CN112051403A - C-reactive protein chemiluminescence immunoassay kit and preparation method and application thereof - Google Patents
C-reactive protein chemiluminescence immunoassay kit and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a C reactive protein chemiluminescence immunoassay kit, a preparation method and application thereof, comprising the following steps: the kit comprises an M reagent, an R reagent, a luminescent substrate solution and a calibrator, wherein the M reagent is a magnetic bead reagent coated with an antibody M, the R reagent is an enzyme labeling reagent connected with an antibody R, both the antibody M and the antibody R can be specifically combined with C reactive protein, and the ratio of affinity constants of the antibody M and the antibody R is 1 multiplied by 10‑7～1×10‑2Or 1X 102～1×107. The invention changes the affinity of the antibody M coated by the magnetic beads and the affinity of the enzyme-labeled antibody R, so that the affinity constants of the antibody M and the antibody R have obvious difference, thereby simultaneously improving the chemical reaction of the C-reactive proteinThe linear range and the sensitivity of the photo-immunoassay kit avoid the problems that the traditional immunoturbidimetry method is low in sensitivity and the chemiluminescence immunoassay method is narrow in linear range, so that the chemiluminescence immunoassay method is limited in clinical use, and the C-reactive protein is accurately detected.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents for immunological detection, and particularly relates to a C-reactive protein chemiluminescence immunoassay kit, and a preparation method and application thereof.
Background
C-reactive protein (CRP) is synthesized by hepatocytes and produced in the fetal stage, non-maternal placental transfer. The generation mechanism is as follows: when the body is infected or the tissues are damaged, macrophages and other white blood cells are activated to produce cytokines such as interleukin-6, interleukin-1, tumor necrosis factor TNF-a and other mediators, and the cytokines and the mediators reach the liver to stimulate the liver cells and the epithelial cells to synthesize CRP. Structurally, CRP contains 5 polypeptide chain subunits, is non-covalently combined into a disk-shaped polymer and has the molecular weight of 11.5-14 ten thousand, and is a typical acute phase protein. Reference values are generally considered to be clinically higher than 10 mg/L. CRP levels in healthy population blood were below 5mg/L and reach approximately 20-500 mg/L in 4-8 hours of acute inflammation under various conditions. According to the CRP for evaluating inflammatory infection, the CRP is 10-50mg/L, which indicates mild inflammation, the CRP content is more than 100mg/L, which indicates more serious bacterial infection, and the viral infection is usually less than or equal to 50mg/L, so that the viral infection can be eliminated, and the diagnosis, treatment and monitoring information of inflammatory diseases can be provided. For cardiovascular disease risk assessment, the CRP content is less than 1.0mg/L, the risk of cardiovascular disease occurrence is low, the CRP content is greater than 3mg/L, the risk is high, and the CRP can be used as an early warning indicator for coronary artery disease or acute coronary syndrome relapse when being matched with traditional clinical diagnosis of acute coronary syndrome, so that higher sensitivity and linear range are clinically needed.
The prior art method for detecting C-reactive protein mainly comprises an immunoturbidimetry method, a fluorescence immunochromatography method, an enzyme-linked immunosorbent assay and a chemiluminescence immunoassay; the common methodology for detecting C-reactive protein on the market is immunoturbidimetry, and chemiluminescence immunoassay is rare, mainly because: although the chemiluminescence immunoassay method has higher sensitivity compared with the immunoturbidimetry method, the linear range is narrow, and the content of the C-reactive protein in a human body is relatively high, so that the chemiluminescence immunoassay method is limited in the clinical detection of the C-reactive protein.
Disclosure of Invention
The invention provides a C-reactive protein chemiluminescence immunoassay kit, a preparation method and application thereof aiming at the defects in the prior art, and the high-affinity antibody and the low-affinity antibody are combined for use, so that the linear range and the sensitivity of the chemiluminescence immunoassay for detecting the C-reactive protein can be improved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a C-reactive protein chemiluminescence immunoassay kit comprises: the kit comprises an M reagent, an R reagent, a luminescent substrate solution and a calibrator, wherein the M reagent is a magnetic bead reagent coated with an antibody M, the R reagent is an enzyme labeling reagent connected with an antibody R, both the antibody M and the antibody R can be specifically combined with C reactive protein, and the ratio of affinity constants of the antibody M and the antibody R is 1 multiplied by 10-7～1×10-2Or 1X 102～1×107。
The invention adopts a chemiluminescence immunoassay double-antibody sandwich method, which has the main principle that: the magnetic beads are coated with a mouse anti-human monoclonal antibody, the mouse anti-human monoclonal antibody is combined with C-reactive protein in human serum or plasma, then a double-antibody sandwich is formed with the mouse anti-human C-reactive protein monoclonal antibody marked by alkaline phosphatase, the alkaline phosphatase catalyzes a substrate to emit light under the condition of existence of a luminescent substrate, and a luminescent signal is detected by a chemiluminescence detection system so as to quantitatively detect the content of the C-reactive protein in a sample. The affinity constant of an antibody represents the affinity between the antibody and an antigenic determinant, namely the binding capacity, wherein the larger the affinity of the antibody is, the higher the sensitivity is, and the corresponding linear range is narrow, while the smaller the affinity of the antibody is, the wider the linear range is, but the sensitivity is low. The invention finds that the affinity constants of the antibody coated by the magnetic beads and the antibody marked by the enzyme are adjusted, so that the affinity constants of the antibody M and the antibody R have difference, and the ratio is 1 multiplied by 10-7～1×10-2Or 1X 102～1×107At the same timeThe linear range and the sensitivity of the detection reagent are obviously improved.
Preferably, the affinity constant of the antibody M is 1X 10-7～1×10-5When the affinity constant of the antibody R is 1X 10-12～1×10-9. That is, the affinity between the antibody M coated with magnetic beads and the antigen is significantly lower than that between the antibody R labeled with enzyme and the antigen.
Preferably, the affinity constant of the antibody M is 1X 10-12～1×10-9When the affinity constant of the antibody R is 1X 10-7～1×10-5. Namely, the affinity between the antibody M coated by the magnetic beads and the antigen is obviously higher than that between the antibody R marked by the enzyme and the antigen.
The invention also provides a preparation method of the chemiluminescence immunoassay kit for the C-reactive protein, which comprises the following steps:
s1, preparation of M reagent: washing and activating the magnetic beads, adding an antibody M and a coupling buffer solution for coupling reaction, then adding a sealing buffer solution for sealing, and diluting with an M diluent to obtain the M reagent;
preparation of S2 and R reagent: respectively activating an antibody R and an enzyme, mixing, carrying out enzyme-linked reaction to obtain an enzyme-labeled antibody compound, and diluting with an R diluent to obtain the R reagent;
s3, preparation of luminescent substrate solution: dissolving a luminescent substrate by using a buffer solution to obtain a luminescent substrate solution;
s4, preparing a calibration product: preparing the C-reactive protein antigen into a plurality of concentrations by using a calibrator diluent to obtain a calibrator;
s5, independently placing the magnetic bead reagent coated with the antibody M, the enzyme labeling reagent connected with the antibody R, the luminescent substrate solution and the calibrator in a packaging container to obtain the chemiluminescence immunoassay kit for the C-reactive protein.
Preferably, step S1 is specifically: (1) taking 10-100 mu L of magnetic beads with the particle size of 1-3 mu m and the concentration of 10-100 mg/mL, carrying out magnetic separation and washing by using 1-3 mL of magnetic bead washing liquid, and then activating for 20-30min at 20-30 ℃ by using 1-3 mL of magnetic bead activating agent;
(2) adding 0.1-0.5 mg of antibody M and 0.5-3 mL of coupling buffer solution into the magnetic beads, and coupling for 2-3h at 20-30 ℃;
(3) continuously adding 0.5-3 mL of closed buffer solution, reacting at 20-30 ℃ for 1-3h, removing supernatant and washing to obtain an antibody coated by magnetic beads;
(4) and (3) diluting the antibody coated by the magnetic beads with M diluent according to the volume ratio of 1: and (5) diluting by 20-60 to obtain the M reagent.
Preferably, step S2 is specifically: (1) taking 0.1-1 mg of antibody R, adding 0.3-1 mL of antibody activator solution, activating at room temperature for 15-30min, and purifying to obtain an activated antibody;
(2) taking 0.1-1 mg of alkaline phosphatase, adding 0.3-1 mL of enzyme activator, reacting at room temperature for 10-20min, and purifying to obtain activated alkaline phosphatase;
(3) mixing the activated antibody and the activated alkaline phosphatase according to the mass ratio of 1: 0.5-1, adding 5-20 mu L of 1M magnesium chloride solution, reacting for 10-15h at the temperature of 2-8 ℃, and purifying to obtain an enzyme-labeled antibody compound;
(4) and (3) diluting the enzyme-labeled antibody complex with an R diluent according to a volume ratio of 1: diluting 100-300 to obtain the R reagent.
Preferably, the pH of the M diluent in the step S1 is 7.0-7.5, and the method comprises the following steps: 20-100 mM buffer solution, 0.1-1% of surfactant, 0.5-3% of protein protective agent and 0.05-0.5% of preservative, wherein the percentages are mass percentages;
the pH of the R diluent is 5.5-7.0, and the R diluent comprises: 20-100 mM buffer solution, 1-10 mM inorganic salt, 1-10% protein protective agent and 0.05-0.5% preservative, wherein the percentage is mass percentage.
Preferably, the buffer is one or more of Tris buffer, HEPES buffer, HEPPSO buffer, MES buffer and phosphate buffer;
the surfactant is one or more of Tween20, Tween-80, EMULGEN A-60, EMULGEN A-90, Brij-35, Triton X-405, Triton X-114 and Triton X-100;
the protein protective agent is one or more of sucrose, pullulan, trehalose, bovine serum albumin, casein, acetaminophen and glycine
The inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride and zinc chloride;
the preservative is one or more of sodium azide, potassium sorbate, sodium benzoate and Proclin series.
The invention also provides application of the kit in detecting the content of the C-reactive protein.
Compared with the prior art, the invention has the beneficial effects that: the method changes the affinity of an antibody M coated by magnetic beads and an antibody R marked by enzyme, so that the affinity constants of the antibody M and the antibody R have obvious difference, and further improves the linear range and sensitivity of the chemiluminescence immunoassay kit for the C-reactive protein, and avoids the problems that the traditional immunoturbidimetry method is wide in linear range but low in sensitivity, and the chemiluminescence immunoassay method is high in sensitivity and narrow in linear range, so that the method is limited in clinical use, and realizes accurate detection of the C-reactive protein.
Drawings
FIG. 1 is a line graph of group 1, group 3 and group 10 in example 2 of the present invention;
FIG. 2 is a line graph correlating the measurements of group 10 agents and Siemens agent in example 2 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood 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 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 invention.
EXAMPLE 1 reagent preparation
(1) Cleaning liquid for magnetic beads: a pH of 5.0 comprising: 100mM MES buffer, 0.05% Tween 20;
(2) magnetic bead activating agent: 10mg/mL of a solution of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) mixed in equal proportions;
(3) coupling buffer: a 100mM boric acid solution having a pH of 8.0 to 8.5;
(4) blocking buffer: pH 7.2, 50-100mM PBS buffer, 1% Bovine Serum Albumin (BSA);
(5) preservation solution: 100mM Tris buffer, 0.1% preservative PC-300;
(6) antibody activator: 100mM Tris buffer, 0.3% NaCl, 5mM EDTA, 10mg/mL 2-IT (2-imino tetrahydrothiophene) cross-linking agent;
(7) enzyme activator: 100mM Tris buffer, 0.3% NaCl, 5mM EDTA, 5mg/mL SMCC crosslinker solubilized with dimethylformamide;
(8) m diluent: a pH of 7.2 comprising: 25mM HEPPSO buffer, 0.5% Tween20, 1% BSA, 1% pullulan, 0.1% PC-300;
(9) r, diluent: a pH of 6.5 comprising: 50mM MES buffer, 1mM ZnCl2，5mM MgCl210mM NaCl, 0.1% PC-300, 5% trehalose, 0.1% glycine, 1% BSA;
(10) and (3) calibrating product diluent: a pH of 7.5 comprising: 50mM Tris buffer, 10mM NaCl, 0.5% Tween20, 5% trehalose, 1% BSA, 0.1% PC-300.
EXAMPLE 2 preparation of the kit
(1) C-reactive protein antigen: purity > 95% and purchased from Wuhan Huamei bioengineering Co.
(2) C-reactive protein antibody: four kinds of mouse anti-human C reactive protein monoclonal antibodies with different affinities are a, b, C and d respectively, the purity is more than 95 percent, the monoclonal antibodies are purchased from Wuhan Huamei bioengineering limited company, and the goods numbers are respectively: CSB-DA402GmN (a), CSB-DA402GmN (b), CSB-DA402GmN (c) and CSB-DA402GmN (d), wherein affinity constants of the four antibodies are shown in the following table:
| c reactive protein antibodies | Affinity constant Kd (mol/L) |
| a | 1.56×10-12 |
| b | 2.47×10-10 |
| c | 5.26×10-6 |
| d | 3.58×10-7 |
Wherein the affinity constant Kd of antibodies a and b is of the order of 10-9～1×10-12Indicates that the affinity of the antibodies is high, and that the affinity constants Kd of the antibodies c and d are in the order of 10-5～1×10-7In between, the low affinity of the antibody is indicated.
The four antibodies are respectively coated with magnetic beads, and simultaneously, the four antibodies are respectively labeled with alkaline phosphatase, wherein the antibodies coated with the magnetic beads are M reagents, the antibodies labeled with the alkaline phosphatase are R reagents, the antibody a is used as the M reagent and labeled with Ma, the antibody a is used as the R reagent and labeled with Ra, and so on, the antibodies can be divided into 12 groups, and the specific steps are as follows:
(3) the specific preparation method for obtaining the M reagent by coating the magnetic beads comprises the following steps:
firstly, taking 100 mu L of magnetic beads with the particle size of 1 mu m and the concentration of 10mg/mL into a centrifuge tube, removing supernatant, carrying out magnetic separation and washing for 3 times by using 1mL of magnetic bead cleaning solution, and removing supernatant;
② adding 1mL of magnetic bead activating agent, activating for 20-30min at 25 ℃, and removing supernatant;
③ adding 0.1mg of antibody and 1mL of coupling buffer solution, carrying out coupling reaction at 25 ℃ for 2-3h, and removing supernatant;
adding 1mL of blocking buffer solution, reacting for 1h at 25 ℃, removing supernatant and washing for 2-3 times;
fifthly, adding 1mL of preservation solution to obtain the antibody M coated by the magnetic beads, preserving one part of the antibody M at the temperature of 2-8 ℃, and diluting the other part of the antibody M with M diluent according to the volume ratio of 1:50 to obtain the M reagent.
(4) The specific preparation method of the reagent R obtained by enzyme labeling comprises the following steps:
taking 0.5mg of antibody, adding 0.3mL of antibody activator solution, activating at room temperature for 20min, and purifying by a molecular sieve chromatography to obtain an activated antibody;
② 0.4mg of alkaline phosphatase is taken, 0.3mL of enzyme activator is added, reaction is carried out for 15min at room temperature, and purification is carried out by molecular sieve chromatography, thus obtaining activated alkaline phosphatase;
thirdly, mixing the activated antibody and the activated alkaline phosphatase according to the mass ratio of 1:0.8, and adding 10 mu L of 1M MgCl2The solution is used as an enzyme protective agent, the reaction is carried out for 12 hours at the temperature of 2-8 ℃, and after the reaction is finished, a molecular sieve is used for purification to obtain an enzyme-labeled antibody compound;
adding glycerol into the enzyme-labeled antibody compound according to the volume ratio of 1:1 to obtain an enzyme-labeled antibody R, and diluting with an R diluent according to the ratio of 1: diluting at a volume ratio of 200 to obtain the R reagent.
(5) Luminescent substrate solution: the method takes a 1, 2-dioxetane compound (AMPPD) as a chemiluminescence substrate of alkaline phosphatase, and specifically comprises the following steps: dissolving 24g Tris, 160g NaCl, 6g KCl, 15mL HCl, 200mL AMPPD and 1mL PC-300 in pure water, and metering to 1L to obtain the luminescence substrate solution.
(6) Preparing a calibration product: the C-reactive protein antigen is prepared into concentration gradients of 0, 1mg/L, 10mg/L, 50mg/L, 200mg/L and 350mg/L by using a calibrator diluent.
Evaluation protocol
The 12 groups of detection kits are used by a conventional method, a full-automatic chemiluminescence analyzer is used for detection, after calibration is carried out by a calibrator, the sensitivity of the groups 1-4 is detected, whether the affinity of the antibody is consistent with the result given by a manufacturer is judged according to the result, the linear range of the groups 1-12 is detected, and the optimal group is screened for other performance evaluation.
The groups 1-12 are evaluated according to the sensitivity and the linear range of the sample, and the evaluation scheme is specifically as follows:
(1) initial judgment of sensitivity: the reagents of groups 1-4 were used to assay low concentration samples (0, 0.01mg/L, 0.05mg/L, 0.10mg/L), each sample was assayed twice, and the sensitivity and the affinity of the antibody were preliminarily determined by the deviation of the assay values from the theoretical values, with the results shown in Table 1.
(2) Linear range: samples with concentrations ranging from 0 to 365mg/L were tested with the reagents of groups 1-12 and the linear correlation coefficients were calculated, with the results shown in Table 2.
(3) Sensitivity: low concentration samples (0, 0.01mg/L, 0.05mg/L) were assayed 20 times using the reagents of groups 5 to 12, and the mean value and the coefficient of variation were calculated, and the results are shown in Table 3.
(4) Precision: the low concentration sample (1mg/L) and the high concentration sample (51.2mg/L) were separately tested 10 times for each sample, and the coefficient of variation CV was calculated to require a CV value of < 10%, with the results shown in Table 4.
(5) Linear range: diluting the high value sample with the low value sample requires: the sample concentration is 0.01mg/L and 10.0mg/L]Within the interval, the linear absolute deviation should be within the range of +/-1.0 mg/L; concentration of sample>At 10.0mg/L, the linear relative deviation should be within + -15%, and the linear correlation coefficient R2> 0.95, and the results are shown in Table 5.
(6) Accuracy: the national standard substances (from China institute of metrology science) were measured and the deviation of the measured value from the target value was calculated, with the deviation required to be < 15%, with the results shown in Table 6.
(7) Sample correlation: samples of different concentrations were tested and compared to the Siemens valueRequire a correlation R2> 0.95, the results are shown in FIG. 2.
TABLE 1 groups 1-4 sensitivity data
As can be seen from the results in Table 1, the measured values of the groups 1 and 2 are more accurate for the samples with low concentrations, while the measured values of the groups 3 and 4 are lower or even undetectable at lower sample concentrations, which indicates that the antibodies used in the groups 1-2 have higher reaction sensitivity than the antibodies used in the groups 3-4, i.e., the antibodies a and b are high affinity antibodies, and the antibodies c and d are low affinity antibodies.
TABLE 2 sets 1-12 Linear Range data
TABLE 3 sensitivity data for groups 5-12
Plotting the linear plots of group 1, group 3 and group 10 according to the linear data of table 2, the results are shown in fig. 1, and combining the data of tables 2-3 and fig. 1, wherein both M and R in groups 1 and 2 are high affinity antibodies, thus the reactivity of the reagent is high, the sensitivity of the low end is high, but the linearity of the high end is poor; both M and R in groups 3 and 4 are low affinity antibodies, so the reagents are low in reactivity, good at the high end, but poor in sensitivity. And after the groups 5-12 combine the high affinity antibody and the low affinity antibody, the linear range is improved compared with the groups 1-2, and the sensitivity is also improved compared with the groups 3-4, which shows that the sensitivity and the linear range of the reagent can be improved by using the combination mode of the high and low affinity antibodies.
Further, as can be seen from the results in Table 2, when M is a low affinity antibody and R is a high affinity antibody (groups 9 to 12), the high-end linear correlation coefficient of the reagent is higher, i.e., better linear, than when M is a high affinity antibody and R is a low affinity antibody (groups 5 to 8); further, the linearity and sensitivity of group 10 were optimized with a linear range of 0-365.14mg/L, a linear correlation coefficient of 0.9999, and a sensitivity of 0.01 mg/L.
The set 10 was then evaluated for other properties.
TABLE 4 set of 10 precision data
TABLE 5 set of 10 Linear Range data
Table 6 set of 10 accuracy data
Combining the results of tables 4-6, wherein the CV values of group 10 were within 5% in both the low and high concentration samples according to the data of Table 4, i.e., the precision of the reagents obtained from group 10 was consistent with the results obtained from group 10The clinical use requirement. From the measurements in Table 5, the linear correlation coefficient R for the group 10 low value samples diluted the high value samples2Is 0.999, and the deviation meets the requirement. According to the measurement results in table 6, the deviation between the detection value of group 10 and the target value was within 10%, and the use requirement was satisfied.
FIG. 2 shows the correlation R between the samples of group 10 and Siemens as a result of the correlation between the samples of different concentrations tested using the reagent of group 10 and the Siemens latex turbidimetric kit, plotting a linear correlation plot of the measured values of the Siemens reagent of the kit of group 10 and calculating the correlation coefficient2The correlation is better if the correlation is 0.9949 and is more than 0.95.
By combining the results, the sensitivity and the linear range of the chemiluminescence detection C-reactive protein can be improved by combining the high-affinity antibody and the low-affinity antibody, the sensitivity can reach 0.01mg/L, and the linear range is 0.01-360mg/L, so that the clinical use requirements are fully met.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (9)
1. A C-reactive protein chemiluminescence immunoassay kit is characterized by comprising: the kit comprises an M reagent, an R reagent, a luminescent substrate solution and a calibrator, wherein the M reagent is a magnetic bead reagent coated with an antibody M, the R reagent is an enzyme labeling reagent connected with an antibody R, both the antibody M and the antibody R can be specifically combined with C reactive protein, and the ratio of affinity constants of the antibody M and the antibody R is 1 multiplied by 10-7～1×10-2Or 1X 102～1×107。
2. The chemiluminescent immunoassay kit for C-reactive protein according to claim 1, wherein the affinity constant of the antibody M is 1 x 10-7～1×10-5When the affinity constant of the antibody R is 1X 10-12～1×10-9。
3. The chemiluminescent immunoassay kit for C-reactive protein according to claim 1, wherein the affinity constant of the antibody M is 1 x 10-12～1×10-9When the affinity constant of the antibody R is 1X 10-7～1×10-5。
4. The method of making a C-reactive protein chemiluminescent immunoassay kit of claim 1, comprising:
s1, preparation of M reagent: washing and activating the magnetic beads, adding an antibody M and a coupling buffer solution for coupling reaction, then adding a sealing buffer solution for sealing, and diluting with an M diluent to obtain the M reagent;
preparation of S2 and R reagent: respectively activating an antibody R and an enzyme, mixing, carrying out enzyme-linked reaction to obtain an enzyme-labeled antibody compound, and diluting with an R diluent to obtain the R reagent;
s3, preparation of luminescent substrate solution: dissolving a luminescent substrate by using a buffer solution to obtain a luminescent substrate solution;
s4, preparing a calibration product: preparing the C-reactive protein antigen into a plurality of concentrations by using a calibrator diluent to obtain a calibrator;
s5, independently placing the magnetic bead reagent coated with the antibody M, the enzyme labeling reagent connected with the antibody R, the luminescent substrate solution and the calibrator in a packaging container to obtain the chemiluminescence immunoassay kit for the C-reactive protein.
5. The method for preparing a chemiluminescent immunoassay kit for C-reactive protein according to claim 4, wherein the step S1 is specifically as follows: (1) taking 10-100 mu L of magnetic beads with the particle size of 1-3 mu m and the concentration of 10-100 mg/mL, carrying out magnetic separation and washing by using 1-3 mL of magnetic bead washing liquid, and then activating for 20-30min at 20-30 ℃ by using 1-3 mL of magnetic bead activating agent;
(2) adding 0.1-0.5 mg of antibody M and 0.5-3 mL of coupling buffer solution into the magnetic beads, and coupling for 2-3h at 20-30 ℃;
(3) continuously adding 0.5-3 mL of closed buffer solution, reacting at 20-30 ℃ for 1-3h, removing supernatant and washing to obtain an antibody coated by magnetic beads;
(4) and (3) diluting the antibody coated by the magnetic beads with M diluent according to the volume ratio of 1: and (5) diluting by 20-60 to obtain the M reagent.
6. The method for preparing a chemiluminescent immunoassay kit for C-reactive protein according to claim 4, wherein the step S2 is specifically as follows: (1) taking 0.1-1 mg of antibody R, adding 0.3-1 mL of antibody activator solution, activating at room temperature for 15-30min, and purifying to obtain an activated antibody;
(2) taking 0.1-1 mg of alkaline phosphatase, adding 0.3-1 mL of enzyme activator, reacting at room temperature for 10-20min, and purifying to obtain activated alkaline phosphatase;
(3) mixing the activated antibody and the activated alkaline phosphatase according to the mass ratio of 1: 0.5-1, adding 5-20 mu L of 1M magnesium chloride solution, reacting for 10-15h at the temperature of 2-8 ℃, and purifying to obtain an enzyme-labeled antibody compound;
(4) and (3) diluting the enzyme-labeled antibody complex with an R diluent according to a volume ratio of 1: diluting 100-300 to obtain the R reagent.
7. The method for preparing the chemiluminescence immunoassay kit for C-reactive protein according to claim 4, wherein the pH of the M diluent in step S1 is 7.0-7.5, and the method comprises the following steps: 20-100 mM buffer solution, 0.1-1% of surfactant, 0.5-3% of protein protective agent and 0.05-0.5% of preservative;
the pH of the R diluent is 5.5-7.0, and the R diluent comprises: 20-100 mM buffer solution, 1-10 mM inorganic salt, 1-10% protein protective agent and 0.05-0.5% preservative.
8. The method for preparing the chemiluminescent immunoassay kit for C-reactive protein according to claim 7, wherein the buffer is one or more of Tris buffer, HEPES buffer, HEPPSO buffer, MES buffer and phosphate buffer;
the surfactant is one or more of Tween20, Tween-80, EMULGEN A-60, EMULGEN A-90, Brij-35, Triton X-405, Triton X-114 and Triton X-100;
the protein protective agent is one or more of sucrose, pullulan, trehalose, bovine serum albumin, casein, acetaminophen and glycine;
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride and zinc chloride;
the preservative is one or more of sodium azide, potassium sorbate, sodium benzoate and Proclin series.
9. Use of the kit of claim 1 for detecting C-reactive protein content.
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