CN114624437A - Detection kit for hypersensitive troponin I - Google Patents

Abstract

The invention is applicable to the technical field of biological detection, and provides a detection kit for hypersensitive troponin I, wherein a test strip comprises: a substrate; the device comprises an NC film arranged on a substrate, wherein a detection line and a quality control line are respectively arranged on the NC film; the detection line comprises a troponin I monoclonal antibody, and the quality control line comprises a goat anti-mouse IgG antibody; a sample pad; a binding pad disposed between the NC membrane and the sample pad, the binding pad having time-resolved fluorescent microspheres linked with specific troponin I antibodies disposed thereon; and the water absorption paper is arranged on one side of the NC membrane, which is far away from the sample pad. The test strip provided by the invention is based on the technical principle of immunochromatography, adopts a double-antibody sandwich method to capture troponin I antigen, can detect picogram-level antigen content in serum within 15 minutes, and can detect the troponin I with lower concentration so as to achieve the purpose of hypersensitive detection of the troponin I.

Description

Detection kit for hypersensitive troponin I

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a detection kit for hypersensitive troponin I.

Background

Cardiac troponin (cTn) is composed of subunits of three different genes: cardiac troponin t (ctn t), cardiac troponin i (ctn i) and troponin c (tnc).

Wherein, troponin I (cTn I) is a specific index of myocardial infarction, the myocardial infarction problems can be found in time when the detection sensitivity is higher, however, the general hypersensitive troponin I detection method is based on a chemiluminescence technology, the report time is long, and the myocardial infarction problems are not easy to be found in time; whereas with immunochromatography results can be obtained within 15 minutes. However, the existing immunochromatography technology can only detect the content of the conventional troponin I, and the sensitivity of the immunochromatography technology cannot meet the requirement of hypersensitivity.

Disclosure of Invention

The embodiment of the invention aims to provide a detection kit for hypersensitive troponin I, aiming at solving the problems in the background art.

The embodiment of the invention is realized in such a way that the detection kit for the hypersensitive troponin I comprises a test card and a calibration card, wherein the test card comprises a test strip, and the test strip comprises:

a substrate;

the device comprises an NC film arranged on a substrate, wherein a detection line and a quality control line are respectively arranged on the NC film; the detection line comprises a troponin I monoclonal antibody, and the quality control line comprises a goat anti-mouse IgG antibody;

a sample pad;

the combination pad is arranged between the NC membrane and the sample pad, and the combination pad is provided with time-resolved fluorescent microspheres connected with specific troponin I antibodies; and

and the water absorption paper is arranged on one side of the NC film, which is far away from the sample pad.

In a preferred embodiment of the present invention, the sample pad is made of glass cellulose.

In another preferred embodiment of the present invention, the bonding pad is made of a polyester film.

As another preferable scheme of the embodiment of the present invention, the preparation method of the test strip includes the following steps:

pasting the non-sample application surface of the NC film on the substrate;

diluting the troponin I monoclonal antibody with an antibody diluent, spraying the diluted troponin I monoclonal antibody on a detection line of an NC membrane, and drying;

diluting a goat anti-mouse IgG antibody with a buffer solution, spraying the diluted goat anti-mouse IgG antibody on a quality control line of an NC membrane, and drying;

soaking the bonding pad with a bonding pad treatment solution, drying, spraying time-resolved fluorescent microspheres connected with specific troponin I antibodies on the bonding pad, and sticking the bonding pad on the NC membrane;

soaking the sample pad with the sample pad treatment solution, drying, and sticking the sample pad on the bonding pad;

and (4) taking absorbent paper, and sticking the absorbent paper on the NC membrane to obtain the test paper strip.

As another preferred embodiment of the present invention, the antibody diluent comprises sucrose and Tris-HCl buffer; in the antibody diluent, the mass concentration of sucrose is 0.5-1.5%; the concentration of Tris-HCl buffer is 0.03-0.07 mol/L, and the pH value is 7.5-8.5.

As another preferable aspect of the embodiment of the present invention, the treatment solution for a conjugate pad comprises the following components per 100 mL: tween-200.1-0.3 g, bovine serum albumin 0.4-0.6 g, polyvinylpyrrolidone 0.4-0.6 g, and the balance of Tris-HCl buffer solution.

As another preferable mode of the embodiment of the present invention, the sample pad treatment solution includes the following components per 100 mL: tween-200.1-0.3 g, bovine serum albumin 0.4-0.6 g, polyvinylpyrrolidone 0.4-0.6 g, rabbit anti-human erythrocyte antibody 0.06-0.1 g, and the balance Tris-HCl buffer solution.

As another preferable embodiment of the present invention, the method for preparing the specific troponin I antibody-linked time-resolved fluorescent microsphere comprises the steps of:

taking carboxyl fluorescent microspheres, and suspending the carboxyl fluorescent microspheres in MES buffer solution to obtain microsphere solution;

adding N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the microsphere solution, and then adding a troponin I monoclonal antibody for reaction to obtain a reaction solution;

and adding bovine serum albumin confining liquid into the reaction liquid for confinement, and then centrifuging and resuspending to obtain the time-resolved fluorescent microsphere connected with the specific troponin I antibody.

The hypersensitive troponin I detection kit provided by the embodiment of the invention is based on the technical principle of immunochromatography, captures troponin I antigen by adopting a double-antibody sandwich method, can detect picogram-level antigen content in serum within 15 minutes, and can detect troponin I with lower concentration so as to achieve the purpose of hypersensitive troponin I detection.

Drawings

Fig. 1 is a schematic structural diagram of a test strip provided in an embodiment of the present invention.

Fig. 2 is a schematic structural diagram of a top view of the test strip provided by the embodiment of the invention.

Fig. 3 is a flowchart of a preparation process of the test strip provided by the embodiment of the invention.

Fig. 4 is a standard curve diagram corresponding to the test strip (hypersensitive cTn I product) prepared in example 2.

FIG. 5 is a linear plot of blank concentration (0-0.1) and T/C values for the test strips prepared in example 2 (hypersensitive cTn I product).

FIG. 6 is a comparison of results for hypersensitivity cTn I + MYO + CKMB triple-card products and hospital-and-hospital hypersensitivity cTn I.

FIG. 7 is a graph comparing results of hypersensitivity cTn I single-card product with hospital-and-hospital hypersensitivity cTn I.

In the figure, 1-NC membrane, 2-sample pad, 3-combination pad, 4-absorbent paper, 5-substrate, T-detection line and C-quality control line.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1

As shown in fig. 1 and 2, this embodiment provides a troponin I hypersensitivity test kit, comprising a test card and a calibration card, wherein the test card comprises a test strip, and the test strip comprises:

a substrate 5;

the device comprises an NC film 1 arranged on a substrate 5, wherein a detection line T and a quality control line C are respectively arranged on the NC film 1; the detection line T comprises a troponin I monoclonal antibody, and the quality control line C comprises a goat anti-mouse IgG antibody;

a sample pad 2;

a binding pad 3 arranged between the NC membrane 1 and the sample pad 2, wherein the binding pad 3 is provided with a time-resolved fluorescent microsphere connected with a specific troponin I antibody; and

and the water absorption paper 4 is arranged on the side, away from the sample pad 2, of the NC film 1.

The assembling method of the test strip specifically comprises the following steps: the bonding pad 3 is adhered above the NC film 1, and the width of the bonding pad covering the NC film 1 is 1 mm; the water absorption paper 4 is adhered above the NC film 1, and the width of the water absorption paper covering the NC film 1 is 2 mm; the sample pad 2 is stuck above the bonding pad 3, and the width of the sample pad covering the bonding pad 3 is 2 mm; the adhered test board is cut into test strips with the width of 4mm in an automatic cutting machine, and the test strips are arranged in a plastic card box to manufacture the test card with a result observation window and a sample adding port.

Specifically, the sample pad 2 is made of glass cellulose; the bonding pad 3 is made of a polyester film. In addition, the specific preparation method of the test strip can refer to the following examples.

Wherein, some raw materials adopted in the following examples are as follows:

carboxyfluorescent microspheres (200nm time-resolved fluorescent microspheres, Sozhou Biotechnology Co., Ltd., F T03, 03C, 10mL, 10mg/mL), cTn I monoclonal antibodies (Shanghai peptide Biotechnology Co., Ltd., 4TC2-20C6), MES (SIGMA, M8250), NHS (SIGMA,130672), EDC (SIGMA, E06), Tris (amresco,0826), BSA (amresco,0332), Proclin300(SIGMA,48912-U), goat anti-mouse IgG (Boehringer Biotechnology Co., Ltd., P200101), PVC substrates (Shanghai Lixin technology Co., Ltd., SMNF31-25, 300X 80mm 29+25+26mm), polyester films (VL78, 300X 200mm), glass fibers (Shanghai Lixin technology Co., SB06, 300X 200mm), Lixin paper (Shanghai Lixin paper, CH 35, Saxisi technology Co., Ltd., NCI, NCV 37, NCT), 1UN14E, 25mm wide), tween-20 (30189397, national drug group chemicals, ltd), PVP (SIGMA, 81420), sucrose (10021492, national drug group chemicals, ltd).

Example 2

As shown in fig. 3, the embodiment provides a preparation method of the test strip for detecting troponin I, which includes the following steps:

and S1, taking the PVC rubber plate as a substrate, cutting the NC film into 25mm by 300mm, and pasting the non-sample-application surface of the NC film on the PVC rubber plate.

S2, diluting the cTn I monoclonal antibody with an antibody diluent until the concentration is 2mg/mL, spraying the diluted cTn I monoclonal antibody on a detection line (the distance between the detection line and one end of a binding pad is 8mm) of an NC membrane by using a scribing and metal spraying instrument at the speed of 100mm/S and the concentration of 1 mu L/cm under the condition that the indoor humidity is 45%, and drying the diluted cTn I monoclonal antibody in an oven at 37 ℃ for 2 hours for later use; similarly, goat anti-mouse IgG antibody was diluted with 1% sucrose in 0.05M Tris-HCl buffer, pH8.0, sprayed onto the NC membrane control line (12 mm from one end of the pad) at 100mm/s and 1. mu.L/cm with room humidity of 45% using a gold-spraying streaking machine, and dried in an oven at 37 ℃ for 2 hours. Wherein the antibody diluent comprises sucrose and Tris-HCl buffer solution; in the antibody diluent, the mass concentration of sucrose is 0.5%; the concentration of Tris-HCl buffer was 0.03mol/L, pH 7.5.

S3, soaking the bonding pad material with polyester film VL78(8mm multiplied by 300mm), and drying the bonding pad material in an oven at 37 ℃ for 4h for later use; then, 50 μ L of the specific troponin I antibody-linked time-resolved fluorescent microspheres stored in the microsphere storage solution were taken, centrifuged at 7500rpm for 20 minutes, the supernatant was discarded, 100 μ L of the microsphere redissolution was used for ultrasonic resuspension, then the microspheres were sprayed on the conjugate pad at a concentration of 100mm/s and 3 μ L/cm using a membrane spraying apparatus, and the conjugate pad was adhered to the top of the NC membrane after drying in a 37 ℃ oven for 3 hours. Wherein, every 100mL of the combined pad treatment solution comprises the following components: tween-200.1 g, bovine serum albumin 0.4g, polyvinylpyrrolidone 0.4g, and the balance Tris-HCl buffer solution; the microsphere storage solution is prepared by weighing 0.2g BSA, adding 100mL Tris-HCl buffer solution with 0.05M, pH-8.0 and 1 ‰ Proclin 300; the microsphere composite solution comprises 0.05MM, Tris-HCl buffer solution with the pH value of 8.0, 10% of sucrose, 1% of BSA and 0.2% of Tween-20.

In addition, the preparation method of the specific troponin I antibody-linked time-resolved fluorescent microsphere comprises the following steps:

(1) taking 100 mu L of carboxyl fluorescent microspheres (10mg/mL), and suspending the microspheres in 800 mu L of MES buffer solution to obtain a microsphere solution; wherein the MES buffer solution is prepared by the following method: weighing 0.976g MES, pouring 98mL deionized water to dissolve completely, detecting the solution concentration by a pH meter, and dropwise adding 1M dilute hydrochloric acid solution to neutralize excessive HO when the pH is more than 6.0^-And when the pH is less than 6.0, adding 1M sodium hydroxide solution dropwise to neutralize excessive H⁺The pH was finally stabilized at 6.0 and finally made to 100mL with deionized water.

(2) NHS (5mg/mL) and EDC (5mg/mL) are prepared by MES buffer solution, 20 mu L of each solution is added into the microsphere solution, the final concentration is NHS (0.1mg/mL) and EDC (0.1mg/mL), the mixture is rotated, mixed and activated for 30 minutes at room temperature (25 ℃), the rotating speed is 10rpm, then the mixture is centrifugally washed (17500rpm is centrifuged to remove the supernatant and is suspended in 900 mu LMES buffer solution (wherein NHS can be mixed with EDC first and then added into microspheres, EDC cannot be added into microspheres first, otherwise microspheres are agglomerated), then 0.1mg of cTn I monoclonal antibody is added into the microsphere solution, the mixture is shaken and mixed evenly, and the reaction solution is rotated and reacted for 2 hours at room temperature (25 ℃) to obtain the reaction solution.

(3) Adding 100. mu.L of BSA blocking solution (20mg/mL) to the reaction solution, blocking the reaction at room temperature (25 ℃) for 2 hours, centrifuging at 17500rpm for 20 minutes, resuspending the mixture with 0.05M Tris-HCl buffer (pH 8.0), and ultrasonically dispersing the mixture for 30 seconds to 1 minute each time; (500 mu L of buffer solution is added firstly, the centrifuge tube is placed into an ultrasonic instrument, the liquid level in the centrifuge tube and the page in the ultrasonic instrument form an angle of 45 degrees, the 500 mu L of buffer solution is added for secondary ultrasonic after the buffer solution is uniformly dispersed, the ultrasonic time is not more than 1 minute each time), the washing is repeated twice, the supernatant is removed after the last centrifugation, and the microsphere preservation solution is used for resuspension; and then centrifuging at 2000rpm for 3min, removing precipitates, and storing at 4 ℃ in a dark place to obtain the time-resolved fluorescent microspheres connected with the specific troponin I antibody. The preparation method of the BSA blocking solution comprises the following steps: 0.2g BSA and 0.075g glycine were weighed and added with water to a volume of 10 mL.

S4, using glass cellulose SB06(30mm multiplied by 300mm) as a sample pad material, soaking the sample pad material by using the sample pad treating solution, drying the sample pad material in an oven at 37 ℃ for 4h, and then pasting the sample pad on the bonding pad. Wherein, every 100mL of the sample pad treatment solution comprises the following components: tween-200.1 g, bovine serum albumin 0.4g, polyvinylpyrrolidone 0.4g, rabbit anti-human erythrocyte antibody 0.06g, and the balance Tris-HCl buffer solution.

S5, taking a piece of 22mm X300 cm absorbent paper, and sticking the absorbent paper on the NC film to obtain the test paper strip.

Example 3

The embodiment provides a preparation method of the test strip for detecting troponin I, which comprises the following steps:

and S1, taking the PVC rubber plate as a substrate, cutting the NC film into 25mm by 300mm, and pasting the non-sample-application surface of the NC film on the PVC rubber plate.

S2, diluting the cTnI monoclonal antibody with an antibody diluent to a concentration of 2mg/mL, spraying the diluted cTnI monoclonal antibody on a detection line (the distance between the detection line and one end of the binding pad is 8mm) of an NC membrane by using a scribing and gold spraying instrument at a speed of 100mm/S and a concentration of 1 mu L/cm under the condition that the indoor humidity is 65%, and drying the detection line in an oven at 37 ℃ for 2h for later use; similarly, goat anti-mouse IgG antibody was diluted with 1% sucrose in 0.05M Tris-HCl buffer, pH8.0, sprayed onto the NC membrane control line (12 mm from one end of the pad) at 100mm/s and 1. mu.L/cm with a gold-spraying streaking device under a room humidity of 65%, and dried in an oven at 37 ℃ for 2 h. Wherein the antibody diluent comprises sucrose and Tris-HCl buffer solution; in the antibody diluent, the mass concentration of sucrose is 1.5%; the concentration of Tris-HCl buffer was 0.07mol/L, pH 8.5.

S3, soaking the bonding pad material with polyester film VL78(8mm multiplied by 300mm), and drying the bonding pad material in an oven at 37 ℃ for 4h for later use; then, 50 μ L of the specific troponin I antibody-linked time-resolved fluorescent microspheres stored in the microsphere storage solution were taken, centrifuged at 7500rpm for 20 minutes, the supernatant was discarded, 100 μ L of the microsphere redissolution was used for ultrasonic resuspension, then the microspheres were sprayed on the conjugate pad at a concentration of 100mm/s and 3 μ L/cm using a membrane spraying apparatus, and the conjugate pad was adhered to the top of the NC membrane after drying in a 37 ℃ oven for 3 hours. Wherein, every 100mL of the combined pad treatment solution comprises the following components: tween-200.3 g, bovine serum albumin 0.6g, polyvinylpyrrolidone 0.6g, and the balance of Tris-HCl buffer solution; the microsphere storage solution is prepared by weighing 0.2g BSA, adding 100mL Tris-HCl buffer solution with 0.05M, pH-8.0 and 1 ‰ Proclin 300; the microsphere composite solution comprises 0.05MM, Tris-HCl buffer solution with the pH value of 8.0, 10% of sucrose, 1% of BSA and 0.2% of Tween-20.

In addition, the preparation method of the specific troponin I antibody-linked time-resolved fluorescent microsphere comprises the following steps:

(1) taking 100 mu L of carboxyl fluorescent microspheres (10mg/mL), and suspending the microspheres in 800 mu L of MES buffer solution to obtain a microsphere solution; wherein the MES buffer solution is prepared by the following method: weighing 0.976g MES, pouring 98mL deionized water to dissolve completely, detecting the solution concentration by a pH meter, and dropwise adding 1M dilute hydrochloric acid solution to neutralize excessive HO when the pH is more than 6.0^-And when the pH is less than 6.0, adding 1M sodium hydroxide solution dropwise to neutralize excessive H⁺The pH was finally stabilized at 6.0 and finally made to 100mL with deionized water.

(2) NHS (5mg/mL) and EDC (5mg/mL) are prepared by MES buffer solution, 20 mu L of each solution is added into the microsphere solution, the final concentration is NHS (0.1mg/mL) and EDC (0.1mg/mL), the mixture is rotated, mixed and activated for 30 minutes at room temperature (25 ℃), the rotating speed is 10rpm, then the mixture is centrifugally washed (17500rpm is centrifuged to remove the supernatant and is suspended in 900 mu LMES buffer solution (wherein NHS can be mixed with EDC first and then added into microspheres, EDC cannot be added into microspheres first, otherwise microspheres are agglomerated), then 0.1mg of cTn I monoclonal antibody is added into the microsphere solution, the mixture is shaken and mixed evenly, and the reaction solution is rotated and reacted for 2 hours at room temperature (25 ℃) to obtain the reaction solution.

(3) Adding 100. mu.L of BSA blocking solution (20mg/mL) to the reaction solution, blocking the reaction at room temperature (25 ℃) for 2 hours, centrifuging at 17500rpm for 20 minutes, resuspending the mixture with 0.05M Tris-HCl buffer (pH 8.0), and ultrasonically dispersing the mixture for 30 seconds to 1 minute each time; (500 mu L of buffer solution is added firstly, the centrifuge tube is placed into an ultrasonic instrument, the liquid level in the centrifuge tube and the page in the ultrasonic instrument form an angle of 45 degrees, the 500 mu L of buffer solution is added for secondary ultrasonic after the buffer solution is uniformly dispersed, the ultrasonic time is not more than 1 minute each time), the washing is repeated twice, the supernatant is removed after the last centrifugation, and the microsphere preservation solution is used for resuspension; and then centrifuging at 2000rpm for 3min, removing precipitates, and storing at 4 ℃ in a dark place to obtain the time-resolved fluorescent microspheres connected with the specific troponin I antibody. The preparation method of the BSA blocking solution comprises the following steps: 0.2g BSA and 0.075g glycine were weighed and added with water to a volume of 10 mL.

S4, using glass cellulose SB06(30mm multiplied by 300mm) as a sample pad material, soaking the sample pad material by using the sample pad treating solution, drying the sample pad material in an oven at 37 ℃ for 4h, and then pasting the sample pad on the bonding pad. Wherein, every 100mL of the sample pad treatment solution comprises the following components: tween-200.3 g, bovine serum albumin 0.6g, polyvinylpyrrolidone 0.6g, rabbit anti-human erythrocyte antibody 0.1g, and the balance Tris-HCl buffer solution.

S5, taking a piece of absorbent paper with the thickness of 22mm multiplied by 300cm, and sticking the absorbent paper on the NC membrane to obtain the test paper strip.

Example 4

The embodiment provides a preparation method of the test strip for detecting troponin I, which comprises the following steps:

and S1, taking the PVC rubber plate as a substrate, cutting the NC film into 25mm by 300mm, and pasting the non-sample-application surface of the NC film on the PVC rubber plate.

S2, diluting the cTn I monoclonal antibody with an antibody diluent until the concentration is 2mg/mL, spraying the diluted cTn I monoclonal antibody on a detection line (the distance between the detection line and one end of a binding pad is 8mm) of an NC membrane by using a scribing and metal spraying instrument at the speed of 100mm/S and the concentration of 1 mu L/cm under the condition that the indoor humidity is 55%, and drying the diluted cTn I monoclonal antibody in an oven at 37 ℃ for 2 hours for later use; similarly, goat anti-mouse IgG antibody was diluted with 1% sucrose in 0.05M Tris-HCl buffer, pH8.0, sprayed onto the NC membrane control line (12 mm from one end of the pad) at 100mm/s and 1. mu.L/cm with a gold-spraying streaking device at 55% room humidity, and dried in an oven at 37 ℃ for 2 h. Wherein the antibody diluent comprises sucrose and Tris-HCl buffer solution; in the antibody diluent, the mass concentration of sucrose is 1%; the concentration of Tris-HCl buffer was 0.05mol/L, pH8.

S3, soaking the bonding pad material with polyester film VL78(8mm multiplied by 300mm), and drying the bonding pad material in an oven at 37 ℃ for 4h for later use; then, 50 μ L of the specific troponin I antibody-linked time-resolved fluorescent microspheres stored in the microsphere storage solution were taken, centrifuged at 7500rpm for 20 minutes, the supernatant was discarded, 100 μ L of the microsphere redissolved solution was ultrasonically resuspended, and then sprayed onto the conjugate pad at a concentration of 100mm/s and 3 μ L/cm using a membrane spraying apparatus, and dried in an oven at 37 ℃ for 3 hours, and then the conjugate pad was adhered onto the NC membrane. Wherein, every 100mL of the combined pad treatment solution comprises the following components: tween-200.2 g, bovine serum albumin 0.5g, polyvinylpyrrolidone 0.5g, and the balance Tris-HCl buffer solution; the microsphere storage solution is prepared by weighing 0.2g BSA, adding 100mL Tris-HCl buffer solution with 0.05M, pH-8.0 and 1 ‰ Proclin 300; the microsphere composite solution comprises 0.05MM, Tris-HCl buffer solution with the pH value of 8.0, 10% of sucrose, 1% of BSA and 0.2% of Tween-20.

In addition, the preparation method of the specific troponin I antibody-linked time-resolved fluorescent microsphere comprises the following steps:

(1) taking 100 mu L of carboxyl fluorescent microspheres (10mg/mL), and suspending the microspheres in 800 mu L of MES buffer solution to obtain a microsphere solution; wherein the MES buffer solution is prepared by the following method: weighing 0.976g MES, pouring 98mL deionized water to dissolve completely, detecting the solution concentration by a pH meter, and adding dropwise 1M dilute hydrochloric acid solution to neutralize excessive HO when the pH is more than 6.0^-And when the pH is less than 6.0, adding 1M sodium hydroxide solution dropwise to neutralize excessive H⁺The pH was finally stabilized at 6.0 and finally made to 100mL with deionized water.

(2) NHS (5mg/mL) and EDC (5mg/mL) are prepared by MES buffer solution, 20 mu L of each solution is added into the microsphere solution, the final concentration is NHS (0.1mg/mL) and EDC (0.1mg/mL), the mixture is rotated, mixed and activated for 30 minutes at room temperature (25 ℃), the rotating speed is 10rpm, then the mixture is centrifugally washed (17500rpm is centrifuged to remove the supernatant and is suspended in 900 mu LMES buffer solution (wherein NHS can be mixed with EDC first and then added into microspheres, EDC cannot be added into microspheres first, otherwise microspheres are agglomerated), then 0.1mg of cTn I monoclonal antibody is added into the microsphere solution, the mixture is shaken and mixed evenly, and the reaction solution is rotated and reacted for 2 hours at room temperature (25 ℃) to obtain the reaction solution.

(3) Adding 100. mu.L of BSA blocking solution (20mg/mL) to the reaction solution, blocking the reaction at room temperature (25 ℃) for 2 hours, centrifuging at 17500rpm for 20 minutes, resuspending the mixture with 0.05M Tris-HCl buffer (pH 8.0), and ultrasonically dispersing the mixture for 30 seconds to 1 minute each time; (500 mu L of buffer solution is added firstly, the centrifuge tube is placed into an ultrasonic instrument, the liquid level in the centrifuge tube and the page in the ultrasonic instrument form an angle of 45 degrees, the 500 mu L of buffer solution is added for secondary ultrasonic after the buffer solution is uniformly dispersed, the ultrasonic time is not more than 1 minute each time), the washing is repeated twice, the supernatant is removed after the last centrifugation, and the microsphere preservation solution is used for resuspension; and then centrifuging at 2000rpm for 3min, removing precipitates, and storing at 4 ℃ in a dark place to obtain the time-resolved fluorescent microspheres connected with the specific troponin I antibody. The preparation method of the BSA blocking solution comprises the following steps: 0.2g BSA and 0.075g glycine were weighed and added with water to a volume of 10 mL.

S4, using glass cellulose SB06(30mm multiplied by 300mm) as a sample pad material, soaking the sample pad material by using the sample pad treating solution, drying the sample pad material in an oven at 37 ℃ for 4h, and then pasting the sample pad on the bonding pad. Wherein, every 100mL of the sample pad treatment solution comprises the following components: tween-200.2 g, bovine serum albumin 0.5g, polyvinylpyrrolidone 0.5g, rabbit anti-human erythrocyte antibody 0.08g, and the balance Tris-HCl buffer solution.

S5, taking a piece of absorbent paper with the thickness of 22mm multiplied by 300cm, and sticking the absorbent paper on the NC membrane to obtain the test paper strip.

The kit can be matched with a dry-type fluorescence immunoassay instrument to carry out hypersensitivity detection on troponin I, and the calibration process is as follows:

1. preparation of calibration solution

Using cTn I negative serum/whole blood or antigen diluent as buffer solution to prepare cTn I calibrator with the following concentration groups: 100/50/10/5/1/0.5/0.1/0.01 ng/mL. Each concentration group was tested in 4 replicates.

2. Drawing of calibration curve

(1) The reagent to be calibrated is extracted, each concentration group is repeatedly detected for 4 times, each test card is loaded with 80 mu L, and the T/C value is detected by a dry fluorescence immunoassay analyzer after waiting for 10 minutes of incubation time.

Note: serum and whole blood need to be tested separately. The sample application time interval of each test card should be consistent with the instrument detection time interval (if the current instrument detection needs 15 seconds, the sample application time interval of each test card is 15 seconds).

(2) Obtaining calibration data: according to the four-parameter fitting equation of the average value of the concentration and the corresponding T/C value, R is required to be more than 0.99.

Wherein, a standard curve chart corresponding to the test strip (hypersensitive cTn I product) prepared in the embodiment 2 is shown in figure 4; the blank concentration (0-0.1) and the linear T/C value of the test strip prepared in example 2 (hypersensitivity cTn I product) are shown in FIG. 5; the comparison graph of results of the hypersensitivity cTn I + MYO + CKMB triple card product and the hospital hypersensitivity cTn I in the province is shown in the attached figure 6; a comparison of results for hypersensitivity cTn I single card products and hospital-and-hospital hypersensitivity cTn I is shown in FIG. 7.

Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.

Claims (8)

1. A detection kit for hypersensitive troponin I, which comprises a test card and a calibration card, wherein the test card comprises a test strip, and the test strip is characterized by comprising:

a substrate;

the device comprises an NC film arranged on a substrate, wherein a detection line and a quality control line are respectively arranged on the NC film; the detection line comprises a troponin I monoclonal antibody, and the quality control line comprises a goat anti-mouse IgG antibody;

a sample pad;

a binding pad disposed between the NC membrane and the sample pad, the binding pad having time-resolved fluorescent microspheres linked with specific troponin I antibodies disposed thereon; and

and the water absorption paper is arranged on one side of the NC film, which is far away from the sample pad.

2. The troponin I detection kit of claim 1, wherein the sample pad is made of glass cellulose.

3. The troponin I measurement kit in accordance with claim 1, wherein the binding pad is made of polyester film.

4. The troponin I detection kit for hypersensitivity according to any one of claims 1 to 3, characterized in that the preparation method of the test strip comprises the following steps:

pasting the non-sample application surface of the NC film on the substrate;

diluting the troponin I monoclonal antibody with an antibody diluent, spraying the diluted troponin I monoclonal antibody on a detection line of an NC membrane, and drying;

diluting a goat anti-mouse IgG antibody with a buffer solution, spraying the diluted goat anti-mouse IgG antibody on a quality control line of an NC membrane, and drying;

soaking the bonding pad with a bonding pad treatment solution, drying, spraying time-resolved fluorescent microspheres connected with specific troponin I antibodies on the bonding pad, and sticking the bonding pad on the NC membrane;

soaking the sample pad with the sample pad treatment solution, drying, and sticking the sample pad on the bonding pad;

and (4) taking absorbent paper, and sticking the absorbent paper on the NC membrane to obtain the test paper strip.

5. The troponin I hypersensitivity assay kit of claim 4, characterized in that said antibody diluent comprises sucrose and Tris-HCl buffer; in the antibody diluent, the mass concentration of sucrose is 0.5-1.5%; the concentration of Tris-HCl buffer is 0.03-0.07 mol/L, and the pH value is 7.5-8.5.

6. The troponin I detection kit of one type of hypersensitivity according to claim 4, characterized in that, every 100mL of the conjugate pad treatment solution comprises the following components: tween-200.1-0.3 g, bovine serum albumin 0.4-0.6 g, polyvinylpyrrolidone 0.4-0.6 g, and the balance of Tris-HCl buffer solution.

7. The troponin I measurement kit of claim 4, wherein each 100mL of the sample pad treatment solution comprises the following components: 200.1-0.3 g of Tween-1, 0.4-0.6 g of bovine serum albumin, 0.4-0.6 g of polyvinylpyrrolidone, 0.06-0.1 g of rabbit anti-human erythrocyte antibody and the balance of Tris-HCl buffer solution.

8. The troponin I hypersensitivity kit according to claim 4, characterized in that the preparation method of the time-resolved fluorescent microspheres linked with the specific troponin I antibody comprises the following steps:

taking carboxyl fluorescent microspheres, and suspending the carboxyl fluorescent microspheres in MES buffer solution to obtain microsphere solution;

adding N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the microsphere solution, and then adding a troponin I monoclonal antibody for reaction to obtain a reaction solution;

and adding bovine serum albumin confining liquid into the reaction liquid for confinement, and then centrifuging and resuspending to obtain the time-resolved fluorescent microsphere connected with the specific troponin I antibody.

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