CN115792223A - Anti-cyclic citrullinated peptide antibody detection kit - Google Patents
Anti-cyclic citrullinated peptide antibody detection kit Download PDFInfo
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Abstract
The invention provides an anti-cyclic citrullinated peptide antibody detection kit, which belongs to the technical field of biology and comprises an anti-cyclic citrullinated peptide antibody detection test strip and a sample pretreatment pipe, wherein a determination liquid S1 is filled in the sample pretreatment pipe, the anti-cyclic citrullinated peptide antibody detection test strip comprises a bottom plate, and a nitrocellulose membrane is arranged on the bottom plate; one end of the nitrocellulose membrane is lapped with a gold-labeled antibody reaction pad, the other end of the nitrocellulose membrane is lapped with absorbent paper, and one end of the gold-labeled antibody reaction pad is lapped with a sample pad; the anti-cyclic citrullinated peptide antibody detection reagent is characterized in that the materials are stirred according to different proportions to form anti-cyclic citrullinated peptide antibody determination liquid, a sample is mixed with the determination liquid S1 in advance before determination, the sample is pretreated, interference components in the sample are conveniently eliminated, the specificity of the anti-cyclic citrullinated peptide antibody detection reagent is improved, and therefore the determination accuracy of the anti-cyclic citrullinated peptide antibody is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an anti-cyclic citrullinated peptide antibody detection kit.
Background
Rheumatoid Arthritis (RA) is a common chronic autoimmune disease with chronic inflammatory response of synovial joints and joint injury as the main clinical manifestations. Rheumatoid arthritis is common clinically and is a multiple autoimmune disease. The disease is mainly characterized by chronic inflammatory hyperplasia of synovial membrane, irreversible progressive destruction of articular cartilage and bone, which can cause symptoms such as joint stiffness, deformity and function loss, and the irreversible joint deformity or destruction can cause the life quality of patients if rheumatoid arthritis is not treated in time. Therefore, an effective method is needed to be found, the accuracy of early diagnosis is improved, intervention is performed as early as possible, the progress of the disease is controlled, and the erosion and damage of joints are reduced.
Currently, there are many methods for clinically diagnosing rheumatoid arthritis, including clinical manifestations, X-rays, rheumatoid factors, etc., and different methods have different effects and have different advantages and disadvantages.
In recent years, attention has been paid to the application effect of serum immunological indicators in the diagnosis of rheumatoid arthritis, and anti-cyclic citrullinated peptide antibodies and the like are common.
The anti-cyclic citrullinated peptide antibody is an autoantibody taking synthesized Cyclic Citrullinated Polypeptide (CCP) as an antigen, has higher sensitivity and specificity on Rheumatoid Arthritis (RA), and is a highly specific index for early diagnosis of RA. The existing determination of the anti-cyclic citrullinated peptide antibody is mostly performed by enzyme linked immunosorbent assay, chemiluminescence assay and the like, and the methods have the disadvantages of long time, complex process and low use efficiency in the determination process.
Disclosure of Invention
The invention aims to overcome the defects of long time and complex determination process in various determination methods of anti-cyclic citrullinated peptide antibodies in the prior art, and provides the anti-cyclic citrullinated peptide antibody detection kit which is simple to operate and can realize family self-detection, and interference competition existing in the reaction process is reduced and the specificity of a reagent is improved by processing a sample to be detected in advance.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: an anti-cyclic citrullinated peptide antibody detection kit comprises an anti-cyclic citrullinated peptide antibody detection test strip and a sample pretreatment tube, wherein a determination liquid S1 is filled in the sample pretreatment tube, the anti-cyclic citrullinated peptide antibody detection test strip comprises a bottom plate, a nitrocellulose membrane is arranged on the bottom plate, and a detection T line coated with 6 different cyclic citrullinated peptide antigens and a quality control C line coated with goat anti-mouse IgG antibody are arranged on the nitrocellulose membrane; one end of the nitrocellulose membrane is lapped with a gold-labeled antibody reaction pad, the other end of the nitrocellulose membrane is lapped with absorbent paper, and one end of the gold-labeled antibody reaction pad is lapped with a sample pad; the determination liquid S1 consists of 90mmol/L PB buffer solution, 15g/L sodium chloride, 18g/L PEG polymeric stabilizer, 0.6g/L PC-300 preservative and 0.6g/L IgG monoclonal antibody blocking agent, the solvent is purified water, and the pH value is 7.2-7.6.
As a further improvement of the technical scheme:
the gold-labeled antibody reaction pad is coated with a mouse anti-human IgG monoclonal antibody-colloidal gold compound.
The particle size of the colloidal gold adopted in the mouse anti-human IgG monoclonal antibody-colloidal gold compound coating coated on the gold-labeled antibody reaction pad is 30nm.
The preparation method of the mouse anti-human IgG monoclonal antibody-colloidal gold compound coating comprises the following steps: preparing colloidal gold by a trisodium citrate reduction method, adding 1mg of mouse anti-human IgG monoclonal antibody into a colloidal gold solution with the particle size of 100mL and 30nm, centrifuging after reaction, removing supernate, adding 5mL of gold redissolving solution, standing for reaction to prepare a mouse anti-human IgG monoclonal antibody-colloidal gold compound, diluting with the gold redissolving solution according to the volume ratio of 1 2 Coating on glass fiber, and drying.
The preparation method of the detection T line and the quality control C line comprises the following steps: diluting the mixed solution of the 6 cyclic citrullinated peptide antigens to 0.8mg/mL and the goat anti-mouse IgG antibody to 1.8mg/mL by using diluent respectively, sequentially spraying the diluted solution on a nitrocellulose membrane, and drying the nitrocellulose membrane to obtain the polypeptide.
The 6 different cyclic citrullinated peptide antigens are CCP-I-CCP-VI and are formed by mixing 6 antigens according to the following ratio of 1.
The diluent is prepared by adding 3wt% of sucrose and 2wt% of trehalose into 1L of 20mmol PBS buffer solution, and the pH value of the PBS buffer solution is 7.2.
The gold-complex solution was composed of 0.05M pH7.4 Tris, 0.05wt% glucose, 0.02wt% trehalose, 0.03wt% Tween-20, 0.02wt% BSA, 0.01wt% NaN 3 Mixing to obtain the final product with pH of 7.0-7.5.
The sample pad is obtained by treating glass fiber with a sample treatment solution and drying.
The sample treatment solution was prepared by mixing 0.5wt% PVA, 0.1wt% S9, 0.02wt% Triton X-100, 0.7mg/mL anti-erythrocyte antibody, 0.4wt% trehalose, and 0.2wt% casein in 1000mL of PB buffer solution having a pH of 8.0 to 8.5.
By adopting the technical scheme, the invention has the following advantages:
(1) The anti-cyclic citrullinated peptide antibody detection reagent is characterized in that the materials are stirred according to different proportions to form anti-cyclic citrullinated peptide antibody determination liquid, a sample is mixed with the determination liquid S1 in advance before determination, the sample is pretreated, interference components in the sample are conveniently eliminated, the specificity of the anti-cyclic citrullinated peptide antibody detection reagent is improved, and therefore the determination accuracy of the anti-cyclic citrullinated peptide antibody is improved.
(2) The sample pad 7 can filter impurities and red blood cells in a sample to be detected after being treated by the sample treatment solution, and is suitable for detecting serum/plasma/whole blood and fingertip blood samples, so that the household self-detection is realized; the sample pad 7 has good permeability, can improve the water absorption speed, quickens the chromatography speed on the NC membrane, and can improve the specificity of the product.
(3) And a quality control C line 4 on the nitrocellulose membrane 2 is used for diluting the cyclic citrullinated peptide antigen mixed solution and the goat anti-mouse IgG antibody by using a diluent containing trehalose and sucrose when the detection T line 3 is coated, so that the stability of the test agent can be improved.
In conclusion, the kit is suitable for various samples, and after the samples are mixed with the determination liquid S1 for pretreatment, the detection of the anti-cyclic citrullinated peptide antibody can be efficiently and accurately completed, the operation is convenient, and the home self-inspection can be realized.
The invention is further described with reference to the following figures and detailed description.
Drawings
FIG. 1 is a schematic structural diagram of an anti-cyclic citrullinated peptide antibody test strip in an embodiment of the present invention.
The kit comprises a base plate 1, a nitrocellulose membrane 2, a detection T line 3, a quality control C line 4, a gold-labeled antibody reaction pad 5, absorbent paper 6 and a sample pad 7.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, in which specific conditions are not specified, and which are performed according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are conventional products which are not indicated by manufacturers and can be obtained by market purchase.
The embodiments described are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without any inventive work belong to the protection scope of the present invention.
Example 1:
as shown in fig. 1, an anti-cyclic citrullinated peptide antibody detection kit comprises an anti-cyclic citrullinated peptide antibody detection test strip and a sample pretreatment tube, wherein a detection liquid S1 is filled in the sample pretreatment tube, the anti-cyclic citrullinated peptide antibody detection test strip comprises a bottom plate 1, a nitrocellulose membrane 2 is arranged on the bottom plate 1, and a detection T line 3 coating 6 different cyclic citrullinated peptide antigens and a quality control C line 4 coating goat anti-mouse IgG antibody are arranged on the nitrocellulose membrane 2; one end of the nitrocellulose membrane 2 is lapped with a gold-labeled antibody reaction pad 5, the other end of the nitrocellulose membrane 2 is lapped with absorbent paper 6, one end of the gold-labeled antibody reaction pad 5 is lapped with a sample pad 7, and the gold-labeled antibody reaction pad 5 is coated with a mouse anti-human IgG monoclonal antibody-colloidal gold compound.
The colloidal gold particle size used in the coating of the mouse anti-human IgG monoclonal antibody-colloidal gold complex coated on the gold-labeled antibody reaction pad 5 was 30nm.
The preparation method of the mouse anti-human IgG monoclonal antibody-colloidal gold compound coating comprises the following steps: preparing colloidal gold by a trisodium citrate reduction method, adding 1mg of mouse anti-human IgG monoclonal antibody into a colloidal gold solution with the particle size of 100mL and 30nm, centrifuging after reaction, removing supernate, adding 5mL of gold redissolving solution, standing for reaction to prepare a mouse anti-human IgG monoclonal antibody-colloidal gold compound, diluting with the gold redissolving solution according to the volume ratio of 1 2 Coating on glass fiber, and drying;
the preparation method of the detection T line 3 and the quality control C line 4 comprises the following steps: diluting the mixed solution of the 6 cyclic citrullinated peptide antigens to 0.8mg/mL and the goat anti-mouse IgG antibody to 1.8mg/mL by using diluent respectively, sequentially spraying the diluted solution on a nitrocellulose membrane 2, and drying the nitrocellulose membrane to obtain the polypeptide.
Wherein, 6 different cyclic citrullinated peptide antigens are CCP-I-CCP-VI, and 6 antigens are mixed according to the following ratio of 1.
The diluted solution was prepared by adding 3wt% of sucrose and 2wt% of trehalose to 1L of 20mmol of PBS buffer solution, which had a pH of 7.2.
Gold complex solution was subjected to 0.05M pH7.4 Tris, 0.05wt% glucose, 0.02wt% trehalose, 0.03wt% Tween-20, 0.02wt% BSA, 0.01wt% NaN 3 Mixing to obtain mixture with pH of 7.0-7.5.
The determination liquid S1 consists of 90mmol/L PB buffer solution, 15g/L sodium chloride, 18g/L PEG polymeric stabilizer, 0.6g/L PC-300 preservative and 0.6g/L IgG monoclonal antibody blocking agent, the solvent is purified water, and the pH value is 7.2-7.6.
The sample pad 7 is obtained by treating glass fiber with a sample treatment solution and drying. The sample treatment solution was prepared by mixing 0.5wt% PVA, 0.1wt% S9, 0.02wt% Triton X-100, 0.7mg/mL anti-erythrocyte antibody (RBC antibody), 0.4wt% trehalose, and 0.2wt% casein in 1000mL of PB buffer solution having a pH of 8.0 to 8.5.
Example 2
The use method of the anti-cyclic citrullinated peptide antibody detection kit comprises the following steps:
(1) Sample collection
a. Serum/plasma/whole blood samples were collected by conventional methods.
b. EDTA, sodium citrate, heparin and the like can be used as the anticoagulant.
c. The serum/plasma sample can be stored for 3 days at the temperature of 2-8 ℃, is frozen and stored at the temperature of more than 3 days-20 ℃, and is repeatedly frozen and thawed for no more than 3 times; the whole blood sample added with the anticoagulant can be stored for 3 days at the temperature of 2-8 ℃ and can not be frozen; whole blood samples without anticoagulant are used immediately (if the sample agglutinates, it can be detected with serum).
d. Taking a fingertip blood sample for use at present;
(2) Detection of
a. Before use, the anti-cyclic citrullinated peptide antibody detection kit and the sample are placed at room temperature for 30 minutes and then are recovered to room temperature (20-30 ℃); b. placing the anti-cyclic citrullinated peptide antibody detection kit on a table board;
c. taking 50-60 mu L of a serum/plasma/whole blood sample to be detected, putting the sample into a sample pretreatment tube, fully reversing and uniformly mixing, and dripping 2 drops of the sample if a disposable plastic straw is used;
d. and opening a sample pretreatment pipe cap, and dripping 80-100 mu L (about 3 drops) of sample mixed liquid at the position of a sample adding hole of the anti-cyclic citrullinated peptide antibody detection kit.
e. The results were interpreted within 8 minutes and after 10 minutes the results were not valid.
(3) Result judgment
a. Positive (+): and a red line appears on each of the quality control line (C) and the detection line (T).
b. Negative (-): only one red line appears on the quality control line (C), and no red line appears on the detection line (T).
c. And (4) invalidation: the position of the quality control line (C) has no line, which indicates misoperation or reagent failure.
Example 3
The examination of the sensitivity, specificity and stability of the anti-cyclic citrullinated peptide antibody detection kit of example 1 of the present invention is as follows:
1. sensitivity study
The positive quantitative samples of the external anti-cyclic citrullinated peptide antibody are diluted to 2000, 1000, 500, 250, 50, 25, 10 and 5RU/mL in a gradient way for sensitivity study, and the results are shown in the following table 1:
TABLE 1
Remarking: "+" represents positive, "+/-" represents weak positive, and "-" represents negative.
The lowest detection limit of the kit for detecting the anti-cyclic citrullinated peptide antibody is 25RU/mL.
2. Investigation of specificity
The anti-cyclic citrullinated peptide antibody detection kit in the embodiment 2 of the invention is adopted to verify the interference and cross substances which are easy to cause the same or similar symptoms in the following table concentration, and the specific detection results are shown in the table 2;
TABLE 2
The results shown in table 2 show that: the interference and cross-reaction substances under the following concentration conditions have no influence on the detection of the kit, which indicates that the kit has good specificity.
3. Stability survey
The anti-cyclic citrullinated peptide antibody detection kit in the embodiment 2 of the invention is placed at 45 ℃ for destructive test, the test period is 6 months, and the stability of the kit is respectively tested on the 1 st day, the 3 rd day, the 7 th day, the 10 th day, the 15 th day, the 30 th day, the 45 th day, the 60 th day, the 90 th day, the 120 th day, the 150 th day and the 180 th day, with the following standards:
(1) Determination of the physical State of the liquid S1
Appearance: colorless, transparent and clear liquid, no particles, floccules, precipitates and no volatility.
(2) Performance index
1) And (3) the negative quality control product conformity rate: and detecting by using 10 parts of anti-cyclic citrullinated peptide antibody negative quality control substances, wherein all detection results are negative.
2) The positive quality control product conformity rate: and (3) detecting by using 10 parts of anti-cyclic citrullinated peptide antibody positive (including strong, medium and weak positive) quality control products, wherein all detection results are positive.
Minimum detection limit: and (4) detecting by using the reference substance S with the lowest detection limit, wherein the result is positive.
Repeatability: the result variation coefficients of the negative reference substance, the positive reference substance and the lowest detection limit from 1 day to 180 days are not more than 10%.
The test result of the stability of the kit is as follows:
the sample diluent physical state stability results are shown in table 3;
TABLE 3
| Time (sky) | 1 | 3 | 7 | 10 | 15 | 30 | 45 | 60 | 90 | 120 | 150 | 180 |
| Physical state | Conform to | Conform to | Conform to | Conform to | Meet with | Conform to | Conform to | Conform to | Conform to | Meet with | Conform to | Conform to |
The results of the 45 ℃ accelerated destruction test of the kit are shown in Table 4;
TABLE 4
Remarking: "+" indicates positive; "+/-" indicates weak positivity; "-" represents negative;
the test proves that the product can be stabilized for at least 60 days at 45 ℃, and according to the stability experiment principle, the Arrhenius formula: d (Ink)/dT = Ea/RT2Ea, is stored for 24 months at normal temperature, is equivalent to 60 days of destruction at 45 ℃, can meet the clinical requirements of hospital inspection departments and clinics, and can also be used for disease diagnosis research of colleges and scientific research institutions.
Example 4
The kit of the invention is used for clinical trial investigation:
1. the kit and the commercially available kit are adopted to simultaneously detect 186 cases of rheumatoid arthritis positive patient samples, and the detection results are shown in the following table 5:
TABLE 5
The results show that: the kit and the commercially available kit have the sensitivity coincidence rate of 98.05 percent, the specificity coincidence rate of 93.75 percent and the total coincidence rate of 97.31 percent.
Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and the embodiments are examples, wherein the details that are not described are all the common general knowledge of those skilled in the art, and the technical solutions described in the foregoing embodiments can be modified or some technical features can be equivalently replaced by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An anti-cyclic citrullinated peptide antibody detection kit is characterized in that: the kit comprises an anti-cyclic citrullinated peptide antibody detection test strip and a sample pretreatment pipe, wherein a determination liquid S1 is filled in the sample pretreatment pipe, the anti-cyclic citrullinated peptide antibody detection test strip comprises a bottom plate, a nitrocellulose membrane is arranged on the bottom plate, and a detection T line coated with 6 different cyclic citrullinated peptide antigens and a quality control C line coated with goat anti-mouse IgG antibodies are arranged on the nitrocellulose membrane; one end of the nitrocellulose membrane is lapped with a gold-labeled antibody reaction pad, the other end of the nitrocellulose membrane is lapped with absorbent paper, and one end of the gold-labeled antibody reaction pad is lapped with a sample pad; the determination liquid S1 consists of 90mmol/L PB buffer solution, 15g/L sodium chloride, 18g/L PEG polymeric stabilizer, 0.6g/L PC-300 preservative and 0.6g/L IgG monoclonal antibody blocking agent, the solvent is purified water, and the pH value is 7.2-7.6.
2. The anti-cyclic citrullinated peptide antibody detection kit according to claim 1, wherein: the gold-labeled antibody reaction pad is coated with a mouse anti-human IgG monoclonal antibody-colloidal gold compound.
3. The anti-cyclic citrullinated peptide antibody detection kit according to claim 2, wherein: the particle size of the colloidal gold adopted in the mouse anti-human IgG monoclonal antibody-colloidal gold compound coating coated on the gold-labeled antibody reaction pad is 30nm.
4. The anti-cyclic citrullinated peptide antibody detection kit according to claim 3, wherein: the preparation method of the mouse anti-human IgG monoclonal antibody-colloidal gold compound coating comprises the following steps: preparing colloidal gold by a trisodium citrate reduction method, adding 1mg of mouse anti-human IgG monoclonal antibody into 100mL of colloidal gold solution with the particle size of 30nm, centrifuging after reaction, removing supernatant, adding 5mL of gold redissolving solution, standing for reaction to prepare a mouse anti-human IgG monoclonal antibody-colloidal gold compound, diluting with the gold redissolving solution according to the volume ratio of 1 2 Coating on glass fiber, and drying.
5. The anti-cyclic citrullinated peptide antibody detection kit according to claim 4, wherein: the preparation method of the detection T line and the quality control C line comprises the following steps: diluting the mixed solution of the 6 cyclic citrullinated peptide antigens to 0.8mg/mL and the goat anti-mouse IgG antibody to 1.8mg/mL by using diluent respectively, sequentially spraying the diluted solution on a nitrocellulose membrane, and drying the nitrocellulose membrane to obtain the polypeptide.
6. The anti-cyclic citrullinated peptide antibody detection kit according to claim 5, wherein: the 6 different cyclic citrullinated peptide antigens are CCP-I-CCP-VI and are formed by mixing 6 antigens according to the following ratio of 1.
7. The anti-cyclic citrullinated peptide antibody detection kit according to claim 6, wherein: the diluent is prepared by adding 3wt% of sucrose and 2wt% of trehalose into 1L of 20mmol PBS buffer solution, and the pH value of the PBS buffer solution is 7.2.
8. The anti-cyclic citrullinated peptide antibody detection kit according to claim 7, wherein: the gold-complex solution was composed of 0.05M pH7.4 Tris, 0.05wt% glucose, 0.02wt% trehalose, 0.03wt% Tween-20, 0.02wt% BSA, 0.01wt% NaN 3 Mixing to obtain the final product with pH of 7.0-7.5.
9. The anti-cyclic citrullinated peptide antibody detection kit according to claim 8, wherein: the sample pad is obtained by treating glass fiber with a sample treatment solution and drying.
10. The anti-cyclic citrullinated peptide antibody detection kit according to claim 9, wherein: the sample treatment solution was prepared by mixing 0.5wt% PVA, 0.1wt% S9, 0.02wt% Triton X-100, 0.7mg/mL anti-erythrocyte antibody, 0.4wt% trehalose, and 0.2wt% casein in 1000mL of PB buffer solution having a pH of 8.0 to 8.5.
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