CN110824160A - Anti-cyclic citrullinated peptide antibody detection kit and preparation method thereof - Google Patents
Anti-cyclic citrullinated peptide antibody detection kit and preparation method thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a detection kit for determining concentration of an anti-cyclic citrullinated peptide antibody and a preparation method thereof, wherein the kit comprises a reagent R1 and a reagent R2, and the reagent R1 comprises: the preservative comprises a first buffer solution, sodium chloride, a first active agent, a first preservative and a first stabilizing agent; the reagent R2 includes: a second buffer, sodium chloride, a second active agent, a second preservative, a second stabilizer, latex microspheres coupled with CCP antigen-BSA. The latex microspheres of the kit adopt the latex microspheres coupled with CCP antigen-BSA, the CCP antigen and the BSA are firstly combined through peptide bonds to form a CCP-BSA coupling compound, and then the CCP-BSA coupling compound is coated on latex particles, so that the problem of low efficiency of directly coupling the CCP antigen with the latex particles is solved. And secondly, because the surface of the BSA molecule contains rich carboxyl groups, 1 BSA molecule can be coupled with a plurality of CCP antigen small molecules, so that the prepared CCP antigen-BSA latex microsphere contains rich CCP antigen sites on the surface and can be used for identifying CCP antibodies, and the detection sensitivity of the kit is improved.
Description
Technical Field
The invention belongs to the field of in-vitro diagnostic reagents, and particularly relates to a detection kit for determining the concentration of anti-cyclic citrullinated peptide by using a latex enhanced immunoturbidimetry technology and a preparation method thereof.
Background
Cyclic citrulline polypeptide antibody (anti-cyclic peptide binding citrulline, anti-CCP): is a polypeptide fragment of cyclic polyguanidine protein, and is an antibody mainly of IgG type. The anti-CCP antibody is spontaneously secreted by B lymphocytes of RA patients, while B lymphocytes of other disease patients and normal people do not spontaneously secrete the anti-CCP antibody. Therefore, anti-CCP antibodies have high specificity for RA.
Rheumatoid Arthritis (RA) is a common systemic autoimmune disease, is mainly characterized clinically by joint synovitis and symmetric and destructive joint lesions, has a long course of disease, is easy to repeat, and causes great pain to patients. The disease has the morbidity of about 0.4 percent in China, the worldwide disease is 0.5 to 1 percent, and the disease onset age of patients is 20 to 50 years old.
Studies have shown that anti-perinuclear factor (APF), anti-keratin antibody (AKA) can be detected in the serum of RA patients, with the antigenic site being Cyclic Citrullinated Polypeptide (CCP). And the detection of the anti-cyclic citrulline polypeptide antibody has certain superiority compared with APF and AKA detection, so the detection of the anti-cyclic citrulline polypeptide antibody has important significance for the diagnosis of RA.
There are many products for measuring anti-cyclic citrullinated peptide antibodies on the market, and the measurement methods adopted by the products mainly comprise: 1. immunochromatography: the detection is rapid, but the detection accuracy and repeatability are not as good as other methods. 2. A chemiluminescence method: the accuracy and reproducibility of detection are optimal, but the price is high. 3. Enzyme linked immunosorbent assay: the detection process is complex and consumes long time. 4. Latex enhanced immunoturbidimetry: the method can realize rapid, accurate and full-automatic detection and controllable cost in the determination of the anti-cyclic citrullinated peptide antibody.
For example, patent CN102507918B discloses an anti-cyclic citrullinated peptide antibody assay kit, which comprises R1 reagent and R2 reagent, wherein R1 contains CCP sensitized nano-microspheres, but the patent does not disclose a preparation method of CCP sensitized nano-microspheres; the anti-CCP secondary antibody contained in R2 is used for improving the detection sensitivity, but the anti-CCP secondary antibody exists in a protein form in the R2 reagent, so that the improvement of the detection sensitivity of the whole reagent is limited. In addition, the kit has the following disadvantages: CCP sensitized nano-microsphere components are prepared in R1, in the detection process, an R1 reagent is firstly incubated with a detection sample, and interference substances in the detection sample in the incubation process easily enable the CCP sensitized nano-microsphere to generate nonspecific reaction and generate a false positive or false negative detection signal.
Patent CN104198725B discloses an anti-cyclic citrullinated peptide antibody detection kit, which comprises a R1 reagent and a R2 reagent, wherein the R1 reagent is a buffer component, and CCP antigen latex particles are contained in the R2 reagent. This patent discloses a process for preparing CCP antigen latex particles and mentions that CCP antigens are directly coupled to the latex particles by activation to prepare CCP antigen latex particles. However, since the CCP antigen is a peptide chain of amino acids having a very small molecular weight (molecular weight of about 4K Da), the efficiency of direct coupling to latex particles with this antigen is extremely low, resulting in a large waste of raw materials. In addition, the CCP antigen is very small in size, so that CCP antibody (150KDa) is difficult to identify, and the reagent has limited detection sensitivity and cannot well detect the anti-cyclic citrullinated peptide antibody.
Patent CN109100515A discloses an anti-cyclic citrullinated peptide antibody detection kit, which comprises a R1 reagent and a R2 reagent, wherein the R1 reagent is a buffer component, and the R2 reagent comprises coupled cyclic citrullinated peptide antigen-biotin-streptavidin-latex particles. The patent discloses a preparation method of cyclic citrullinated peptide antigen-biotin-streptavidin-latex particles, and solves the problem of low efficiency of directly coupling cyclic citrullinated peptide antigen with latex particles. However, one streptavidin molecule can only bind to a maximum of 4 biotinylated cyclic citrullinated peptide antigens, resulting in a limited amount of antigen coupled to latex particles and poor reagent detection sensitivity.
In summary, the existing latex enhanced immunoturbidimetry kit for determining the cyclic citrullinated peptide antibody has the following problems: 1) inefficient direct coupling of cyclic citrullinated peptide antigens to latex particles; 2) coupling the latex particles by adopting a biotin-streptavidin grafting mode, wherein the coverage of the cyclic citrullinated peptide antigen is limited; 3) the detection sensitivity of the currently disclosed anti-cyclic citrullinated peptide antibody detection kit is low.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an improved anti-cyclic citrullinated peptide antibody detection kit and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight:
the reagent R2 comprises the following components in percentage by weight:
according to some embodiments of the invention, the latex microspheres are prepared by first binding a CCP antigen to BSA (bovine serum albumin) via a peptide bond to form a CCP antigen-BSA conjugate, and then coating the CCP antigen-BSA conjugate on latex particles.
According to some embodiments of the invention, the method of preparing the latex microspheres comprises the steps of:
(1) preparation of CCP antigen-BSA conjugates
Preparing a BSA solution, a CCP antigen solution and an EDC solution by using MES buffer solution respectively, and then mixing the BSA solution, the CCP antigen solution and the EDC solution according to a volume ratio of 1: 1-3: 0.5-1.5, uniformly mixing in a centrifuge tube, incubating at constant temperature for 1-3 hours, transferring the incubated reagent into an ultrafiltration tube for centrifugation, washing with PBS (phosphate buffer solution), inverting and centrifuging the ultrafiltration tube after washing, collecting a concentrated solution, and diluting the concentrated solution with the PBS to obtain a CCP antigen-BSA coupler reagent;
(2) preparation of latex microspheres coupled with CCP antigen-BSA
Preparing a latex particle solution by using a PBS (phosphate buffer solution), uniformly mixing the latex particle solution and a CCP (glycidyl methacrylate) -BSA (bovine serum albumin) conjugate reagent in a centrifugal tube, incubating at constant temperature for 0.5-2 hours, adding an EDC solution prepared by using an MES (maleic anhydride) buffer solution, uniformly mixing, and incubating at constant temperature for 0.5-2 hours for activation; and adding a BSA solution prepared by using a PBS buffer solution, incubating for 0.5-2 hours at constant temperature for sealing, centrifuging after sealing, removing supernatant, and washing by using the PBS buffer solution to prepare the latex microspheres coupled with the CCP antigen-BSA.
According to some example aspects of this invention, the method of preparing the latex microspheres comprises the steps of:
(1) preparation of CCP antigen-BSA conjugates
A. Respectively preparing 0.5% BSA solution, 0.5mg/mL CCP antigen solution and 10mg/mLEDC solution by using 25mM MES buffer solution;
B. uniformly mixing 1 part of BSA solution, 2 parts of CCP antigen solution and 1 part of EDC solution with a centrifuge tube, placing the mixture in a constant temperature shaking table for incubation for 2 hours at the incubation temperature of 37 ℃, transferring the incubated reagent into an ultrafiltration tube for centrifugation, washing the ultrafiltration tube for 5 times by using 10mM PBS buffer solution, inverting and centrifuging the ultrafiltration tube after washing, collecting concentrated solution, and diluting the concentrated solution to 4 parts by volume by using 10mM PBS buffer solution to obtain CCP antigen-BSA reagent;
(2) preparation of latex microspheres coupled with CCP antigen-BSA
A. Preparing 1 part of 1% latex particle solution by using 10mM PBS buffer solution, placing the 1 part of 1% latex particle solution in a centrifuge tube, then adding an equal amount of CCP antigen-BSA reagent, uniformly mixing, placing the mixture in a constant temperature shaking table for incubation for 1 hour, wherein the incubation temperature is 37 ℃, and obtaining a latex-CCP antigen-BSA solution;
B. preparing 10mg/mL EDC solution by using 25mM MES buffer solution, then adding 0.1 part of EDC solution into latex-CCP antigen-BSA solution, mixing uniformly, placing in a constant temperature shaking table, incubating for 1 hour for activation, and incubating at 37 ℃;
C. preparing a 2% BSA solution by using 10mM PBS buffer solution, then adding 0.1 part of BSA solution into the latex-CCP antigen-BSA solution for uniformly mixing, placing the mixture in a constant temperature shaking table for incubation for 1 hour for sealing, wherein the incubation temperature is 37 ℃, placing a centrifugal tube in a centrifugal machine for centrifugation after sealing is finished, removing supernatant, and washing by using 10mM PBS buffer solution to prepare the latex microspheres coupled with the CCP antigen-BSA.
Preferably, the particle size of the latex microsphere is 50-500 nm.
Preferably, the latex particles are polystyrene latex particles or other latex particles that contain carboxyl groups on their surface.
Preferably, the first buffer solution and the second buffer solution are respectively and independently selected from one of 10-100mM MES buffer solution, 10-100mM HEPES buffer solution, 10-50mM PB buffer solution, 10-100mM Tris buffer solution and 20-100mM glycine buffer solution.
Preferably, the first active agent and the second active agent are respectively and independently selected from one or a combination of more of Tween20, Tween80, PEG4000, PEG6000, PVP and PVA.
Preferably, the first preservative and the second preservative are respectively and independently selected from one or a combination of several of sodium azide, Proclin-300 and Proclin.
Preferably, the first stabilizer and the second stabilizer are respectively and independently selected from one or a combination of casein, bovine serum albumin, sucrose, trehalose and mannitol.
The invention adopts another technical scheme that: a preparation method of the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody comprises the following steps:
1) preparation of reagent R1
Uniformly mixing water, a first buffer solution, sodium chloride, a first active agent, a first preservative and a first stabilizer to prepare a reagent R1;
2) preparation of storage solution for reagent R2
Uniformly mixing water, a second buffer solution, sodium chloride, a second active agent, a second preservative and a second stabilizer to prepare a preservation solution of the reagent R2;
3) preparation of reagent R2
The reagent R2 was prepared by dissolving the CCP antigen-BSA-coupled latex microspheres in a storage solution of the reagent R2.
Preferably, the preparation of the reagent R1 is specifically as follows: adding a proper amount of purified water and a magnetic stirrer into a beaker, placing the beaker on magnetic stirring for magnetic stirring, adding a first buffer solution while stirring, and stirring for 2-10 minutes until the materials are completely dissolved; then adding sodium chloride while stirring until the materials are completely dissolved; then adding a first active agent while stirring until the materials are completely dissolved; then adding a first preservative while stirring until the materials are completely dissolved; then adding the first stabilizer while stirring until the materials are completely dissolved. And finally, fixing the volume to the final volume and marking.
Preferably, the preparation of the preservation solution of the reagent R2 is specifically as follows: adding a proper amount of purified water and a magnetic stirrer into a beaker, placing the beaker on magnetic stirring for magnetic stirring, adding a second buffer solution while stirring, and stirring for 2-10 minutes until the materials are completely dissolved; then adding sodium chloride while stirring until the materials are completely dissolved; then adding a second active agent while stirring until the materials are completely dissolved; then adding a second preservative while stirring until the materials are completely dissolved; then adding a second stabilizing agent while stirring until the materials are completely dissolved. And finally, fixing the volume to the final volume and marking.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
the latex microspheres in the kit adopt the latex microspheres coupled with CCP antigen-BSA, wherein CCP antigen small molecules and BSA macromolecules are firstly combined through peptide bonds to form a CCP-BSA coupling compound, and then the CCP-BSA coupling compound is coated on latex particles, so that the problem of low efficiency of the CCP antigen direct coupling of the latex particles is solved. And secondly, because the surface of BSA macromolecule contains abundant carboxyl groups, 1 BSA molecule can be coupled with a plurality of CCP antigen micromolecules, so that the prepared CCP antigen-BSA latex microsphere contains abundant CCP antigen sites on the surface and can be used for identifying CCP antibodies, and the detection sensitivity of the kit is improved.
Detailed Description
Specific embodiments of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1
The detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody provided by the embodiment comprises a reagent R1 and a reagent R2, wherein,
the components and concentrations of the reagent R1 were as follows: the first buffer is 25mM MES buffer; sodium chloride, at a concentration of 9%; the first active agent is Tween20 with the concentration of 0.2%; the first preservative is Proclin, and the concentration of the first preservative is 0.05%; the first stabilizer was bovine serum albumin at a concentration of 5%.
The components and concentrations of the reagent R2 were as follows: the second buffer solution is 25mM MES buffer solution; sodium chloride, at a concentration of 5%; the second active agent is Tween20 with the concentration of 0.5%; the second preservative is Proclin with the concentration of 0.05%; the second stabilizer is bovine serum albumin, the concentration of which is 0.5 percent; CCP-BSA coupled latex microspheres at 1% concentration.
The preparation method of the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody comprises the following steps:
(1) preparation of reagent R1
Adding a proper amount of purified water and a magnetic stirrer into a beaker, placing the beaker on magnetic stirring for magnetic stirring, adding a first buffer solution while stirring, and stirring for 5 minutes until the materials are completely dissolved; then adding sodium chloride while stirring until the materials are completely dissolved; then adding a first active agent while stirring until the materials are completely dissolved; then adding a first preservative while stirring until the materials are completely dissolved; then adding the first stabilizer while stirring until the materials are completely dissolved. And finally, fixing the volume to the final volume and marking.
(2) Preparation of reagent R2
21) Preparation of reagent R2 preservation solution
Adding a proper amount of purified water and a magnetic stirrer into a beaker, placing the beaker on magnetic stirring for magnetic stirring, adding a second buffer solution while stirring, and stirring for 5 minutes until the materials are completely dissolved; then adding sodium chloride while stirring until the materials are completely dissolved; then adding a second active agent while stirring until the materials are completely dissolved; then adding a second preservative while stirring until the materials are completely dissolved; then adding a second stabilizing agent while stirring until the materials are completely dissolved. And finally, fixing the volume to the final volume and marking.
22) Preparation of CCP antigen-BSA conjugates
0.5% BSA solution in 25mM MES buffer; preparing 0.5mg/mL CCP antigen solution by using 25mM MES buffer solution; preparing 10mg/mL EDC solution by using 25mM MES buffer solution; uniformly mixing 1 part of BSA solution, 2 parts of CCP antigen solution and 1 part of EDC solution in a centrifuge tube, and placing the centrifuge tube in a constant temperature shaking table for incubation for 2 hours at the incubation temperature of 37 ℃; transferring the incubated reagent to an ultrafiltration tube for centrifugation, and washing 5 times with 10mM PBS; after washing, inverting and centrifuging the ultrafiltration tube, and collecting a concentrated solution; the concentrate was diluted to 4 volumes with 10mM PBS buffer to obtain CCP antigen-BSA coupler reagent.
23) Preparation of latex microspheres coupled with CCP antigen-BSA
1 part of 1% latex particle solution is prepared by 10mM PBS buffer solution and is put in a centrifuge tube; uniformly mixing an equivalent CCP antigen-BSA reagent with the latex particle solution, and placing the mixture in a constant temperature shaking table for incubation for 1 hour, wherein the incubation temperature is 37 ℃; preparing 10mg/mL EDC solution by using 25mM MES buffer solution; adding 0.1 part of EDC solution into the latex-CCP antigen-BSA solution, mixing uniformly, and placing the mixture in a constant temperature shaking table to incubate for 1 hour for activation, wherein the incubation temperature is 37 ℃; preparing 2% BSA solution with 10mM PBS buffer; adding 0.1 part of BSA solution into the latex-CCP antigen-BSA solution, mixing uniformly, placing the mixture in a constant temperature shaking table, and incubating for 1 hour for sealing, wherein the incubation temperature is 37 ℃; after completion of blocking, the centrifuge tube was centrifuged in a centrifuge, and the supernatant was discarded and washed once with 10mM PBS buffer to obtain latex microspheres to which CCP antigen-BSA was coupled.
24) Preparation of reagent R2
And (3) decoupling the latex microspheres linked with CCP antigen-BSA by using 1 part of a preservation solution of the reagent R2 to obtain the reagent R2.
In this example, the latex particles used were 300nm in diameter.
Example 2
The difference between the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody provided in this example and the detection kit provided in example 1 is that: when preparing the CCP antigen-BSA conjugate, uniformly mixing a BSA solution, a CCP antigen solution and an EDC solution, and incubating the mixture in a volume ratio of 1: 1: 1.
example 3
The difference between the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody provided in this example and the detection kit provided in example 1 is that: the latex particles are 200nm in diameter.
Comparative example 1
The difference between the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody provided in this example and the detection kit provided in example 1 is that: the reagent R2 was prepared by direct coupling of CCP antigen to latex particles. The components and concentrations of the reagent R2 were as follows: the second buffer solution is 25mM MES buffer solution; sodium chloride, at a concentration of 5%; the second active agent is Tween20 with the concentration of 0.5%; the second preservative is Proclin with the concentration of 0.05%; the second stabilizer is bovine serum albumin, the concentration of which is 0.5 percent; CCP coupled latex microspheres at a concentration of 1%.
Preparation of reagent R2
1) Preparation of reagent R2 preservation solution
Adding a proper amount of purified water and a magnetic stirrer into a beaker, placing the beaker on magnetic stirring for magnetic stirring, adding a second buffer solution while stirring, and stirring for 5 minutes until the materials are completely dissolved; then adding sodium chloride while stirring until the materials are completely dissolved; then adding a second active agent while stirring until the materials are completely dissolved; then adding a second preservative while stirring until the materials are completely dissolved; then adding a second stabilizing agent while stirring until the materials are completely dissolved. And finally, fixing the volume to the final volume and marking.
2) Preparation of CCP antigen coupled latex microspheres
1 part of 1% latex particle solution is prepared by 10mM PBS buffer solution and is put in a centrifuge tube; uniformly mixing the CCP antigen with the same amount of 0.5mg/mL and the latex particle solution, and placing the mixture in a constant temperature shaking table for incubation for 1 hour, wherein the incubation temperature is 37 ℃; preparing 10mg/mL EDC solution by using 25mM MES buffer solution; adding 0.1 part of EDC solution into the latex-CCP antigen solution, mixing uniformly, and placing the mixture in a constant temperature shaking table to incubate for 1 hour for activation, wherein the incubation temperature is 37 ℃; preparing 2% BSA solution with 10mM PBS buffer; adding 0.1 part of BSA solution into the latex-CCP antigen solution, mixing uniformly, placing the mixture in a constant temperature shaking table, and incubating for 1 hour for sealing, wherein the incubation temperature is 37 ℃; after the completion of the blocking, the centrifuge tube was centrifuged in a centrifuge, and the supernatant was discarded and washed once with 10mM PBS buffer to obtain latex microspheres to which CCP antigen was coupled.
3) Preparation of reagent R2
And (3) decoupling the latex microspheres connected with the CCP antigen by using 1 part of a preservation solution of the reagent R2 to obtain the reagent R2.
In this example, the latex particles used were 300nm in diameter.
Comparative example 2
The difference between the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody provided in this example and the detection kit provided in example 1 is that: the R2 reagent was prepared by coating streptavidin-conjugated latex particles with a biotin-conjugated CCP antigen. The components and concentrations of the reagent R2 were as follows: the second buffer solution is 25mM MES buffer solution; sodium chloride, at a concentration of 5%; the second active agent is Tween20 with the concentration of 0.5%; the second preservative is Proclin with the concentration of 0.05%; the second stabilizer is bovine serum albumin, the concentration of which is 0.5 percent; CCP-biotin-streptavidin coupled latex microspheres at a concentration of 1%.
Preparation of reagent R2
1) Preparation of reagent R2 preservation solution
Adding a proper amount of purified water and a magnetic stirrer into a beaker, placing the beaker on magnetic stirring for magnetic stirring, adding a second buffer solution while stirring, and stirring for 5 minutes until the materials are completely dissolved; then adding sodium chloride while stirring until the materials are completely dissolved; then adding a second active agent while stirring until the materials are completely dissolved; then adding a second preservative while stirring until the materials are completely dissolved; then adding a second stabilizing agent while stirring until the materials are completely dissolved. And finally, fixing the volume to the final volume and marking.
2) Preparation of CCP antigen-coupled biotin
Preparing 0.5mg/mL biotin-NHS solution by using 25mM MES buffer solution; preparing 0.5mg/mL CCP antigen solution by using 25mM MES buffer solution; uniformly mixing 1 part of biotin-NHS solution and 1 part of CCP antigen solution in a centrifuge tube, and placing the centrifuge tube and the CCP antigen solution in a constant temperature shaking table for incubation for 2 hours, wherein the incubation temperature is 37 ℃; transferring the incubated reagent to an ultrafiltration tube for centrifugation, and washing 5 times with 10mM PBS; after washing, inverting and centrifuging the ultrafiltration tube, and collecting a concentrated solution; the concentrate was diluted to 4 volumes with 10mM PBS buffer to obtain CCP antigen-biotin conjugate reagent.
3) Preparation of streptavidin coupled latex microspheres
1 part of 1% latex particle solution is prepared by 10mM PBS buffer solution and is put in a centrifuge tube; uniformly mixing the equivalent 0.5mg/mL streptavidin and the latex particle solution, and placing the mixture in a constant-temperature shaking table to incubate for 1 hour at the incubation temperature of 37 ℃; preparing 10mg/mL EDC solution by using 25mM MES buffer solution; adding 0.1 part of EDC solution into the latex-streptavidin solution, mixing uniformly, placing the mixture in a constant temperature shaking table to incubate for 1 hour for activation, wherein the incubation temperature is 37 ℃; preparing 2% BSA solution with 10mM PBS buffer; adding 0.1 part of BSA solution into the latex-streptavidin solution, mixing uniformly, placing the mixture in a constant-temperature shaking table, incubating for 1 hour, and sealing, wherein the incubation temperature is 37 ℃; after blocking, the tube was centrifuged in a centrifuge, the supernatant was discarded and washed once with 10mM PBS buffer to obtain streptavidin-coupled latex microspheres.
4) Preparation of CCP antigen-biotin-streptavidin latex microspheres
Uniformly mixing 1 part of CCP antigen-biotin coupling reagent and 1 part of streptavidin-latex particle coupling reagent in a centrifuge tube, and placing the mixture in a constant temperature shaking table for incubation for 1 hour, wherein the incubation temperature is 37 ℃; preparing 2% BSA solution with 10mM PBS buffer; adding 0.1 part of BSA solution into CCP antigen-biotin-streptavidin latex microsphere solution, mixing uniformly, placing in a constant temperature shaking table, incubating for 1 hour, and sealing, wherein the incubation temperature is 37 ℃; and after the sealing is finished, placing the centrifugal tube in a centrifugal machine for centrifugation, discarding the supernatant, and washing the supernatant once by using 10mMPBS buffer solution to obtain the latex microspheres coupled with the CCP antigen-biotin-streptavidin.
5) Preparation of reagent R2
And (3) decoupling the latex microspheres linked with the CCP antigen-biotin-streptavidin by using 1 part of a preservation solution of the reagent R2 to obtain the reagent R2.
In this example, the latex particles used were 300nm in diameter.
Clinical sample detection and data analysis
The anti-cyclic citrullinated peptide antibody detection kit prepared by the method is suitable for various types of full-automatic biochemical analyzers, taking Hitachi 7170 full-automatic biochemical analyzer as an example, the detection process of clinical samples is as follows:
computer-operating parameters:
and (3) calibrating by adopting a self-prepared calibration standard:
the above calibration data analysis shows that the reactivity of example 1 is the highest when the same concentration of calibration sample is detected, which indicates that the concentration of the anti-CCP antibody-CCP antigen latex particle complex generated by the reaction is the highest and the sensitivity of the kit prepared in example 1 is the highest.
Test results of 20 clinical samples:
the analysis of the detection data of the clinical samples shows that the probability of zero occurrence is lowest and the detection sensitivity is optimal when the kit prepared in the embodiment 1 is used for detecting low-value clinical samples. Meanwhile, the kit prepared in the embodiment 1 is adopted to detect 20 clinical samples, and the correlation between the result and the Roche electrochemiluminescence detection result is the best.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody comprises a reagent R1 and a reagent R2, and is characterized in that the reagent R1 comprises the following components in percentage by weight:
0.8-25 g/L of first buffer solution;
0.5-15 g/L of sodium chloride;
0.1% -5% of a first active agent;
0.01% -0.1% of first preservative;
0.1 to 10 percent of first stabilizer;
the reagent R2 comprises the following components in percentage by weight:
the second buffer solution is 0.8-25 g/L;
0.5-15 g/L of sodium chloride;
0.1% -5% of a second active agent;
0.01 to 0.1 percent of second preservative;
0.1% -10% of a second stabilizer;
0.05% -1% latex microspheres coupled with CCP antigen-BSA.
2. The detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody according to claim 1, characterized in that: the latex microsphere is prepared by combining CCP antigen and BSA through peptide bonds to form a CCP antigen-BSA coupler and then coating the CCP antigen-BSA coupler on latex particles.
3. The detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody according to claim 2, wherein the preparation method of the latex microsphere comprises the following steps:
(1) preparation of CCP antigen-BSA conjugates
Preparing a BSA solution, a CCP antigen solution and an EDC solution by using MES buffer solution respectively, and then mixing the BSA solution, the CCP antigen solution and the EDC solution according to a volume ratio of 1: 1-3: 0.5-1.5, uniformly mixing in a centrifuge tube, incubating at constant temperature for 1-3 hours, transferring the incubated reagent into an ultrafiltration tube for centrifugation, washing with PBS (phosphate buffer solution), inverting and centrifuging the ultrafiltration tube after washing, collecting a concentrated solution, and diluting the concentrated solution with the PBS to obtain the cyclic citrullinated peptide-BSA (bovine serum albumin) conjugate reagent;
(2) preparation of latex microspheres coupled with CCP antigen-BSA
Preparing a latex particle solution by using a PBS (phosphate buffer solution), uniformly mixing the latex particle solution and a cyclic citrullinated peptide-BSA (bovine serum albumin) coupler reagent in a centrifugal tube, incubating at constant temperature for 0.5-2 hours, adding an EDC solution prepared by using an MES (methyl methacrylate) buffer solution, uniformly mixing, and incubating at constant temperature for 0.5-2 hours for activation; and adding a BSA solution prepared by using a PBS buffer solution, incubating for 0.5-2 hours at constant temperature for sealing, centrifuging after sealing, removing supernatant, and washing by using the PBS buffer solution to prepare the latex microspheres coupled with the CCP antigen-BSA.
4. The detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody according to claim 3, wherein the preparation method of the latex microsphere comprises the following steps:
(1) preparation of CCP antigen-BSA conjugates
A. Respectively preparing 0.5% BSA solution, 0.5mg/mL CCP antigen solution and 10mg/mL EDC solution by using 25mM MES buffer solution;
B. uniformly mixing 1 part of BSA solution, 2 parts of CCP antigen solution and 1 part of EDC solution with a centrifuge tube, placing the mixture in a constant temperature shaking table for incubation for 2 hours at the incubation temperature of 37 ℃, transferring the incubated reagent into an ultrafiltration tube for centrifugation, washing the ultrafiltration tube for 5 times by using 10mM PBS buffer solution, inverting and centrifuging the ultrafiltration tube after washing, collecting concentrated solution, and diluting the concentrated solution to 4 parts by volume by using the 10mM PBS buffer solution to obtain CCP antigen-BSA reagent;
(2) preparation of latex microspheres coupled with CCP antigen-BSA
A. Preparing 1 part of 1% latex particle solution by using 10mM PBS buffer solution, placing the 1 part of 1% latex particle solution in a centrifuge tube, then adding an equal amount of CCP antigen-BSA reagent, uniformly mixing, placing the mixture in a constant temperature shaking table for incubation for 1 hour, wherein the incubation temperature is 37 ℃, and obtaining a latex-CCP antigen-BSA solution;
B. preparing 10mg/mL EDC solution by using 25mM MES buffer solution, then adding 0.1 part of EDC solution into latex-CCP antigen-BSA solution, mixing uniformly, placing in a constant temperature shaking table, incubating for 1 hour for activation, and incubating at 37 ℃;
C. preparing a 2% BSA solution by using 10mM PBS buffer solution, then adding 0.1 part of BSA solution into the latex-CCP antigen-BSA solution for uniformly mixing, placing the mixture in a constant temperature shaking table for incubation for 1 hour for sealing, wherein the incubation temperature is 37 ℃, placing a centrifugal tube in a centrifugal machine for centrifugation after sealing is finished, removing supernatant, and washing by using 10mM PBS buffer solution to prepare the latex microspheres coupled with the CCP antigen-BSA.
5. The detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody according to claim 1, characterized in that: the particle size of the latex microspheres is 50-500 nanometers.
6. The detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody according to claim 1, characterized in that: the first buffer solution and the second buffer solution are respectively and independently selected from one of 10-100mM MES buffer solution, 10-100mM HEPES buffer solution, 10-50mM PB buffer solution, 10-100mM Tris buffer solution and 20-100mM glycine buffer solution.
7. The detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody according to claim 1, characterized in that: the first active agent and the second active agent are respectively and independently selected from one or a combination of more of Tween20, Tween80, PEG4000, PEG6000, PVP and PVA.
8. The detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody according to claim 1, characterized in that: the first preservative and the second preservative are respectively and independently selected from one or a combination of more of sodium azide, Proclin-300 and Proclin.
9. The detection kit for determining the concentration of an anti-cyclic citrullinated peptide antibody according to claim 1, characterized in that: the first stabilizer and the second stabilizer are respectively and independently selected from one or a combination of more of casein, bovine serum albumin, sucrose, trehalose and mannitol.
10. A method for preparing the detection kit for determining the concentration of the anti-cyclic citrullinated peptide antibody according to any one of claims 1 to 9, wherein the method comprises the following steps:
1) preparation of reagent R1
Uniformly mixing water, a first buffer solution, sodium chloride, a first active agent, a first preservative and a first stabilizer to prepare a reagent R1;
2) preparation of storage solution for reagent R2
Uniformly mixing water, a second buffer solution, sodium chloride, a second active agent, a second preservative and a second stabilizer to prepare a preservation solution of the reagent R2;
3) preparation of reagent R2
The reagent R2 was prepared by dissolving the CCP antigen-BSA-coupled latex microspheres in a storage solution of the reagent R2.
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