid or sodium hydroxide; The magnetic bead mother solution was diluted with respective magnetic bead diluents to a magnetic bead concentration of 0.25 mg / ml, thereby obtaining the first reagent.

[0024] 2. Preparation of the second reagent: The labeled antibody is the self-produced antibody 2 (Cat. No. YM0228), which is labeled according to the labeling ratio (40-100ugNHS-SA-acridine / 1mg antibody), and the labeling buffer is a phosphate buffer with a pH of 8.0. Incubate at 25-37°C for 2h, purify with molecular sieves to obtain the labeled antibody solution, and dilute the antibody concentration to 0.1-0.4ug / ml with an acridine diluent, which is 50mM MES, 0.15M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein, to obtain the second reagent.

[0025] Specifically, the application of the kit is: adding the first reagent to the sample and performing the first incubation, performing magnetic separation and washing after the first incubation, then adding the second reagent and performing the second incubation, performing magnetic separation and washing after the second incubation, then adding the pre-excitation solution and the excitation solution and reading the luminescent signal value, and calculating the concentration of the plasmin-α2 plasmin inhibitor complex (PIC) according to the luminescent intensity against the standard curve.

[0026] An application example of the kit is: the instrument obtains 10ul of sample, adds 50ul of the first reagent to the sample, and then incubates for 2 minutes. After the incubation, it is magnetically separated and washed 3 times. After the washing, 50ul of the second reagent is added, and then incubated for another 2 minutes. After the incubation, it is magnetically separated and washed 3 times. Then, 100ul of pre-excitation solution and 100ul of excitation solution are added, and the luminescent signal value is read. According to the luminescent intensity, the concentration of plasmin-α2 plasmin inhibitor complex (PIC) is calculated by comparing the standard curve.

[0027] In combination with the above preparation method, the difference between the control kit and the example kit is that the R1 component of the control kit does not contain a plasmin inhibitor, while the R1 component of the example kit contains 10 mM 6-aminocaproic acid. It should be noted that the above-mentioned stimulating solution, pre-stimulating solution and cleaning solution were purchased from Feipeng Biological Company.

[0028] Repeatability test: The plasma samples with measured values ​​of 0.82ug / mL and 21.35ug / mL of Sysmex were tested 10 times respectively using the kit of the embodiment to test the repeatability performance of the kit. The repeatability test results are shown in Table 1 below.

[0029] Table 1 For plasma samples with detection values ​​of 0.82ug / mL and 21.35ug / mL of the Sysmex test kit, the kit of Example was used for 10 repeated tests, with the average values ​​of 0.83ug / mL and 21.42ug / mL, and the CVs of 3.61% and 3.69%, respectively. The repeatability of the kit of Example meets clinical requirements.

[0030] Linear range: A high-value plasma sample with a measured value of 38.25 ug / mL of Sysmex was diluted into 6 samples with plasma without PIC analyte, and the linearity of the kit was tested using the kit of the embodiment and the kit of the control example. The average value of the test luminescence value and the theoretical concentration were linearly fitted using the least squares method to obtain the regression equation and correlation coefficient R. The linear range test results of the embodiment are shown in Table 2 below, and the linear range test results of the control example are shown in Table 3 below.

[0031] Table 2 Table 3 Table 2 and Table 3 are plotted Figure 1 and Figure 2 Linear graph shown. Figure 1 , Figure 2The results show that the correlation coefficient R of the control kit is 0.8331, and a high-dose hook effect appears at the high end; the correlation coefficient R of the example kit is 0.9998, and the example kit is reliable within the detection range of 0.05-40ug / mL. Adding a plasmin inhibitor to the kit can improve the linearity of the kit.

[0032] Clinical relevance: The kit of Example was used to test 40 clinical samples that were quantified by the Sysmex PIC kit. The clinical relevance test results are shown in Table 4 below.

[0033] Table 4 Table 4 is obtained by mapping Figure 3 Combining Table 4 and Figure 3 The results showed that the correlation R-squared between the Example Kit and the Sysmex test value was 0.987, and k was 0.98. The two determination methods have a high correlation. The reason for the high correlation between the two may be that the plasmin inhibitor in the Example Kit and the PIC in the sample are bound in a concentration gradient, that is, the percentage of PIC in the sample that is opened by the plasmin inhibitor is fixed. The coated antibody can only recognize PIC that has not opened the non-covalent bond, so the Example Kit and the Sysmex PIC Kit have a high correlation.

[0034] In summary, the technical effect achieved by the present application is that the above-mentioned kit does not need to be diluted when testing the sample, and it has a significant correlation with the commercially available Sysmex PIC kit. The preparation method of the above-mentioned kit can reduce the amount of antibody used and simplify the testing procedure, and has the advantages of simple operation, low cost and short time to produce results.

[0035] The above-described embodiments only express one or more implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A kit, characterized in that: The kit is used to detect the content of plasmin-α2 plasmin inhibitor complex, and comprises: a first reagent and a second reagent, wherein the first reagent comprises: magnetic particles coated with plasmin-α2 plasmin inhibitor complex antibody and a magnetic particle buffer, and the second reagent comprises: plasmin-α2 plasmin inhibitor complex antibody labeled with a marker and an acridine diluent.

2. The kit according to claim 1, characterized in that The magnetic particle buffer and the acridine diluent contain a buffer system, a stabilizer and a preservative.

3. The kit according to claim 1, characterized in that The magnetic particle buffer contains a plasmin inhibitor.

4. The kit according to claim 3, characterized in that The plasmin inhibitor is lysine or a lysine analog.

5. The kit according to claim 4, characterized in that The lysine analogs include: 6-aminocaproic acid, tranexamic acid, and aminobenzoic acid.

6. The kit according to claim 3, characterized in that The concentration of the plasmin inhibitor is 1-50 mM.

7. The kit according to claim 3, characterized in that The concentration of the plasmin inhibitor was 10 mM.

8. A preparation method for preparing the kit according to any one of claims 1 to 7, characterized in that: The antibody of plasmin-α2 plasmin inhibitor complex is referred to as antibody for short; the first reagent is prepared by coupling the antibody with tosyl magnetic beads, removing the excess uncoupled antibody after coupling, and then blocking the uncoupled tosyl magnetic beads with blocking night, washing with magnetic bead preservation solution after blocking and re-dissolving into mother liquor, and diluting the mother liquor with magnetic particle buffer to obtain the first reagent; The second reagent is prepared by labeling, incubating and purifying the antibody to obtain a labeled antibody solution, and diluting the antibody solution with an acridine diluent to obtain the second reagent.

9. The preparation method according to claim 8, characterized in that: Also includes: To prepare the magnetic particle buffer, add plasmin inhibitor to the magnetic bead storage solution, and then use hydrochloric acid or sodium hydroxide to adjust the pH of the solution to 7.3-7.

5.

10. A use of a kit, using the kit according to any one of claims 1 to 7, characterized in that: The first reagent is added to the sample and the first incubation is performed. After the first incubation, magnetic separation and cleaning are performed. Then, the second reagent is added and the second incubation is performed. After the second incubation, magnetic separation and cleaning are performed. Then, pre-excitation solution and excitation solution are added and the luminescent signal value is read. The luminescent intensity is compared with the standard curve and the concentration of the plasmin-α2 plasmin inhibitor complex is calculated.

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