CN117723533A

CN117723533A - Activating reagent, chromogenic reagent, plasminogen activity detection reagent and/or kit and application thereof

Info

Publication number: CN117723533A
Application number: CN202311754728.9A
Authority: CN
Inventors: 张潇镱; 徐丽秀; 刘鹏; 穆文超
Original assignee: Chuang Ning Biotechnology Beijing Co ltd
Current assignee: Chuang Ning Biotechnology Beijing Co ltd
Priority date: 2023-12-19
Filing date: 2023-12-19
Publication date: 2024-03-19

Abstract

The invention relates to the field of biological detection, in particular to an activating reagent, a chromogenic reagent, a plasminogen activity detection reagent and/or a kit and application thereof. The present invention provides an activating agent comprising: 150-400U/mL of streptokinase, 0.2-0.8 w/v% of bovine serum albumin, 1.0-4.0 w/v% of glycine and 5.0-9.0 w/v% of mannitol. The invention provides a plasminogen activity assay kit and a preparation method thereof, which are a kit for quantitatively detecting the plasminogen activity based on interaction of plasmin and a specific chromogenic substrate.

Description

Activating reagent, chromogenic reagent, plasminogen activity detection reagent and/or kit and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an activating reagent, a chromogenic reagent, a plasminogen activity detection reagent and/or a kit and application thereof.

Background

The fibrinolytic system is an important component of the coagulation system and plays a vital role in maintaining the fluidity of blood and integrity of blood vessels. Plasminogen (hereinafter referred to as PLG) is one of the key precursors in the processes of fibrinolysis and thrombolysis, and is activated by a plasminogen activator to form plasmin, thereby exerting fibrinolysis and thrombolysis effects. Under physiological conditions, plasminogen is synthesized by the liver and exists in human body in two forms, namely glutamate-type plasminogen (Glu-PLG) with glutamate as a starting amino acid and lysine-type plasminogen (Lys-PLG) with lysine as a starting amino acid, is a single-chain serine protease consisting of 791 amino acids and has a relative molecular weight of 84000-92000 and comprises 7 structural domains: an N-terminal polypeptide region, 5 loop domains, and a serine protease domain. PLG activators are classified into two types, one is endogenous, including tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator, which are proteases that exist in blood and cleave Arg 561-Val 562 peptide bonds of PLG to form a heavy chain and a light chain linked by disulfide bonds, i.e., active plasmin in a double-chain form, and this activator is extremely easily adsorbed by fibrin clots, which is unfavorable for dissolution of thrombus. The other is exogenous activating factors, such as streptokinase, staphylokinase, etc., produced by bacteria, and their activating mechanism is different from t-PA. Exogenous activating factors are not proteases per se, do not cleave the cleavage of the plasmin peptide chain, but rather must be bound to PLG in a 1:1 molecular ratio to form a complex, both of which bind rapidly and stably, thereby exposing the PLG active site for conversion to active plasmin. Plasmin plays an important role in dissolving fibrin, thrombolysis, and the like.

Studies have shown that plasminogen deficiency includes genetic deficiency and acquired deficiency. Hereditary plasminogen deficiency is divided into type I and type II, with reduced levels and activities of plasminogen antigen in type I patients; type II patients have normal levels of plasminogen antigen, but some of them lose their active function and their activity is reduced. There are various causes of acquired plasminogen deficiency, such as reduced PLG synthesis caused by liver disease; PLG loss due to kidney disease; excessive PLG consumption caused by disseminated intravascular coagulation, sepsis or thrombolytic therapy, etc., and increased PLG concentration caused by tumor and diabetes. Both hereditary and acquired plasminogen deficiencies can lead to thrombosis, and in addition, increased plasminogen concentrations, once activated, can lead to the risk of bleeding, so that it is necessary to conduct plasma PLG assays in clinic or to observe changes dynamically, with a certain reference value for clinical diagnosis, estimated prognosis and efficacy observation.

At present, two methods for detecting the plasminogen are available, one is to quantitatively detect the content of PLG antigen, such as an immunoassay method, and the method is specific and sensitive, but has complex operation, time consumption and incapability of detecting type II PLG deficiency; another method is to quantitatively detect PLG activity, such as chromogenic substrate method. At present, the similar product kits sold in the market all adopt a chromogenic substrate method for measuring the plasma PLG activity, and the method has the characteristics of high sensitivity, good accuracy, short detection time and the like, can be suitable for various automatic analytical instruments, and is accepted by hospitals as a first-choice screening experiment for detecting the plasminogen activity. In addition, the product kit is an imported product and is high in price, so that the wide clinical application of the product kit is limited.

Principle of chromogenic substrate method: the plasmin circulates in plasma in the form of inactive zymogen, under the activation of plasmin activator, the plasmin is activated and converted into active plasmin, the plasmin acts on specific chromogenic substrate, the substrate is cracked to generate 4-nitroaniline (pNA) chromogenic group, the amount of 4-nitroaniline is positively correlated with the change of absorbance at 405nm, so the amount of 4-nitroaniline generated is positively correlated with the plasmin activity. Thus, the activity level of plasminogen in plasma can be calculated by measuring the change in absorbance at 405nm.

In order to solve the problem that the project is difficult to popularize and apply in hospitals, based on a chromogenic substrate method, it is particularly important to develop a plasminogen activity assay kit and a preparation method thereof.

Disclosure of Invention

In view of this, the present invention provides activating reagents, chromogenic reagents, plasminogen activity assay reagents and/or kits and uses thereof. The kit disclosed by the invention is simple in composition, easy to prepare and low in production cost. The kit aims at the problem that the streptokinase and the chromogenic substrate are generally unstable in aqueous solution and cannot be stored for a long time, and the stability of the streptokinase and the chromogenic substrate in the aqueous solution can be effectively improved by adopting the specific stabilizer, and the kit is more beneficial to enhancing the stability of the streptokinase and the chromogenic substrate in a dry powder type appearance form.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides an activating agent comprising:

in some embodiments of the invention, the activating agent comprises:

in some embodiments of the invention, the concentration in the activating agent is the final concentration.

The present invention also provides a chromogenic reagent comprising:

1.0-3.0 mg/mL of chromogenic substrate

Mannitol 5.0-9.0 w/v%.

In some embodiments of the invention, the above-described chromogenic agent comprises:

chromogenic substrate 2.0mg/mL or 3.0mg/mL

Mannitol 5.0w/v%, 7.0w/v% or 9.0w/v%.

In some embodiments of the invention, the concentration in the above-described chromogenic reagent is the final concentration.

The invention also provides a reagent for detecting the activity of plasminogen, comprising: the activating reagent and/or the developing reagent.

In some embodiments of the present invention, the detection reagent further comprises: 50-100 mmol/LHEPES-NaOH buffer.

In some embodiments of the present invention, the detection reagent further comprises: 30mmol/L, 50mmol/L, 80mmol/L or 100mmol/L HEPES-NaOH buffer.

In some embodiments of the present invention, in the above detection reagent, the pH of the buffer is 7.2 to 8.0.

In some embodiments of the invention, in the above detection reagent, the pH of the buffer is 7.2.

In some embodiments of the invention, the detection reagent described above comprises:

and/or

1.0-3.0 mg/mL of chromogenic substrate

Mannitol 5.0-9.0 w/v%

30mmol/L buffer balance.

The invention also provides a preparation method of the detection reagent, which comprises the steps of respectively mixing the activating reagent and the chromogenic reagent with the buffer solution, and freeze-drying to obtain the detection reagent.

The invention also provides application of the activating reagent, the chromogenic reagent, the detection reagent and/or the detection reagent obtained by the preparation method in preparation of a kit for detecting the activity of the plasminogen.

In some embodiments of the invention, in the above application, the detecting comprises the steps of: mixing the pretreated sample with the activating reagent, mixing the pretreated sample with the chromogenic reagent, and obtaining the activity of the plasminogen according to an OD value after a chromogenic reaction.

In some embodiments of the invention, in the above application, the volume ratio of the activating reagent to the chromogenic reagent is: 1:1.

In some embodiments of the invention, in the above application, the pretreatment comprises: mixing with a diluent; the volume ratio of the sample to the diluent is: 1:30; the diluent comprises: physiological saline.

In some embodiments of the invention, in the above application, the time of mixing with the activating agent is 4 minutes.

In some embodiments of the invention, in the above application, the time of the color reaction is 90s and the wavelength is 405nm.

In some embodiments of the invention, in the above application, the OD value is a number from 15s to 75 s.

The invention also provides a plasminogen activity assay kit comprising: the activating reagent, the chromogenic reagent, the detection reagent and/or the detection reagent obtained by the preparation method, and acceptable auxiliary agents or carriers.

The invention also provides the application of the activating reagent, the color reagent, the detection reagent obtained by the preparation method and/or the kit in preparation of the kit for detecting the activity of the fibrinolytic system.

The present invention provides an activating agent comprising:

the beneficial effects of the invention include:

(1) The kit has simple composition, easy preparation and lower production cost;

(2) Aiming at the problem that the streptokinase and the chromogenic substrate are generally unstable in the aqueous solution and cannot be stored for a long time, the kit can effectively improve the stability of the streptokinase and the chromogenic substrate in the aqueous solution by adopting a specific stabilizer, and the kit exists in a dry powder type appearance form, which is more beneficial to enhancing the stability of the streptokinase and the chromogenic substrate;

(3) Experiments show that the plasminogen activity assay kit has excellent performance, and is stable for 24 months at the temperature of 2-8 ℃ with the relative deviation not exceeding +/-10%; precision CV value: no more than 8%; inter-vial differential CV value: no more than 5%; linear range of [5, 170 ]]% of the fitting degree R ² 0.99; the kit can be stably maintained for 3 days at 37 ℃ after being redissolved, and can be at least stable for 60 days at 2-8 ℃; the stability is high;

(4) The invention provides a plasminogen activity assay kit and a preparation method thereof, which are a kit for quantitatively detecting the plasminogen activity based on interaction of plasmin and a specific chromogenic substrate.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows calibration curves for streptokinase and urokinase at the same concentration;

FIG. 2 shows calibration curves for different concentrations of streptokinase;

FIG. 3 shows calibration curves for example 6, example 7 and commercially available cognate kits;

FIG. 4 shows the correlation and relative bias of example 6 with commercially available kits of the same type; wherein: the correlation of example 6 with commercially available kits of the same type is shown above; the relative deviation of example 6 from a commercially available homogeneous kit is shown below;

FIG. 5 shows the linearity of the kit of example 6;

FIG. 6 shows model differences of the kit of example 6 on different instruments; wherein: the model differences of the example 6 kit on acltop CTS and CS 5100 instruments are shown above; the model differences of the example 6 kit on the ZK-600 and CS 5100 instruments are shown below.

Detailed Description

The invention discloses an activating reagent, a chromogenic reagent, a plasminogen activity detection reagent and/or a kit and application thereof.

It should be understood that the expression "one or more of … …" individually includes each of the objects recited after the expression and various combinations of two or more of the recited objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

The invention provides a plasminogen activity assay kit, which comprises a diluent, an activating reagent and a chromogenic reagent.

The diluent is normal saline;

the activating reagent comprises streptokinase, bovine serum albumin, glycine, mannitol and buffer solution;

the chromogenic reagent comprises a chromogenic substrate, mannitol and a buffer solution;

based on chromogenic substrate method, streptokinase can activate plasmin in blood plasma sample fast and effectively to convert it into active plasmin, which acts on specific substrate to dissociate chromogenic group. Compared with tissue type plasminogen activator (t-PA), the streptokinase can activate the plasminogen more rapidly and fully, shortens the reaction time, and improves the sample detection rate and the accuracy of detecting the activity of the plasminogen.

Preferably, the diluent is physiological saline.

Preferably, the concentration of each component in the activating reagent is: 150U/mL-400U/mL of streptokinase, 0.2 w/v-0.8 w/v of bovine serum albumin, 1.0 w/v-4.0 w/v of glycine, 5.0 w/v-9.0 w/v of mannitol and the balance of buffer solution.

Preferably, the concentrations of the components of the color reagent are as follows: 1.0 mg/L-3.0 mg/mL of chromogenic substrate, 5.0 w/v-9.0 w/v of mannitol and the balance of buffer solution.

Preferably, the buffer is 30mmol/L to 100mmol/L, pH 7.2-8.0N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid-sodium hydroxide (HEPES-NaOH) buffer.

The invention also provides a preparation method of the kit, which comprises the steps of respectively preparing diluent, activating reagent and developing reagent, and freeze-drying the activating reagent and the developing reagent.

The invention provides a plasminogen activity assay kit and a preparation method thereof. The preparation raw materials of the kit comprise diluent, an activating reagent and a color reagent; the diluent is normal saline; activating agents include streptokinase, bovine serum albumin, glycine, mannitol and buffers; the chromogenic reagent comprises chromogenic substrate, mannitol and buffer. The invention has the following advantages:

the kit has simple composition, easy preparation and lower production cost;

aiming at the problem that the streptokinase and the chromogenic substrate are generally unstable in the aqueous solution and cannot be stored for a long time, the kit adopts the specific stabilizer, can effectively improve the stability of the streptokinase and the chromogenic substrate in the aqueous solution, and is in a dry powder type appearance form, thereby being more beneficial to enhancing the stability of the streptokinase and the chromogenic substrate;

experiments show that the plasminogen activity assay kit has excellent performance, and is stable for 24 months at the temperature of 2-8 ℃ with the relative deviation not exceeding +/-10%; precision CV value: no more than 8%; inter-vial differential CV value: no more than 5%; linear range of [5, 170 ]]% of the fitting degree R ² 0.99; the kit can be stably maintained for 3 days at 37 ℃ after being redissolved, and can be at least stable for 60 days at 2-8 ℃; the stability is high.

In examples 1 to 9 and verification examples 1 to 4 of the present invention, the raw materials and reagents used were commercially available.

The commercial homogeneous kits mentioned in the examples of the present invention are: ceemen medical diagnostic products, germany, plasmin Activity assay kit (chromogenic substrate method), lot number: 00944.

the invention is further illustrated by the following examples:

example 1

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are proportioned to 100U/mL streptokinase, 0.1w/v% bovine serum albumin, 0.5w/v% glycine, 4w/v% mannitol and 50mmol/L pH7.4 HEPES-NaOH buffer solution.

Preparation of a color reagent: the ratio of each component is 2mg/L chromogenic substrate S2403, 5w/v% mannitol, 30mmol/L pH7.2 HEPES-NaOH buffer solution.

After the preparation of the activating reagent and the color-developing reagent is completed, the activating reagent and the color-developing reagent are packaged and freeze-dried to prepare freeze-dried powder. Example 1 serves as a comparative example.

Example 2

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are 150U/mL streptokinase, 0.5w/v% bovine serum albumin, 2.0w/v% glycine, 7w/v% mannitol and 50mmol/L pH7.2 HEPES-NaOH buffer solution.

After the preparation of the activating reagent and the color-developing reagent is completed, the activating reagent and the color-developing reagent are packaged and freeze-dried to prepare freeze-dried powder.

Example 3

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are 200U/mL streptokinase, 0.5w/v% bovine serum albumin, 2.0w/v% glycine, 7w/v% mannitol and 80mmol/L HEPES-NaOH buffer solution with pH of 7.4.

Example 4

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are 300UL streptokinase, 0.5w/v% bovine serum albumin, 2.0w/v% glycine, 7w/v% mannitol and 100mmol/L HEPES-NaOH buffer solution with pH of 7.8.

Example 5

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are proportioned to 400U/mL streptokinase, 0.5w/v% bovine serum albumin, 2.0w/v% glycine, 9w/v% mannitol and 100mmol/L HEPES-NaOH buffer solution with pH of 8.0.

Example 6

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are mixed with 250U/mL streptokinase, 0.2w/v% bovine serum albumin, 1.0w/v% glycine, 5w/v% mannitol and 50mmol/L pH7.4 HEPES-NaOH buffer solution.

Preparation of a color reagent: the ratio of each component is 2mg/L chromogenic substrate S2403, 7w/v% mannitol, 30mmol/L pH7.2 HEPES-NaOH buffer solution.

Example 7

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are 450U/mL streptokinase, 0.8w/v% bovine serum albumin, 4.0w/v% glycine, 12w/v% mannitol and 100mmol/L pH7.4 HEPES-NaOH buffer solution.

Preparation of a color reagent: the ratio of each component is 3mg/L chromogenic substrate S2403, 9w/v% mannitol, 30mmol/L pH7.2 HEPES-NaOH buffer solution.

After the preparation of the activating reagent and the color-developing reagent is completed, the activating reagent and the color-developing reagent are packaged and freeze-dried to prepare freeze-dried powder. Example 7 serves as a comparative example.

Example 8

Preparation of the diluent: 100mL of physiological saline.

Preparation of the activating reagent: the components are mixed with urokinase 150U/mL, bovine serum albumin 0.5w/v%, glycine 2.0w/v%, mannitol 7w/v% and HEPES-NaOH buffer solution 60mmol/L pH 7.4.

After the preparation of the activating reagent and the color-developing reagent is completed, the activating reagent and the color-developing reagent are packaged and freeze-dried to prepare freeze-dried powder. Example 8 serves as a comparative example.

EXAMPLE 9 operation of the plasminogen Activity assay

The plasminogen activity detection kit of the invention is stood for 10min after redissolution, and a Sysmex5100 full-automatic coagulation analyzer is adopted to set parameters according to the specification provided by the manufacturer: mixing a sample (a calibrator, a quality control product or plasma) with a diluent according to a ratio of 1:30, taking out 50 mu L of the mixed solution, adding an equal volume of an activating reagent, mixing and incubating for 4min, adding an equal volume of a chromogenic reagent, mixing, reacting for 90s under the irradiation of 405nm wavelength, and reading the signal intensity (absorbance OD value) of 15-75 s in a sample to be detected; finally, the activity of the plasminogen in the plasma is obtained by calculating the signal intensity and comparing the signal intensity with a standard curve of a standard substance with known concentration.

FIGS. 1 and 2 show the performance evaluation of standard curves of the kits for measuring the activity of plasminogen according to the present invention.

Verification example 1 screening for activators (streptokinase and urokinase)

The universal calibrator with the known plasminogen activity of 98% is re-dissolved, and is operated according to an automatic calibration program in an instrument operation manual, and a series of concentrations (1/1, 1/2, 1/4, 1/8 and 0/1) are set for calibration and calibration in a multi-point calibration and calibration mode. A concentration of activator (streptokinase and urokinase) was prepared, and the absorbance OD corresponding to the activity of the universal calibrator, plasminogen, was measured using the kit of example 2 and example 8, and each sample was repeatedly tested 2 times to average, and the calibration curve of each example was drawn, and the results are shown in fig. 1.

As shown in fig. 1, the scaling curve equations of example 2 and example 8 are y=0.0022x+0.0014, r, respectively ² =0.9998 and y=0.0012x+0.0006, r ² =0.9984, and the absorbance OD corresponding to the activity of the plasminogen of the assay standard of the kit of example 2 is significantly higher than that of the kit of example 8, i.e. the sensitivity of the streptokinase to activate plasminogen is higher than that of the kit of example 8Urokinase, preferably streptokinase, is used as an activator.

Verification of the Effect of streptokinase concentration on calibration Curve

The universal calibrator with the known plasminogen activity of 98% is re-dissolved, and is operated according to an automatic calibration program in an instrument operation manual, and a series of concentrations (1/1, 1/2, 1/4, 1/8 and 0/1) are set for calibration and calibration in a multi-point calibration and calibration mode. A series of concentration activators were prepared, and the kit of example 1, example 2, example 3, example 4 and example 5 was used to detect the absorbance OD value corresponding to the activity of plasminogen of the universal calibrator, and each sample was repeatedly tested 2 times to average, and the calibration curve of each example was drawn, and the results are shown in fig. 2.

As shown in fig. 2, example 1, example 2, example 3, example 4 and example 5 scale the linear R curve ² All are larger than 0.998, and as the streptokinase activator increases, the absorbance OD value corresponding to the activity of the plasminogen of the detection standard substance is larger, and the absorbance OD values of the embodiment 2, the embodiment 3, the embodiment 4 and the embodiment 5 are consistent with each other to a high degree, namely, the activation efficiency of the plasminogen is highest and sufficient.

Verification of the Effect of the stabilizer composition on the stability of streptokinase

The kits of example 1, example 2, example 3, example 4, example 5, example 6 and example 7 were divided into three groups, respectively, reconstituted, and calibrated as shown in FIGS. 2 and 3 according to the automatic calibration procedure in the instrument operating manual. The kits of example 1, example 2, example 3, example 4, example 5, example 6 and example 7 were respectively divided into three groups, and placed at 2℃to 8℃and 25℃and 37℃respectively, and the normal quality control plasma was examined by the same operator or a group of operators on the same instrument at a predetermined time interval according to the operation method of the measurement of the plasminogen activity, and the change in the plasminogen activity was recorded as a result of the examination, and it was judged whether or not the activation of streptokinase of the kit was decreased, as shown in tables 1 to 3:

TABLE 1 stability of plasminogen Activity assay kit at 2℃to 8 ℃

TABLE 2 stability of plasminogen Activity assay kit at 25℃

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TABLE 3 stability of plasminogen Activity assay kit at 37℃

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FIGS. 2 and 3 show that the calibration curves for the kits of examples 2, 3, 4, 5, 6 and 7 are substantially identical, i.e., the absorbance OD values are exactly identical and higher than that of example 1, and that the concentrations of the components of the kits of the invention are optimized. The results in tables 1-3 show that the relative deviation of the kits of example 2, example 3, example 4, example 5, example 6 and example 7, which are placed at different temperatures, is within + -4% compared with day 0, so that the performance of the kits of example 2, example 3, example 4, example 5, example 6 and example 7 is not significantly lost at least 60 days at 2-8 ℃ after the re-dissolution of the reagents of example 2, example 3, example 4, example 5, example 6 and example 7; can be stably stored for 30 days at 25 ℃; the activity can be stably maintained for 3 days at 37 ℃, and the stability of the kit is consistent, which shows that the composition (bovine serum albumin, mannitol and glycine) provided by the invention can maintain the stability of streptokinase for a long time. In addition, the high concentration of the active reagent component of the kit of example 7 results in longer lyophilization process times and increased costs.

Verification example 4 analytical performance assessment of the kit of the present invention

1. Example 6 drawing of calibration Standard Curve for similar kits on the market

The universal calibrator with the known plasminogen activity of 98% is re-dissolved, the operation is carried out according to an automatic calibration program in an instrument operation manual, a multi-point calibration mode is adopted, a series of concentrations (1/1, 1/2, 1/4, 1/8 and 0/1) are set for respectively calibrating and calibrating the kit and the commercial similar kit, the OD value of the calibrator is detected on a Sysmex5100 coagulation analyzer, the OD value is taken as an ordinate, the corresponding plasminogen activity value is taken as an abscissa, and a standard curve is drawn, as shown in figure 3.

As shown in FIG. 3, based on the chromogenic substrate method, the absorbance OD value of the detection calibrator of the kit and the commercial homogeneous kit according to the present invention is defined as Y=0.0022X+0.0017, R, respectively, as a linear function of the corresponding plasminogen activity ² =0.9997 and y=0.0013X-0.0033, r ² = 0.9977. Therefore, compared with the commercial similar kits, the kit provided by the invention has good linear relationship and high sensitivity.

2. Example 6 detection of precision with commercially available similar kits

The average, standard deviation and coefficient of variation CV of the measured values were calculated by the same or a group of operators on the same instrument, 10 times on the repeated sides of normal and abnormal quality control plasma in example 6 and the commercially available homogeneous kit according to the operation method of the plasminogen activity assay, as shown in Table 4:

TABLE 4 precision of plasminogen Activity assay kit

Coefficient of variation, CV, is generally used to measure the precision of a measurement method, with smaller CV values indicating better precision of the results of the measurement method. The results in Table 4 show that the CV values of the kit of the invention and the commercial similar kits are respectively 0.84% and 2.19% when the normal quality control plasma plasminogen activity is detected; the CV is 2.75% and 4.57% respectively when the activity of plasma plasminogen is controlled by abnormal quality, which shows that the precision of the kit is obviously superior to that of the commercial similar kit.

3. Example 6 detection of differences between batches

Taking out three batches of the kit, re-dissolving, and respectively detecting normal quality control plasma and abnormal quality control plasma 10 times by the same operator or a group of operators on the same instrument according to the operation method of measuring the activity of the plasminogen, and calculating the average value, standard deviation and variation coefficient CV of the measured values, wherein the average value, standard deviation and variation coefficient CV are shown in tables 5-6:

TABLE 5 detection of the batch to batch differences in normal quality control plasma by the plasminogen Activity assay kit

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TABLE 6 detection of differences between batches of abnormal quality control plasma by plasminogen Activity assay kit

Coefficient of variation, CV, is generally used to measure the precision of a measurement method, with smaller CV values indicating better precision of the results of the measurement method. The results in tables 5 and 6 show that the precision (CV) values of the detection of normal and abnormal quality control plasma by the kit of the present invention are 0.97% and 2.91%, respectively, indicating that the kit of the present invention has excellent inter-batch differences.

4. Example 6 detection of intra-batch differences

The same batch of 10 kits of the invention with different numbers are used for re-dissolution, and according to the operation method of the activity measurement of the plasminogen, the same operator or a group of operators respectively perform one-bottle measurement on normal quality control plasma and abnormal quality control plasma for 10 times and 10 bottles for 1 time, and the average value, standard deviation and variation coefficient CV of the measured values are calculated as shown in tables 7 to 8:

TABLE 7 detection of in-batch differences in normal quality control plasma by plasminogen Activity assay kit

TABLE 8 detection of in-batch differences in abnormal quality control plasma by plasminogen Activity assay kit

The results in tables 7 and 8 show that the precision (CV) values of the detection of normal and abnormal quality control plasma by the kit of the present invention are 0.87% and 3.91%, respectively, indicating that the kit of the present invention has excellent intra-batch differences.

5. Example 6 detection of correlation with commercially available homogeneous kits

The kit of the invention and the commercial similar kit are respectively subjected to detection of plasminogen activity in a Sysmex5100 full-automatic hemagglutination analyzer on 40 fresh plasma samples (including normal and abnormal samples), and correlation analysis is carried out on the measured values (the result is shown in FIG. 4, the X axis represents the measured value of the commercial kit, and the Y axis represents the measured value of the kit of the invention or the relative deviation from the measured value of the commercial similar kit).

FIG. 4 shows that the linear equation for the kit of the present invention versus the commercial homogeneous kit is:y=0.9895x+0.8133, correlation coefficient: r is R ² The relative deviation is within +/-10 percent, the detection results are basically consistent, and the results show that the kit has good correlation with the commercial similar kit.

6. Example 6 detection of Linear Range

Plasma samples with a plasmin activity of approximately 170% and gradients of 1, 0.8, 0.6, 0.5, 0.4, 0.2, 0.1, 0.05 and 0.025 were each repeated 2 times to calculate the mean value of the measurement results for each sample. The linear regression equation and the correlation coefficient R are obtained by taking the dilution factor as an independent variable and the average value of the measurement results as a dependent variable as shown in FIG. 5, the dilution concentration is substituted into the linear regression equation, and the estimated value and the relative deviation or absolute deviation between the average value of the measurement results and the estimated value are calculated as shown in Table 9:

TABLE 9 linearity of the plasminogen Activity assay kit

As shown in fig. 5 and table 9, the linear regression equation y=172.99x+0.7803, r ² =0.9998, the relative deviation is between-4.03% and 4.80%, so the linearity range of the kit of the invention: 5% -170%.

7. Example 6 detection of model Difference

Calibration was performed on Sysmex5100, ACL TOP CTS and ZK-6000 fully automatic hemagglutination analyzers using the kit of example 6, and 40 plasma samples were tested for plasminogen activity, ranging from [20, 160]%, based on the calibration curves on the respective instruments, with the results of the tests being shown in Table 10. In order to analyze the model difference of the plasmin sample plasmin activity detected by the kit of the invention on the three instruments, the result of detecting the plasmin activity by a Sysmex5100 instrument is taken as an abscissa, the result of detecting by an ACL TOP CTS or ZK-6000 instrument is taken as an ordinate, and a model difference curve graph 6 is drawn.

TABLE 10 model differences of plasminogen Activity assay kit

Table 10 shows that the inventive kit compares the results of detecting the plasmin activity of the plasma samples on Sysmex5100, ACL TOP CTS and ZK-6000 fully automatic hemagglutination analyzers, respectively, with relative deviations of substantially + -8%, and FIG. 6 reflects the correlation R of the inventive kit for detecting the plasmin activity of the plasma samples on these three instruments ² All are larger than 0.99, so the kit has small model difference and can be applied to various types of hemagglutination analyzers.

8. Interference immunity of example 6

Fresh pooled plasma samples (plasminogen activity approximately 100% pooled samples) were collected and pooled with bilirubin solution (800 mg/dL), chyle (8000 mg/dL) and hemoglobin solution (4000 mg/dL), respectively, to give 4 gradients (10, 20, 40, 80 mg/dL) of bilirubin pooled plasma, 4 gradients (100, 200, 400, 800 mg/dL) of chyle pooled plasma and 4 gradients (50, 100, 200, 400 mg/dL) of hemoglobin pooled plasma, and deionized water of placebo plasma. The plasma sample plasmin activity was measured by selecting the kit of example 6 and the commercially available similar kit, and the average value was measured 2 times for each sample, and the relative deviation between the measurement result of the plasmin activity with the interfering substance added and the measurement result of the plasmin without the interfering substance added was plotted, and the results are shown in tables 11 to 13:

TABLE 11 interference resistance to bilirubin by the example 6 kit and commercially available cognate kits

TABLE 12 interference resistance to chyle by example 6 and commercially available kits of the same type

TABLE 13 interference resistance to hemoglobin for example 6 and commercially available homogeneous kits

As can be seen from tables 11 to 13, the results of example 6, in which the results of the plasmin activity of the plasma with the addition of the interfering substances (80 mg/dL bilirubin, 800mg/dL chyle, 200mg/dL hemoglobin) and the results of the plasmin activity of the plasma without the addition of the interfering substances were both within.+ -. 10%, were significantly superior to the commercially available similar kits. Therefore, the kit has stronger anti-interference performance on interference substances bilirubin, chyle and hemoglobin when detecting the activity of plasma plasminogen.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An activating reagent, comprising:

2. a chromogenic reagent, comprising:

1.0-3.0 mg/mL of chromogenic substrate

Mannitol 5.0-9.0 w/v%.

3. A reagent for detecting plasminogen activity, comprising: an activating reagent according to claim 1 and/or a chromogenic reagent according to claim 2.

4. The detection reagent of claim 3, further comprising: 30-100 mmol/L HEPES-NaOH buffer.

5. The method for preparing a detection reagent according to claim 4, wherein the detection reagent is obtained by mixing the activating reagent and the developing reagent with the buffer solution, respectively, and freeze-drying the mixture.

6. Use of an activating reagent according to claim 1, a chromogenic reagent according to claim 2, a detection reagent according to claim 3 or 4 and/or a detection reagent obtained by a preparation method according to claim 5 for preparing a kit for detecting plasminogen activity.

7. The use of claim 6, wherein the detecting comprises the steps of: mixing the pretreated sample with the activating reagent, mixing the pretreated sample with the chromogenic reagent, and obtaining the activity of the plasminogen according to an OD value after a chromogenic reaction.

8. The use according to claim 7, wherein the volume ratio of the activating reagent to the chromogenic reagent is: 1:1.

9. The use according to claim 7 or 8, wherein the pre-treatment comprises: mixing with a diluent; the volume ratio of the sample to the diluent is: 1:30; the diluent comprises: physiological saline.

10. A plasminogen activity assay kit comprising: an activating reagent according to claim 1, a chromogenic reagent according to claim 2, a detection reagent according to claim 3 or 4 and/or a detection reagent obtainable by a method of preparation according to claim 5 and an acceptable adjuvant or carrier.