CN118311272A - Soluble growth stimulation expressed gene 2 protein detection kit, preparation method and detection method thereof - Google Patents

Abstract

The invention relates to the technical field of immunodiagnosis, and particularly discloses a soluble growth stimulation expression gene 2 protein detection kit, a preparation method and a detection method thereof, wherein the kit comprises a reagent detection card, the reagent detection card comprises an immunochromatography test strip and a reagent card shell, the immunochromatography test strip comprises a PVC bottom plate, a release pad, absorbent paper and a nitrocellulose membrane are arranged on the PVC bottom plate, a ST2 antibody indirectly marked by fluorescent quantum dots is fixed on the release pad, a detection line and a quality control line are coated on the nitrocellulose membrane, the absorbent paper and the release pad are respectively contacted with two ends of the nitrocellulose membrane, a sample pad is arranged on the release pad, the reagent card shell comprises an upper cover and a lower cover, the immunochromatography test strip is clamped in the lower cover, and the upper cover is clamped on the lower cover; solves the technical problems that the existing method for measuring the soluble growth stimulation expressed gene 2 protein has higher cost, longer detection time, complex operation and relatively complex operation, and is difficult to meet the requirements of timely diagnosis and rapid acquisition of detection results of emergency and clinical patients.

Description

Soluble growth stimulation expressed gene 2 protein detection kit, preparation method and detection method thereof

Technical Field

The invention relates to the technical field of immunodiagnosis, and particularly discloses a soluble growth stimulation expressed gene 2 protein detection kit, a preparation method and a detection method thereof.

Background

Soluble growth stimulatory expressed gene 2 protein (ST 2) is a member of the interleukin 1 (IL-1) receptor family, consisting essentially of two isoforms: 1. ST2 or sST2 in soluble form; 2. ST2, in the form of a membrane-bound receptor, is referred to as ST2 receptor or ST2L. Wherein, the soluble ST2 is a substance secreted when heart diseases or myocardial cells are damaged, and when the concentration of the soluble ST2 in blood is low, ST2L is combined with IL-33 to play a role in protecting the heart and maintaining the heart function; when the concentration of soluble ST2 in blood is high, it can competitively bind with IL-33, resulting in that ST2L/IL-33 signal channel is cut off, thus possibly inducing inflammatory reaction of myocardial cells, causing myocardial fibrosis, myocardial function decline and the like, and leading to or accelerating heart failure. Therefore, the detection of the soluble growth stimulation expressed gene 2 protein has great significance for diagnosis and prognosis evaluation of heart failure patients.

The currently known method for measuring soluble growth stimulation expressed gene 2 protein in the market mainly comprises an enzyme-linked immunosorbent assay (ELISA) method and a chemiluminescence method, wherein the ELISA method is characterized in that antibodies are adsorbed on the surface of a solid phase carrier, when an added sample contains an antigen to be measured, the antibodies are combined with the antibodies fixed on the surface of the carrier, then another enzyme-labeled antibody is added as a detection antibody, so that a solid phase antibody-antigen-enzyme-labeled antibody compound is formed, after an enzyme reaction substrate is added, the substrate is catalyzed by enzyme to generate a color reaction, and the concentration of the antigen in the sample to be measured can be judged by detecting the color depth of the color product, so that qualitative or quantitative detection is realized. The principle of the chemiluminescence method is basically the same as that of the ELISA method, but the enzyme-labeled antibody is changed into a chemiluminescent substance-labeled antibody, and the chemiluminescent intensity in the reaction system is detected by a chemiluminescent instrument so as to determine the content of the object to be detected. However, both methods have the defects of higher cost, longer detection time, complicated operation, relatively more complex operation and the like, and are difficult to meet the requirements of timely diagnosis and rapid acquisition of detection results of emergency treatment and clinical patients.

Disclosure of Invention

In view of the above, the invention aims to provide a soluble growth stimulation expressed gene 2 protein detection kit, a preparation method and a detection method thereof, so as to solve the technical problems that the existing soluble growth stimulation expressed gene 2 protein detection method is high in cost, long in detection time, complex in operation and relatively complex, and can not meet the requirements of timely diagnosis of emergency and clinical patients and rapidly obtain detection results.

In order to achieve the above purpose, the present invention provides the following technical solutions: the utility model provides a soluble growth stimulus expresses gene 2 protein detection kit, includes the reagent detection card, the reagent detection card contains immunochromatography test paper strip and reagent cassette, the immunochromatography test paper strip includes the PVC bottom plate, be provided with release liner, absorbent paper and nitrocellulose membrane on the PVC bottom plate, be fixed with the indirect marked ST2 antibody of fluorescence quantum dot on the release liner, the last detection line and the matter control line of being coated of nitrocellulose membrane, absorbent paper and release liner contact with nitrocellulose membrane both ends respectively, be provided with the sample pad on the release liner. When the device is used, a sample to be detected is dripped on the sample pad, the sample to be detected moves from the sample pad to the release pad through capillary phenomenon, then moves from the release pad to the nitrocellulose membrane, and is detected through the detection line and the quality control line, and finally is adsorbed by the absorbent paper.

Further, the reagent card shell comprises an upper cover and a lower cover, the immunochromatographic test strip is clamped in the lower cover, the upper cover is clamped on the lower cover, the immunochromatographic test strip is positioned between the upper cover and the lower cover, the upper cover is provided with a sample adding hole, and the sample adding hole is positioned above the sample pad. The reagent card shell is used for protecting the immunochromatographic test strip, and is convenient for a user to use, and the user can drop the sample to be tested from the sample adding hole.

Further, the preparation method of the soluble growth stimulation expressed gene 2 protein detection kit comprises the following steps:

S1, preparing an ST2 quality control product, wherein the ST2 quality control product comprises an ST2 antigen and an antigen dilution buffer, the ST2 antigen is diluted by the antigen dilution buffer and then freeze-dried by a freeze dryer, the antigen dilution buffer comprises 1-10% of protein excipient, 1-5% of protein stabilizer, 0.05-1% of preservative and 1-100mM buffer with pH of 6.0-8.0, the protein excipient is selected from mannitol, sorbitol, polyethylene glycol and polyvinylpyrrolidone, the protein stabilizer is selected from glucose, sucrose, trehalose, glycerol, casein and bovine serum albumin, the preservative is selected from sodium azide, sodium benzoate, gentamycin sulfate, sodium nitrite, methylisothiazolinone and Proclin300, and the buffer is selected from PB buffer, PBS buffer and Tris buffer.

S2, preparing a sample pad: soaking an untreated initial sample pad in a sample pad treatment liquid, and drying to obtain a sample pad; the sample pad treatment solution comprises 1-100mM phosphate buffer solution, 0.1-2% bovine serum albumin and 1% -5% sucrose.

S3, preparing a release pad, uniformly mixing N-hydroxysuccinimide activated biotin and an ST2 antibody solution according to a molar ratio of 1-20:1 at 2-8 ℃ or room temperature for reaction for 0.5-2 hours, and removing redundant biotin after dialysis to obtain a biotin-labeled mouse (or rabbit or sheep) ST2 antibody; diluting the streptavidin coupled fluorescent quantum dots and the biotin-labeled ST2 antibody with PBS or Tris buffer solution at a ratio of 1:1-10, mixing the diluted fluorescent quantum dots and the ST2 antibody at a ratio of 1:0.1-1, and reacting for 1-3 hours to obtain the ST2 antibody indirectly labeled by the fluorescent quantum dots; spraying the ST2 antibody solution indirectly marked by the fluorescent quantum dots on an initial release pad, and drying to obtain the release pad.

S4, preparing a nitrocellulose membrane, diluting a detection line coated antibody by using a coating diluent to obtain a detection line coated antibody solution with specific concentration, wherein the coating diluent comprises 1% -5% of trehalose, 1% -5% of BSA, 0.05% -1% of Proclin300 and 1-100mM phosphate buffer with pH of 6.0-8.0, the detection line coated antibody is a mouse (or rabbit or sheep) ST2 antibody, the coating concentration of the detection line coated antibody is 0.3-1.2mg/mL, then diluting the quality control line coated antibody to obtain a quality control line coated antibody solution with specific concentration, the quality control line coated antibody is an anti-mouse (or anti-rabbit or anti-sheep) IgG antibody, the coating concentration of the quality control line coated antibody solution is 0.5-1.5mg/mL, and then scribing the detection line coated antibody solution and the quality control line coated antibody solution on an untreated initial nitrocellulose membrane by a film metal spraying instrument respectively to obtain the nitrocellulose membrane.

S5, assembling, namely sequentially pasting the treated sample pad, the release pad, the nitrocellulose membrane and the absorbent paper on a PVC bottom plate, firstly pasting the nitrocellulose membrane on the PVC bottom plate, then pasting the release pad and the absorbent paper on the PVC plate, respectively contacting the release pad and the absorbent paper with two ends of the nitrocellulose membrane, pasting the sample pad on the release pad, cutting the sample pad into an immunochromatographic test strip with the width of 3.5mm after the pasting is completed, then loading the immunochromatographic test strip into a groove of a lower cover, and tightly clamping and then covering an upper cover, so that the upper cover and the lower cover are tightly combined, thereby preparing the reagent detection card.

Further, in step S1, the protein excipient is the mannitol. Mannitol is selected as the protein excipient with the best effect.

Further, in step S1, the protein stabilizer is the trehalose. The protein stabilizer has the best effect of trehalose.

Further, in step S1, the preservative is the Proclin300. The preservative is Proclin300 with the best effect.

Further, in step S1, the buffer is the PBS buffer. The buffer solution is PBS buffer solution with optimal effect.

Further, in step S1, the ST2 antibody is a monoclonal antibody, a polyclonal antibody, a monoclonal antibody Fab fragment or a polyclonal antibody Fab fragment of an outsourced mouse (or rabbit or sheep).

Further, the detection method of the soluble growth stimulation expressed gene 2 protein detection kit comprises the following steps:

S1: taking out the SD card, and scanning a two-dimensional code on the SD card by using a code scanning gun connected with the dry-type fluorescence immunoassay analyzer to read standard curve information;

S2, adding 500 mu L of pure water into a freeze-drying bottle with an ST2 quality control product, fully and uniformly mixing and dissolving for 1-2min, then adding 80 mu L of uniformly mixed liquid into a sample adding hole of a detection reagent card, reacting for 10min, inserting the reagent card into a dry type fluorescence immunoassay instrument, clicking Test to obtain a measured value result, wherein the measured value result indicates that the reagent card is normal within the target value range of the quality control product, and the method can be used for normal detection of a sample;

S3: and taking 80 mu L of a blood sample to be detected, adding the blood sample into a sample adding hole of a detection reagent card, reacting for 10min, inserting the reagent card into a dry type fluorescence immunoassay instrument, clicking a Test for detection, and reading a detection result.

The blood sample to be detected can be rapidly detected, clinical diagnosis of patients can be timely and efficiently met, the detection kit is small and exquisite, the detection steps are simple, and the patients can carry out home self-detection.

The working principle and the beneficial effects of the scheme are as follows:

The kit and the detection method are simple and quick to operate, the detection time of a sample is shortened to 10min, and compared with 20-30min or even more than 30min required by a common enzyme-linked immunosorbent assay or a chemiluminescence assay, the detection time is greatly shortened; the kit has high specificity and accurate detection result, and compared with the detection result of the commercial ST2 kit, the correlation coefficient can reach more than 0.99; and the device does not depend on a large instrument, has low cost, can be produced in a large scale, is convenient to carry, is timely and efficient to detect, and can better meet the requirements of timely clinical diagnosis or home self-test of patients and quick acquisition of detection results.

Drawings

FIG. 1 is a schematic structural diagram of an immunochromatographic test strip in an embodiment;

FIG. 2 is a schematic diagram of a reagent cartridge according to an embodiment;

FIG. 3 is a comparison of the results of 100 serum samples using the soluble growth stimulation expressed gene 2 protein assay kit of the example and a commercially available ST2 assay kit;

fig. 4 is a process flow diagram of the quality control product preparation of example ST 2.

The figures are marked as follows: the device comprises a PVC base plate 1, a detection line 2, a quality control line 3, a sample pad 4, a release pad 5, a nitrocellulose membrane 6, absorbent paper 7, a lower cover 8, an upper cover 9 and a sample adding hole 10.

Detailed Description

The following is a further detailed description of the embodiments:

Example 1

As shown in fig. 1 to 4, a soluble growth stimulation expressed gene 2 protein detection kit is disclosed, comprising an ST2 quality control, an SD card and a reagent detection card; the ST2 quality control product is prepared by diluting ST2 antigen to a specific concentration by using an antigen dilution buffer solution and freeze-drying by a freeze dryer; the two-dimensional code of the reagent kit detection card is stuck on the SD card, and information such as a standard curve of the reagent detection card is contained in the two-dimensional code; the reagent detection card comprises a reagent card shell and an immunochromatographic test strip, wherein the reagent card shell comprises an upper cover 9 and a lower cover 8, the lower cover 8 is provided with the immunochromatographic test strip, the upper cover 9 is provided on the lower cover 8, the immunochromatographic test strip is positioned between the upper cover 9 and the lower cover 8 of the reagent card shell, the upper cover 9 is provided with a sample adding hole 10, the immunochromatographic test strip comprises a sample pad 4, a release pad 5, a nitrocellulose membrane 6, a water absorbing paper 7 and a PVC bottom plate 1, the nitrocellulose membrane 6 is stuck on the PVC bottom plate 1, the two ends of the nitrocellulose membrane 6 are stuck with the water absorbing paper 7 and the release pad 5, the release pad 5 is stuck with the sample pad 4, and the sample pad 4 is aligned with the sample adding hole 10.

The preparation method of the soluble growth stimulation expressed gene 2 protein detection kit comprises the following steps:

s1, preparing an ST2 quality control product, namely adding 1% mannitol, 1% trehalose and 0.1% Proclin300 into 20mM PBS buffer solution, fully stirring and uniformly mixing, regulating the pH to 7.1-7.3, preparing an ST2 antigen dilution buffer solution, diluting 1mg/mL of ST2 antigen to 50ng/mL by using the dilution buffer solution, then split charging into freeze-drying bottles, covering a rubber stopper, and freeze-drying in a freeze dryer to obtain the ST2 quality control product.

S2, preparing a sample pad 4, namely adding 2% bovine serum albumin and 1% sucrose into 20mM PBS buffer solution, fully stirring and dissolving, adjusting the pH to 7.1-7.3 to prepare a sample pad treatment solution, soaking an untreated initial sample pad 4 in the sample pad treatment solution for 30min, taking out, and drying in a 37 ℃ drying oven for 5-10h to obtain the sample pad 4.

S3, preparation of biotin-labeled ST2 antibody solution: adding 0.5mg of ST2 antibody into a proper amount of 50mMPBS buffer solution to dilute the antibody to 1mg/mL, then adding the solution into a dialysis bag for dialysis, changing the dialysis solution once every 2-3 hours for 4-5 times, and transferring the ST2 antibody solution into a centrifuge tube after the last dialysis; then weighing 0.5 mgN-hydroxysuccinimide activated Biotin (NHS-Biotin), and adding pure water to dilute the concentration to 1mg/mL to obtain an NHS-Biotin aqueous solution; adding NHS-Biotin aqueous solution into ST2 antibody solution, uniformly mixing ST2 antibody and NHS-Biotin according to the mol ratio of 1:5 at room temperature for reaction for 2 hours, transferring the mixed reaction solution to a dialysis bag to be dialyzed in 100mMPBS buffer solution, changing the dialysis solution once every 2-3 hours, changing the dialysis solution for 4-5 times, and obtaining Biotin-labeled ST2 antibody solution after dialysis.

S4: preparation of streptavidin-coupled fluorescent quantum dot and biotin-labeled antibody mixed solution: diluting streptavidin-coupled fluorescent quantum dots and biotin-labeled ST2 antibody with PBS buffer solution at a ratio of 1:10, mixing the diluted fluorescent quantum dots and ST2 antibody at a ratio of 1:0.2, supplementing 70 mu L with the PBS buffer solution, fully and uniformly mixing, and reacting for 1h to obtain a mixed solution of the streptavidin-coupled fluorescent quantum dots and the biotin-labeled antibody; the mixture was sprayed onto an initial release pad at a rate of 2. Mu.L/cm, and dried to give a release pad 5.

S5: preparation of nitrocellulose membrane 6, namely adding 1% trehalose, 5% BSA and 0.1% Proclin300 into 20mM PBS buffer solution, fully stirring and dissolving, and then adjusting pH to 7.1-7.3 to prepare coating diluent; then, diluting the coated antibody of the detection line 2, namely the mouse ST2 antibody by using coating diluent to ensure that the coating concentration is 0.5mg/mL; diluting the coating antibody of the quality control line 3, namely the goat anti-mouse IgG antibody, so that the coating concentration is 1mg/mL; then scribing is carried out by using a scribing metal spraying instrument, wherein the scribing amount is 1 mu L/cm.

S6: the test paper strip is assembled, firstly, a nitrocellulose membrane 6 is stuck on a PVC bottom plate 1, then a release pad 5 and a water absorbing paper 7 are stuck on the PVC bottom plate 1, meanwhile, the release pad 5 and the water absorbing paper 7 are respectively stuck at two ends of the cellulose acetate membrane 6 and are contacted with the cellulose acetate membrane 6, then a sample pad 4 is stuck on the release pad 5, and the assembled PVC plate is cut into an immunochromatographic test paper strip with the width of 3.5mm by a slitter, as shown in figure 1; then the immunochromatographic test strip is put into a groove of a lower cover 8 of the reagent card case, an upper cover 9 is covered after clamping, a sample adding hole 10 is positioned above a sample pad 4, then the sample adding hole is placed on a card pressing machine, the height of the card pressing machine is adjusted to be 4.62mm, and the upper cover 9 and the lower cover 8 of the reagent card case are tightly combined to prepare the reagent detection card.

The detection method of the soluble growth stimulation expressed gene 2 protein detection kit comprises the following steps:

S1, taking out an SD card, and scanning a two-dimensional code on the SD card by using a code scanning gun connected with a dry-type fluorescence immunoassay analyzer to read standard curve information;

S2, adding 500 mu L of pure water into a freeze-drying bottle with an ST2 quality control product, fully and uniformly mixing and dissolving for 1-2min, then adding 80 mu L of liquid into a sample adding hole 10 of a detection reagent card, reacting for 10min, and inserting the reagent card into a dry type fluorescence immunoassay analyzer to obtain a measured value result, wherein the measured value result indicates that the reagent card is normal within the target value range of the quality control product, and the method can be used for normal detection of a sample;

and S3, taking 80 mu L of a blood sample to be detected, adding the blood sample into the sample adding hole 10 of the detection reagent card, reacting for 10min, inserting the reagent card into the dry type fluorescence immunoassay instrument, clicking a Test for detection, and reading a detection result.

Embodiment two:

The difference from the first embodiment is that: the buffer was changed to PB buffer.

Embodiment III:

the difference from the first embodiment is that: the buffer was changed to Tris buffer.

Embodiment four:

the difference from the first embodiment is that: the protein excipient was changed to sorbitol.

Fifth embodiment:

the difference from the first embodiment is that: the protein excipient is modified to polyethylene glycol.

Example six:

the difference from the first embodiment is that: the protein excipient is modified to polyvinylpyrrolidone.

Embodiment seven:

the difference from the first embodiment is that: the protein stabilizer is modified to glucose.

Example eight:

The difference from the first embodiment is that: the protein stabilizer is modified to sucrose.

Example nine:

the difference from the first embodiment is that: the protein stabilizer is changed into bovine serum albumin.

Example ten:

the difference from the first embodiment is that: the protein stabilizer is modified to glycerol.

Example eleven:

the difference from the first embodiment is that: the protein stabilizer is changed to casein.

Embodiment twelve:

The difference from the first embodiment is that: the preservative is modified to sodium azide.

Embodiment thirteen:

The difference from the first embodiment is that: the preservative is changed to sodium benzoate.

Fourteen examples:

the difference from the first embodiment is that: the preservative is changed to sodium nitrite.

Example fifteen:

The difference from the first embodiment is that: the preservative is changed into methylisothiazolinone.

Example sixteen:

the difference from the first embodiment is that: the preservative is changed into gentamicin sulfate.

Test example:

Test example one: a percent buffer signal retention assay; test example two: protein excipient signal retention percentage assay; test example three: protein stabilizer signal retention percentage detection assay; test example four: a preservative signal retention percentage detection assay; test example five: accuracy evaluation experiments; test example six: linear range assessment experiments; test example seven: sensitivity evaluation experiments; test example eight: performing precision evaluation experiments; test example nine: clinical evaluation test;

Test example one: percent Signal retention assay for buffers

TABLE 1ST2 antigen dilution buffer base buffer screening

As can be seen from Table 1, the percent signal retention of PBS buffer is best over PB buffer and Tris buffer, so PBS buffer is chosen for the buffers.

Test example two: protein excipient Signal retention percent assay

TABLE 2ST2 antigen dilution buffer protein excipient screening

As can be seen from Table 2, mannitol is the best signal retention percentage, which is superior to sorbitol, polyethylene glycol and polyvinylpyrrolidone, so mannitol is used as the protein excipient.

Test example three: protein stabilizer signal retention percentage assay

TABLE 3ST2 antigen dilution buffer protein stabilizer screening

As can be seen from Table 3, the percent signal retention of trehalose was best compared to glucose, sucrose, bovine serum albumin, glycerol and casein, so trehalose was used as the protein stabilizer.

Test example four: preservative Signal Retention percentage test

TABLE 4ST2 antigen dilution buffer preservative screening

As can be seen from Table 4, the percent signal retention of Proclin300 is best, and is superior to sodium azide, sodium benzoate, sodium nitrite, methylisothiazolinone and gentamicin sulfate, so Proclin300 is selected as the preservative.

The results of the first to fourth test examples show that the stability of the measurement results of the first embodiment, that is, the quality control effect is optimal, and the first embodiment is optimal in the first to sixteenth embodiments, so that the kit of the application can obtain more accurate detection results.

Test example five: accuracy assessment experiment

ST2 enterprise standard substances of 20ng/mL, 60ng/mL and 180ng/mL are respectively prepared by using PBS buffer solution containing 50% calf serum, the standard substances of each concentration are detected for 3 times, then the concentration values are respectively read on a dry type fluorescence immunoassay analyzer, and the relative deviation from the marked concentration is calculated, wherein the detection result is shown in Table 5.

TABLE 5 accuracy test results

The result shows that the relative deviation between the test results and the marked concentration of the three standard substances of the kit is not more than +/-10%.

Test example six: linear range assessment experiment

10 Gradient concentration soluble growth stimulation expressed gene 2 protein reference substances are respectively prepared by using a PBS buffer solution containing 50% calf serum, then the reference substances with each concentration are repeatedly detected for 3 times by using the kit, the concentration values of the reference substances are respectively read on a dry type fluorescence immunoassay analyzer, the average value of 3 times of test results is calculated, the correlation coefficient between the average value of the results and the marked concentration is calculated, the correlation coefficient R is more than or equal to 0.99, and the detection results are shown in Table 6.

TABLE 6 Linear Range detection results

As can be seen from the results in Table 6, the correlation coefficient r of the kit of the present application was 0.9990 in the concentration range of 1.00-460.80ng/mL, and thus the linearity of the kit was 1.00-460.80ng/mL.

Test example seven: sensitivity evaluation experiment

Standards of soluble growth stimulation expressed gene 2 protein were prepared to 6.40ng/mL, 3.20ng/mL, 1.60ng/mL, 0.80ng/mL and 0.40ng/mL respectively by PBS containing 50% calf serum, then these 5 standards were tested using the kit of the present application, each standard was tested 10 times, its concentration value was read by a dry fluorescence analyzer, the average value and coefficient of variation CV were calculated, and when it measured the lowest concentration and the coefficient of variation was less than 10%, the sensitivity that could be achieved for the test was considered, and the test results are shown in table 7.

TABLE 7 sensitivity test results

The result shows that the detection sensitivity of the kit is 0.70ng/mL.

Test example eight: precision evaluation experiment

Two ST2 reference products of 20ng/mL and 40ng/mL are respectively prepared by using PBS containing 50% calf serum, then the two reference products are detected by using the kit, each reference product is repeatedly tested for 10 times, the value of each reference product is read on a dry type fluorescence immunoassay analyzer, the average value, standard deviation and variation coefficient CV of 10 times of test results are calculated, the variation coefficient CV is smaller than 15%, and the detection results are shown in Table 8.

TABLE 8 precision test results

As can be seen from Table 8, the coefficient of variation CV for the two references is 5.6% and 7.0%, respectively, indicating that the precision of the kit of the present application is within 10%.

Test example nine: clinical evaluation test

The kit and the determination method thereof are used as evaluation reagents to be tested in clinical evaluation tests, a certain ST2 detection kit approved for marketing is selected as a contrast reagent, 100 clinical samples are detected and subjected to contrast test research, the clinical application performance of the kit and the determination method is evaluated by analyzing the correlation between the test results of the ST2 kit and the commercial ST2 kit, and the contrast results are shown in figure 3. The results show that the correlation coefficient between the ST2 kit and the measuring method and the commercial ST2 detection kit can reach 0.9939.

The methods of detection of the percent buffer signal retention assay, the percent protein excipient signal retention assay, the percent protein stabilizer signal retention assay, and the percent preservative signal retention assay are all well known to those skilled in the art and are not described in detail herein.

The test cases five to nine are all the soluble growth stimulation expression gene 2 protein (ST 2) detection kits prepared in the first embodiment, and the results show that the first embodiment has high sensitivity, high precision and accurate detection results; the whole operation time can be shortened to 10min, and meanwhile, the correlation coefficient of the clinical performance can reach more than 0.99 compared with the detection result of the commercial ST2 kit; and the device does not depend on a large instrument, has low cost, can be produced in a large scale, is convenient to carry, is timely and efficient to detect, and can better meet the requirements of timely clinical diagnosis or home self-test of patients and quick acquisition of detection results.

The foregoing is merely exemplary embodiments of the present invention, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the present invention.

Claims (9)

1. A soluble growth stimulation expressed gene 2 protein detection kit, which is characterized in that: including the reagent detection card, the reagent detection card contains immunochromatography test strip and reagent cassette, the immunochromatography test strip includes the PVC bottom plate, be provided with release liner, absorbent paper and nitrocellulose membrane on the PVC bottom plate, be fixed with the indirect marked ST2 antibody of fluorescence quantum dot on the release liner, nitrocellulose membrane is last to be coated with detection line and matter control line, absorbent paper and release liner contact with nitrocellulose membrane both ends respectively, be provided with the sample pad on the release liner.

2. The soluble growth stimulation expression gene 2 protein assay kit of claim 1, wherein: the reagent card shell comprises an upper cover and a lower cover, the immunochromatographic test strip is clamped in the lower cover, the upper cover is clamped on the lower cover, the immunochromatographic test strip is positioned between the upper cover and the lower cover, the upper cover is provided with a sample adding hole, and the sample adding hole is positioned above the sample pad.

3. The method for preparing a soluble growth stimulation expression gene 2 protein assay kit according to claim 2, comprising the steps of:

S1, preparing an ST2 quality control product, wherein the ST2 quality control product comprises an ST2 antigen and an antigen dilution buffer, the ST2 antigen is diluted by the antigen dilution buffer and then freeze-dried by a freeze dryer, the antigen dilution buffer comprises 1-10% of protein excipient, 1-5% of protein stabilizer, 0.05-1% of preservative and 1-100mM buffer with pH of 6.0-8.0, the protein excipient is selected from mannitol, sorbitol, polyethylene glycol and polyvinylpyrrolidone, the protein stabilizer is selected from glucose, sucrose, trehalose, glycerol, casein and bovine serum albumin, the preservative is selected from sodium azide, sodium benzoate, gentamycin sulfate, sodium nitrite, methylisothiazolinone and Proclin300, and the buffer is selected from PB buffer, PBS buffer and Tris buffer;

S2, preparing a sample pad: soaking an untreated initial sample pad in a sample pad treatment liquid, and drying to obtain a sample pad; the sample pad treatment solution comprises 1-100mM phosphate buffer solution, 0.1-2% bovine serum albumin and 1-5% sucrose;

S3, preparing a release pad, uniformly mixing N-hydroxysuccinimide activated biotin and an ST2 antibody solution according to a molar ratio of 1-20:1 at 2-8 ℃ or room temperature for reaction for 0.5-2 hours, and removing redundant biotin after dialysis to obtain a biotin-labeled mouse (or rabbit or sheep) ST2 antibody; diluting the streptavidin coupled fluorescent quantum dots and the biotin-labeled ST2 antibody with PBS or Tris buffer solution at a ratio of 1:1-10, mixing the diluted fluorescent quantum dots and the ST2 antibody at a ratio of 1:0.1-1, and reacting for 1-3 hours to obtain the ST2 antibody indirectly labeled by the fluorescent quantum dots; spraying the ST2 antibody solution indirectly marked by the fluorescent quantum dots on an initial release pad, and drying to obtain the release pad;

S4, preparing a nitrocellulose membrane, diluting a detection line coated antibody by using a coating diluent to obtain a detection line coated antibody solution with a specific concentration, wherein the coating diluent comprises 1% -5% of trehalose, 1% -5% of BSA, 0.05% -1% of Proclin300 and 1-100mM phosphate buffer with the pH of 6.0-8.0, the detection line coated antibody is a mouse (or rabbit or sheep) ST2 antibody, the coating concentration of the detection line coated antibody is 0.3-1.2mg/mL, then diluting the quality control line coated antibody to obtain a quality control line coated antibody solution with the specific concentration, the quality control line coated antibody is an anti-mouse (or anti-rabbit or anti-sheep) IgG antibody, the coating concentration of the quality control line coated antibody solution is 0.5-1.5mg/mL, and then scribing the detection line coated antibody solution and the quality control line coated antibody solution on an untreated initial nitrocellulose membrane by a film metal spraying instrument respectively to obtain the nitrocellulose membrane;

S5, assembling, namely sequentially pasting the treated sample pad, the release pad, the nitrocellulose membrane and the absorbent paper on a PVC bottom plate, firstly pasting the nitrocellulose membrane on the PVC bottom plate, then pasting the release pad and the absorbent paper on the PVC plate, respectively contacting the release pad and the absorbent paper with two ends of the nitrocellulose membrane, pasting the sample pad on the release pad, cutting the sample pad into an immunochromatographic test strip with the width of 3.5mm after the pasting is completed, then loading the immunochromatographic test strip into a groove of a lower cover, and tightly clamping and then covering an upper cover, so that the upper cover and the lower cover are tightly combined, thereby preparing the reagent detection card.

4. A method of preparation according to claim 3, characterized in that: in step S1, the protein excipient is the mannitol.

5. The method of manufacturing according to claim 4, wherein: in step S1, the protein stabilizer is the trehalose.

6. The method of manufacturing according to claim 5, wherein: in step S1, the preservative is the Proclin300.

7. The method of manufacturing according to claim 6, wherein: in step S1, the buffer is the PBS buffer.

8. The method of manufacturing according to claim 7, wherein: in step S1, the ST2 antibody is a monoclonal antibody, a polyclonal antibody, a monoclonal antibody Fab fragment or a polyclonal antibody Fab fragment of an outsourced mouse (or rabbit or sheep).

9. The method for detecting a soluble growth stimulation expressed gene 2 protein detection kit according to claim 2, comprising the steps of:

S1: taking out the SD card, and scanning a two-dimensional code on the SD card by using a code scanning gun connected with the dry-type fluorescence immunoassay analyzer to read standard curve information;

S2, adding 500 mu L of pure water into a freeze-drying bottle with an ST2 quality control product, fully and uniformly mixing and dissolving for 1-2min, then adding 80 mu L of uniformly mixed liquid into a sample adding hole of a detection reagent card, reacting for 10min, inserting the reagent card into a dry type fluorescence immunoassay instrument, clicking Test to obtain a measured value result, wherein the measured value result indicates that the reagent card is normal within the target value range of the quality control product, and the method can be used for normal detection of a sample;

S3: and taking 80 mu L of a blood sample to be detected, adding the blood sample into a sample adding hole of a detection reagent card, reacting for 10min, inserting the reagent card into a dry type fluorescence immunoassay instrument, clicking a Test for detection, and reading a detection result.