CN115420899A

CN115420899A - Preparation method of fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA

Info

Publication number: CN115420899A
Application number: CN202211343967.0A
Authority: CN
Inventors: 姚辉; 张瑜; 陶慧云
Original assignee: Dialab Zhangjiagang Biotechnology Co ltd
Current assignee: Dialab Zhangjiagang Biotechnology Co ltd
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2022-12-02
Anticipated expiration: 2042-10-31
Also published as: CN115420899B

Abstract

The invention provides a preparation method of a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA. The kit comprises an immunochromatographic test strip and a sample diluent, wherein the immunochromatographic test strip is obtained by assembling a sample pad treated by a first buffer solution, a combination pad which is respectively combined with a rheumatoid factor antigen-fluorescent microsphere conjugate treated by a second buffer solution and a DNP-BSA-fluorescent microsphere conjugate, a nitrocellulose membrane, absorbent paper and a base plate. The sample diluent is a buffer solution with the pH value of 7-8 and containing 0.5-1.5 wt% of Tween-20, 0.4-0.8 wt% of sodium dodecyl sulfate and 100-400 mmol/L of LNaCl. The kit prepared by the method can detect and quantify three types of rheumatoid factors at one time, has good comprehensive performance and particularly has high sensitivity.

Description

Preparation method of fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA

Technical Field

The invention relates to the technical field of immunofluorescence detection, and particularly relates to a preparation method of a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA.

Background

Rheumatoid Factor (RF) is an antibody against an antigenic determinant of Fc fragment of human or animal IgG molecules, is an autoantibody targeting denatured IgG, and is mainly present in serum of patients with rheumatoid arthritis. RF positivity supports a predisposing diagnosis of early Rheumatoid Arthritis (RA). Wherein, igG type rheumatoid factor (RF-IgG), igM type rheumatoid factor (RF-IgM) and IgA type rheumatoid factor (RF-IgA) have detection significance.

Detection significance of RF-IgG: the appearance of IgG-type RF in the serum or synovial fluid of RA patients is closely related to the symptoms of synovitis, vasculitis and joints in patients.

The detection significance of RF-IgM: the increase of the concentration level of the RF-IgM can cause more inflammatory reactions of joints, and meanwhile, the positive rate of rheumatoid arthritis patients is the highest and reaches about 80 percent, and the positive rate is one of the important serological indexes for diagnosing RA. Poor prognosis is often indicated in patients with RA who have serum RF-IgM concentrations >80IU/ml with severe joint dysfunction.

The detection significance of RF-IgA is as follows: RF-IgA is found in RA, scleroderma, fisher-Tropsch syndrome (Felty syndrome) and SLE and is an index of clinical activity in RA, and RF-IgA is related to bone destruction, and early RF-IgA rise often indicates severe disease and poor prognosis.

And simultaneously, the detection of IgG type rheumatoid factors (RF-IgG), igM type rheumatoid factors (RF-IgM) and IgA type rheumatoid factors (RF-IgA) can improve the detection rate and specificity of RF in rheumatoid arthritis.

The most common detection methods at present are colloidal gold, enzyme-linked immunosorbent assay and chemiluminescence assay. For example, in the prior patent CN102707050A, colloidal gold-labeled human denatured IgG is coated on a gold-labeled fiber pad, and anti-mu chain IgM and human denatured IgG are coated on a nitrocellulose membrane, so that the problems that the sensitivity is not high enough, the detection result is not accurate, and the accurate quantification cannot be realized exist, and the contents of three types of RF-IgG, RF-IgM and RF-IgA cannot be obtained through one-time detection in the patent. The prior patent CN101871940A adopts an enzyme-linked immunosorbent assay method, which has complex operation, higher requirement on inspection personnel and longer time consumption. The existing patent CN109406796B develops a chemiluminescence immunoassay kit based on a chemiluminescence platform, and has the disadvantages of more related reagents and inconvenient operation. The fluorescence immunochromatography can realize the typing of a plurality of antibodies which are detected at one time, and is simple and convenient to operate, but aiming at different antigen antibodies, the preparation method of the fluorescence immunochromatography test paper, the formula of sample diluent and the like are required to be continuously adjusted, the comprehensive performance of the kit is ensured, and particularly, the sensitivity and the accuracy are improved.

Disclosure of Invention

The invention aims to provide a preparation method of a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA.

The invention also aims to provide the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which is prepared by the preparation method.

The invention further aims to provide a RF-IgG/RF-IgM/RF-IgA combined quantitative detection system.

In order to solve the technical problems, the invention adopts the following technical scheme:

the preparation method of the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA comprises an immunochromatographic test strip and a sample diluent, and the preparation method of the immunochromatographic test strip comprises the following steps:

a) Treating the first glass fiber membrane with a first buffer solution to obtain a sample pad,

b) Treating the rheumatoid factor antigen-fluorescent microsphere conjugate and the DNP-BSA-fluorescent microsphere conjugate by using a second buffer solution, then respectively combining the treated rheumatoid factor antigen-fluorescent microsphere conjugate and the DNP-BSA-fluorescent microsphere conjugate to a second glass fiber membrane to prepare a combination pad,

c) And assembling the sample pad, the combination pad, the nitrocellulose membrane, the absorbent paper and the base plate to obtain the fluorescence immunochromatographic test strip.

The pH value of the first buffer solution is 8.0-9.0, the first buffer solution comprises 0.5-1.5 wt% of Tween-20, 3-8 wt% of trehalose and 0.1-0.6 wt% of casein, the pH value of the second buffer solution is 5.5-6.5, the second buffer solution comprises 0.5-1.5 wt% of Tween-20, 5-15 wt% of trehalose and 0.1-0.6 wt% of casein, and an IgG detection line, an IgM, igA and a quality control line are arranged on the nitrocellulose membrane.

The sample diluent is a third buffer solution with the pH value of 7.0-8.0, and comprises 0.5-1.5 wt% of Tween-20, 0.4-0.8 wt% of sodium dodecyl sulfate and 100-400 mmol/L of NaCl.

Preferably, the first buffer, the second buffer and the third buffer are each independently selected from PBS buffer, BBS buffer, MES buffer, tris buffer or citrate buffer.

Further preferably, the first buffer is Tris buffer.

Still more preferably, the concentration of the Tris buffer is 0.05mmol/L to 0.1mmol/L, such as 0.05mmol/L,0.06mmol/L,0.07mmol/L,0.08mmol/L,0.09mmol/L,0.1mmol/L.

The second buffer is MES buffer.

Still more preferably, the concentration of the MES buffer is 0.05mol/L to 0.2mol/L, such as 0.05mol/L,0.06mol/L,0.08mol/L,0.1mol/L,0.12mol/L,0.14mol/L,0.16mol/L,0.18mol/L,0.2mol/L.

The third buffer solution is PBS buffer solution.

Still more preferably, the concentration of the PBS buffer is 5mmol/L to 20mmol/L, such as 5mmol/L,6mmol/L,7mmol/L,8mmol/L,9mmol/L,10mmol/L,11mmol/L,12mmol/L,13mmol/L,14mmol/L,15mmol/L,16mmol/L,17mmol/L,18mmol/L,19mmol/L,20mmol/L.

Preferably, the pH value of the first buffer is 8.5 to 9.0, such as 8.5,8.6,8.7,8.8,8.9,9.0.

Preferably, the pH of the second buffer is 5.5 to 6.0, e.g., 5.5,5.6,5.7,5.8,5.9,6.0.

Preferably, the pH of the third buffer is 7.0 to 7.5, e.g., 7.0,7.1,7.2,7.3,7.4,7.5.

Further preferably, the first buffer solution is a Tris buffer solution with the pH value of 8.0-9.0 and the concentration of 0.01 mmol/L-0.1 mmol/L, and comprises 0.5wt% -1.5 wt% of Tween-20, 3wt% -8 wt% of trehalose and 0.1wt% -0.6 wt% of casein.

Still further preferably, the first buffer solution is a Tris buffer solution with the pH value of 8.4-8.6 and the concentration of 0.02-0.06 mmol/L, and comprises 0.8-1.2 wt% of Tween-20, 3-8 wt% of trehalose and 0.4-0.6 wt% of casein.

Further preferably, the pH value of the second buffer solution is 5.5-6.5, the MES buffer solution with the concentration of 0.05-0.2 mol/L comprises 0.5-1.5 wt% of Tween-20, 5-15 wt% of trehalose and 0.1-0.6 wt% of casein.

Still further preferably, the pH value of the second buffer solution is 5.8-6.2, the concentration of the MES buffer solution is 0.08-0.15 mol/L, 0.8-1.2 wt% of Tween-20, 8-12 wt% of trehalose and 0.4-0.6 wt% of casein.

Further preferably, the sample diluent is a PBS buffer solution with a pH value of 7.0 to 7.5 and a concentration of 8mmol/L to 15mmol/L, and the PBS buffer solution contains 0.5wt% to 1.5wt% of Tween-20, 0.4wt% to 0.6wt% of sodium dodecyl sulfate and 200mmol/L to 300mmol/L of NaCl.

Preferably, the sample pad is soaked in a first buffer solution for 1min to 3min, and the sample pad is obtained by drying at 30 ℃ to 60 ℃ in an environment with the humidity of less than 30% and the temperature of 18 ℃ to 28 ℃.

Further preferably, the sample pad is soaked in a first buffer solution for 1min to 1.5min, and dried at the temperature of between 30 and 40 ℃ in the environment with the humidity of less than 30 percent and the temperature of between 18 and 28 ℃ to obtain the sample pad.

Preferably, the rheumatoid factor antigen-fluorescent microsphere conjugate and the second buffer solution are mixed to obtain a rheumatoid factor antigen-fluorescent microsphere conjugate solution with a microsphere content of 0.05wt% -0.5 wt%, the DNP-BSA-fluorescent microsphere conjugate and the second buffer solution are mixed to obtain a DNP-BSA-fluorescent microsphere conjugate solution with a microsphere content of 0.05wt% -0.5 wt%, the mixture of the rheumatoid factor antigen-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution is sprayed onto the bonding pad according to a volume ratio of (15 to 18): 1, and the bonding pad is dried at 30-60 ℃ in an environment with a humidity of less than 30% and a temperature of 18-28 ℃ to obtain the bonding pad.

The preparation methods of the rheumatoid factor antigen-fluorescent microsphere conjugate and the DNP-BSA-fluorescent microsphere conjugate are not particularly limited, and conventional methods in the field can be selected.

Preferably, the preparation method of the rheumatoid factor antigen-fluorescent microsphere conjugate comprises the following steps: adding biotin 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and fluorescent microspheres into a coupling buffer solution, incubating for 10min to 20min at room temperature, then adding rheumatoid factor antigen, and incubating for 1.5h to 2.5h at room temperature, wherein in a coupling system, the dosage of the biotin 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) is 10 to 15 mu g/mL, the volume of the fluorescent microspheres is 8 to 12 percent of the total coupling volume, and the concentration of the rheumatoid factor antigen is 0.05mg/mL to 0.5mg/mL. And after coupling, mixing the rheumatoid factor antigen-fluorescent microsphere conjugate with the second buffer solution to prepare a rheumatoid factor antigen-fluorescent microsphere conjugate solution. The coupling buffer solution is preferably MES buffer solution with the pH value of 5.5-6.5 and the concentration of 0.05 mol/L-0.5 mol/L.

Specifically, the preparation method of the rheumatoid factor antigen-fluorescent microsphere conjugate or the DNP-BSA-fluorescent microsphere conjugate comprises the following steps:

1) Activation and coupling of microspheres: adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and fluorescent microspheres into a coupling buffer solution, carrying out shaking table incubation at room temperature for 10min to 2 min, then adding rheumatoid factor antigen or DNP-BSA protein, carrying out shaking table incubation at room temperature for 1.5h to 2.5h, wherein in a coupling system, the using amount of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) is 10 to 15 mu g/mL, the volume of the fluorescent microspheres is 8 to 12 percent of the total coupling volume, and the concentration of the rheumatoid factor antigen or DNP-BSA protein is 0.05mg/mL to 0.5mg/mL. The coupling buffer solution is preferably MES buffer solution with the pH value of 5.5-6.5 and the concentration of 0.05 mol/L-0.5 mol/L.

2) Sealing the microspheres: and incubating at room temperature for 1.5h to 2.5h, adding a closed buffer solution with the volume of 8-12% of the total volume of the coupling system, and incubating at room temperature for 0.5h to 1.5h by a shaking table. The blocking buffer is preferably BSA buffer with the concentration of 5% -15%.

3) Cleaning the microspheres: and after the sealing is finished, centrifuging to remove the supernatant, redissolving by using a coupling buffer solution with the volume of 150-250% of the total coupling volume, performing ultrasonic treatment, centrifuging and discarding the supernatant to obtain the rheumatoid factor antigen-fluorescent microsphere conjugate or DNP-BSA-fluorescent microsphere conjugate.

Preferably, a first coating solution is obtained by mixing a mouse anti-human IgG antibody and a fourth buffer solution, the first coating solution is used for scribing on the nitrocellulose membrane, and the IgG detection line is formed by drying at 30-60 ℃ in an environment with the humidity of less than 30% and the temperature of 18-28 ℃. Further preferably drying at 30-40 ℃.

Preferably, a mouse anti-human IgM antibody is mixed with a fourth buffer solution to obtain a second coating solution, the second coating solution is used for scribing on the nitrocellulose membrane, and the IgM detection line is formed by drying at 30 to 60 ℃ in an environment with the humidity of less than 30% and the temperature of 18 to 28 ℃. Further preferably drying at 30-40 ℃.

Preferably, a third coating solution is obtained by mixing a mouse anti-human IgA antibody with a fourth buffer solution, the third coating solution is used for scribing on the nitrocellulose membrane, and the IgA detection line is formed by drying at 30-60 ℃ in an environment with the humidity of less than 30% and the temperature of 18-28 ℃. Further preferably drying at 30-40 ℃.

Preferably, mixing the DNP monoclonal antibody with a fourth buffer solution to obtain a fourth coating solution, scribing on the nitrocellulose membrane by using the fourth coating solution, and drying at 30 to 60 ℃ in an environment with the humidity of less than 30% and the temperature of 18 to 28 ℃ to form the quality control line. Further preferably drying at 30-40 ℃.

Preferably, the concentrations of the antibodies in the first, second, third and fourth coating solutions are independently 1mg/mL to 5mg/mL, such as 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL.

Preferably, the pH of the fourth buffer is 7 to 7.5, for example 7, 7.1,7.2,7.3,7.4,7.5.

Preferably, the fourth buffer solution is PBS buffer solution with the concentration of 5 mmol/L-20 mmol/L, and comprises 2wt% -8 wt% of trehalose and 2wt% -8 wt% of methanol.

Further preferably, the fourth buffer solution is a PBS buffer solution with a concentration of 8mmol/L to 12mmol/L, and comprises 4wt% to 6wt% of trehalose and 4wt% to 6wt% of methanol.

Preferably, the sample pad and the combination pad are made of glass fiber films or polyester fiber films.

Preferably, the bottom plate is made of PVC.

Preferably, the fluorescent microsphere is a europium-chelated fluorescent microsphere.

Preferably, the surface of the fluorescent microsphere is provided with a modifying group, and the modifying group is an amino group and/or a carboxyl group.

Preferably, the fluorescent microspheres are fluorescent microspheres activated with N-hydroxysuccinimide ester and/or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.

Preferably, the diameter of the fluorescent microsphere is 0.1-0.3 μm.

Preferably, the excitation wavelength of the fluorescent microsphere is 320nm to 350nm, and the emission wavelength of the fluorescent microsphere is 600nm to 620nm.

Preferably, the rheumatoid factor antigen of the rheumatoid factor antigen-fluorescent microsphere conjugate is an artificially synthesized antigen.

Preferably, the IgG detection line is coated with a mouse anti-human IgG antibody, the IgM detection line is coated with a mouse anti-human IgM antibody, the IgA detection line is coated with a mouse anti-human IgA antibody, and the quality control line is coated with a DNP monoclonal antibody.

The invention also provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which is prepared by the preparation method.

The invention also provides an RF-IgG/RF-IgM/RF-IgA combined quantitative detection system which comprises the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, a fluorescence immunoassay analyzer and an ID card arranged on the fluorescence immunochromatographic test strip, wherein calibration data and/or a standard curve are/is stored in the ID card.

The detection method of the RF-IgG/RF-IgM/RF-IgA combined quantitative detection system comprises the following steps:

(1) Inserting the ID card into the fluorescence immunoassay analyzer, and reading calibration data and/or standard curves related to the RF-IgG/RF-IgM/RF-IgA combined detection item stored in the ID card;

(2) Diluting a sample to be detected by using a sample diluent, dripping the diluted sample to be detected onto the fluorescence immunochromatographic test strip, and sequentially passing the diluted sample to be detected through the treated sample pad, the treated combination pad, the nitrocellulose membrane and the absorbent paper;

(3) After waiting for 5min to 15min, inserting the fluorescence immunochromatographic test strip into the fluorescence immunoassay analyzer again, reading the fluorescence intensity of the IgG detection line, the IgM detection line, the IgA detection line and the quality control line, and calculating and outputting the RF-IgG content, the RF-IgM content and the RF-IgA content of the sample to be detected according to the calibration data and/or the standard curve.

In the invention, the sample detected by the combined detection RF-IgG/RF-IgM/RF-IgA fluorescence immunochromatographic kit and the combined RF-IgG/RF-IgM/RF-IgA quantitative detection system is a serum sample.

Compared with the prior art, the invention has the following advantages:

the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which is prepared by the method, has the advantages of high sensitivity, high accuracy, good stability, good repeatability, simplicity, easiness in operation and the like, and under the premise of extremely less clinical sample usage and only one fluorescence immunoassay analyzer, the RF-IgG content, the RF-IgM content and the RF-IgA content can be obtained simultaneously by one detection, so that the application prospect is wide.

Drawings

FIG. 1 is a schematic structural diagram of a fluorescence immunochromatographic test strip in the fluorescence immunochromatographic kit of the present invention,

wherein, 1, a bottom plate; 2. a sample pad; 3. a bonding pad; 4. a nitrocellulose membrane; 5, absorbent paper; 6. an IgA detection line; 7. an IgM detection line; 8. an IgG detection line; 9. and (4) quality control line.

Detailed Description

The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

At present, in domestic laboratories and clinics, a colloidal gold method, an enzyme linked immunosorbent assay and a chemiluminescence method are mainly adopted to detect rheumatoid factors, or the problems of poor sensitivity and accuracy, complex operation, high production cost and the like exist. The fluorescence immunochromatography can realize the typing of simultaneously detecting RF-IgG/RF-IgM/RF-IgA at one time, is simple and convenient to operate, but has high development difficulty, and no precedent for preparing a test strip for detecting RF-IgG/RF-IgM/RF-IgA on a fluorescence immunochromatography platform exists.

Specifically, the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA comprises an immunochromatographic test strip and a sample diluent, and the preparation method of the immunochromatographic test strip comprises the following steps:

c) And assembling the sample pad, the combination pad, the nitrocellulose membrane, the absorbent paper and the bottom plate to obtain the fluorescence immunochromatographic test strip.

The pH value of the first buffer solution ranges from 8 to 9, the first buffer solution comprises 0.5wt% to 1.5wt% of Tween-20, 3wt% to 8wt% of trehalose and 0.1wt% to 0.6wt% of casein, the pH value of the second buffer solution ranges from 5.5 to 6.5, the second buffer solution comprises 0.5wt% to 1.5wt% of Tween-20, 5wt% to 15wt% of trehalose and 0.1wt% to 0.6wt% of casein, and an IgG detection line, an IgM detection line, an IgA detection line and a quality control line are arranged on the nitrocellulose membrane.

The sample diluent is a third buffer solution with the pH value of 7-8, and comprises 0.5-1.5 wt% of Tween-20, 0.4-0.8 wt% of sodium dodecyl sulfate and 100-400 mmol/L of NaCl.

According to the invention, the sample pad treated by the first buffer solution, the rheumatoid factor antigen-fluorescent microsphere conjugate and the DNP-BSA-fluorescent microsphere conjugate treated by the second buffer solution are used, so that the detection sensitivity and stability are ensured, and the accuracy of a detection result is improved and the repeatability is good when the sample is diluted by matching with a sample diluent for detection. In addition, the immunofluorescence test strip has the advantages of simple structure, short production period, low equipment requirement and easy realization of mass production, and the kit provided by the invention also has the advantages of short detection time, simplicity in operation, convenience in use, low requirement on the relevant technical level of personnel, wide application scene and the like.

The technical scheme of the invention is further illustrated by combining specific examples and comparative examples.

In the following examples and comparative examples, the raw materials, reagents and the like used were all conventional commercially available products unless otherwise specified.

In the following examples and comparative examples, the rheumatoid factor antigen used was an artificially synthesized antigen obtained from bioengineering, inc. of Huamei, wuhan, etc. Dinitrophenol-conjugated bovine serum albumin (DNP-BSA) and DNP monoclonal antibodies were purchased from Biotech GmbH, gmbH. The fluorescent microspheres are europium-chelated fluorescent microspheres purchased from Thermo corporation of Seimer Feishell science and technology (China), the surface of the dyed particles is provided with carboxyl groups, the particle size of the fluorescent microspheres is about 0.2 mu m, the excitation wavelength is 333nm, and the emission wavelength is 613nm. Mouse anti-human IgM antibody, mouse anti-human IgG antibody, and mouse anti-human IgA antibody were purchased from Beijing Borxi technologies, inc. Calibrators and references for RF-IgG, RF-IgM, RF-IgA were purchased from Fermat Biotechnology, inc., shanghai.

In the following examples and comparative examples, the dry fluoroimmunoassay analyzer DL300 manufactured by Biotechnology Ltd of Laibo (Zhang Home harbor) was used as a fluorescence immunoassay analyzer.

Example 1

The embodiment provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which comprises an immunochromatographic test strip and a sample diluent. The immunochromatography test strip has a structure shown in figure 1, and comprises a bottom plate 1, and a sample pad 2, a combination pad 3, a nitrocellulose membrane 4 and absorbent paper 5 which are sequentially lapped on the bottom plate 1, wherein the nitrocellulose membrane 4 is provided with an IgA detection line 6, an IgM detection line 7, an IgG detection line 8 and a quality control line 9. Wherein, the material of bottom plate is PVC, and the material of sample pad 2 and combination pad 3 is glass cellulose membrane. The immunochromatographic test strip is provided with a sample adding hole (not shown in the figure), and the sample adding hole is arranged by the conventional technical means in the field.

The preparation method of the fluorescence immunochromatographic kit for joint detection of RF-IgG/RF-IgM/RF-IgA of the embodiment comprises the following steps:

1. preparation of sample pad 2:

1.1 Preparing a first buffer solution: tris buffer with pH value of 8.5 and concentration of 0.05mmol/L, which contains 1wt% of Tween-20, 5wt% of trehalose and 0.5wt% of casein.

1.2 Immersing the glass cellulose membrane into the first buffer solution for 1min, taking out, placing in an environment with the humidity of less than 30% and the temperature of 18-28 ℃, drying for 24 hours at 37 ℃, and cutting to obtain the required specification for later use.

2. Preparation of the conjugate pad 3:

2.1 Preparing a second buffer solution: MES buffer with pH value of 6 and concentration of 0.1mol/L, which contains 1wt% Tween-20, 10wt% trehalose and 0.5wt% casein.

2.2 Preparing a rheumatoid factor antigen-fluorescent microsphere conjugate solution and a DNP-BSA-fluorescent microsphere conjugate solution:

1) Activation and coupling of microspheres: adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and fluorescent microspheres into a coupling buffer solution, placing the coupling buffer solution in a shaking table, incubating at 25 ℃ for 15min at 250r/min, then carrying out 16000rpm at 10 ℃, centrifuging for 15min, removing supernatant, and re-dissolving by using MES buffer solution with the pH value of 6 and the concentration of 0.1 mol/L; then adding rheumatoid factor antigen, placing in a shaking table, incubating at 25 ℃ for 2h at 250 r/min.

In the coupling system, the dosage of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) is 12.5 mu g/mL, the volume of the fluorescent microsphere is 10 percent of the total coupling volume, and the final concentration of the rheumatoid factor antigen is 0.1mg/mL.

2) Sealing of the microspheres: after 2h of shake-bed incubation, blocking buffer solution with the volume of 10% of the total coupling volume is added into the coupling system, and the coupling system is placed in a shake bed and incubated for 1h at 25 ℃ and 250 r/min. The blocking buffer was 10% BSA buffer.

3) Cleaning the microspheres: and after the sealing is finished, centrifuging to remove the supernatant to obtain the rheumatoid factor antigen-fluorescent microsphere conjugate.

4) And mixing the rheumatoid factor antigen with a second buffer solution to obtain a rheumatoid factor antigen-fluorescent microsphere conjugate solution with the concentration of 0.1 wt%.

The preparation method of the DNP-BSA-fluorescent microsphere conjugate solution is basically the same as that of the rheumatoid factor antigen-fluorescent microsphere conjugate, and only the rheumatoid factor antigen with the final concentration of 0.1mg/mL is replaced by DNP-BSA protein with the final concentration of 0.1mg/mL.

2.3 And cutting the glass cellulose membrane to obtain the required specification. Mixing the rheumatoid factor antigen-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution according to the volume ratio of 16 to obtain a gold spraying solution, uniformly spraying the gold spraying solution onto a glass cellulose membrane by using a gold spraying instrument, placing the glass cellulose membrane in an environment with the humidity of less than 30% and the temperature of 18-28 ℃, and drying the glass cellulose membrane for 2 hours at 37 ℃.

3. Preparation of nitrocellulose membrane 4:

3.1 preparing a fourth buffer: PBS buffer with pH 7.4 and concentration of 10mmol/L, containing 5wt% trehalose and 5wt% methanol.

3.2 And taking a proper amount of fourth buffer solution, adding a mouse anti-human IgG antibody, and mixing to obtain a first coating solution, wherein the concentration of the mouse anti-human IgM antibody in the first coating solution is 2mg/mL.

Taking a proper amount of fourth buffer solution, adding a mouse anti-human IgM antibody, and mixing to obtain a second coating solution, wherein the concentration of the mouse anti-human IgG antibody in the second coating solution is 2mg/mL.

And taking a proper amount of fourth buffer solution, adding a mouse anti-human IgA antibody, and mixing to obtain a third coating solution, wherein the concentration of the mouse anti-human IgG antibody in the third coating solution is 2mg/mL.

And taking a proper amount of fourth buffer solution, adding the DNP monoclonal antibody, and mixing to obtain a fourth coating solution, wherein the concentration of the DNP monoclonal antibody in the fourth coating solution is 2mg/mL.

3.3 Coating the first coating solution on the position of an IgG detection line 8 at a uniform coating speed of 1 muL/cm by using a film scratching instrument, coating the second coating solution on the position of an IgM detection line 7 at a uniform coating speed of 1 muL/cm, coating the third coating solution on the position of an IgA detection line 6 at a uniform coating speed of 1 muL/cm, coating the fourth coating solution on the position of a quality control line 9 at a uniform coating speed of 1 muL/cm, placing the coated liquid in an environment with the humidity of less than 30% and the temperature of 18-28 ℃, and drying the coated liquid at 37 ℃ for 24 hours.

4. Assembling a fluorescence immunochromatographic test strip: and sequentially overlapping the sample pad 2, the combination pad 3, the nitrocellulose membrane 4 and the absorbent paper 5 at a specific position on the bottom plate 1, and cutting the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper at a fixed width of 3.9mm after assembly to obtain the fluorescence immunochromatographic test strip.

5. Preparing a sample diluent: PBS buffer at pH 7.4 and a concentration of 10mmol/L, containing 0.5wt% Tween-20, 0.5wt% sodium dodecyl sulfate and 300mmol/L NaCl.

The application method of the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA comprises the following steps:

uniformly mixing 5 mu L of sample to be detected with 95 mu L of sample diluent, and adding 80 mu L of mixed solution into a sample adding hole of a fluorescence immunochromatographic test strip;

after waiting for 15min, inserting a fluorescence immunoassay analyzer to test the fluorescence intensity of an IgG detection line 8 and record the fluorescence intensity of an IgM detection line 7 and the fluorescence intensity of an IgA detection line 6 as T1 values, and the fluorescence intensity of a quality control line 9 as C values, and calculating T1/C values, T2/C values and T3/C values, wherein the T1/C values correspond to RF-IgG, the T2/C values correspond to RF-IgM, and the T3/C values correspond to RF-IgA.

The embodiment also provides an RF-IgG/RF-IgM/RF-IgA joint quantitative detection system, which comprises the fluorescence immunochromatographic test strip prepared in the embodiment, a fluorescence immunoassay analyzer and an ID card, wherein the ID card stores a standard curve.

6. Preparing an ID card:

RF-IgG standard curve:

the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA in the embodiment is adopted to carry out quantitative detection on 6 concentrations of RF-IgG calibrator, wherein the concentration of IgG type rheumatoid factor in the calibrator is 0AU/mL, 12AU/mL, 30AU/mL, 60AU/mL, 120AU/mL and 240AU/mL, and each concentration calibrator is tested for 6 times to obtain a fitted standard curve of RF-IgG concentration and T/C: y = (A-D)/[ 1+ (x/C ^ B)]+ D, wherein a =6.12124, b = -2.10707, C = -107.21651, D = -0.33637, where y represents T/C value, x represents RF concentration value, R represents ² =0.99684。

RF-IgM standard curve:

the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA in the embodiment is adopted to carry out quantitative detection on 6 concentrations of RF-IgM calibrator, wherein the IgM type rheumatoid factor concentration of the calibrator is 0IU/mL, 12IU/mL, 30IU/mL, 60IU/mL, 120IU/mL and 240IU/mL, each concentration calibrator is tested repeatedly for 6 times to obtain a fitting standard curve of RF-IgM concentration and T/C: y = (A-D)/[ 1+ (x/C ^ B)]+ D, wherein A =7.30203, B = -1.41240, C = -110.53665, D = -0.04382, where y represents a T/C value, x represents an RF concentration value, R represents ² =0.99647。

RF-IgA standard curve:

the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA of the embodiment is adopted to carry out quantitative detection on 6 concentrations of RF-IgA calibrators, the IgA type rheumatoid factor concentrations of the calibrators are 0RU/mL, 12RU/mL, 30RU/mL, 60RU/mL, 120RU/mL and 240RU/mL, each concentration calibrator is tested repeatedly for 6 times, and a fitting standard curve of the RF IgA concentration and T/C is obtained: y = (A-D)/[ 1+ (x/C ^ B)]+ D, where A =8.05859, B = -1.55437, C =199.97765, D =0.03911, where y denotes T =C value, x represents RF concentration value, R ² =0.99975。

And writing the RF-IgG standard curve, the RF-IgM standard curve and the RF-IgA standard curve into an ID card, putting the ID card into a fluorescence immunochromatographic test strip, and enabling a processor of the fluorescence immunoassay analyzer to read the content of the ID card and process a sample detection result.

The RF-IgG/RF-IgM/RF-IgA combined quantitative detection system of the embodiment is used as follows:

taking 5 mu L of a sample to be detected, uniformly mixing with 95 mu L of sample diluent, and adding 80 mu L of mixed solution into a sample adding hole of a fluorescence immunochromatographic test strip;

after waiting for 15min, inserting a fluorescence immunoassay analyzer to test the fluorescence intensity of an IgG detection line 8 and record the fluorescence intensity as a T1 value, testing the fluorescence intensity of an IgM detection line 7 and record the fluorescence intensity as a T2 value, testing the fluorescence intensity of an IgA detection line 6 and record the fluorescence intensity as a T3 value, testing a quality control line 9 and record the fluorescence intensity as a C value, calculating the T1/C value, the T2/C value and the T3/C value, and automatically converting the T1/C value into the RF-IgG content, converting the T2/C value into the RF-IgM content and converting the T3/C value into the RF-IgA content through calibration curve information in an ID card.

Comparative example 1

The comparative example provides a fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA and a RF-IgG/RF-IgM/RF-IgA combined quantitative detection system, the structure of the kit is the same as that of example 1, the preparation method of the kit is basically the same as that of example 1, and the difference is that sample diluent does not contain sodium dodecyl sulfate.

Comparative example 2

The comparative example provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which has the same structure as that of example 1, and the preparation method is basically the same as that of example 1, except that NaCl is not contained in the sample diluent, and the amount of sodium dodecyl sulfate used is 1%.

Comparative example 3

This comparative example provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which has the same structure as example 1 and the preparation method basically as example 1, except that the amount of sodium dodecyl sulfate in the sample diluent is 0.25wt% and the amount of Tween-20 is 2wt%.

Comparative example 4

The comparative example provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, the structure of which is the same as that in example 1, and the preparation method of which is basically the same as that in example 1, except that the amount of Tween-20 in the sample diluent is 1wt%, and the amount of NaCl is 500mmol/L.

Comparative example 5

This comparative example provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which has the same structure as example 1 and is prepared substantially in the same manner as example 1 except that the second buffer is PBS buffer with pH of 7.4 and concentration of 10 mmol/L.

Comparative example 6

The comparative example provides a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA, which has the same structure as that of example 1, and the preparation method is basically the same as that of example 1, except that the casein in the first buffer solution and the second buffer solution is replaced by bovine serum albumin.

Performance testing

1. Sensitivity testing

Taking 9 concentrations of reference substances of RF-IgG, RF-gM and RF-IgA, respectively using the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA of example 1 and comparative examples 1 to 6 to verify the signal intensity, wherein the concentrations of the reference substances of RF-IgG are 0AU/mL, 2.5AU/mL, 5AU/mL, 10AU/mL, 25AU/mL, 50AU/mL, 100AU/mL, 150AU/mL, 200AU/mL, the concentrations of the reference substances of RF-IgM are 0IU/mL, 2.5IU/mL, 5IU/mL, 10IU/mL, 25IU/mL, 50IU/mL, 100IU/mL, 150IU/mL, 200IU/mL, the RF-IgA reference substance concentration is 0RU/mL, 2.5RU/mL, 5RU/mL, 10RU/mL, 25RU/mL, 50RU/mL, 100RU/mL, 150RU/mL, 200RU/mL, each concentration reference substance is repeatedly tested for 2 times, and the corresponding mean values of T1 value, T2 value, T3 value and C value, and the corresponding mean values of T1/C value, T2/C value and T3/C value are recorded, the RF-IgG test result is shown in Table 1, the RF-gM test result is shown in Table 2, and the RF-IgA test result is shown in Table 3.

And (4) conclusion: the sensitivity test result shows that when the reference substance with the same concentration is detected, the three typing signals of the fluorescence immunochromatographic kit for combined detection of the rheumatoid factors IgG/IgM/IgA, which is prepared according to the example 1, are the highest, which indicates that the sensitivity of the example 1 is the highest.

2. Accuracy test

RF-IgG reference: the concentration is 12AU/mL and 80AU/mL; RF-IgM reference: the concentration is 12IU/mL and 80IU/mL; RF-IgA reference: the concentrations were 12RU/mL and 80RU/mL.

The fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA in example 1 was used for accuracy testing, RF enterprise reference substances at the above concentrations were taken as samples for testing, the testing was repeated 3 times, the test results were recorded, and the relative deviations were respectively calculated, and the results are shown in Table 4.

The fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA in example 1 has the accuracy performance of three types, and the relative deviation of the accuracy performance of the three types and the corresponding enterprise reference products is within the range of +/-15%.

3. Repeatability test

The fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA in example 1 was used for repetitive tests, RF enterprise reference substances at the above concentrations were taken as samples for testing, the measurements were repeated 10 times, the test results were recorded, and the average (M), standard Deviation (SD) and coefficient of variation CV (%) of the test results were calculated, and the results are shown in Table 5.

And (4) conclusion: the fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA in example 1 has the repeatability performance and the Coefficient of Variation (CV) of all three types within the range of +/-15%.

4. Stability test

The test strips of example 1 were placed in a 37 ℃ oven for accelerated thermal stability validation. The test strips for 4 days, 7 days, 10 days and 14 days were taken out in sequence according to the baking time, and after preparing all test strips required for detection, the test strips were used to detect the concentrations of the reference substances, 10 replicates were set for each treatment, the standard curve of the unbaked test strips was used for concentration calculation, and the deviation of the average values of the test strips baked for 4 days, 7 days, 10 days and 14 days from the average values of the unbaked test strips was counted to determine the stability of the test strips of example 1. Wherein the RF-IgG reference: the concentration is 12AU/mL and 80AU/mL; RF-IgM reference: the concentration is 12IU/mL and 80IU/mL; RF-IgA reference: the concentrations were 12RU/mL and 80RU/mL. The stability test results are shown in table 6.

The fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA in example 1 has three types of stability performance, and the deviation change of two reference concentrations is tested to be within the range of +/-15% after being accelerated at 37 ℃.

5. Testing of yin-yang coincidence rate of serum sample

Clinical serum samples were detected using the combined RF-IgG/RF-IgM/RF-IgA detection kit of example 1 and the industry-approved rheumatoid factor (IgG class) detection kit of osmund medical diagnosis (china) ltd (enzyme-linked immunosorbent assay), rheumatoid factor (IgM class) detection kit (enzyme-linked immunosorbent assay), and rheumatoid factor (IgA class) detection kit (enzyme-linked immunosorbent assay), respectively. The detection method of the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA is as described above, and the description of the detection method of the rheumatoid factor (IgG) detection kit (enzyme-linked immunosorbent assay), the rheumatoid factor (IgM) detection kit (enzyme-linked immunosorbent assay), and the rheumatoid factor (IgA) detection kit (enzyme-linked immunosorbent assay) of the European Mongolian medical diagnosis (China) Co. The test results of the fluorescence immunochromatographic test strip and the indirect immunofluorescence kit of example 1 were analyzed for the yin-yang coincidence rate, the test results are shown in table 7, and the yin-yang coincidence rate is shown in table 8.

The results in tables 7 and 8 show that the three typing negative and positive results obtained by the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA in example 1 are very high in compliance rate with the negative and positive results measured by the European Mongolian ELISA kit generally accepted in the market, and meet the acceptance standard.

The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.

Claims

1. The preparation method of the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA is characterized in that the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA comprises an immunochromatographic test strip and a sample diluent,

the preparation method of the immunochromatographic test strip comprises the following steps:

b) Treating the rheumatoid factor antigen-fluorescent microsphere conjugate and the DNP-BSA-fluorescent microsphere conjugate by adopting a second buffer solution, then respectively combining the treated rheumatoid factor antigen-fluorescent microsphere conjugate and the DNP-BSA-fluorescent microsphere conjugate to a second glass fiber membrane to prepare a combined pad,

c) Assembling the sample pad, the combination pad, the nitrocellulose membrane, the absorbent paper and the bottom plate to obtain the fluorescence immunochromatographic test strip,

wherein the pH value of the first buffer solution is 8.0-9.0, and the first buffer solution comprises 0.5-1.5 wt% of Tween-20, 3-8 wt% of trehalose and 0.1-0.6 wt% of casein,

the pH value of the second buffer solution is 5.5 to 6.5, and the second buffer solution comprises 0.5wt% to 1.5wt% of Tween-20, 5wt% to 15wt% of trehalose and 0.1wt% to 0.6wt% of casein,

the nitrocellulose membrane is provided with an IgG detection line, an IgM detection line, an IgA detection line and a quality control line,

2. The method for preparing a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA according to claim 1, wherein the first buffer, the second buffer and the third buffer are each independently selected from PBS buffer, BBS buffer, MES buffer, tris buffer or citric acid buffer.

3. The preparation method of the fluorescence immunochromatography kit for joint detection of RF-IgG/RF-IgM/RF-IgA according to claim 2, wherein the first buffer solution is a Tris buffer solution with a concentration of 0.05mmol/L to 0.1 mmol/L; and/or the second buffer solution is MES buffer solution with the concentration of 0.05-0.2 mol/L; and/or the third buffer solution is PBS buffer solution with the concentration of 5 mmol/L-20 mmol/L.

4. The preparation method of the fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA according to claim 1, wherein the pH value of the first buffer is 8.5 to 9.0; and/or the pH value of the second buffer solution is 5.5 to 6.0; and/or the pH value of the third buffer solution is 7.0-7.5.

5. The preparation method of the fluorescence immunochromatographic kit for joint detection of RF-IgG/RF-IgM/RF-IgA according to claim 1, wherein the sample pad is obtained by infiltrating the sample pad with a first buffer solution for 1min to 3min and drying at 30 ℃ to 60 ℃.

6. The preparation method of the fluorescence immunochromatographic kit for joint detection of RF-IgG/RF-IgM/RF-IgA according to claim 1, wherein the rheumatoid factor antigen-fluorescent microsphere conjugate and the second buffer solution are mixed to obtain a rheumatoid factor antigen-fluorescent microsphere conjugate solution with a microsphere content of 0.05wt% to 0.5wt%, the DNP-BSA-fluorescent microsphere conjugate and the second buffer solution are mixed to obtain a DNP-BSA-fluorescent microsphere conjugate solution with a microsphere content of 0.05wt% to 0.5wt%, and then the DNP-BSA-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution are mixed according to a volume ratio of (15 to 18): 1, sprayed onto the binding pad, and dried at 30 ℃ to 60 ℃ to obtain the binding pad.

7. The preparation method of the fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA according to claim 1, wherein a first coating solution is obtained by mixing a mouse anti-human IgG antibody with a fourth buffer solution, the first coating solution is used for scribing on the nitrocellulose membrane, and drying is performed at 30-60 ℃ to form the IgG detection line;

mixing a mouse anti-human IgM antibody with a fourth buffer solution to obtain a second coating solution, scribing on the nitrocellulose membrane by using the second coating solution, and drying at 30-60 ℃ to form the IgM detection line;

mixing a mouse anti-human IgA antibody with a fourth buffer solution to obtain a third coating solution, scribing on the nitrocellulose membrane by using the third coating solution, and drying at 30-60 ℃ to form the IgA detection line;

mixing the DNP monoclonal antibody with a fourth buffer solution to obtain a fourth coating solution, scribing on the nitrocellulose membrane by using the fourth coating solution, drying at 30-60 ℃ to form the quality control line,

the concentrations of antibodies in the first coating solution, the second coating solution, the third coating solution and the fourth coating solution are respectively and independently 1 mg/mL-5 mg/mL, the pH value of the fourth buffer solution is 7-7.5, the fourth buffer solution is a PBS buffer solution with the concentration of 5 mmol/L-20 mmol/L, and the PBS buffer solution comprises 2wt% -8 wt% of trehalose and 2wt% -8 wt% of methanol.

8. The method for preparing a fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA according to claim 1, wherein the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent paper are carried on the base plate in this order;

and/or the fluorescent microspheres are europium-chelated fluorescent microspheres;

and/or the surface of the fluorescent microsphere is provided with a modifying group, the modifying group is amino and/or carboxyl, and the fluorescent microsphere is activated by N-hydroxysuccinimide ester and/or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;

and/or the diameter of the fluorescent microsphere is 0.1-0.3 μm, the excitation wavelength of the fluorescent microsphere is 320nm-350nm, and the emission wavelength of the fluorescent microsphere is 600nm-620nm;

and/or the rheumatoid factor antigen of the rheumatoid factor antigen-fluorescent microsphere conjugate is an artificially synthesized antigen;

and/or a mouse anti-human IgG antibody is coated on the IgG detection line, a mouse anti-human IgM antibody is coated on the IgM detection line, a mouse anti-human IgA antibody is coated on the IgA detection line, and a DNP monoclonal antibody is coated on the quality control line.

9. A fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA is characterized in that: the fluorescence immunochromatographic kit prepared by the preparation method of any one of claims 1 to 8.

10. An RF-IgG/RF-IgM/RF-IgA combined quantitative detection system is characterized in that: comprising the fluorescence immunochromatography kit for combined detection of RF-IgG/RF-IgM/RF-IgA according to claim 9, a fluorescence immunoassay analyzer, and an ID card provided on the fluorescence immunochromatography test strip, wherein calibration data and/or a standard curve are stored in the ID card,

(2) Diluting a sample to be detected by using the sample diluent, dripping the diluted sample to be detected onto the fluorescence immunochromatographic test strip, and sequentially passing the diluted sample to be detected through the processed sample pad, the processed combination pad, the nitrocellulose membrane and the absorbent paper;