CN112505332A - High-sensitivity cTnI detection method and kit thereof - Google Patents
High-sensitivity cTnI detection method and kit thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cTnI detection methods, and particularly relates to a high-sensitivity cTnI detection method and a kit thereof. According to the detection method provided by the invention, a homogeneous phase chemiluminescence platform technology is utilized to introduce a dissociation agent such as trisodium citrate and Ethylene Diamine Tetraacetic Acid (EDTA) to dissociate the cTnI-cTnC dimer into single cTnI, so that the cTnI in a detection sample is ensured to be in a free state, the epitope is completely exposed, and the false negative risk is reduced; so that the cTnI content in the sample is increased; thereby improving the detection sensitivity. Compared with the traditional method, the detection method provided by the invention has the advantages of short immunoreaction time, low instrument requirement and strong reagent flexibility, and is beneficial to the transportation, storage and operation of products.
Description
Technical Field
The invention belongs to the technical field of cTnI detection methods, and particularly relates to a high-sensitivity cTnI detection method and a kit thereof.
Background
Currently, common detection techniques for cTnI include: colloidal gold method, enzyme linked immunosorbent assay (ELISA), fluorescence immunochromatography, chemiluminescence method, and the like. The colloidal gold method is a novel immunolabeling technology which applies colloidal gold as a tracer marker to antigen and antibody, and the colloidal gold can be combined with a plurality of other biomacromolecules, such as SPA, PNA, ConA and the like, besides being combined with protein; ELISA has become the leading topic in the field of analytical chemistry at present, is a special reagent analysis method, and is a novel immunoassay technology developed on the basis of an immunoenzyme technology; the fluorescence immunochromatography is a novel membrane detection technology based on antigen-antibody specific immunoreaction, takes strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) as a stationary phase, takes a test solution as a mobile phase, fixes a fluorescence labeling antibody or antigen on a connecting pad, and enables an analyte to move on the chromatography strip through capillary action; the chemiluminescence method is a method for measuring a reactant, a catalyst, a sensitizer and an inhibitor of a chemiluminescence reaction, and a reactant, a catalyst and a sensitizer in a coupling reaction by using chemiluminescence.
Because various influence factors exist in cTnI detection, the detection sensitivity of reagents of different platforms is uneven, and detection omission occurs sometimes;
(1) cTnI poor stability: researches find that cTnI is unstable and is easily acted by proteolytic enzyme, different parts are affected differently, and the N end and the C end are easily degraded;
(2) cTnI exists in a variety of forms: the spatial conformation of the epitope of the cTnI in free or combined form may be different, and when the cTnI in a sample to be detected exists in a combined form, the epitope of the cTnI is easily shielded, so that the concentration of the cTnI which can be detected in the sample is reduced (the cTnI is mainly in the combined form, and more than 90 percent of cTnI-cTnC dimers);
the traditional chemiluminescence method has relatively high sensitivity, overcomes the influence factors of signal interference and manual operation, greatly improves the detection sensitivity, still has multiple steps such as washing separation and the like, thereby causing long detection time and failing to meet the requirement of rapid detection, and the detection sensitivity still needs to be improved for the hypersensitive requirement of cTnI at the present stage.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a high-sensitivity cTnI detection method and a kit thereof. According to the detection method provided by the invention, the dissociation reagent is introduced, so that the cTnI content in the sample is increased, the cTnI in the detection sample can be ensured to be in a free state, the epitope is completely exposed, the false negative risk is reduced, and the detection sensitivity is improved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a cTnI detection kit comprises an acceptor microsphere-antibody compound, a donor microsphere-avidin compound, a biotinylated antibody compound and a reagent storage buffer solution.
Preferably, the preparation method of the receptor microsphere-antibody complex comprises the following steps:
(1) pretreatment of the acceptor microspheres: sucking microsphere suspension with mass concentration of 10mg/mL, placing in a high-speed centrifuge, and centrifuging to remove supernatant; adding purified water with 2 times of the volume of the microsphere suspension; ultrasonic mixing, cleaning, and centrifuging at high speed to remove supernatant; then suspending the mixture to 10mg/mL by using 0.05M PH5.0 MES buffer solution for later use;
(2) and (2) mixing the microspheres in the step (1) according to the mass ratio of the microspheres to EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) to NHS (N-hydroxysuccinimide) of 2:1:1, suspending the microspheres to 5mg/mL by using the MES buffer solution, carrying out rotary reaction at 30 ℃ for 30min, and washing twice by using purified water; suspending in 0.05M HEPES buffer solution (pH 8.0) to 10mg/mL for use;
(3) labeling the microspheres and the receptor microspheres in the step (2) with corresponding receptor microsphere labeled antibodies, and mixing the labeled antibodies with a mixture of 20:1, suspending the microspheres to 5mg/mL with the HEPES buffer solution, performing rotary incubation for 2h at 30 ℃ in the dark, adding 0.01% BSA, performing further incubation for 30min, centrifuging to remove the supernatant, washing twice with PBST (pH 7.4, 0.5% Tween 20), and suspending to 20mg/mL with 0.05M pH7.4 TRIS buffer solution at 2-8 ℃ for storage.
Preferably, the specific operation of high speed centrifugation in step (1) of the preparation method of the receptor microsphere-antibody complex is centrifugation at 12000rpm for 20 min.
Preferably, the preparation method of the donor microsphere-avidin complex comprises the following steps:
(1) pretreatment of donor microspheres: sucking microsphere suspension with the mass concentration of 10mg/mL into a high-speed centrifuge, and centrifuging to remove supernatant; adding purified water with 2 times of the volume of the microsphere suspension; ultrasonic mixing, cleaning, and centrifuging at high speed to remove supernatant; then suspending the mixture to 10mg/mL by using 0.05M PH5.0 MES buffer solution for later use;
(2) mixing the microspheres in the step (1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide according to a mass ratio of 2:1:1, suspending the microspheres to 5mg/ml by using MES buffer solution, carrying out rotary reaction at 30 ℃ for 30min, and washing twice by using purified water; suspending in 0.05M buffer pH 9.0Na2HPO4 to 10 mg/mL;
(3) mixing the microspheres in the step (2) and streptavidin in a mass ratio of 20:1, and using the Na2HPO4Suspending the microspheres to 5mg/mL in buffer, rotary incubating at 30 ℃ for 4h in the dark, adding 0.01% BSA, incubating for 30min, centrifuging to remove supernatant, washing twice with PBST (pH 7.4, 0.5% Tween 20), suspending in 0.05M pH7.4 TRIS buffer to 20mg/mL, and storing at 2-8 ℃ for further use.
Preferably, the high speed centrifugation in step (1) of the preparation method of the donor microsphere-avidin complex is specifically operated to centrifuge at 12000rpm for 20 min.
Preferably, the preparation method of the biotinylated antibody complex comprises the following steps:
(1) preparing biotin: redissolving the purchased biotin freeze-dried powder to 1mg/mL by using DMSO;
(2) the method comprises the following steps of (1) marking an antibody of a receptor microsphere and biotin according to the mass ratio of 20:1 into 0.01M PBS buffer solution with pH7.4, and rotary incubating for 2h at 25 ℃; dialyzing the incubated antibody biotin solution with 0.01M PBS buffer solution with pH7.4 at 2-8 deg.C for 4h, repeating the dialysis for 3 times, and finally storing the biotinylated antibody complex at 2-8 deg.C to a final concentration of 0.4 mg/mL.
Preferably, the reagent preservation buffer is used for preserving the acceptor microsphere-antibody complex, donor microsphere-avidin complex, biotinylated antibody complex.
Preferably, the reagent preservation buffer comprises TRIS, a NaCl solution, a BSA solution, a sucrose solution, a bovine gamma-globulin solution, Tween 20, Proclin300, a dissociation agent and water; the reagent preservation buffer solution is characterized in that the final pH value is 7.4, the adding amount of TRIS is 50mM, the final mass percentage concentration of the NaCl solution is 0.9%, the final mass percentage concentration of the BSA solution is 1.5%, the final mass percentage concentration of the sucrose solution is 1.0%, the final mass percentage concentration of the bovine gamma-globulin solution is 0.5%, the final mass percentage concentration of the Tween 20 solution is 0.2%, the final mass percentage concentration of the Proclin300 solution is 0.05%, and the dissociation agent contains one or two of an EDTA solution with the final mass percentage concentration of 0.1-1.0% and a trisodium citrate solution with the final mass percentage concentration of 0.2-1.5%.
Preferably, the reagent preservation buffer solution can also comprise one of a Tween 80 solution with a final mass percentage concentration of 0.5% and a TritonX-100 solution with a final mass percentage concentration of 0.5%.
The invention also provides a method for detecting cTnI by using the cTnI detection kit, which comprises the following steps: adding 20 mu L of sample into a sample hole, then adding 50 mu L of each of the receptor microsphere-antibody complex, the donor microsphere-avidin complex and the biotinylated antibody complex, placing at 37 ℃ and incubating for 15min, and carrying out signal detection; the working solution concentrations of the acceptor microsphere-antibody complex, the donor microsphere-avidin complex and the biotinylated antibody complex are respectively diluted to 0.1mg/mL, 0.1mg/mL and 1 mu g/mL by using reagent preservation buffer solution.
In the research process, the inventor finds that in the actual detection process, the adding sequence of three reagents, namely a biotinylated antibody (reagent A), an acceptor microsphere-antibody complex (reagent B) and a donor microsphere-avidin complex (reagent C) is not unique, and after the sample is added, 50 mu L of each of the reagent A, the reagent B and the reagent C can be added; reagent A + reagent B can be added, and 100 μ L of mixed reagent and 50 μ L of reagent C can be added; the reagent A50 μ L and the reagent B + reagent C100 μ L can be added; reagent A + reagent B + reagent C may be added to mix with 150. mu.L of reagent; and the sequence of addition does not have great influence on the final detection result.
Meanwhile, when the reagent storage buffer is prepared, a dissociation agent is added to dissociate the cTnI-cTnI dimer, so that the cTnI content in a sample is increased, and the detection sensitivity can be improved, and the inventor finds that good dissociation effect can be achieved by adding EDTA and trisodium citrate either or by mixing the EDTA and the trisodium citrate when selecting the dissociation agent: when the EDTA is singly added, the concentration range of the EDTA is 0.1 to 1.0 percent; the concentration range of the trisodium citrate is 0.5% -1.5%; when mixed, the concentration range of EDTA is 0% -0.2%, and the concentration range of trisodium citrate is 0.2% -0.5%.
Compared with the prior art, the cTnI detection method and the kit thereof provided by the invention have the following advantages:
(1) according to the cTnI detection method provided by the invention, in the detection process, the dissociating agent is added, so that the combined cTnI is converted into the free type, the sample test result is more accurate, the sensitivity is higher, and the missed detection risk is reduced;
(2) the detection method provided by the invention omits a plurality of steps such as washing, separation and the like, thereby shortening the detection time, improving the detection efficiency, being flexible to use, and realizing the test of a single sample and the test of a plurality of samples;
(3) the cTnI detection kit provided by the invention can change three reagents in the kit into 2 or 1 (mixed according to different forms), has high flexibility, and is beneficial to transportation, storage and operation of products.
Drawings
FIG. 1 shows the comparison of four-parameter fitting curves of different reagent-preserving buffers;
FIG. 2 is a comparison of TritonX-100 in the reagent storage buffer described in example 7 at different concentrations with a four-parameter fit curve;
FIG. 3 shows the comparison results of four-parameter fitting curves when reagent A, reagent B and reagent C are added in different ways;
FIG. 4 is a comparison of four parameter fit curves using different dissociating agents;
FIG. 5 shows the comparison results of four-parameter fitting curves at different EDTA concentrations;
FIG. 6 shows the comparison of four-parameter fitting curves at different concentrations of trisodium citrate;
FIG. 7 is a comparison of four parameter fit curves using different combinations of dissociating agents;
FIG. 8 is a schematic diagram of the detection of cTnI ligation by the double antibody sandwich method;
fig. 9 is a schematic diagram of the signal generation when cTnI is present in the system.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The corresponding antibodies are receptor microsphere labeled antibodies and biotin labeled antibodies, and can be purchased from Fengcheng Biotechnology Limited, receptor microsphere labeled antibodies, cargo number: CTNI-MCAB-29; biotin-labeled antibody, cat No.: CTNI-MCAB-30; the streptavidin may be purchased from Sigma reagent, cat # S4762; the biotin lyophilized powder can be purchased from Sigma reagent company, Cat code No. B3295; the homogeneous phase luminometer is a Baotai biological 12-channel luminometer, and can be purchased from Baotai biotechnology limited company, model: BIO-FG 12; the Yapei instrument is available from Yapei, USA, and has model number of i-2000; the HEPES buffer can be purchased from Sigma reagent company, CAS number: 7365-45-9; the TRIS buffer can be purchased from Sigma reagent company, CAS No.: 77-86-1; the MES buffer was purchased from Sigma reagent company, CAS No.: 145224-94-8; the cTnI sample is an antigen reference substance which is diluted by a cTnI antigen (cTnI antigen) with an antigen diluent to different concentration gradients, and the cTnI antigen manufacturer: xiamen-heart company, cat #: ZKP 13.
EXAMPLE 1A reagent storage buffer
The reagent preservation buffer solution comprises TRIS, a NaCl solution, a BSA solution, a sucrose solution, a bovine gamma-globulin solution, Tween 20, Proclin300, a dissociation agent and water; the final pH value of the reagent preservation buffer solution is 7.4, the addition amount of TRIS is 50mM, the final mass percentage concentration of the NaCl solution is 0.9%, the final mass percentage concentration of the BSA solution is 1.5%, the final mass percentage concentration of the sucrose solution is 1.0%, the final mass percentage concentration of the bovine gamma-globulin solution is 0.5%, the final mass percentage concentration of the Tween 20 solution is 0.2%, the final mass percentage concentration of the Proclin300 solution is 0.05%, and the dissociation agent is an EDTA solution with the final mass percentage concentration of 0.5%.
Example 2A reagent storage buffer
The reagent preservation buffer solution comprises TRIS, a NaCl solution, a BSA solution, a sucrose solution, a bovine gamma-globulin solution, Tween 20, Proclin300, a Tween 80 solution, a dissociation agent and water; the final pH value of the reagent preservation buffer solution is 7.4, the addition amount of TRIS is 50mM, the final mass percentage concentration of the NaCl solution is 0.9%, the final mass percentage concentration of the BSA solution is 1.5%, the final mass percentage concentration of the sucrose solution is 1.0%, the final mass percentage concentration of the bovine gamma-globulin solution is 0.5%, the final mass percentage concentration of the Tween 20 solution is 0.2%, the final mass percentage concentration of the Proclin300 is 0.05%, the final mass percentage concentration of the Tween 80 solution is 0.5%, and the dissociation agent is a trisodium citrate solution with the final mass percentage concentration of 0.5%.
EXAMPLE 3A reagent storage buffer
The reagent preservation buffer solution comprises TRIS, a NaCl solution, a BSA solution, a sucrose solution, a bovine gamma-globulin solution, Tween 20, Proclin300, a TritonX-100 solution, a dissociation agent and water; the final pH value of the reagent preservation buffer solution is 7.4, the addition amount of TRIS is 50mM, the final mass percentage concentration of the NaCl solution is 0.9%, the final mass percentage concentration of the BSA solution is 1.5%, the final mass percentage concentration of the sucrose solution is 1.0%, the final mass percentage concentration of the bovine gamma-globulin solution is 0.5%, the final mass percentage concentration of the Tween 20 solution is 0.2%, the final mass percentage concentration of the Proclin300 is 0.05%, the final mass percentage concentration of the TritonX-100 solution is 0.5%, and the dissociation agent comprises an EDTA solution with the final mass percentage concentration of 0.2% and a trisodium citrate solution with the final mass percentage concentration of 0.5%.
Example 4 a cTnI assay kit
The cTnI detection kit consists of a biotinylated antibody complex (reagent A), an acceptor microsphere-antibody complex (reagent B), a donor microsphere-avidin complex (reagent C) and a reagent preservation buffer solution;
the preparation method of the receptor microsphere-antibody compound comprises the following steps:
(1) pretreatment of the acceptor microspheres: sucking microsphere suspension with mass concentration of 10mg/mL, placing in a high-speed centrifuge, centrifuging at 12000rpm for 20min, and removing supernatant; adding purified water with 2 times of the volume of the microsphere suspension; ultrasonically mixing uniformly and cleaning, then placing in a high-speed centrifuge with 12000rpm for centrifuging for 20min, and removing the supernatant; then suspending the mixture to 10mg/mL by using 0.05M PH5.0 MES buffer solution for later use;
(2) and (2) mixing the microspheres obtained in the step (1) according to the mass ratio of microspheres to EDC to NHS of 2:1:1, suspending the microspheres to 5mg/mL by using MES buffer solution, carrying out rotary reaction at 30 ℃ for 30min, and washing twice by using purified water; suspending in 0.05M HEPES buffer solution (pH 8.0) to 10mg/mL for use;
(3) mixing the microspheres obtained in the step (2) with corresponding antibodies in a ratio of 20:1, suspending the microspheres with the HEPES buffer solution to 5mg/mL, performing rotary incubation for 2h at 30 ℃ in a dark condition, adding 0.01% BSA, performing further incubation for 30min, centrifuging to remove supernatant, washing with PBST (pH 7.4, 0.5% Tween 20) twice, suspending with 0.05M pH7.4 TRIS buffer solution to 20mg/mL, and storing at 2-8 ℃ for later use;
the preparation method of the donor microsphere-avidin compound comprises the following steps:
(1) pretreatment of donor microspheres: sucking microsphere suspension with mass concentration of 10mg/mL into a high-speed centrifuge, centrifuging at 12000rpm for 20min, and removing supernatant; adding purified water with 2 times of the volume of the microsphere suspension; ultrasonically mixing and cleaning, centrifuging in a high-speed centrifuge at 12000rpm for 20min, and removing supernatant; then suspending the mixture to 10mg/mL by using 0.05M PH5.0 MES buffer solution for later use;
(2) mixing the microspheres, EDC and NHS in the step (1) according to a mass ratio of 2:1:1, suspending the microspheres to 5mg/ml by using MES buffer solution, carrying out rotary reaction at 30 ℃ for 30min, and washing with purified water twice; using 0.05M pH 9.0Na2HPO4Suspending the buffer solution in10mg/mL for standby;
(3) mixing the microspheres in the step (2) and streptavidin in a mass ratio of 20:1, and using the Na2HPO4The microspheres were spun in buffer to 5mg/mL for 4h at 30 ℃ in the dark, then 0.01% BSA was added for another 30min, the supernatant was centrifuged off, washed twice with PBST (pH 7.4, 0.5% Tween 20), suspended in 0.05M pH7.4 TRIS buffer to 20mg/mL and stored at 2-8 ℃ for further use.
The preparation method of the biotinylated antibody complex comprises the following steps:
(1) preparing biotin: redissolving the purchased biotin freeze-dried powder to 1mg/mL by using DMSO;
(2) and (3) mixing the antibody biotin according to a mass ratio of 20:1 into 0.01M PBS buffer solution with pH7.4, and rotary incubating for 2h at 25 ℃; dialyzing the incubated antibody biotin solution with 0.01M PBS buffer solution with pH7.4 at 2-8 deg.C for 4h, repeating the dialysis for 3 times, and finally storing the biotinylated antibody complex at 2-8 deg.C to a final concentration of 0.4 mg/mL.
The reagent storage buffer was prepared using the formulation described in example 1.
Example 5 a cTnI assay kit
The cTnI detection kit is similar to example 4;
the difference from example 4 is that the reagent storage buffer of example 5 was prepared using the formulation described in example 2.
Example 6 cTnI detection kit
The cTnI detection kit is similar to example 4;
the difference from example 4 is that the reagent storage buffer of example 6 was prepared using the formulation described in example 3.
The cTnI detection method comprises the following operation processes: the biotinylated antibody (reagent A), the acceptor microsphere-antibody complex (reagent B) and the donor microsphere-avidin complex (reagent C) were diluted to 1. mu.g/mL, 0.1mg/mL and 0.1mg/mL respectively with the reagent storage buffer described in example 1, 20. mu.L of the cTnI sample was taken, 50. mu.L each of the reagent A, the reagent B and the reagent C was added thereto in sequence, and the mixture was incubated on a homogeneous luminescence apparatus of Baotai Biotech Co., Ltd for 15min to detect a light signal.
The cTnI detection method comprises the following operation processes: the biotinylated antibody (reagent A), the acceptor microsphere-antibody complex (reagent B) and the donor microsphere-avidin complex (reagent C) were diluted to 1. mu.g/mL, 0.1mg/mL and 0.1mg/mL respectively with the reagent storage buffer described in example 2, 20. mu.L of the cTnI sample was taken, the reagent A and the reagent B were mixed uniformly, 100. mu.L of the cTnI sample solution was added, 50. mu.L of the reagent C was then added, and the mixture was incubated on a homogeneous luminescence apparatus of Baotai Biotech Co., Ltd for 15min to detect the optical signal.
The cTnI detection method comprises the following operation processes: the biotinylated antibody (reagent A), the acceptor microsphere-antibody complex (reagent B) and the donor microsphere-avidin complex (reagent C) were diluted to 1. mu.g/mL, 0.1mg/mL and 0.1mg/mL respectively with the reagent storage buffer described in example 3, 20. mu.L of the cTnI sample was taken, the reagent B and the reagent C were mixed uniformly, 100. mu.L of the cTnI sample solution was added, 50. mu.L of the reagent A was then added, and the mixture was incubated on a homogeneous luminescence apparatus of Baotai Biotech Co., Ltd for 15min to detect the optical signal.
Embodiment 10 a cTnI detection method
The cTnI detection method comprises the following operation processes: the biotinylated antibody (reagent A), the acceptor microsphere-antibody complex (reagent B) and the donor microsphere-avidin complex (reagent C) were diluted to 1. mu.g/mL, 0.1mg/mL and 0.1mg/mL respectively with the reagent storage buffer described in example 3, 20. mu.L of the cTnI sample was taken, the reagent A, the reagent B and the reagent C were mixed uniformly, 150. mu.L of the mixed solution was added to the cTnI sample solution, and the light signal was detected after incubation for 15min on a homogeneous luminescence instrument of Baotai Biotech Co., Ltd.
Experimental example 1 Effect of different reagent buffers on the test results
1. Test samples: the reagent buffer prepared in examples 1-3 of the present invention is prepared according to formula 1 (reference: development of free chorionic gonadotropin and pregnancy related serum protein photoluminescence immunoassay reagent): 25mM HEPES, 10mM DTPA, 2mg/mL Dextran-500, 50mM NaCl, 0.5% BSA, 0.02% bovine gamma-globulin, 0.1% Tween 20, 0.01% Proclin300, 0.01% gentamicin (pH-7.4); reference recipe 2: 50mM TRIS, 50mM NaCl, 0.2% Tween 20, 0.2% PEG6000, 0.05% Proclin300, the formulation being referenced to the relevant turbidimetric reagent assay buffer.
2. The test method comprises the following steps: when the concentration of cTnI is detected, the reagent buffer solution is respectively used for diluting the reagent storage buffer solution to the concentration of the invention, after dilution, the detection method of the invention embodiment 7 is adopted for detection, and the linearity and the stability of the detection result are measured.
3. And (3) test results: the results of the five buffer linear comparison tests are shown in table 1 and fig. 1; the stability of the five reagent buffer solutions is shown in tables 2-6, respectively.
TABLE 1 Linear comparison of different reagent buffers
As is clear from the test results in Table 1 and FIG. 1, there was no significant difference in the linear comparison results and signal values between the five reagent buffers.
Table 2 stability results for reference formulation 1
Table 3 stability results for reference formulation 2
Table 4 stability results of reagent buffer prepared in example 1
Table 5 stability results of reagent buffer prepared in example 2
Table 6 stability results of reagent buffer prepared in example 2
Therefore, the deviation of the five groups of buffer solution, Baotai self-made formula 3 is basically within +/-10% when the temperature is accelerated to 14d and is compared with 3d at 37 ℃, and the stability is good; the other groups are in descending trend from 3d to 14d all the time, and the stability is poor; from this, it was determined that the key component in the stability buffer formulation was TritonX-100.
Test example 2 screening of TritonX-100 concentration in buffer
1. Test samples: TritonX-100 with the concentration of 0.05%, TritonX-100 with the concentration of 0.1%, TritonX-100 with the concentration of 0.5%, TritonX-100 with the concentration of 1% and TritonX-100 with the concentration of 2%, and the other reagents are respectively the same as the reagent buffer solution in the embodiment 3 of the invention.
2. The test method comprises the following steps: the concentration of triton x-100 in the reagent buffer of example 3 was changed to the concentration described for the above test sample, and the other components were diluted to the working solution concentration described in the present invention, and then the stability of the test results was tested by the method described in example 7.
3. And (3) test results: the linear comparison results of TritonX-100 with different concentrations are shown in Table 7 and FIG. 2, and the stability results are shown in tables 8-12.
TABLE 7 TritonX-100 Linear comparison results for different concentrations
As can be seen from Table 7 and FIG. 2, the linearity and signal value of the final buffer solution were not significantly different when TritonX-100 was added at different concentrations.
TABLE 8 stability of buffer with 0.05% Triton X-100 addition
TABLE 9 stability of buffer with 0.1% Triton X-100 addition
TABLE 10 stability of buffer with 0.5% Triton X-100 addition
TABLE 11 stability of buffer solution with 1.0% Triton X-100 added
TABLE 12 stability of buffer with 2.0% Triton X-100 addition
Therefore, TritonX-100 with the concentration of 0.1% -1.0% in the five groups of buffer solutions is accelerated to 14d and 3d at 37 ℃ and the comparative deviation is within +/-10, so that the stability is good; the other groups are in descending trend from 3d to 14d all the time, and the stability is poor; from this, it was determined that the optimum concentration of TritonX-100 in example 3 was 0.1% to 1.0%.
Test example 3 comparison of different addition sequences of reagents
1. Test samples: the kit prepared in example 6;
2. the test method comprises the following steps: in the actual detection process, reagents A, B and C in the kit are mixed according to different sequences and are stored separately for A, B, C; mixing the reagent A and the reagent B, and storing the reagent C separately; storing the reagent A separately, and mixing the reagent B and the reagent C; and mixing the reagent A, the reagent B and the reagent C, and detecting the cTnI by using the detection method.
3. And (3) test results: the results of the linear comparison of the kits for different reagent composition modes are shown in Table 13 and FIG. 3.
TABLE 13 Linear comparison results of kits with different reagent composition modes
As is clear from Table 7 and FIG. 3, the linearity results and the signal value detection results of the kit stored in these 4 modes were not clearly different from each other.
TABLE 14 stability of the kit when reagent composition is A, B, C
TABLE 15 stability of the kit when the reagent composition is A + B, C
TABLE 16 stability of the kit at reagent composition A, B + C
TABLE 17 stability of the kit when the reagent composition is A + B + C
It is clear from this that the 4 reagent composition patterns are in equilibrium with the deviation of the 14d and 3d accelerated at 37 ℃ being within ± 10%, and therefore the 4 reagent composition patterns are all good in the stability of the kit.
Test example 4 screening of different dissociating Agents
1. Test samples: a 0.5% dissociation agent EDTA was added to the reagent buffer (containing no dissociation agent and other components) prepared in example 3 of the present invention; adding trisodium citrate serving as a dissociating agent with the concentration of 0.5% into the reagent buffer solution prepared in the embodiment 3 of the invention; adding a detergent dissociating agent-Gly-HCl + 7% TritonX-100+ 5% CNAP into the reagent buffer solution prepared in the embodiment 3 of the invention;
2. the test method comprises the following steps: and (3) replacing the dissociating agents in the reagent buffer solution with the test samples, keeping other components in the buffer solution unchanged, diluting the reagents A, B and C to working solution concentration by using the reagent buffer solution respectively, detecting by using the method disclosed by the embodiment 7 of the invention after dilution, and judging the stability of the detection result.
3. And (3) test results: the specific comparison results of different dissociation agents are shown in Table 18 and FIG. 4, and in FIG. 4, signal-1 to signal-4 represent the signals obtained when no dissociation agent, the dissociation agent in the literature, EDTA at a concentration of 0.5% according to the present invention as the dissociation agent, and trisodium citrate at a concentration of 0.5% according to the present invention as the dissociation agent, respectively; the Yapei concentration is cTnI concentration detected by a Yapei instrument, and the Yapei concentration sample source is as follows: cTnI antigen (cTnI antigen) antigen reference samples of different concentration gradients diluted with antigen diluent, cTnI antigen manufacturer: xiamen-heart company, cat #: ZKP13, Yapei concentration samples involved in the subsequent steps are consistent with the samples at this stage.
(note: antigen dilution PBS + 10% newborn bovine serum, newborn bovine serum manufacturer: Dening biology, specification: 500 mL/bottle, PBS formulation (1L as an example):potassium dihydrogen phosphate(KH2PO4)：0.24g，Disodium hydrogen phosphate(Na2HPO4)：1.44g，Sodium chloride(NaCl): 8g, and finally adjusting the pH value to 7.4; the pharmaceutical manufacturer: sigma, specification: 500 g/bottle).
TABLE 18 results of specific comparison of different dissociating agents
Thus, compared with the control non-dissociating agent linear R2-0.8580, the dissociating agent linear R2-0.8500, 0.5% EDTA R2-0.9485, 0.5% trisodium citrate R2-0.9688 in the literature have poor dissociating effect, and 0.5% EDTA and 0.5% trisodium citrate have better dissociating effect.
Test example 5 concentration screening of dissociating agent EDTA
1. Test samples: EDTA solutions of different concentrations were added to the reagent buffers described in example 3 (without a dissociation agent), and the concentrations of the added EDTA were 0.1%, 0.2%, 0.5%, 1.0%, and 1.5%, respectively, to prepare 5 different reagent buffers;
2. the test method comprises the following steps: and replacing the final concentration of the EDTA solution in the reagent buffer solution with the concentration of the test sample, diluting the reagent A, the reagent B and the reagent C to the concentration of the working solution, and detecting the cTnI by adopting the method in the embodiment 7 after dilution, wherein the specificity and the stability of the detection result are detected.
3. And (3) test results: the results of comparison of the EDTA specificities at different concentrations are shown in Table 19 and FIG. 5, and in FIG. 5, signals 1 to 6 represent signals obtained when EDTA was used at a concentration of 0.1%, 0.2%, 0.5%, 1.0% and 1.5%, respectively, without a dissociating agent; the stability results of EDTA at each concentration are shown in tables 20-25.
TABLE 19 comparison of buffer specificity at different concentrations of EDTA
As shown by the comparison of R2 in each group in Table 19 and FIG. 4, the EDTA concentration in the range of 0.2% to 1.0% has better dissociation effect.
TABLE 20 stability of buffer without dissociating agent
Table 21: stability of buffer when EDTA was added at a concentration of 0.1%
Table 22: stability of buffer when EDTA was added at a concentration of 0.2%
Table 23: stability of buffer when EDTA was added at a concentration of 0.5%
Table 24: stability of buffer when EDTA was added at a concentration of 1.0%
Table 25: stability of buffer when EDTA was added at a concentration of 1.5%
Therefore, compared with the group without the dissociator, the EDTA concentration is accelerated to be within +/-10% at 37 ℃ until the 14d and 3d are compared and deviated, and the EDTA is in an equilibrium state and has good stability; when the EDTA concentration is 1.5%, the reduction amplitude is continuously increased along with the increase of the acceleration time, and the EDTA is in an unstable state, so that the EDTA use concentration is 0.1-1.0%.
Test example 6 dissociating agent trisodium citrate concentration screening
1. Test samples: to the reagent buffer solution (not containing the dissociation agent) described in example 3, trisodium citrate solutions of different concentrations were added, and the concentrations of the added trisodium citrate were 0.2%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively, to prepare 6 different reagent buffer solutions;
2. the test method comprises the following steps: and replacing the final concentration of the trisodium citrate solution in the reagent buffer solution with the concentration of the test sample, diluting the reagent A, the reagent B and the reagent C to the concentration of a working solution, and detecting the specificity and the stability of the detection result by adopting the method in the embodiment 7 after dilution.
3. And (3) test results: specific comparison results of trisodium citrate with different concentrations are shown in tables 26 and 6, and in FIG. 6, signals 1 to 6 represent signals of trisodium citrate with a concentration of 0.2%, trisodium citrate with a concentration of 0.5%, trisodium citrate with a concentration of 1.0%, trisodium citrate with a concentration of 1.5%, and trisodium citrate with a concentration of 2.0%, respectively, without a dissociating agent, and the stability results of trisodium citrate with each concentration are shown in tables 27 to 31.
TABLE 26 specific comparison of trisodium citrate at different concentrations
Thus, the comparison of R2 in each group in Table 26 and FIG. 5 shows that the trisodium citrate concentration is better at 0.5% -1.5% dissociation.
TABLE 27 stability of buffer when adding trisodium citrate as dissociating agent at a concentration of 0.2%
TABLE 28 stability of buffer with addition of trisodium citrate as dissociating agent at a concentration of 0.5%
TABLE 29 stability of buffer when trisodium citrate, a dissociating agent, is added at a concentration of 1.0%
TABLE 30 stability of buffer when adding trisodium citrate as dissociating agent at a concentration of 1.5%
TABLE 31 stability of buffer when adding trisodium citrate as dissociating agent at a concentration of 2.0%
Therefore, compared with the group without the dissociator, the concentration of the trisodium citrate is accelerated to be within +/-10% from 0.2-1.5% at 37 ℃ until the comparison deviation between 14d and 3d is within +/-10%, and the trisodium citrate is in a balanced state and has good stability; when the concentration of the trisodium citrate is 2.0%, the amplitude of the trisodium citrate is continuously increased along with the increase of the acceleration time, and the trisodium citrate is in an unstable state; trisodium citrate is used in a concentration of 0.2% to 1.5%.
Test example 7 dissociating agent combination screening
1. Test samples: to the reagent buffer prepared in example 3 (without the dissociating agent) was added the dissociating agents of the combinations described in table 32 below, respectively;
TABLE 32 different debonder combinations
2. The test method comprises the following steps: the dissociation agents in the reagent buffer solutions were replaced with the reagents and concentrations described in table 32 above, and then used to dilute the reagent a, reagent B, and reagent C to working solution concentrations, after dilution, the cTnI was detected by the method described in example 7, and the specificity and stability of the detection results were determined.
3. And (3) test results: the results of linear comparisons of buffers with different combinations of the dissociating agents are shown in Table 33 and FIG. 7, in FIG. 7, signals 1 to 10 represent signals obtained when the dissociating agents were added to no dissociating agent, combination 1, combination 2, combination 3, combination 4, combination 5, combination 6, combination 7, combination 8, combination 9 and combination 10, respectively, and the results of stability of buffers with different combinations of the dissociating agents are shown in tables 34 to 44.
TABLE 33 Linear comparison of buffers with different combinations of dissociating agents
From this, it can be seen from the comparison of group R2 in FIG. 7 and Table 33 that the range of concentrations of the combinations, EDTA: 0% -0.2%, trisodium citrate: 0.2 to 0.5 percent.
TABLE 34 stability of buffer without addition of dissociating agent
TABLE 35 stability of buffer when adding the dissociation agent described in combination 1
Table 36: stability of buffer when the dissociation agent described in combination 2 was added
Table 37: stability of buffer when the dissociation agent described in combination 3 was added
Table 38: stability of buffer when the dissociation agent described in combination 4 was added
Table 39: stability of buffer when the dissociation agent described in combination 5 was added
Table 40: stability of buffer when the dissociation agent described in combination 6 was added
Table 41: stability of buffer when the dissociation agent described in combination 7 was added
Table 42: stability of buffer when the dissociation agent described in combination 8 was added
Table 43: stability of buffer when the dissociation agent described in combination 9 was added
Therefore, the combination of the dissociation agents with different concentrations accelerates to 14d and has the comparative deviation of 3d within +/-10% at 37 ℃, is in an equilibrium state and has good stability; combination range of dissociation agents, EDTA: 0% -0.2%, trisodium citrate: 0.2 to 0.5 percent.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The cTnI detection kit is characterized by consisting of an acceptor microsphere-antibody complex, a donor microsphere-avidin complex, a biotinylated antibody complex and a reagent storage buffer.
2. The cTnI detection kit of claim 1, wherein the method for preparing the receptor microsphere-antibody complex comprises the steps of:
(1) pretreatment of the acceptor microspheres: sucking microsphere suspension with mass concentration of 10mg/mL, placing in a high-speed centrifuge, and centrifuging to remove supernatant; adding purified water with 2 times of the volume of the microsphere suspension; ultrasonic mixing, cleaning, and centrifuging at high speed to remove supernatant; then suspending the mixture to 10mg/mL by using 0.05M PH5.0 MES buffer solution for later use;
(2) mixing the microspheres obtained in the step (1) with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 2:1:1, suspending the microspheres to 5mg/mL by using the MES buffer solution, carrying out rotary reaction at 30 ℃ for 30min, and washing twice by using purified water; suspending the mixture to 10mg/mL by using 0.05M PH8.0HEPES buffer solution for later use;
(3) labeling the microspheres and the receptor microspheres in the step (2) with corresponding receptor microsphere labeled antibodies, and mixing the labeled antibodies with a mixture of 20:1, suspending the microspheres to 5mg/mL with the HEPES buffer solution, performing rotary incubation for 2h at 30 ℃ in the dark, adding 0.01% BSA, performing further incubation for 30min, centrifuging to remove the supernatant, washing twice with PBST (pH 7.4, 0.5% Tween 20), and suspending to 20mg/mL with 0.05M pH7.4 TRIS buffer solution at 2-8 ℃ for storage.
3. The cTnI detection kit of claim 2, wherein the high speed centrifugation in step (1) of the method for preparing the acceptor microsphere-antibody complex is performed at 12000rpm for 20 min.
4. The cTnI detection kit of claim 1, wherein the method of preparing the donor microsphere-avidin complex comprises the steps of:
(1) pretreatment of donor microspheres: sucking microsphere suspension with the mass concentration of 10mg/mL into a high-speed centrifuge, and centrifuging to remove supernatant; adding purified water with 2 times of the volume of the microsphere suspension; ultrasonic mixing, cleaning, and centrifuging at high speed to remove supernatant; then suspending the mixture to 10mg/mL by using 0.05M PH5.0 MES buffer solution for later use;
(2) mixing the microspheres in the step (1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide according to a mass ratio of 2:1:1, suspending the microspheres to 5mg/ml by using MES buffer solution, carrying out rotary reaction at 30 ℃ for 30min, and washing twice by using purified water; using 0.05M pH 9.0Na2HPO4Suspending the buffer solution to 10mg/mL for later use;
(3) mixing the microspheres in the step (2) and streptavidin in a mass ratio of 20:1, and using the Na2HPO4Suspending the microspheres to 5mg/mL in buffer, rotary incubating at 30 ℃ for 4h in the dark, adding 0.01% BSA, incubating for 30min, centrifuging to remove supernatant, washing twice with PBST (pH 7.4, 0.5% Tween 20), suspending in 0.05M pH7.4 TRIS buffer to 20mg/mL, and storing at 2-8 ℃ for further use.
5. The cTnI detection kit of claim 4, wherein the high speed centrifugation operation of step (1) in the preparation method of the donor microsphere-avidin complex is centrifugation at 12000rpm for 20 min.
6. The cTnI detection kit of claim 1, wherein the biotinylated antibody complex is prepared by a method comprising the steps of:
(1) preparing biotin: redissolving the purchased biotin freeze-dried powder to 1mg/mL by using DMSO;
(2) and (2) labeling the receptor microsphere with an antibody and biotin according to a mass ratio of 20:1 into 0.01M PBS buffer solution with pH7.4, and rotary incubating for 2h at 25 ℃; dialyzing the incubated antibody biotin solution with 0.01M PBS buffer solution with pH7.4 at 2-8 deg.C for 4h, repeating the dialysis for 3 times, and finally storing the biotinylated antibody complex at 2-8 deg.C to a final concentration of 0.4 mg/mL.
7. The cTnI detection kit of claim 1, wherein the reagent storage buffer is used to store the acceptor microsphere-antibody complex, donor microsphere-avidin complex, biotinylated antibody complex.
8. The cTnI detection kit of claim 7, wherein the reagents storage buffer comprises TRIS, NaCl solution, BSA solution, sucrose solution, bovine gamma-globulin solution, tween 20, Proclin300, a dissociating agent, water; the reagent preservation buffer solution is characterized in that the final pH value is 7.4, the adding amount of TRIS is 50mM, the final mass percentage concentration of the NaCl solution is 0.9%, the final mass percentage concentration of the BSA solution is 1.5%, the final mass percentage concentration of the sucrose solution is 1.0%, the final mass percentage concentration of the bovine gamma-globulin solution is 0.5%, the final mass percentage concentration of the Tween 20 solution is 0.2%, the final mass percentage concentration of the Proclin300 solution is 0.05%, and the dissociation agent contains one or two of an EDTA solution with the final mass percentage concentration of 0.1-1.0% and a trisodium citrate solution with the final mass percentage concentration of 0.2-1.5%.
9. The cTnI detection kit of claim 8, wherein the reagent storage buffer may further comprise one of a final wt.% tween 80 solution, a final wt.% concentration of 0.5% and a final wt.% concentration of triton x-100 solution.
10. A method for detecting cTnI using the cTnI detection kit of claim 1, wherein the detection process comprises: adding 20 mu L of sample into a sample hole, then adding 50 mu L of each of the receptor microsphere-antibody complex, the donor microsphere-avidin complex and the biotinylated antibody complex, placing at 37 ℃ and incubating for 15min, and carrying out signal detection; the working solution concentrations of the acceptor microsphere-antibody complex, the donor microsphere-avidin complex and the biotinylated antibody complex are respectively diluted to 0.1mg/mL, 0.1mg/mL and 1 mu g/mL by using reagent preservation buffer solution.
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