CN114216897A - sST2 chemiluminescence detection kit and detection method thereof - Google Patents
sST2 chemiluminescence detection kit and detection method thereof Download PDFInfo
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Abstract
The application relates to the technical field of biology, in particular to an sST2 chemiluminescence detection kit and a detection method thereof. The kit comprises an Ra component and an Rb component, wherein the Ra component comprises a biotin-labeled antibody-streptavidin-coated magnetic bead, and the Rb component comprises an alkaline phosphatase-labeled antibody. The antigen in the sample to be detected and the biotin-labeled antibody-streptavidin-coated magnetic bead in the kit are combined to form a compound, namely the antigen-biotin-labeled antibody-streptavidin-coated magnetic bead is combined with the alkaline phosphatase-labeled antibody to generate chemiluminescence, and a chemiluminescence immunoassay analyzer can be used for detecting the luminescence condition to judge the antigen condition in the sample to be detected to achieve the purpose of detection.
Description
Technical Field
The application relates to the technical field of biology, in particular to an sST2 chemiluminescence detection kit and a detection method thereof.
Background
There are two ST2 proteins that directly affect the progression of heart disease: soluble ST2 and transmembrane form of ST 2L. Soluble growth-stimulating expression gene 2 protein (ST2) is one of the members of the interleukin i receptor family. When the concentration of soluble ST2 is reduced, the ligand IL-33 of ST2 can be combined with ST2L to have protective effect on the heart; when soluble ST2 levels are high, soluble ST2 can competitively bind to IL-33, resulting in reduced binding of IL-33 to ST2L, thereby disabling IL-33 from entering the cardioprotective signal transduction pathway. Thus, the heart continues to be stressed, resulting in cell death and tissue fibrosis, reducing heart function, and increasing disease progression.
The detection technology applied in China comprises an enzyme-linked immunosorbent assay, a colloidal gold immunochromatography and a chemiluminescence method. The ELISA technology is mature (publication number: CN201510665932.2), but the sensitivity is poor, the sample needs to be diluted before use and then is measured, the steps are complex, the time consumption is high, the professional requirement on detection personnel is high, and the requirements of rapid and high-throughput diagnosis at present cannot be met; the colloidal gold immunochromatography method has the advantages of rapid detection (publication number: CN113533716A), low cost and visual detection result, but the difference between products is large, and the antibody is easy to fall off from the surface of gold particles by adopting the physical adsorption method for combination, so that the reproducibility is poor; the chemiluminescence immunoassay technology has high accuracy and specificity, is one of the most important technologies in the detection method, has the advantages of high sensitivity, wide detection range, simple and quick operation, good stability of a marker, no pollution, simple and economical instruments and the like, and the glow chemiluminescence based on the acridinium ester (publication No. CN112433048A) has the advantages of quick reaction, high intensity and low background, but the acridinium ester is unstable in a buffer solution and is easy to hydrolyze, and meanwhile, the required instruments and equipment are expensive and are not beneficial to popularization of a primary medical institution, while the glow chemiluminescence based on the alkaline phosphatase has the advantages of long duration of a light signal, high sensitivity, wide linear range and the like, and is outstanding in the detection method of the existing in-vitro diagnostic reagent.
Disclosure of Invention
The application provides a chemical luminescence detection kit for sST2 and a detection method thereof, which aim to solve the technical problem of poor precision of the existing kit for a fluorescein isothiocyanate amplification system.
In a first aspect, the application provides a chemiluminescent detection kit for sST2, the kit comprises an Ra component and an Rb component, the Ra component comprises a biotin-labeled antibody-streptavidin-coated magnetic bead, and the Rb component comprises an alkaline phosphatase-labeled antibody.
Optionally, in the biotin-labeled antibody-streptavidin-coated magnetic beads, the molar ratio of the biotin label to the magnetic beads is 1: 1-100.
Optionally, in the biotin label, the molar ratio of biotin to the sST2 antibody is 1-500: 1.
Optionally, the preparation method of the biotin-labeled antibody-streptavidin-coated magnetic bead includes:
washing and resuspending magnetic beads by using a buffer solution to obtain magnetic bead mother liquor, wherein the magnetic beads comprise streptavidin;
obtaining biotin mother liquor dissolved by an organic solvent;
obtaining sST2 antibody resuspended in the buffer;
mixing and marking the biotin mother liquor and an antibody solution to obtain a biotin marker, wherein the biotin marker is an antibody marked by biotin;
and mixing and reacting the biotin labeling substance with the magnetic bead mother liquor, then washing with the buffer solution, and adding a protective agent to obtain the biotin-labeled antibody-streptavidin-coated magnetic bead.
Optionally, the Ra component further comprises a biotin-labeled antibody-streptavidin coated magnetic bead diluent to stabilize and protect the biotin-labeled antibody-streptavidin coated magnetic bead; the biotin-labeled antibody-streptavidin coated magnetic bead diluent comprises biotinylated bovine serum albumin.
Optionally, the preparation method of the alkaline phosphatase labeled antibody comprises the following steps:
mixing and reacting an sST2 antibody with an activation solution containing an activating agent to obtain an activated antibody;
mixing and reacting an SMCC solution containing succinimide 4- (N-maleimide methyl) cyclohexane-1-carboxylate with an alkaline phosphatase solution to obtain activated alkaline phosphatase;
mixing and reacting the activated antibody and the activated alkaline phosphatase to obtain an alkaline phosphatase labeled antibody.
Optionally, the molar ratio of the succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate to the alkaline phosphatase in the alkaline phosphatase solution is 1: 1-1000.
Optionally, the molar ratio of the activated antibody to the activated alkaline phosphatase is 1: 0.8-2.
Optionally, the Rb component further comprises an enzyme label diluent to stabilize the alkaline phosphatase-labeled antibody.
In a second aspect, the present application provides a method of using the kit of the first aspect, the method comprising: and mixing and reacting the Ra component and the Rb component with sST2 antigen in sequence, adding the mixture, and detecting by using a chemiluminescence immunoassay analyzer to realize the detection of sST2 protein.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the kit provided by the embodiment of the application, an antigen in a sample to be detected and a biotin-labeled antibody-streptavidin-coated magnetic bead in the kit are combined into a compound, namely the antigen-biotin-labeled antibody-streptavidin-coated magnetic bead is combined with an alkaline phosphatase-labeled antibody, chemiluminescence can be generated, a chemiluminescence immune analyzer can be used for detecting the luminescence condition, so that the antigen condition in the sample to be detected is judged to achieve the purpose of detection, the particle size of the biotin-labeled antibody-streptavidin-coated magnetic bead is large or small, and the problems that the existing small-particle-size magnetic bead is weak and the large-particle-size magnetic bead is poor in precision are solved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a schematic flow chart of a method for preparing a magnetic bead coated with a biotin-labeled antibody-streptavidin according to an embodiment of the present disclosure;
FIG. 2 is a schematic flow chart of a method for preparing an alkaline phosphatase-labeled antibody according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In a first aspect, the application provides a chemiluminescent detection kit for sST2, the kit comprising an Ra component and an Rb component, the Ra component comprising a biotin-labeled antibody-streptavidin-coated magnetic bead, and the Rb component comprising an alkaline phosphatase-labeled antibody.
In the embodiment of the application, the streptavidin-biotin amplification reaction system of the biotin-labeled antibody-streptavidin-coated magnetic beads and the alkaline phosphatase-labeled antibody is used, so that the defects of the existing fluorescein isothiocyanate amplification system are overcome, and the kit is high in stability and specificity and does not need to be stored in a dark place.
In some embodiments, the biotin-labeled antibody-streptavidin coated magnetic beads have a molar ratio of biotin label to magnetic beads of 1: 1-100.
In the embodiment of the application, the molar ratio of the biotin label to the magnetic beads is controlled to be 1: the reason for 1-100 is that the ratio of the biotin label to the magnetic beads is kept reasonable, if the ratio is more than 1:1, the adverse effect of surplus biotin label and low titer is caused, and if the ratio is less than 1:100, the adverse effect of waste of magnetic beads is caused.
In some embodiments, the biotin label comprises a molar ratio of biotin to sST2 antibody of 1-500: 1.
In the examples of the present application, biotin is vitamin H, and the reason why the molar ratio of biotin to sST2 antibody is controlled to 1-500:1 is that if the ratio is greater than 1:1, the sST2 antibody is not sufficiently labeled, resulting in the adverse effect of waste, and if the ratio is less than 1:500, resulting in the adverse effect of low titer of labeled scouring articles.
In some embodiments, the method for preparing the biotin-labeled antibody-streptavidin-coated magnetic beads, as shown in fig. 1, comprises the following steps:
s1, cleaning and resuspending magnetic beads by using a buffer solution to obtain magnetic bead mother liquor, wherein the magnetic beads comprise streptavidin;
s2, obtaining a biotin mother liquor dissolved by an organic solvent,
s3, obtaining an sST2 antibody resuspended by the buffer solution;
s4, mixing and marking the biotin mother liquor and an antibody solution to obtain a biotin marker, wherein the biotin marker is an antibody marked by biotin;
and S5, mixing and reacting the biotin marker with the magnetic bead mother liquor, then washing with the buffer solution, and adding a protective agent to obtain the biotin-marked antibody-streptavidin-coated magnetic beads.
Specifically, the magnetic beads are streptavidin-containing magnetic beads which can be purchased from Saimeisha fly, the particle size is 1-5 μm, and the concentration of the mother liquor of the SA magnetic beads can be 10 mg/mL.
Specifically, the organic solvent may be N, N-dimethylformamide or DMSO. The buffer solution can be PBS buffer solution with pH7.0-7.8; the buffer carried by the antibody can be replaced by Phosphate Buffer (PB) with pH7.4 and 50mM before labeling,
in some embodiments, the Ra fraction further comprises a dilution of biotin-labeled antibody-streptavidin coated magnetic beads to stabilize and protect the biotin-labeled antibody-streptavidin coated magnetic beads; the biotin-labeled antibody-streptavidin coated magnetic bead diluent comprises biotinylated bovine serum albumin.
In the embodiment of the application, biotinylated bovine serum albumin is newly added into a biotin-labeled antibody-streptavidin-coated magnetic bead component to seal free SA magnetic beads which are not combined with a biotin antibody, so that biotin interference possibly existing in a sample is reduced, and the detection accuracy is improved; the sensitivity is improved by using streptavidin magnetic beads, and high-concentration sodium chloride is added into an enzyme marker diluent, so that non-specific adsorption is reduced, and the detection accuracy is improved.
In some embodiments, the method for preparing the alkaline phosphatase-labeled antibody, as shown in fig. 2, comprises the steps of:
s11, mixing and reacting an sST2 antibody with an activating solution containing an activating agent to obtain an activated antibody;
s12, mixing and reacting an SMCC solution containing succinimide 4- (N-maleimide methyl) cyclohexane-1-carboxylate with an alkaline phosphatase solution to obtain activated alkaline phosphatase;
s13, mixing and reacting the activated antibody and the activated alkaline phosphatase to obtain an alkaline phosphatase labeled antibody.
In some embodiments, the molar ratio of succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate to alkaline phosphatase in the alkaline phosphatase solution is 1:1 to 1000.
In the examples of the present application, the reason why the molar ratio of the succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate to the alkaline phosphatase is controlled to be 1:1-1000 is that if the ratio is more than 1:1, the adverse effect of increasing the difficulty of purification in the subsequent step is caused, and if the ratio is less than 1:1000, the adverse effect of insufficient reaction of the alkaline phosphatase is caused. The activator may be 2IT (2-iminosulfane hydrochloride).
In some embodiments, the molar ratio of the activated antibody to the activated alkaline phosphatase is 1: 0.8-2.
In the examples of the present application, the reason why the molar ratio of the activated antibody to the activated alkaline phosphatase is controlled to be 1:0.8-2 is that if the ratio is more than 1:0.8, the adverse effect of causing an excess of reducing antibody to waste is caused, and if the ratio is less than 1:2, the adverse effect of causing low titer of the final marker and affecting stability is caused.
In some embodiments, the Rb component further comprises an enzyme label diluent to stabilize the alkaline phosphatase-labeled antibody.
In the embodiment of the application, through adding biotinylation bovine serum albumin in the magnetic bead diluent, can effectively seal the SA magnetic bead that does not combine with the biotin antibody, reduce the biotin interference that probably exists in the sample, improve detection accuracy.
In a second aspect, the present application provides a method of using the kit of the first aspect, the method comprising: and mixing and reacting the Ra component and the Rb component with sST2 antigen in sequence, and detecting by using a chemiluminescence immunoassay analyzer to realize the detection of sST2 protein.
The process of the present invention will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1
The embodiment provides a preparation method of a biotin-labeled antibody-streptavidin-coated magnetic bead, which comprises the following steps:
s1, cleaning and resuspending magnetic beads by using a buffer solution to obtain magnetic bead mother liquor, wherein the magnetic beads comprise streptavidin; specifically, the supernatant is replaced before use, washed with PBS buffer solution with pH7.4 for three times, and then the magnetic beads with the particle sizes are mixed according to a certain proportion and then resuspended into a proper amount of basic buffer solution to prepare magnetic bead mother solution (the concentration of the magnetic bead mother solution is 10 mg/mL);
s2, obtaining a biotin mother liquor dissolved by an organic solvent; specifically, Phosphate Buffer (PB) with pH of 7.450mM is used for replacing buffer carried by the antibody before labeling, and anhydrous N, N-dimethylformamide is used for dissolving biotin into biotin mother liquor with concentration of 0.05-200 mg/mL;
s3, obtaining an sST2 antibody resuspended by the buffer solution;
s4, mixing and marking the biotin mother liquor and an antibody solution to obtain a biotin marker, wherein the biotin marker is an antibody marked by biotin; under the dark condition, the antibody of the displaced buffer solution and the biotin mother liquor are taken to be marked according to the molar concentration of 1 (1-500), the marking buffer solution is pH7.450mMPB, the suspension mixing marking is carried out for 0.5-6 hours under the dark condition, a protein purifier is used for purification after the reaction is finished, the biotin marked antibody is obtained, an ultramicro spectrophotometer is used for measuring the protein concentration, and the buffer solution added with a protective agent is used for diluting to 0.5 mg/mL;
s5, mixing and reacting the biotin marker with the magnetic bead mother liquor, then washing with the buffer solution, and adding a protective agent to obtain biotin-labeled antibody-streptavidin-coated magnetic beads; specifically, the biotin label and the mother solution of the magnetic beads are mixed according to a certain proportion and reacted for 30 minutes, then washed three times by PBS buffer solution with pH7.4, and finally the buffer solution added with the protective agent is used to reach 10 mg/mL.
The embodiment provides a preparation method of an alkaline phosphatase labeled antibody, which comprises the following steps:
s11, mixing and reacting an sST2 antibody with an activating solution containing an activating agent to obtain an activated antibody; specifically, a certain mass of antibody activator 2IT (2-iminothiolane hydrochloride) is taken, dissolved to 10mg/ml by using an aqueous solution containing 50mM Tris, 0.1M NaCl and 0.005M EDTA and having the pH value of 8.5 +/-0.05, a certain amount of sST2 monoclonal antibody is accurately measured, the antibody concentration in the solution is 3.5mg/ml by concentration or volume fixing, and the solvent is placed at the bottom of a reaction test tube by using the solvent of the 2IT solution; adding 2IT solution with the volume ratio of 1/20 into a test tube containing 3.5mg/mlsST2 monoclonal antibody solution for activation, mixing uniformly, reacting at room temperature for 20 minutes, and removing excessive 2IT in the solution by using a molecular sieve chromatography mode after the reaction is finished to obtain an activated antibody;
s12, mixing and reacting a solution containing succinimide 4- (N-maleimide methyl) cyclohexane-1-carboxylate with an alkaline phosphatase solution to obtain activated alkaline phosphatase; specifically, a certain amount of alkaline phosphatase is accurately measured and placed at the bottom of a reaction test tube; weighing appropriate amount of (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester, and dissolving with Dimethylformamide (DMF) to 5 mg/ml; adding an SMCC solution with the volume of 1/20 into a test tube containing alkaline phosphatase, uniformly mixing, reacting at room temperature for 15 minutes, and removing excessive SMCC in the solution by using molecular sieve chromatography after the reaction is finished to obtain activated alkaline phosphatase;
s13, mixing and reacting the activated antibody and the activated alkaline phosphatase to obtain an alkaline phosphatase labeled antibody. Antibody activation: specifically, the activated antibody and the activated alkaline phosphatase are mixed according to the mass ratio of 1:0.8, 6 mu L of 1MMgCl2 solution is added, the mixture is uniformly mixed and reacts for 24 hours at the temperature of 2-8 ℃, after the reaction is finished, the final alkaline phosphatase labeled antibody is obtained by purification through a molecular sieve chromatography method, the protein concentration is measured through a BCA method, and then the final alkaline phosphatase labeled antibody is diluted to 0.5mg/mL through a buffer solution added with a protective agent.
The application provides an sST2 chemiluminescence detection kit, which comprises an Ra component and an Rb component, wherein the Ra component comprises biotin-labeled antibody-streptavidin-coated magnetic beads and biotin-labeled antibody-streptavidin-coated magnetic bead diluent, and the Rb component comprises alkaline phosphatase-labeled antibody and enzyme-labeled diluent; the kit also comprises an sST2 calibrator and an sST2 quality control product.
The biotin-labeled antibody-streptavidin coated magnetic bead diluent comprises the following components: morpholine ethanesulfonic acid (MES): 50-200mmol/L buffer system, casein: 0.5% -2%, tween 20: 0.05% -0.2%, TritonX-114: 0.1%, biotinylated bovine serum albumin: 1%, sodium azide: 0.05 percent of biotin-labeled antibody-streptavidin coated magnetic bead diluent, filtering the diluent by using a 0.2 mu m filter membrane, and storing the diluent at 2-8 ℃ for later use, wherein the pH of the diluent is 6.5;
and (3) coating the prepared biotin-labeled antibody-streptavidin magnetic beads, measuring a proper amount of mother liquor by using a pipettor, adding the mother liquor into a clean centrifugal tube, adding the mother liquor into a magnetic bead coating diluent, and uniformly mixing for later use.
The components of the enzyme marker diluent comprise: MOPSO: 50-100mmol/L, NaCl: 3% -5% of ZnCl2：0.1mM-0.5mM、MgCl2: 1mM-5mM, BSA: 0.5% -2%, PVP: 0.05% -0.2%, fetal bovine serum: 2% and PC 300: 0.05 percent, the pH value of the enzyme marker diluent is 6.5, and the enzyme marker diluent is filtered by a 0.5 mu m filter membrane and stored for standby at the temperature of 2-8 ℃;
and (3) measuring a proper amount of mother liquor into a clean centrifuge tube by using a pipette, adding the mother liquor into an enzyme marker diluent, and uniformly mixing for later use.
A method of using the kit, the method comprising: and mixing and reacting the Ra component and the Rb component with sST2 antigen in sequence, and detecting by using a chemiluminescence immunoassay analyzer to realize the detection of sST2 protein.
Example 2
On the basis of the example 1, 1% mannitol is added into the magnetic bead coating diluent, and other components and processes are the same as those of the example 1;
example 3
On the basis of example 1, 3% glucose is added into the magnetic bead coating diluent, and other components and processes are the same as those in example 1;
example 4
3% Bio-TergeaS40 was added to the enzyme-labeled diluent in addition to example 1, and the other components and processes were the same as in example 1.
Example 5
On the basis of example 1, 3% sorbitol was added to the enzyme labeling diluent, and the other components and processes were the same as in example 1.
Example 6
On the basis of example 1, 1.5% potassium chloride was added to the enzyme-labeled diluent, and the other components and processes were the same as in example 1.
Control group 1
Based on example 1, the coating diluent of magnetic beads is not added with 1% of biotinylated bovine serum albumin, and other components and processes are the same as example 1.
Control group 2
In addition to example 1, SA magnetic beads with a particle size of 3 μm were not added in the pretreatment of the magnetic beads, and other components and processes were the same as those of example 1.
Control group 3
On the basis of example 1, the magnetic beads of SA are replaced by the magnetic beads of anti-fluorescein isothiocyanate antibodies, biotin is replaced by fluorescein isothiocyanate, and other components and processes are the same as those in example 1.
Experimental detection
The reagents prepared in the embodiments 1-6 and the comparison groups 1-3 are placed on a full-automatic chemiluminescence immunoassay analyzer, an sST2 calibrator is selected according to the detection process of the analyzer for calibration, a quality control product is tested to observe whether the concentration of the calibrator is within the range of the labeled concentration, if so, the next step of testing can be carried out, and if not, a new reagent, a matched calibrator and a quality control product are replaced, and the steps are repeated.
And testing performance indexes such as sensitivity, linearity and the like, wherein the evaluation method comprises the following steps:
evaluation of sensitivity: blank samples and low-concentration samples with the concentrations of sST2 of 0.5ng/mL, 1.0ng/mL and 1.5ng/mL are measured, each sample is measured for 10 times, and the mean value, the deviation and the coefficient of variation are calculated, wherein CV is required to be less than 10% and the deviation is required to be less than 10%. As shown in tables 1 and 2; CV is the estimation of standard deviation based on a sample, which reflects the degree of dispersion of a value from a mean value (mean). The calculation formula of the relative deviation is as follows: (mean-concentration of sST 2)/concentration of sST 2.
And (3) linear evaluation: the high concentration sample near the upper limit of the linear zone is diluted with the low concentration sample near the lower limit of the linear zone and mixed to at least 6 diluted concentrations. The samples were measured 3 times for each dilution concentration using the reagents prepared in examples 1 to 6 and controls 1 to 3, respectively, and the results of each dilution concentration measurement were averaged. And (3) calculating a linear regression equation by taking the dilution concentration as an independent variable and the mean value of the detection result as a dependent variable, and calculating a correlation coefficient (r) of the linear regression, wherein the r is required to be more than 0.99. With reference to the CRP criteria of criteria YYT 1513 and 2017,
accelerated stability of the reagent: examples 1-6 and controls 1-3 were allowed to stand at 37 ℃ for 1 day, 3 days, 7 days, and 14 days, respectively, to verify the accelerated stability of the reagents.
Long-term stability of the reagent: the reagents of examples 1-6 and control groups 1-3 were stored at 2-8 deg.C for 0 month, 3 months, 6 months, 9 months and 12 months, respectively, and tested to verify the long-term stability of the reagents.
Sample correlation evaluation: 60 fresh serum samples with linear range of 3.1-200ng/ml are selected, and the correlation of the detection results of the kit of the invention and the soluble growth stimulation expression gene 2 protein detection kit (Presage ST2assay kit) of intensive care diagnostic GmbH is compared, as required by the technical standards YYT 1580-.
Evaluation of anti-Biotin interference Capacity: preparing biotin interference negative serum (control) and biotin interference positive serum (prepared by adding biotin into the biotin interference negative serum, wherein the content of biotin is 100ng/mL), repeatedly measuring the two samples for 3 times respectively, and calculating concentration deviation;
table 1 examples 1-6 sensitivity data.
Table 2 control 1-3 sensitivity data.
As can be seen from Table 1-2, in the examples 1-6, the sensitivity test has a low background measured value, the ratio of the measured value of the low-value sample to the measured value of the blank sample is more than 3 times, and when the sensitivity is 1.5ng/mL, the deviation and CV are within 10% to meet the requirements; the contrast groups 1-3 are subjected to sensitivity test, the background measured value is higher, the ratio of the measured value of the low-value sample to the measured value of the blank sample is less than 2 times, the deviation and CV are higher, and the requirements are not met.
Table 3 examples 1-6 linearity data.
Table 4 control 1-3 linear data.
As can be seen from tables 3-4, the linear correlation coefficient is greater than 0.9900 and the relative deviation is less than 10% when the linear tests are carried out in the examples 1-6, so that the requirements are met; the control group 2 is subjected to linear test, the linear correlation coefficient is greater than 0.9900, but the relative deviation is greater than 10%, and the linear correlation coefficient is not satisfactory, while the control group 1/3 is subjected to linear test, the linear correlation coefficient is less than 0.9900, and the relative deviation is greater than 10%, and the linear correlation coefficient does not satisfy the experimental requirements.
Table 5 accelerated stability data.
As can be seen from Table 5, the control group 3, which was subjected to the 14-day accelerated stability test, had an activity retention of less than 90%, which did not meet the requirements; the examples 1-6 and the control groups 1-2 are subjected to accelerated stability tests, the activity retention rate is greater than 90%, the requirements are met, and the streptavidin system is better in stability compared with the isothiocyanate system.
Table 6 long term stability data.
| Accelerated reagent stability | Month 0 | Month 3 | Month 6 | Month 9 | Month 12 |
| Example 1 | 99％ | 98％ | 93％ | 90％ | 85％ |
| Example 2 | 98％ | 93％ | 92％ | 91％ | 86％ |
| Example 3 | 97％ | 98％ | 91％ | 90％ | 88％ |
| Example 4 | 98％ | 93％ | 93％ | 91％ | 88％ |
| Example 5 | 99％ | 91％ | 88％ | 84％ | 82％ |
| Example 6 | 99％ | 96％ | 95％ | 94％ | 91％ |
| Control group 1 | 96％ | 91％ | 81％ | 80％ | 75％ |
| Control group 2 | 99％ | 97％ | 92％ | 88％ | 86％ |
| Control group 3 | 98％ | 92％ | 90％ | 85％ | 79％ |
As can be seen from Table 6, the 12-month long-term stability tests of examples 1-6 and control groups 1-3 showed that the 12 th-month retention rate of control groups 1 and 3 was less than 80%, which did not meet the requirements; and the 12 th month retention rate of the implementation 1-6 and the control group 2 is higher than 80 percent, thereby meeting the requirements.
Table 7 correlation of samples from examples 1-6, controls 1-3 and Presage test kit.
The data show that 60 fresh clinical samples are tested, the clinical relevance of the examples 1-6 is better, R is larger than 0.9900, and the correlation coefficient of the control groups 1-3 is worse and smaller than 0.9900, which indicates that the system is favorable for improving the relevance of the reagent samples.
Table 8 biotin interference bias data.
As can be seen from Table 8, the control group 1 did not add 1% biotinylated BSA, and the apparent measurement deviation of the biotin-detected sample showed poor anti-biotin interference ability, indicating that the addition of 1% biotinylated BSA can effectively improve the anti-biotin interference ability of the reagent.
In summary, the soluble growth stimulation expression gene 2 protein kit prepared by the invention based on the alkaline phosphatase-biotin-streptavidin magnetic bead system has the advantages of good stability, high sensitivity, strong anti-interference capability and other properties capable of meeting the clinical use requirements.
It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The sST2 chemiluminescence detection kit is characterized by comprising an Ra component and an Rb component, wherein the Ra component comprises a biotin-labeled antibody-streptavidin-coated magnetic bead, and the Rb component comprises an alkaline phosphatase-labeled antibody.
2. The kit of claim 1, wherein the biotin-labeled antibody-streptavidin coated magnetic beads are coated with a biotin label at a molar ratio of 1: 1-100.
3. The kit of claim 2, wherein the biotin label is present in a molar ratio of biotin to sST2 antibody of 1-500: 1.
4. The kit according to any one of claims 1 to 3, wherein the method for preparing the biotin-labeled antibody-streptavidin-coated magnetic beads comprises:
washing and resuspending magnetic beads by using a buffer solution to obtain magnetic bead mother liquor, wherein the magnetic beads comprise streptavidin;
obtaining biotin mother liquor dissolved by an organic solvent;
obtaining sST2 antibody resuspended in the buffer;
mixing and marking the biotin mother liquor and an antibody solution to obtain a biotin marker, wherein the biotin marker is an antibody marked by biotin;
and mixing and reacting the biotin labeling substance with the magnetic bead mother liquor, then washing with the buffer solution, and adding a protective agent to obtain the biotin-labeled antibody-streptavidin-coated magnetic bead.
5. The kit of claim 1, wherein the Ra fraction further comprises a dilution of biotin-labeled antibody-streptavidin coated magnetic beads to stabilize and protect the biotin-labeled antibody-streptavidin coated magnetic beads; the biotin-labeled antibody-streptavidin coated magnetic bead diluent comprises biotinylated bovine serum albumin.
6. The kit of claim 1, wherein the alkaline phosphatase-labeled antibody is prepared by a method comprising:
mixing and reacting an sST2 antibody with an activation solution containing an activating agent to obtain an activated antibody;
mixing and reacting an SMCC solution containing succinimide 4- (N-maleimide methyl) cyclohexane-1-carboxylate with an alkaline phosphatase solution to obtain activated alkaline phosphatase;
mixing and reacting the activated antibody and the activated alkaline phosphatase to obtain an alkaline phosphatase labeled antibody.
7. The kit of claim 6, wherein the molar ratio of succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate to alkaline phosphatase in the alkaline phosphatase solution is 1: 1-1000.
8. The kit of claim 6, wherein the molar ratio of said activated antibody to said activated alkaline phosphatase is 1: 0.8-2.
9. The kit of claim 6, wherein said Rb component further comprises an enzyme label diluent to stabilize said alkaline phosphatase-labeled antibody.
10. A method of using the kit of any one of claims 1 to 9, the method comprising: and mixing and reacting the Ra component and the Rb component with sST2 antigen in sequence, and detecting by using a chemiluminescence immunoassay analyzer to realize the detection of sST2 protein.
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| CN117405874A (en) * | 2023-12-15 | 2024-01-16 | 迪亚莱博（张家港）生物科技有限公司 | Preparation method of PIC antibody magnetic bead reagent and kit |
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