CN115201486A

CN115201486A - Kit for detecting activity of vitamin K-dependent protein S

Info

Publication number: CN115201486A
Application number: CN202210754251.3A
Authority: CN
Inventors: 杨军京
Original assignee: Beijing Zongci Technology Development Co ltd
Current assignee: Affiliated Hospital Of Qingdao University Pingdu; Beijing Zongci Technology Development Co ltd
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2022-10-18

Abstract

The invention discloses a kit for detecting activity of vitamin K-dependent protein S, and relates to the technical field of medical and biological detection. The reagent R1 is a plasma freeze-dried product lacking protein S activity; the reagent R2 is a freeze-dried product containing an activator, phospholipid, calcium chloride, activated protein C and a stabilizer required by blood coagulation reaction. The invention only comprises two reagents of a reagent R1 and a reagent R2, the reagent kit has the advantages of easily obtained raw materials, simple preparation steps, simple and convenient operation and low preparation cost, can detect the activity of the protein S in a sample on a full-automatic blood coagulation analyzer, can quickly and accurately obtain a result, and has excellent reagent performance and wide application prospect.

Description

Kit for detecting activity of vitamin K-dependent protein S

Technical Field

The invention relates to the technical field of medical and biological detection, in particular to a kit for detecting activity of vitamin K-dependent protein S.

Background

Protein S (PS) is a vitamin K-dependent single-chain glycoprotein, is a cofactor of Activated Protein C (APC), and can enhance the affinity of APC and phospholipid and improve the activity of activated protein C. The activated protein C can eliminate the protective effect of the blood coagulation factor X alpha on the blood coagulation factors V alpha and IX alpha on the blood coagulation factor VIII alpha, so that the blood coagulation factors V alpha and IX alpha are hydrolyzed. Protein S, protein C inhibitor and thrombomodulin jointly form an important anticoagulation system-protein C anticoagulation system in the organism, and play an important balance role in the blood coagulation and fibrinolysis processes.

Protein S is present in plasma in two forms, free PS (FPS) and complement-bound PS (C4 bp-PS), and it is believed that only FPS has anticoagulant activity. Currently, the detection of PS can be divided into two categories: one is the measurement of the concentration of the antigen and the other is the measurement of its activity. Because the antigen detection can only detect the content of protein S in blood plasma, the function of the blood plasma can not be truly and accurately evaluated, and the function of the blood plasma can be better evaluated by adopting the detection of the activity of the blood plasma. The protein S activity is detected by mainly adopting a coagulation method to measure the FPS activity. Many common diseases, such as liver disease, kidney disease, cardiovascular and cerebrovascular diseases, affect the content of protein S in blood, so the determination of protein S activity in blood plasma is of great value for prevention and diagnosis, disease observation and prognosis judgment of the diseases.

At present, protein S activity detection kits on the market are imported from foreign countries and are expensive, so that the development of the protein S activity detection kits in domestic hospitals at all levels is limited. In order to solve the above problems, it is particularly necessary to develop a kit for detecting the activity of vitamin K-dependent protein S.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the kit for detecting the activity of the vitamin K-dependent protein S, which has the advantages of simple preparation steps, simple and convenient operation, low cost, capability of detecting the activity of the protein S in a sample on a full-automatic blood coagulation analyzer, quick and accurate result output and easy popularization and use.

In order to achieve the purpose, the invention is realized by the following technical scheme: a kit for detecting activity of vitamin K-dependent protein S comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a lyophilized plasma product lacking activity of the protein S; the reagent R2 is a freeze-dried product containing an activator, phospholipid, calcium chloride, activated protein C and a stabilizer required by blood coagulation reaction.

Preferably, the reagent R1 adopts a magnetic bead affinity purification method to prepare plasma lacking protein S activity, and the preparation method comprises the following steps: firstly, an anti-protein S antibody is combined on a magnetic bead through a chemical coupling method, then the magnetic bead coupled with the anti-protein S antibody is fully mixed with sodium citrate anticoagulated plasma, protein S in the plasma is adsorbed on the magnetic bead, the magnetic bead in the plasma is removed through a magnetic frame, and the remaining plasma is the plasma with the protein S removed.

Preferably, the magnetic bead has a particle size of 1.0 to 5.0 μm, and one group selected from a carboxyl group, an amino group and a tosyl group is modified on the surface of the magnetic bead.

Preferably, the anti-protein S antibody comprises a monoclonal antibody, a polyclonal antibody or an antibody Fab fragment, and the source of the antibody comprises mouse, rat, rabbit, sheep or chicken.

Preferably, the plasma from which the protein S is removed is supplemented with an amount of fibrinogen, including human or animal sources, to supplement the fibrinogen content of the plasma.

Preferably, the plasma from which protein S is removed is supplemented with a certain amount of factor va to complement the level of factor va in the plasma, and the source of factor va includes human or animal sources.

Preferably, a certain amount of stabilizer is added into the plasma from which the protein S is removed, wherein the stabilizer is one or two of sucrose, trehalose or mannitol.

The magnetic beads adsorbed with the protein S can be washed away with a regeneration buffer solution, and the regenerated magnetic beads can be stored in a storage solution at 2-8 ℃ for a long time and can be reused.

Preferably, the reagent R2 contains an activating agent capable of activating a blood coagulation waterfall reaction, and the activating agent adopts one of viper venom RVV, recombinant tissue factor or kaolin.

Preferably, the phospholipid contained in the reagent R2 is animal cephalin, plant phospholipid or synthetic phospholipid; the synthetic phospholipid is a mixture of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine.

Preferably, the stabilizer contained in the reagent R2 is one or two of sucrose, trehalose and mannitol.

The invention has the beneficial effects that: the kit only comprises two reagents, namely a reagent R1 and a reagent R2, the kit has the advantages of easily available raw materials, simple preparation steps, simplicity and convenience in operation and low preparation cost, can be used for detecting the activity of the protein S in a sample on a full-automatic blood coagulation analyzer, can quickly and accurately obtain a result, has the same performance as that of an imported reagent, and has a wide application prospect.

Drawings

The invention is described in detail below with reference to the drawings and the detailed description;

FIG. 1 is a graph showing the results of comparative testing of plasma samples in example 3 of the present invention;

FIG. 2 is a linear range evaluation scattergram in example 3 of the present invention.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained by combining the specific embodiments.

Referring to fig. 1-2, the following technical solutions are adopted in the present embodiment: a kit for detecting activity of vitamin K-dependent protein S comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a lyophilized plasma product lacking activity of the protein S; the reagent R2 is a freeze-dried product containing an activator, phospholipid, calcium chloride, activated protein C and a stabilizer required by blood coagulation reaction.

It is noted that the reagent R1 is prepared by magnetic bead affinity purification method to plasma lacking protein S activity, and the preparation method is as follows: firstly, an anti-protein S antibody is combined on a magnetic bead through a chemical coupling method, then the magnetic bead coupled with the anti-protein S antibody is fully mixed with sodium citrate anticoagulated plasma, protein S in the plasma is adsorbed on the magnetic bead, the magnetic bead in the plasma is removed through a magnetic frame, and the remaining plasma is the plasma with the protein S removed.

The particle size of the magnetic bead is 1.0-5.0 μm, and one group of carboxyl, amino or tosyl is modified on the surface of the magnetic bead; the anti-protein S antibody comprises a monoclonal antibody, a polyclonal antibody or an antibody Fab fragment, and the source of the antibody comprises a mouse, a rat, a rabbit, a sheep or a chicken. Adding a certain amount of fibrinogen, coagulation factor Va and stabilizer into the plasma without protein S, wherein the fibrinogen is used for complementing the fibrinogen content in the plasma, and the fibrinogen source comprises human source or animal source; the coagulation factor Va is used for complementing the content of the coagulation factor Va in plasma, and the source of the coagulation factor Va comprises a human source or an animal source; the stabilizer is one or two of sucrose, trehalose or mannitol.

The magnetic beads adsorbed with the protein S can be washed away with a regeneration buffer solution, and the regenerated magnetic beads can be stored in a storage solution at 2-8 ℃ for a long time and can be reused. And packaging the reagent R1 into glass bottles, freeze-drying, and storing in a refrigerator at 2-8 ℃.

In addition, the reagent R2 contains an activating agent capable of activating blood coagulation waterfall reaction, and the activating agent adopts one of viper venom RVV, recombinant tissue factor or kaolin. The phospholipid contained in the reagent R2 is animal cephalin, plant phospholipid or synthetic phospholipid; the synthetic phospholipid is a mixture of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine. The stabilizer contained in the reagent R2 is one or two of sucrose, trehalose and mannitol. And packaging the reagent R2 into glass bottles, freeze-drying, and storing in a refrigerator at 2-8 ℃.

The specific embodiment only comprises two reagents, namely a reagent R1 and a reagent R2, wherein the reagent R1 adopts a magnetic bead affinity purification method to prepare the protein S activity spent factor plasma, the operation is simple and rapid, the purification efficiency is high, and the affinity purification magnetic beads can be repeatedly used, so that the preparation cost is greatly saved; the reagent R2 has the advantages of easily obtained raw materials, simple preparation steps and low production cost. The kit can be used for detecting the activity of the protein S in a sample on a full-automatic coagulation analyzer, is simple to operate, has a quick result and has a wide market application prospect.

Example 1: a kit for detecting activity of vitamin K-dependent protein S, preparation of a reagent R1 protein S activity-poor factor plasma, comprises the following steps:

(1) Preparing protein S affinity purification magnetic beads: and (3) cleaning magnetic beads: putting 500 μ l of magnetic beads with particle size of 1.5-3.0 μm and concentration of 10% in a 10ml centrifuge tube, adding 3ml of washing solution (20mM MES pH 6.0), mixing, putting on a magnetic frame, adsorbing the magnetic beads on the centrifuge tube wall by the magnetic frame, sucking away the liquid, adding 3ml of washing solution (20mM MES pH 6.0), taking off the centrifuge tube from the magnetic frame, mixing the magnetic beads, putting on the magnetic frame again, adsorbing the magnetic beads on the centrifuge tube wall by the magnetic frame, and sucking away the liquid. The magnetic beads were mixed by removing 4ml of a washing reagent (20mM MES pH 6.0) from the centrifuge tube and then removing the centrifuge tube from the magnetic rack.

Anti-protein S antibody labeling of magnetic beads: to the above centrifuge tube, 350. Mu.l of EDC (prepared with 20mM MES pH6.0 to a final concentration of 1 mg/ml) was added, shaking reaction was performed at room temperature for 10 minutes, 1mg of anti-protein S antibody was added (diluted with 20mM MES pH6.0 to a final concentration of 0.5 mg/ml), shaking reaction was performed at room temperature for 2 hours, and 3ml of 10% BSA aqueous solution was added, shaking reaction was performed at room temperature and overnight; the centrifugal tube is placed on the magnetic frame, and the magnetic beads are adsorbed on the wall of the centrifugal tube by the magnetic frame to suck away liquid. After washing the beads 3 times with PBST, the beads were resuspended in 5ml PBS.

(2) Preparation of protein S-poor factor plasma: adding 1ml of the above affinity magnetic beads into a 5ml centrifuge tube, placing the centrifuge tube on a magnetic frame, adsorbing the affinity magnetic beads on the wall of the centrifuge tube by the magnetic frame, and sucking away liquid. Taking the centrifugal tube off the magnetic frame, adding 3ml of sodium citrate anticoagulated plasma, and rotating and mixing uniformly at 2-8 ℃ for 15-20 minutes to fully mix the magnetic beads and the plasma. The centrifuge tube was placed on a magnetic rack, the magnetic beads were adsorbed onto the centrifuge tube wall by the magnetic rack, and the plasma was aspirated off and placed in a new 5ml centrifuge tube. Adding fibrinogen to remove protein S to obtain final concentration of 1mg/ml, adding blood coagulation factor Va to obtain final concentration of 10 μ g/ml, adding mannitol to obtain final concentration of 2mg/ml, packaging into glass bottles with volume of 1ml, lyophilizing, and storing at 2-8 deg.C.

(3) Regeneration of protein S affinity purification magnetic beads: the magnetic beads having the protein S adsorbed thereon were placed in 3ml of a regeneration buffer (100 mM glycine pH 2.7), mixed by shaking for 1 minute, and adsorbed onto the wall of the centrifuge tube by a magnetic holder, and the liquid was removed. This step was repeated 2 times. Finally, the magnetic beads are stored in 1ml of storage solution (PBS contains 0.02 percent of sodium azide, pH7.4), and can be stored for a long time at the temperature of 2-8 ℃ for repeated use.

Example 2: a kit for detecting activity of vitamin K dependent protein S, preparation of a reagent R2, comprises the following steps: weighing 0.596g of HEPES, 2g of sucrose, 2g of trehalose and 0.138g of calcium chloride, adding 40ml of deionized water, stirring to dissolve, adding 10ul Proclin300, adding 0.5g of BSA, stirring to dissolve, adding 0.3mg of activated protein C, stirring to dissolve, adding RVV 150U, stirring to dissolve, adding 200mg of rabbit brain phospholipid, stirring to dissolve, adjusting the pH to 7.5 by using NaOH, fixing the volume of the deionized water to 50ml, subpackaging into glass bottles, wherein each bottle is 1ml, freeze-drying and storing in a refrigerator at the temperature of 2-8 ℃.

Example 3: the performance analysis of the kit for detecting the activity of the vitamin K-dependent protein S comprises the following detection principles: in the RVV-activated blood coagulation cascade, factor xa, under the action of factor va, causes prothrombin to form thrombin, which ultimately converts fibrinogen to fibrin. The activated protein C can decompose blood coagulation factors Va and VIII a under the action of phospholipid and calcium ions, and inhibit the coagulation of blood, thereby showing stronger anticoagulation. In this reaction, protein S acts as a cofactor that accelerates the reaction, resulting in prolonged clotting time of the sample, and is proportional to protein S activity. Reagent R1 is plasma lacking protein S activity, and can make reaction contain enough fibrinogen, coagulation factor V and other necessary coagulation factors besides protein S.

The detection method comprises the following steps: before the assay, the reaction mixture was diluted with 100mM imidazole buffer pH7.4 at a ratio of 1: plasma samples were diluted 5-fold. Mu.l of plasma sample was added to the reaction cuvette, 50. Mu.l of R1 reagent was added, incubated at 37 ℃ for 2 minutes, 50. Mu.l of R2 reagent was added, a timer on the coagulometer was immediately started, and the clotting time was started. The analytical results were as follows:

(1) correlation and identity analysis with existing commercial reagents:

30 plasma samples, 17 normal samples and 13 protein S activity abnormal samples, were tested by using the kit of this example, and the correlation between the two sets of data was evaluated by linear regression using the existing commercially available Siemens protein S activity assay kit. The detection result is shown in fig. 1, the correlation coefficient of the two kits is r =0.987, and the linear regression equation y =0.9961x-1.8968, which indicates that the kit of the present embodiment has better correlation between the clinical sample and the imported reagent.

(2) And (3) precision evaluation:

the kit of this example was used to detect normal and abnormal plasma samples, respectively, and the detection was repeated 20 times for each sample, and the mean, standard Deviation (SD) and Coefficient of Variation (CV) in batch were calculated, respectively. The results are shown in Table 1:

TABLE 1 detection of Normal and abnormal values for plasma samples

The results in this table show that the CV value of the normal value sample was 3.08%, and the CV value of the abnormal value sample was 7.79%, indicating that the precision of the kit of this example is good.

(3) Evaluation of Linear Range:

the high and low samples for linear range evaluation in this example were clinical plasma samples, one higher sample (H, measured 132.6%) and one lower sample (L, measured 12.7%) were selected, diluted to 7 points at the ratio of H, 5H + L, 4H +2L, 3H +3L, 2H +4L, H +5L, and the measurements were repeated three times per dilution, and the average value was calculated. And eliminating outliers after data inspection, judging repeatability, and finally comparing the average value of the measurement result with a theoretical value to perform linear regression analysis. Theoretical value = (CL × VL + CH × VH)/(VL + VH). The linear range evaluation results are shown in fig. 2, and the detection data are shown in table 2:

TABLE 2 Linear Range evaluation test data

It can be known that there is no significant outlier in the above detection results, and the obtained regression equation is: y = 0.9781x-0.4902 ² =0.9978, indicating a linear range of about 13% -130%.

(4) Evaluation of stability:

the protein S activity was measured in each of the normal and abnormal control plasma using the kit of this example, and the measured values obtained were initial measured values. The measurement kit was placed in an environment of 37 ℃ for thermal destruction, protein S activity was measured in each of the two quality-controlled plasma samples on the 1 st day, 3 rd day, 7 th day, 10 th day and 14 th day of thermal destruction, and the relative deviation between the measured value after thermal destruction and the initial measured value was calculated and was regarded as stable within. + -. 10%, and the results are shown in Table 3.

TABLE 3 calculation of relative deviation of Normal versus abnormal values for quality control of plasma for protein S activity

As can be seen from the above table, after 14 days of thermal destruction, the relative deviation of the quality control plasma of the normal value and the abnormal value is within + -10%, which indicates that the stability of the kit of the embodiment is qualified.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A kit for detecting activity of vitamin K-dependent protein S is characterized by comprising a reagent R1 and a reagent R2, wherein the reagent R1 is a lyophilized plasma product lacking the activity of the protein S; the reagent R2 is a freeze-dried product containing an activator, phospholipid, calcium chloride, activated protein C and a stabilizer; the reagent R1 adopts a magnetic bead affinity purification method to prepare plasma lacking protein S activity, fibrinogen, a coagulation factor Va and a stabilizer are added into the reagent R1, the fibrinogen source comprises human or animals, the coagulation factor Va source comprises human or animals, and the stabilizer in the reagent R1 is one of sucrose, trehalose or mannitol.

2. The kit for detecting activity of vitamin K dependent protein S according to claim 1, wherein the reagent R2 contains an activator capable of activating blood coagulation waterfall reaction, and the activator adopts one of viper venom RVV, recombinant tissue factor or kaolin.

3. The kit for detecting the activity of vitamin K dependent protein S according to claim 1, wherein the phospholipid contained in the reagent R2 is animal cephalin, plant phospholipid or synthetic phospholipid.

4. The kit for detecting activity of vitamin K-dependent protein S according to claim 3, wherein the synthetic phospholipid is a mixture of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine.

5. The kit for detecting activity of vitamin K dependent protein S according to claim 1, wherein the stabilizing agent contained in the reagent R2 is one or two of sucrose, trehalose and mannitol.