CN113030456A - Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody - Google Patents
Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody Download PDFInfo
- Publication number
- CN113030456A CN113030456A CN202110358940.8A CN202110358940A CN113030456A CN 113030456 A CN113030456 A CN 113030456A CN 202110358940 A CN202110358940 A CN 202110358940A CN 113030456 A CN113030456 A CN 113030456A
- Authority
- CN
- China
- Prior art keywords
- line
- antibody
- fluorescent antibody
- citrullinated peptide
- cyclic citrullinated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a detection test strip for an anti-cyclic citrullinated peptide antibody, which comprises a nitrocellulose membrane, wherein the nitrocellulose membrane is coated with a closed line, an anti-cyclic citrullinated peptide antigen detection line and a goat anti-mouse IgG quality control line which are separated from each other and sequentially distributed, a fluorescent antibody line is arranged on a solid phase on the closed line, and the closed line and the fluorescent antibody line are sprayed in the following modes: spraying the prepared sealing liquid on the position, corresponding to the sealing line, on the nitrocellulose membrane, drying for 0.5h to prepare the sealing line, diluting the fluorescent antibody with diluent, uniformly mixing, and spraying the fluorescent antibody at the middle position of the dried sealing line to prepare the fluorescent antibody line. The problem that the fluorescent antibody is coated on the nitrocellulose membrane and is difficult to release after being stored for a long time is solved, the production cost is reduced, the problem that the fluorescent antibody cannot be stored conventionally by a wet immunofluorescence chromatography method is solved, the stability of the fluorescent antibody is improved, the transportation and the storage of the kit are facilitated, and the quality guarantee period of the kit is prolonged.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection test strip, a detection card and a kit for an anti-cyclic citrullinated peptide antibody.
Background
Rheumatoid Arthritis (RA) is one of the most common autoimmune and frequently chronic inflammatory joint diseases, and is suffered by about 1% and about 75% of women worldwide. The pathological manifestations are chronic inflammation of joint synovium, pannus formation, cartilage and bone destruction of joints, and finally joint deformity and function loss can be caused if timely diagnosis and regular treatment are not available. Early, reliable diagnosis of RA is important to adopt appropriate treatment to control disease progression and avoid irreversible joint damage.
Early onset of RA disease appears to be diverse, and the most common serological test for suspected RA patients is Rheumatoid Factor (RF) in addition to the conventional inflammatory parameters. The RF is an anti-gamma globulin antibody (mainly an IgM antibody), and the positive rate in RA is 60-80%. Since RF can be detected in healthy people and patients with various infectious diseases and other autoimmune diseases (such as systemic lupus erythematosus, Sjogren syndrome, scleroderma and the like), the specificity is poor, and the early diagnosis is not facilitated.
In recent years, it has been found that an anti-cyclic citrullinated peptide antibody (anti-cyclic citrullinated peptide) is a polypeptide fragment of cyclic polyserine, has good sensitivity and specificity to RA, can be positive in the early stage of RA, has a high positive predictive value, and can be used to predict the severity of RA.
Research shows that anti-nuclear factor (APF) and anti-keratin antibody (AKA) can be detected in the serum of RA patients, the antigenic sites of the antibodies are cyclic citrullinated polypeptide, the anti-cyclic citrullinated peptide antibodies (anti-cyclic citrullinated peptide) are mainly IgG antibodies, the specificity to RA is 95%, and 70-80% of patients have anti-cyclic citrullinated peptide antibodies (anti-cyclic citrullinated peptide) in serum and synovial fluid at the very early stage of the disease, so that the detection of the anti-cyclic citrullinated peptide antibodies (anti-cyclic citrullinated peptide) has important significance for early diagnosis and prognosis of RA.
At present, a plurality of methods for detecting an anti-cyclic citrullinated peptide antibody (anti-cyclic citrullinated peptide) exist, and enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, immunoturbidimetry, chemiluminescence, radioimmunoassay and other methods are used for determination, but a rapid, simple and reliable detection method is lacked. The enzyme-linked immunosorbent assay has low automation degree, long detection time and is greatly influenced by human factors; the colloidal gold immunochromatography method is simple and convenient to operate, but the interpretation result has human errors and only can be qualitatively detected; the immunoturbidimetry is influenced by the contents of the sample, and the result has poor grading and high omission ratio; the radioimmunoassay has the problem of environmental pollution; while the chemiluminescence method has high sensitivity, but has a small measurement linear range and high detection cost, and needs a specific chemiluminescence detector, so that the application range of the chemiluminescence detector is small.
Compared with other methods, the fluorescence immunochromatography method for detecting the anti-cyclic citrullinated peptide antibody (anti-cyclic citrullinated peptide) in serum has the remarkable characteristics of simple operation, short time, high sensitivity, wide linear range, no pollution, automatic result display of an instrument, quantitative detection and the like, so the fluorescence immunochromatography method has great research value. At present, the fluorescence immunochromatography is mostly in a liquid phase reaction mode, and the kit needs to be placed at 2-8 ℃, so that the storage and transportation of the kit are inconvenient.
Disclosure of Invention
Aiming at the defects, the invention provides the detection test strip for the anti-cyclic citrullinated peptide antibody, which has the advantages of high detection sensitivity, good specificity, and quick and accurate detection.
Also provides a detection reagent card of the anti-cyclic citrullinated peptide antibody, which is convenient to carry and can detect quickly and accurately.
The kit is convenient to use, simple to operate and convenient to store and transport.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the detection test strip for the anti-cyclic citrullinated peptide antibody comprises a nitrocellulose membrane, wherein a closed line, an anti-cyclic citrullinated peptide antigen detection line and a goat anti-mouse IgG quality control line which are separated from each other and distributed in sequence are coated on the nitrocellulose membrane, a fluorescent antibody line is arranged on a solid phase on the closed line, and the closed line and the fluorescent antibody line are sprayed in the following modes:
spraying the prepared sealing liquid at the position corresponding to the sealing line on the nitrocellulose membrane by the spraying amount of 1.5 mu L/cm, then drying at 37 ℃ for 0.5h to prepare the sealing line, diluting the fluorescent antibody by using fluorescent antibody diluent according to the proportion of 1:10, uniformly mixing, and spraying the fluorescent antibody at the middle position of the dried sealing line by the spraying amount of 0.5 mu L/cm to prepare the fluorescent antibody line.
The method comprises the steps of firstly spraying a sealing liquid on a nitrocellulose membrane, firstly sealing the nitrocellulose membrane, and then carrying out solid-phase fluorescence antibody spraying, so that the problem that the fluorescence antibody is coated on the nitrocellulose membrane and is difficult to release after being stored for a long time is solved, the solid phase of the fluorescence antibody is on the nitrocellulose membrane instead of on an independent combination pad, the production cost is reduced, the problem that the fluorescence antibody cannot be stored conventionally by a wet immunofluorescence chromatography method is solved, the stability of the fluorescence antibody is improved, the transportation and the storage of a kit are facilitated, and the quality guarantee period of the kit is prolonged.
As an optimization scheme, the preparation method of the fluorescent antibody comprises the following steps:
labeling of fluorescent antibody: diluting the mouse anti-human IgG antibody to the final concentration of 2-10 mg/mL by PBS buffer solution, and preparing NaHCO with pure water3The concentration is 1mol/L, NaHCO is added into the diluted mouse anti-human IgG antibody3，NaHCO3The final concentration of the fluorescent dye is 0.1-1 mol/L, the residual volume is filled with PBS buffer solution, the fluorescent dye is added, the final concentration of the fluorescent dye is 1-10 mmol/L, the mixture is mixed evenly, a micro-oscillator is started, and the mixture is marked at the dark room temperatureThe reaction is carried out for 15-60 min.
Purification of fluorescent antibody: and (4) taking out the labeled fluorescent antibody, and separating and purifying the fluorescent antibody through a sephadex column.
As an optimized scheme, the sealing liquid comprises the following components:
20-100 mM Tris; 0.1-1.5 g of NaCL; 0.5-3 g of bovine serum albumin; 0.1-0.5 g of casein; 0.1-5 g of PVP; 1-10 g of sucrose; 1-3 g of trehalose; tween 0.01-0.1 mL, and the pH value of the Tris buffer solution is 8.0-8.5.
As an optimized scheme, the fluorescent antibody diluent comprises the following components:
20-100 mM Tris; 0.1-1.5 g of NaCL; 0.5-3 g of bovine serum albumin; 0.1-5 g of PVP; 1-10 g of sucrose; 1-3 g of trehalose; the pH value of the Tris buffer solution is 8.0-8.5.
As an optimized scheme, the coating method of the anti-cyclic citrullinated peptide antigen detection line is as follows:
coupling the anti-cyclic citrullinated peptide antigen with carrier protein, and carrying out thermal destruction treatment on the carrier protein at 55 ℃, wherein the carrier protein is bovine serum albumin.
And diluting the coupled anti-cyclic citrullinated peptide antigen to 0.5mg/mL by using an antigen diluent, and spraying the antigen to a corresponding antigen detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm.
The coating method of the goat anti-mouse IgG quality control line comprises the following steps:
the goat anti-mouse IgG is diluted to 1.0mg/mL by PBS buffer solution, and sprayed on the corresponding quality control detection line of the nitrocellulose membrane by a spray amount of 1.0 mu L/cm.
As an optimized scheme, the antigen diluent is prepared by the following steps:
60.5mg Tris, 0.9g sodium chloride and 3g trehalose are dissolved in 100mL distilled water, 10mL glycerol is added for even mixing, and the mixture is filtered and filled into a wide-mouth bottle for later use.
The utility model provides an anti cyclic citrullinated peptide antibody's detection card, the detection card is including the upper cover and the lower cover of lock, cover down and have the recess, foretell test paper strip has been placed in the recess.
A detection kit for an anti-cyclic citrullinated peptide antibody comprises the detection card and a sample diluent, wherein the sample diluent comprises 20-100 mM Tris, 0.1-1.5 g of NaCL, 0.1-0.5 g of casein, 0.1-5 g of PEG, 0.05-0.5 g of gelatin, 90.1-1 mL of S, 0.1-1 mL of Tween and 0.1-1 mL of PC 3000.1-1 mL, and the pH of a Tris buffer solution is 8.0-8.5.
By adopting the technical scheme, the invention has the following advantages:
(1) the kit adopts a dry immunofluorescence chromatography method, firstly sprays the sealing liquid on the nitrocellulose membrane, firstly seals the nitrocellulose membrane, and then carries out solid phase fluorescence antibody in a spraying mode, thereby being beneficial to releasing the fluorescence antibody, solving the problem that the fluorescence antibody is coated on the nitrocellulose membrane and is difficult to release after being stored for a long time, reducing the production cost because the fluorescence antibody is in a solid phase on the nitrocellulose membrane instead of in a solid phase on an independent combination pad, simultaneously solving the problem that the fluorescence antibody can not be stored conventionally in a wet immunofluorescence chromatography method, improving the stability of the fluorescence antibody, being beneficial to the transportation and storage of the kit, and prolonging the quality guarantee period of the kit.
(2) The fluorescent antibody is separated and purified through a sephadex column, and compared with a conventional dialysis method, the fluorescent antibody and free fluorescence are completely separated according to the molecular sieve effect, so that the base line background of the detection process is reduced, and the specificity of the result is increased.
(3) The trehalose with a certain concentration is added into the antigen diluent to enhance the stability of an antigen structure, the hydrophilicity is increased by adding the glycerol, the contact reaction of the antigen and the antibody is accelerated, the detection sensitivity of the kit is improved, meanwhile, the fluorescent antibody combined at an antigen line is more uniform, and the problem of repeatability caused by nonuniform fluorescence absorption of an instrument due to a hollow effect is avoided.
(4) The antigen of the short peptide is coupled with the macromolecular substance bovine serum albumin, the difficult problem that the antigen is not easily coated on the nitrocellulose membrane due to the undersize molecular weight in the chromatography process is solved, the coupling agent bovine serum albumin is subjected to thermal destruction treatment, and the coupling rate of the antigen is improved.
(5) Casein is added into the sample buffer solution, so that a certain blocking effect is achieved on non-specific binding reaction, and the background is reduced; the sensitivity is improved by adding the macromolecular substance PEG; by adding a certain amount of gelatin, positive color development is more obvious, negative color is more white and clean, the discrimination of the instrument is improved, and the judgment of suspicious samples is reduced. By adding the surfactant with proper concentration, the problem of repeatability caused by the fact that after the anti-cyclic citrullinated peptide antigen is coupled with the bovine serum albumin damaged at 55 ℃, the coupled antigen is combined with a membrane for a long time and the wettability is poor is solved, meanwhile, the problem of false positive caused by overlong reaction time of an antibody in a sample at a detection line is solved, and the specificity of detection is improved.
(6) The kit is used for in vitro qualitative or semi-quantitative detection of anti-cyclic citrullinated peptide (anti-cyclic citrullinated peptide) antibodies in human serum, plasma and whole blood. In the actual operation process, only 1 mu L of serum, plasma or 2 mu L of whole blood is needed, the vein blood taking or the collection of peripheral blood can meet the requirements, the micro sample adding operation reduces the pain of a patient in the blood taking process, particularly the problem of blood taking for children is solved, after the serum, the plasma or the whole blood and a sample buffer solution are mixed uniformly, the result is about to be measured in 5-10 min, the operation is convenient and fast, and the waiting process is shortened.
(7) The kit has the advantages of long quality guarantee period, good specificity, high sensitivity, small sample demand, convenient and simple operation, high detection speed, direct result display of an instrument, accuracy and reliability. Can meet the clinical requirements of hospital clinics and health quarantine departments, and can also be used for disease diagnosis research of colleges and universities and scientific research institutions.
The invention is further described with reference to the following figures and detailed description.
Drawings
FIG. 1 is a schematic diagram of the structure of the detection card of the present invention.
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 not indicated by manufacturers, and are all conventional products available on the market.
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 the attached drawing 1, the detection test strip for the anti-cyclic citrullinated peptide antibody comprises a base plate 8, wherein the base plate 8 is a PVC plate, a whole blood filter pad 9, a nitrocellulose membrane 10 and absorbent paper 11 which are sequentially overlapped are adhered on the base plate 8, one end of the whole blood filter pad 9 presses one end of the nitrocellulose membrane 10 for 1mm, one end of the absorbent paper 11 presses the other end of the nitrocellulose membrane 10 for 1mm, the prepared nitrocellulose membrane 10 is adhered at the middle position of the base plate 8, the whole blood filter pad 9 and the absorbent paper 11 are adhered above the position of the nitrocellulose membrane 10 fixed on the base plate 8, and a product obtained by the steps is flattened and then cut into test strips with the width of 4.0 mm.
The nitrocellulose membrane 10 is coated with a closed line 12, an anti-cyclic citrullinated peptide antigen detection line 5 and a goat anti-mouse IgG quality control line 6 which are separated from each other and sequentially distributed, a fluorescent antibody line 13 is arranged on the closed line 12 in a solid phase manner, the closed line 12 is 4mm away from the left end of the nitrocellulose membrane 10, the width of the closed line is 1.5mm, and the fluorescent antibody line 13 is 4.5mm away from the left end of the nitrocellulose membrane 10, and the width of the fluorescent antibody line is 0.5 mm.
The spraying mode of the closed line 12 and the fluorescent antibody line 13 is as follows:
spraying the prepared sealing liquid at the position corresponding to the sealing line on the nitrocellulose membrane by the spraying amount of 1.5 mu L/cm, then drying at 37 ℃ for 0.5h to obtain the sealing line 12, diluting the fluorescent antibody with the fluorescent antibody diluent according to the proportion of 1:10, uniformly mixing, and spraying the fluorescent antibody at the middle position of the dried sealing line 12 by the spraying amount of 0.5 mu L/cm to obtain the fluorescent antibody line 13.
Because the nitrocellulose membrane has stronger adsorption force to protein, before the solid-phase fluorescent antibody, the membrane needs to be sealed at the position of the solid-phase fluorescent antibody, and then the solid-phase fluorescent antibody is beneficial to the release of the fluorescent antibody.
The dry fluorescence immunochromatography method is adopted, the fluorescent antibody is directly immobilized on the nitrocellulose membrane instead of the conventional solid phase on the whole blood filter pad 9, and the method is different from the wet fluorescence immunochromatography method in that the fluorescent antibody is separately stored in a specific environment of 2-8 ℃, so that the fluorescence stability is improved. Because the nitrocellulose membrane has stronger adsorption force to protein, before the solid-phase fluorescent antibody, the membrane is firstly sealed by using a sealing liquid, and then the solid-phase fluorescent antibody is favorable for releasing the fluorescent antibody.
The preparation method of the fluorescent antibody comprises the following steps:
labeling of fluorescent antibody: the mouse anti-human IgG antibody was diluted to a final concentration of 2mg/mL with PBS buffer, and NaHCO was prepared with purified water3The concentration is 1mol/L, NaHCO is added into the diluted mouse anti-human IgG antibody3，NaHCO3The final concentration of the fluorescent dye is 0.1mol/L, the residual volume is filled with PBS buffer solution, the fluorescent dye is added, the final concentration of the fluorescent dye is 1mmol/L, the mixture is mixed evenly, a micro-oscillator is started, and the mixture is labeled and reacted for 15min at the room temperature in a dark place.
Purification of fluorescent antibody: taking out the labeled fluorescent antibody, and separating and purifying through a sephadex column; according to the molecular sieve effect, the complete separation between the free fluorescent dye and the labeled fluorescent antibody is realized.
The labeled fluorescent antibody is separated and purified through the sephadex column, compared with a conventional dialysis method, the fluorescent antibody and free fluorescence are completely separated, the base line background of the detection process is reduced, and the specificity of the result is increased.
Composition of the confining liquid: buffer systems, protein protectants, macromolecular substances, surfactants, and proteins.
The sealing liquid comprises the following components:
20mM Tris; 0.1g of NaCL; bovine serum albumin 0.5 g; 0.1g of casein; 0.1g of PVP; 1g of sucrose; 1g of trehalose; tween 0.01mL, Tris buffer pH 8.0.
The fluorescent antibody needs to be diluted to a proper concentration by using a fluorescent antibody diluent, and the composition of the fluorescent antibody diluent is as follows: buffer systems, protein protectants, macromolecular substances, and proteins.
The fluorescent antibody diluent comprises the following components:
20mM Tris; 0.1g of NaCL; bovine serum albumin 0.5 g; 0.1g of PVP; 1g of sucrose; 1g of trehalose; the pH of the Tris buffer was 8.0.
The coating method of the anti-cyclic citrullinated peptide antigen detection line comprises the following steps:
coupling the anti-cyclic citrullinated peptide antigen with carrier protein, and carrying out thermal destruction treatment on the carrier protein at 55 ℃, wherein the carrier protein is bovine serum albumin.
And diluting the coupled anti-cyclic citrullinated peptide antigen to 0.5mg/mL by using an antigen diluent, and spraying the antigen to a corresponding antigen detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm.
According to the conventional method, an anti-cyclic citrullinated peptide antigen is directly coated on a membrane, but the anti-cyclic citrullinated peptide antigen is a short peptide with 12-20 AA, and is not easy to coat.
The coating method of the goat anti-mouse IgG quality control line comprises the following steps:
the goat anti-mouse IgG is diluted to 1.0mg/mL by PBS buffer solution, and sprayed on the corresponding quality control detection line of the nitrocellulose membrane by a spray amount of 1.0 mu L/cm.
The preparation method of the antigen diluent comprises the following steps:
60.5mg Tris, 0.9g sodium chloride and 3g trehalose are dissolved in 100mL distilled water, 10mL glycerol is added for even mixing, and the mixture is filtered and filled into a wide-mouth bottle for later use.
And (3) drying the nitrocellulose membrane, after spraying a fluorescent antibody line, an anti-cyclic citrullinated peptide antigen detection line and a goat anti-mouse IgG quality control line, and balancing the nitrocellulose membrane for 2 hours at the temperature of 37 ℃ and under the humidity of less than 40%.
Example 2: as shown in the attached drawing 1, the detection test strip for the anti-cyclic citrullinated peptide antibody comprises a base plate 8, wherein the base plate 8 is a PVC plate, a whole blood filter pad 9, a nitrocellulose membrane 10 and absorbent paper 11 which are sequentially overlapped are adhered on the base plate 8, one end of the whole blood filter pad 9 presses one end of the nitrocellulose membrane 10 for 1mm, one end of the absorbent paper 11 presses the other end of the nitrocellulose membrane 10 for 1mm, the prepared nitrocellulose membrane 10 is adhered at the middle position of the base plate 8, the whole blood filter pad 9 and the absorbent paper 11 are adhered above the position of the nitrocellulose membrane 10 fixed on the base plate 8, and a product obtained by the steps is flattened and then cut into test strips with the width of 4.0 mm.
The nitrocellulose membrane 10 is coated with a closed line 12, an anti-cyclic citrullinated peptide antigen detection line 5 and a goat anti-mouse IgG quality control line 6 which are separated from each other and sequentially distributed, a fluorescent antibody line 13 is arranged on the closed line 12 in a solid phase manner, the closed line 12 is 4mm away from the left end of the nitrocellulose membrane 10, the width of the closed line is 1.5mm, and the fluorescent antibody line 13 is 4.5mm away from the left end of the nitrocellulose membrane 10, and the width of the fluorescent antibody line is 0.5 mm.
The spraying mode of the closed line 12 and the fluorescent antibody line 13 is as follows:
spraying the prepared sealing liquid at the position corresponding to the sealing line on the nitrocellulose membrane by the spraying amount of 1.5 mu L/cm, then drying at 37 ℃ for 0.5h to obtain the sealing line 12, diluting the fluorescent antibody with the fluorescent antibody diluent according to the proportion of 1:10, uniformly mixing, and spraying the fluorescent antibody at the middle position of the dried sealing line 12 by the spraying amount of 0.5 mu L/cm to obtain the fluorescent antibody line 13.
Because the nitrocellulose membrane has stronger adsorption force to protein, before the solid-phase fluorescent antibody, the membrane needs to be sealed at the position of the solid-phase fluorescent antibody, and then the solid-phase fluorescent antibody is beneficial to the release of the fluorescent antibody.
The dry fluorescence immunochromatography method is adopted, the fluorescent antibody is directly immobilized on the nitrocellulose membrane instead of the conventional solid phase on the whole blood filter pad 9, and the method is different from the wet fluorescence immunochromatography method in that the fluorescent antibody is separately stored in a specific environment of 2-8 ℃, so that the fluorescence stability is improved. Because the nitrocellulose membrane has stronger adsorption force to protein, before the solid-phase fluorescent antibody, the membrane is firstly sealed by using a sealing liquid, and then the solid-phase fluorescent antibody is favorable for releasing the fluorescent antibody.
The preparation method of the fluorescent antibody comprises the following steps:
labeling of fluorescent antibody: the mouse anti-human IgG antibody was diluted to a final concentration of 10mg/mL with PBS buffer, and NaHCO was prepared with purified water3Concentration ofAt 1mol/L, NaHCO was added to the diluted murine anti-human IgG antibody3，NaHCO3The final concentration of the fluorescent dye is 1mol/L, the residual volume is filled with PBS buffer solution, the fluorescent dye with the final concentration of 10mmol/L is added, the mixture is mixed evenly, a micro-oscillator is started, and the mixture is marked and reacted for 60min at room temperature in a dark place.
Purification of fluorescent antibody: taking out the labeled fluorescent antibody, and separating and purifying through a sephadex column; according to the molecular sieve effect, the complete separation between the free fluorescent dye and the labeled fluorescent antibody is realized.
The labeled fluorescent antibody is separated and purified through the sephadex column, compared with a conventional dialysis method, the fluorescent antibody and free fluorescence are completely separated, the base line background of the detection process is reduced, and the specificity of the result is increased.
Composition of the confining liquid: buffer systems, protein protectants, macromolecular substances, surfactants, and proteins.
The sealing liquid comprises the following components:
100mM Tris; 1.5g of NaCL; 3g of bovine serum albumin; 0.5g of casein; 5g of PVP; 10g of cane sugar; 3g of trehalose; tween 0.1mL, Tris buffer pH 8.5.
The fluorescent antibody needs to be diluted to a proper concentration by using a fluorescent antibody diluent, and the composition of the fluorescent antibody diluent is as follows: buffer systems, protein protectants, macromolecular substances, and proteins.
The fluorescent antibody diluent comprises the following components:
100mM Tris; 1.5g of NaCL; 3g of bovine serum albumin; 5g of PVP; 10g of cane sugar; 3g of trehalose; the pH of the Tris buffer was 8.5.
The coating method of the anti-cyclic citrullinated peptide antigen detection line comprises the following steps:
coupling the anti-cyclic citrullinated peptide antigen with carrier protein, and carrying out thermal destruction treatment on the carrier protein at 55 ℃, wherein the carrier protein is bovine serum albumin.
And diluting the coupled anti-cyclic citrullinated peptide antigen to 0.5mg/mL by using an antigen diluent, and spraying the antigen to a corresponding antigen detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm.
According to the conventional method, an anti-cyclic citrullinated peptide antigen is directly coated on a membrane, but the anti-cyclic citrullinated peptide antigen is a short peptide with 12-20 AA, and is not easy to coat.
The coating method of the goat anti-mouse IgG quality control line comprises the following steps:
the goat anti-mouse IgG is diluted to 1.0mg/mL by PBS buffer solution, and sprayed on the corresponding quality control detection line of the nitrocellulose membrane by a spray amount of 1.0 mu L/cm.
The preparation method of the antigen diluent comprises the following steps:
60.5mg Tris, 0.9g sodium chloride and 3g trehalose are dissolved in 100mL distilled water, 10mL glycerol is added for even mixing, and the mixture is filtered and filled into a wide-mouth bottle for later use.
And (3) drying the nitrocellulose membrane, after spraying a fluorescent antibody line, an anti-cyclic citrullinated peptide antigen detection line and a goat anti-mouse IgG quality control line, and balancing the nitrocellulose membrane for 4 hours at 37 ℃ under the environment with the humidity of less than 40%.
Example 3: as shown in the attached drawing 1, the detection test strip for the anti-cyclic citrullinated peptide antibody comprises a base plate 8, wherein the base plate 8 is a PVC plate, a whole blood filter pad 9, a nitrocellulose membrane 10 and absorbent paper 11 which are sequentially overlapped are adhered on the base plate 8, one end of the whole blood filter pad 9 presses one end of the nitrocellulose membrane 10 for 1mm, one end of the absorbent paper 11 presses the other end of the nitrocellulose membrane 10 for 1mm, the prepared nitrocellulose membrane 10 is adhered at the middle position of the base plate 8, the whole blood filter pad 9 and the absorbent paper 11 are adhered above the position of the nitrocellulose membrane 10 fixed on the base plate 8, and a product obtained by the steps is flattened and then cut into test strips with the width of 4.0 mm.
The nitrocellulose membrane 10 is coated with a closed line 12, an anti-cyclic citrullinated peptide antigen detection line 5 and a goat anti-mouse IgG quality control line 6 which are separated from each other and sequentially distributed, a fluorescent antibody line 13 is arranged on the closed line 12 in a solid phase manner, the closed line 12 is 4mm away from the left end of the nitrocellulose membrane 10, the width of the closed line is 1.5mm, and the fluorescent antibody line 13 is 4.5mm away from the left end of the nitrocellulose membrane 10, and the width of the fluorescent antibody line is 0.5 mm.
The spraying mode of the closed line 12 and the fluorescent antibody line 13 is as follows:
spraying the prepared sealing liquid at the position corresponding to the sealing line on the nitrocellulose membrane by the spraying amount of 1.5 mu L/cm, then drying at 37 ℃ for 0.5h to obtain the sealing line 12, diluting the fluorescent antibody with the fluorescent antibody diluent according to the proportion of 1:10, uniformly mixing, and spraying the fluorescent antibody at the middle position of the dried sealing line 12 by the spraying amount of 0.5 mu L/cm to obtain the fluorescent antibody line 13.
Because the nitrocellulose membrane has stronger adsorption force to protein, before the solid-phase fluorescent antibody, the membrane needs to be sealed at the position of the solid-phase fluorescent antibody, and then the solid-phase fluorescent antibody is beneficial to the release of the fluorescent antibody.
The dry fluorescence immunochromatography method is adopted, the fluorescent antibody is directly immobilized on the nitrocellulose membrane instead of the conventional solid phase on the whole blood filter pad 9, and the method is different from the wet fluorescence immunochromatography method in that the fluorescent antibody is separately stored in a specific environment at 2-8 ℃, so that the fluorescence stability is improved. Because the nitrocellulose membrane has stronger adsorption force to protein, before the solid-phase fluorescent antibody, the membrane is firstly sealed by using a sealing liquid, and then the solid-phase fluorescent antibody is favorable for releasing the fluorescent antibody.
The preparation method of the fluorescent antibody comprises the following steps:
labeling of fluorescent antibody: the mouse anti-human IgG antibody was diluted to a final concentration of 6mg/mL with PBS buffer, and NaHCO was prepared with purified water3The concentration is 1mol/L, NaHCO is added into the diluted mouse anti-human IgG antibody3，NaHCO3The final concentration of the fluorescent dye is 0.5mol/L, the residual volume is filled with PBS buffer solution, the fluorescent dye is added, the final concentration of the fluorescent dye is 5mmol/L, the mixture is mixed evenly, a micro-oscillator is started, and the mixture is labeled and reacted for 38min at room temperature in a dark place.
Purification of fluorescent antibody: taking out the labeled fluorescent antibody, and separating and purifying through a sephadex column; according to the molecular sieve effect, the complete separation between the free fluorescent dye and the labeled fluorescent antibody is realized.
The labeled fluorescent antibody is separated and purified through the sephadex column, compared with a conventional dialysis method, the fluorescent antibody and free fluorescence are completely separated, the base line background of the detection process is reduced, and the specificity of the result is increased.
Composition of the confining liquid: buffer systems, protein protectants, macromolecular substances, surfactants, and proteins.
The sealing liquid comprises the following components:
60mM Tris; 0.9g of NaCL; 1g of bovine serum albumin; 0.3g of casein; 2.5g of PVP; 5g of cane sugar; 2g of seaweed; tween 0.05mL, Tris buffer pH 8.2.
The fluorescent antibody needs to be diluted to a proper concentration by using a fluorescent antibody diluent, and the composition of the fluorescent antibody diluent is as follows: buffer systems, protein protectants, macromolecular substances, and proteins.
The fluorescent antibody diluent comprises the following components:
60mM Tris; 0.9g of NaCL; 2g of bovine serum albumin; 2.5g of PVP; 5g of cane sugar; 2g of trehalose; the pH of the Tris buffer was 8.2.
The coating method of the anti-cyclic citrullinated peptide antigen detection line comprises the following steps:
coupling the anti-cyclic citrullinated peptide antigen with carrier protein, and carrying out thermal destruction treatment on the carrier protein at 55 ℃, wherein the carrier protein is bovine serum albumin.
And diluting the coupled anti-cyclic citrullinated peptide antigen to 0.5mg/mL by using an antigen diluent, and spraying the antigen to a corresponding antigen detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm.
According to the conventional method, an anti-cyclic citrullinated peptide antigen is directly coated on a membrane, but the anti-cyclic citrullinated peptide antigen is a short peptide with 12-20 AA, and is not easy to coat.
The coating method of the goat anti-mouse IgG quality control line comprises the following steps:
the goat anti-mouse IgG is diluted to 1.0mg/mL by PBS buffer solution, and sprayed on the corresponding quality control detection line of the nitrocellulose membrane by a spray amount of 1.0 mu L/cm.
The preparation method of the antigen diluent comprises the following steps:
60.5mg Tris, 0.9g sodium chloride and 3g trehalose are dissolved in 100mL distilled water, 10mL glycerol is added for even mixing, and the mixture is filtered and filled into a wide-mouth bottle for later use.
And (3) drying the nitrocellulose membrane, after spraying a fluorescent antibody line, an anti-cyclic citrullinated peptide antigen detection line and a goat anti-mouse IgG quality control line, and balancing the nitrocellulose membrane for 3 hours at 37 ℃ under the environment with the humidity of less than 40%.
Example 4: as shown in figure 1, the detection card for the anti-cyclic citrullinated peptide antibody comprises an upper cover 1 and a lower cover 2 which are buckled with each other, wherein a groove 7 is formed in the lower cover 2, and the test paper strips in any one of embodiments 1-3 are placed in the groove 7.
Have application hole 3 and detection window 4 on the upper cover 1, the position in application hole 3 corresponds the position at whole blood filter pad 9 place, makes the sample can the dropwise add on whole blood filter pad 9, and the position of detection window 4 corresponds anti cyclic citrullinated peptide antigen detection line 5 and sheep anti mouse IgG matter accuse line 6 position, and fluorescence antibody line 13 needs to be protected from light, is sheltered from by upper cover 1, and upper cover 1 and lower cover 2 are plastic material.
Example 5: a detection kit of an anti-cyclic citrullinated peptide antibody comprises the detection card described in embodiment 4 and a sample diluent, wherein the sample diluent comprises 20mM Tris, NaCL 0.1g, casein 0.1g, PEG 0.1g, gelatin 0.05g, S90.1mL, Tween 0.1mL and PC3000.1mL, and the pH of a Tris buffer solution is 8.0.
The method comprises the steps of sequentially coating a fluorescence-labeled mouse anti-human IgG antibody, a detection line coated anti-cyclic citrullinated peptide antigen and a quality control line coated goat anti-mouse IgG antibody on a nitrocellulose membrane, adding serum to be detected, combining the anti-cyclic citrullinated peptide antibody in the serum to be detected with the fluorescence-labeled mouse anti-human IgG antibody, combining with the anti-cyclic citrullinated peptide antigen coated at the detection line under the action of chromatography to form a compound, combining with the goat anti-mouse IgG antibody to form the compound when the fluorescence-labeled mouse anti-human IgG antibody continuously migrates to the quality control line, and detecting the relative content of the anti-cyclic citrullinated peptide antibody through a fluorescence immunoassay analyzer.
In the actual operation detection process, a micro sample injector is used for sucking 1 mu L of serum or 2 mu L of whole blood sample, the serum or the whole blood sample is added into a sample diluent tube and mixed evenly to obtain sample liquid to be detected, 75 mu L of sample liquid to be detected is sucked and added into a sample adding hole, the detection result of an instrument is detected for 5-L0 min, and the result is directly judged according to the numerical value displayed by the instrument.
Example 6: a detection kit for an anti-cyclic citrullinated peptide antibody comprises the detection card described in embodiment 4 and a sample diluent, wherein the sample diluent comprises 100mM Tris, NaCL 1.5g, casein 0.5g, PEG 5g, gelatin 0.5g, S91 mL, Tween 1mL, PC 3001 mL, and the pH of Tris buffer is 8.5.
The method comprises the steps of sequentially coating a fluorescence-labeled mouse anti-human IgG antibody, a detection line coated anti-cyclic citrullinated peptide antigen and a quality control line coated goat anti-mouse IgG antibody on a nitrocellulose membrane, adding serum to be detected, combining the anti-cyclic citrullinated peptide antibody in the serum to be detected with the fluorescence-labeled mouse anti-human IgG antibody, combining with the anti-cyclic citrullinated peptide antigen coated at the detection line under the action of chromatography to form a compound, combining with the goat anti-mouse IgG antibody to form the compound when the fluorescence-labeled mouse anti-human IgG antibody continuously migrates to the quality control line, and detecting the relative content of the anti-cyclic citrullinated peptide antibody through a fluorescence immunoassay analyzer.
In the actual operation detection process, a micro sample injector is used for sucking 1 mu L of serum or 2 mu L of whole blood sample, the serum or the whole blood sample is added into a sample diluent tube and mixed evenly to obtain sample liquid to be detected, 75 mu L of sample liquid to be detected is sucked and added into a sample adding hole, the detection result of an instrument is detected for 5-L0 min, and the result is directly judged according to the numerical value displayed by the instrument.
Example 7: a detection kit for an anti-cyclic citrullinated peptide antibody comprises the detection card described in embodiment 4 and a sample diluent, wherein the sample diluent comprises 60mM Tris, NaCL 0.8g, casein 0.3g, PEG 2.5g, gelatin 0.25g, S90.5mL, Tween 0.5mL and PC3000.5 mL, and the pH of Tris buffer is 8.2.
The method comprises the steps of sequentially coating a fluorescence-labeled mouse anti-human IgG antibody, a detection line coated anti-cyclic citrullinated peptide antigen and a quality control line coated goat anti-mouse IgG antibody on a nitrocellulose membrane, adding serum to be detected, combining the anti-cyclic citrullinated peptide antibody in the serum to be detected with the fluorescence-labeled mouse anti-human IgG antibody, combining with the anti-cyclic citrullinated peptide antigen coated at the detection line under the action of chromatography to form a compound, combining with the goat anti-mouse IgG antibody to form the compound when the fluorescence-labeled mouse anti-human IgG antibody continuously migrates to the quality control line, and detecting the relative content of the anti-cyclic citrullinated peptide antibody through a fluorescence immunoassay analyzer.
In the actual operation detection process, a micro sample injector is used for sucking 1 mu L of serum or 2 mu L of whole blood sample, the serum or the whole blood sample is added into a sample diluent tube and mixed evenly to obtain sample liquid to be detected, 75 mu L of sample liquid to be detected is sucked and added into a sample adding hole, the detection result of an instrument is detected for 5-L0 min, and the result is directly judged according to the numerical value displayed by the instrument.
Experimental data:
1. and (5) quality control product data.
The test strips of examples 1-3 were used for the detection of internal quality control products (negative quality control product, positive quality control product, minimum detection limit) of an enterprise, each quality control product was repeated 3 times, and the detection results are shown in the following table.
The test results of the following table on the internal quality control products of different enterprises show that the test strips in the embodiments 1, 2 and 3 have better detection effects.
2. Specificity and sensitivity data.
Comparative example 1 is a method of dialyzing and separating the labeled fluorescent antibody through PBS to separate the free fluorescent dye from the fluorescent antibody, which is different from example 1 in that separation and purification are not performed through a glucose gel column.
The fluorescent antibody of example 1 and the fluorescent antibody of comparative example 1 were used to detect different samples, and the detection results are shown in the following table:
according to the detection results of different types of samples, the invention can be found that the labeled fluorescent antibody is separated and purified by the glucose gel column, so that the separation of free fluorescent dye and the fluorescent antibody is realized, and the specificity and the sensitivity of the detection result are increased.
3. Fluorescence relative intensity data.
Comparative example 2 bovine serum albumin was not subjected to thermal destruction treatment and then subjected to coupling. The prepared anti-cyclic citrullinated peptide antigen is respectively diluted to 0.5, 0.6, 0.75 and 1.0mg/ml by using an antigen coating solution, sprayed to the corresponding antigen detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm, diluted to 1.0mg/ml by using PBS buffer solution, and sprayed to the corresponding quality control detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm.
The fluorescence of the embodiment 1 and the fluorescence of the comparative example 2 are respectively used for detecting the internal quality control products of different enterprises, the relative signal intensity of the fluorescence is checked, and the detection results are shown in the following table:
the fluorescence relative intensity can be detected by performing different coating concentrations on the prepared anti-cyclic citrullinated peptide antigen through the table, and the result can be found that the coupling efficiency can be greatly improved by performing thermal destruction treatment on bovine serum albumin and then coupling the bovine serum albumin with the anti-cyclic citrullinated peptide antigen; the relative intensity of fluorescence of the coating concentration of 0.5mg/ml after damage is consistent with that of fluorescence of the coating concentration of 0.75mg/ml without damage, the marking efficiency is improved by 50 percent, the raw materials are saved, and the production cost is reduced.
4. Accuracy data
Comparative example 3 differs from example 1 in that:
diluting the fluorescent antibody with a fluorescent antibody diluent according to a ratio of 1:10, uniformly mixing, and directly spraying the fluorescent antibody on a nitrocellulose membrane in a spraying amount of 0.5 mu L/cm without spraying a closed line.
The residue of the fluorescent antibody was examined by accelerated destruction at 37 ℃ in example 1 and comparative example 3, and the results are shown in the following table:
the detection of the residual fluorescence intensity through the table above shows that the fluorescent antibody is directly solid-phase without spraying the confining liquid, the residual fluorescent antibody is gradually increased along with the prolonging of the destruction time at 37 ℃, the residual fluorescence participating in the reaction is gradually reduced, and the result is greatly influenced. According to the arrhenius formula: d (Ink)/dT = Ea/RT2 Ea, destruction at 37 ℃ for 6 months is equivalent to storage at normal temperature for 2 years; the kit disclosed by the invention is sprayed with the confining liquid firstly and then the fluorescent antibody, so that the residue of the fluorescent antibody is little and is not increased along with the prolonging of the destruction time within 2 years of the validity period of the kit, the detection result is not influenced at all, and the accuracy of the kit is ensured.
5. The linear range of the anti-cyclic citrullinated peptide antibody dry immunofluorescence chromatography detection kit.
The conjugate carrier macromolecular substance bovine serum albumin was subjected to thermal destruction treatment, and then coupled with an anti-cyclic citrullinated peptide antigen to prepare a detection plate by the method described in example 2. Diluting high concentration sample (640R μm/ml) of anti-cyclic citrullinated peptide near the upper limit of linear range to 1024 times with negative serum fold ratio, preparing into 10 solutions with different concentration, repeating the determination for 3 times per concentration, performing linear regression analysis on the average value of the determined concentration and theoretical concentration, and calculating regression equation to be y =0.9998x +0.0249 with correlation coefficient R20.9998, indicating that the kit of the present invention has a good correlation in the linear range of 1.25R μm/ml to 640R μm/ml.
6. And (3) carrying out stability experimental investigation on the anti-cyclic citrullinated peptide antibody dry immunofluorescence chromatography detection kit.
The kit provided by the invention is placed at 37 ℃ 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 1 month, the 45 th day, the 2 months, the 3 months, the 4 months, the 5 months and the 6 months, and the standard is as follows:
(1) physical state of dilution
Appearance: colorless transparent clear liquid without particles, floccules and precipitates.
(2) Performance index
Negative coincidence rate: the negative reference products N1-N3 are used for detection, and the results are negative (negative coincidence rate: 3/3).
Positive compliance rate: the positive reference products P1-P5 are used for detection, and the results are positive (positive coincidence rate: 5/5).
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 6 months are not more than 15%.
The test result of the stability of the kit is as follows:
sample dilution physical stability results
Test result of 37 ℃ accelerated destruction of kit
Note: the concentration of the negative reference substance is less than or equal to 10R mu m/ml, and the concentration of the positive reference substance is more than 10R mu m/ml.
The test proves that the product is stable for at least half a year at 37 ℃. According to the arrhenius formula: d (Ink)/dT = Ea/RT2 Ea, the destruction at 37 ℃ for 6 months is equivalent to the storage at normal temperature for 2 years, can meet the clinical requirements of hospital clinics and health quarantine departments, and can also be used for disease diagnosis research of colleges and universities and scientific research institutions.
7. The coincidence rate of the anti-cyclic citrullinated peptide antibody dry immunofluorescence chromatography detection kit and a Shanghai new enzyme immunoassay detection kit of a company on the market.
300 parts of serum for confirming to be RA patients and 200 parts of serum for healthy physical examination are collected from clinic, the anti-cyclic citrullinated peptide antibody dry immunofluorescence chromatography detection kit and the Shanghai new enzyme immunoassay detection kit are used for simultaneous detection, and the specificity and the detection rate are as follows:
comparison of specificity and detection rate of two kits with different sera
The total compliance is as follows:
total coincidence rate of two kits for 500 samples
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 (8)
1. A test strip for detecting an anti-cyclic citrullinated peptide antibody is characterized in that: the kit comprises a nitrocellulose membrane, wherein the nitrocellulose membrane is coated with a closed line, an anti-cyclic citrullinated peptide antigen detection line and a goat anti-mouse IgG quality control line which are separated from each other and distributed in sequence, a fluorescent antibody line is arranged on a solid phase on the closed line, and the closed line and the fluorescent antibody line are sprayed in the following modes:
spraying the prepared sealing liquid at the position corresponding to the sealing line on the nitrocellulose membrane by the spraying amount of 1.5 mu L/cm, then drying at 37 ℃ for 0.5h to prepare the sealing line, diluting the fluorescent antibody by using fluorescent antibody diluent according to the proportion of 1:10, uniformly mixing, and spraying the fluorescent antibody at the middle position of the dried sealing line by the spraying amount of 0.5 mu L/cm to prepare the fluorescent antibody line.
2. The test strip for detecting the anti-cyclic citrullinated peptide antibody according to claim 1, wherein: the preparation method of the fluorescent antibody comprises the following steps:
labeling of fluorescent antibody: diluting the mouse anti-human IgG antibody to the final concentration of 2-10 mg/mL by PBS buffer solution, and preparing NaHCO with pure water3The concentration is 1mol/L, NaHCO is added into the diluted mouse anti-human IgG antibody3，NaHCO3The final concentration of the fluorescent dye is 0.1-1 mol/L, the residual volume is filled with PBS buffer solution, the fluorescent dye is added, the final concentration of the fluorescent dye is 1-10 mmol/L, the mixture is uniformly mixed, a micro-oscillator is started, and the labeling reaction is carried out for 15-60 min at the room temperature in a dark place;
purification of fluorescent antibody: and (4) taking out the labeled fluorescent antibody, and separating and purifying the fluorescent antibody through a sephadex column.
3. The test strip for detecting an anti-cyclic citrullinated peptide antibody according to claim 1 or 2, wherein: the sealing liquid comprises the following components:
20-100 mM Tris; 0.1-1.5 g of NaCL; 0.5-3 g of bovine serum albumin; 0.1-0.5 g of casein; 0.1-5 g of PVP; 1-10 g of sucrose; 1-3 g of trehalose; tween 0.01-0.1 mL, and the pH value of the Tris buffer solution is 8.0-8.5.
4. The test strip for detecting an anti-cyclic citrullinated peptide antibody according to claim 1 or 2, wherein: the fluorescent antibody diluent comprises the following components:
20-100 mM Tris; 0.1-1.5 g of NaCL; 0.5-3 g of bovine serum albumin; 0.1-5 g of PVP; 1-10 g of sucrose; 1-3 g of trehalose; the pH value of the Tris buffer solution is 8.0-8.5.
5. The test strip for detecting an anti-cyclic citrullinated peptide antibody according to claim 1 or 2, wherein: the coating method of the anti-cyclic citrullinated peptide antigen detection line comprises the following steps:
coupling an anti-cyclic citrullinated peptide antigen with a carrier protein, wherein the carrier protein is subjected to 55 ℃ thermal destruction treatment, and is bovine serum albumin;
diluting the coupled anti-cyclic citrullinated peptide antigen to 0.5mg/mL by using an antigen diluent, and spraying the antigen to a corresponding antigen detection line of the nitrocellulose membrane by the spraying amount of 1.0 mu L/cm;
the coating method of the goat anti-mouse IgG quality control line comprises the following steps:
the goat anti-mouse IgG is diluted to 1.0mg/mL by PBS buffer solution, and sprayed on the corresponding quality control detection line of the nitrocellulose membrane by a spray amount of 1.0 mu L/cm.
6. The test strip for detecting the anti-cyclic citrullinated peptide antibody according to claim 5, wherein: the preparation method of the antigen diluent comprises the following steps:
60.5mg Tris, 0.9g sodium chloride and 3g trehalose are dissolved in 100mL distilled water, 10mL glycerol is added for even mixing, and the mixture is filtered and filled into a wide-mouth bottle for later use.
7. A detection card of an anti-cyclic citrullinated peptide antibody is characterized in that: the detection card comprises an upper cover and a lower cover which are buckled, wherein a groove is formed in the lower cover, and the test strip of any one of claims 1 to 6 is placed in the groove.
8. A detection kit for an anti-cyclic citrullinated peptide antibody is characterized in that: the kit comprises the detection card of claim 7 and a sample diluent, wherein the sample diluent comprises 20-100 mM Tris, 0.1-1.5 g of NaCL, 0.1-0.5 g of casein, 0.1-5 g of PEG, 0.05-0.5 g of gelatin, 90.1-1 mL of S, 0.1-1 mL of Tween and 0. 3000.1-1 mL of PC, and the pH of a Tris buffer solution is 8.0-8.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110358940.8A CN113030456A (en) | 2021-04-02 | 2021-04-02 | Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110358940.8A CN113030456A (en) | 2021-04-02 | 2021-04-02 | Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113030456A true CN113030456A (en) | 2021-06-25 |
Family
ID=76453468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110358940.8A Pending CN113030456A (en) | 2021-04-02 | 2021-04-02 | Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113030456A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113834929A (en) * | 2021-09-27 | 2021-12-24 | 山东康华生物医疗科技股份有限公司 | Detection membrane strip, detection card and detection kit for allergen detection |
| CN114062666A (en) * | 2021-11-24 | 2022-02-18 | 山东康华生物医疗科技股份有限公司 | Preparation method of detection plate for quantitatively detecting RF, ASO, CRP and CCP in one card mode |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87107982A (en) * | 1986-11-26 | 1988-06-15 | 伯伦格·曼海姆有限公司 | Method for measuring substance capable of specifically binding |
| US20040241767A1 (en) * | 2001-06-13 | 2004-12-02 | Brigitte Incaurgarat | Method for detecting rheumatoid arthritis-specific autoantibodies |
| CN101187665A (en) * | 2007-12-27 | 2008-05-28 | 北京博晖创新光电技术股份有限公司 | Enterovirus immunofluorescence chromatographic assay test paper and its preparation method |
| CN101865913A (en) * | 2009-04-16 | 2010-10-20 | 上海荣盛生物药业有限公司 | Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography |
| CN101988924A (en) * | 2009-07-31 | 2011-03-23 | 上海科新生物技术股份有限公司 | Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method |
| CN102023219A (en) * | 2009-09-22 | 2011-04-20 | 上海荣盛生物药业有限公司 | Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography |
| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
| CN106645738A (en) * | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | Anti-cyclic citrullinated peptide antibody chemiluminescence immune detection kit and preparation method thereof |
| CN108196073A (en) * | 2018-03-13 | 2018-06-22 | 江苏浩欧博生物医药股份有限公司 | A kind of kit for measuring cyclic citrullinated peptid and its application |
-
2021
- 2021-04-02 CN CN202110358940.8A patent/CN113030456A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87107982A (en) * | 1986-11-26 | 1988-06-15 | 伯伦格·曼海姆有限公司 | Method for measuring substance capable of specifically binding |
| US20040241767A1 (en) * | 2001-06-13 | 2004-12-02 | Brigitte Incaurgarat | Method for detecting rheumatoid arthritis-specific autoantibodies |
| CN101187665A (en) * | 2007-12-27 | 2008-05-28 | 北京博晖创新光电技术股份有限公司 | Enterovirus immunofluorescence chromatographic assay test paper and its preparation method |
| CN101865913A (en) * | 2009-04-16 | 2010-10-20 | 上海荣盛生物药业有限公司 | Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography |
| CN101988924A (en) * | 2009-07-31 | 2011-03-23 | 上海科新生物技术股份有限公司 | Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method |
| CN102023219A (en) * | 2009-09-22 | 2011-04-20 | 上海荣盛生物药业有限公司 | Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography |
| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
| CN106645738A (en) * | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | Anti-cyclic citrullinated peptide antibody chemiluminescence immune detection kit and preparation method thereof |
| CN108196073A (en) * | 2018-03-13 | 2018-06-22 | 江苏浩欧博生物医药股份有限公司 | A kind of kit for measuring cyclic citrullinated peptid and its application |
Non-Patent Citations (1)
| Title |
|---|
| 郭利宁 等: "一种基于量子点检测抗CCP抗体的免疫荧光层析法", 中国生物化学与分子生物学报, vol. 29, no. 4, pages 389 - 395 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113834929A (en) * | 2021-09-27 | 2021-12-24 | 山东康华生物医疗科技股份有限公司 | Detection membrane strip, detection card and detection kit for allergen detection |
| CN114062666A (en) * | 2021-11-24 | 2022-02-18 | 山东康华生物医疗科技股份有限公司 | Preparation method of detection plate for quantitatively detecting RF, ASO, CRP and CCP in one card mode |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111426831B (en) | Kit for combined detection of 2019 novel coronavirus IgM and IgG antibodies and preparation method thereof | |
| CN111398588A (en) | Use method of immunochromatography kit for rapidly detecting novel coronavirus N protein | |
| CN111273001A (en) | System for rapidly detecting new coronavirus 2019-nCoV in blood sample and preparation method thereof | |
| CN111337673B (en) | Synthetic polypeptide composition for novel coronavirus immunodetection and application | |
| CN111896730B (en) | Dry immunofluorescence quantitative Heparin Binding Protein (HBP) detection kit | |
| CN113030456A (en) | Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody | |
| CA1340973C (en) | Immunometric assay kit and method applicable to whole cells | |
| JP3451091B2 (en) | Analytical cuvette used for quantification of an analyte present in a whole blood sample, quantification method, and diagnostic test kit | |
| CN204989196U (en) | Qualitative test paper strip of immunity chromatography is united to six antinuclear antibodiess | |
| GB2181840A (en) | Method for the immunoassay of a macromolecular analyte | |
| CN113933502B (en) | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography | |
| CN114686592A (en) | Kit for multi-type target combined detection, preparation method and use method | |
| US20210247392A1 (en) | Direct immunoassay measurement of autoantibodies | |
| CN107144686A (en) | A kind of accurate fluorescence quantitative detecting method | |
| JP2824794B2 (en) | A method to search for and identify erythrocyte antibodies by solid-phase method | |
| JP2003279577A (en) | Composition for flow through type inspection, and kit using the same, and inspection method | |
| CN103713121A (en) | Human vasculitis diagnostic kit and preparation method thereof | |
| JP2553852B2 (en) | Immunological assay for biological substances in samples | |
| CN111562392A (en) | Novel coronavirus IgG antibody fluorescence immunoassay detection kit | |
| CN111505314A (en) | Novel combined detection kit for coronavirus antibody and application thereof | |
| CN105911274A (en) | Immunochromatography device for synchronously and quantitatively detecting different molecular forms of human neutrophil lipocalin (HNL) and preparation method thereof | |
| CN106546729B (en) | Novel process method for removing serum matrix effect in dry immunofluorescence quantitative detection | |
| CN212483600U (en) | IgA antibody detection test strip, detection card and detection kit for multiple respiratory pathogens | |
| CN111735962B (en) | Novel coronavirus IgM/IgG antibody joint detection immunochromatography test strip | |
| CN204514933U (en) | B2M immunochromatographiassay assay quantitative detection test paper |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |