Fluorescent immunochromatographic test strip for combined detection of Anti-mcv antibody, Anti-ccp antibody and RF

Abstract

The present application relates to a fluorescent immunochromatographic test strip for detecting rheumatoid arthritis. The test strip comprises a sample loading area, a detection area and a water absorption area, the detection area comprising a first sub-detection area having affixed a mutant citrullinated vimentin, a second sub-detection area having affixed a cyclic citrullinated peptide, and a third sub-detection having affixed a denatured human IgG.

Description

Fluorescent immunochromatographic test strip for combined detection of anti-MCV antibodies, anti-CCP antibodies, and RF

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of Chinese Application No. 202310870599.3, filed on July 14, 2023, the entire contents of which are incorporated herein for all purposes. Technical Field

[0003] The present application relates to the field of medical testing technology, specifically to a fluorescent immunochromatographic test strip for simultaneously detecting anti-mutant citrullinated vimentin antibodies (Anti-MCV), anti-cyclic citrullinated peptide antibodies (Anti-CCP) and rheumatoid factor (RF) and its application. Background Art

[0004] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. In addition to arthritis and surrounding tissue involvement, extra-articular manifestations such as subcutaneous nodules, anemia, interstitial lung disease, and vasculitis may also occur. Untreated RA can lead to significant joint loss and progress to limb disability and functional loss, imposing a significant economic burden on patients, their families, and society. With the deepening understanding of RA pathogenesis and the development of numerous high-quality clinical trials, RA treatment strategies have been continuously updated, and diagnostic and therapeutic levels have rapidly improved. Autoantibodies can be produced before RA patients exhibit typical clinical symptoms and have a certain predictive value, thus playing an important role in the early diagnosis, treatment monitoring, and prognostic assessment of RA. Among them, rheumatoid factor (RF) and anti-citrullinated peptide / protein antibodies (ACPA) were included in the 2010 American College of Rheumatology (ACR), European League Against Rheumatism (EULAR), and Chinese RA classification criteria due to their excellent diagnostic performance. They are now widely used in clinical practice and have become one of the most common autoantibody tests in clinical laboratories. Other antibodies have certain supplementary diagnostic value for RA and can be used as combined diagnostic indicators in clinical practice.

[0005] RF was the first serological marker used to diagnose RA and is currently the most commonly used clinical marker. It has high sensitivity but poor specificity, and a certain positive rate is also observed in patients with other autoimmune diseases, tumors, infections, and even in healthy individuals. ACPA is a class of autoantibodies targeting citrullinated proteins, including anti-perinuclear factor (APF), anti-keratin antibodies (AKA), anti-cyclic citrullinated peptide antibodies (Anti-CCP), and anti-mutant citrullinated vimentin antibodies (Anti-MCV). Although APF and AKA have high specificity, they require immunofluorescence detection, which is susceptible to subjective interpretation and has low sensitivity. Anti-CCP and Anti-MCV have high specificity and sensitivity. There are reports of Anti-MCV being detected in the serum of Anti-CCP-negative RA patients, suggesting that Anti-MCV has slightly higher specificity than Anti-CCP, but lower sensitivity. The sensitivity of RF, Anti-CCP and Anti-MCV in diagnosing RA is Anti-CCP>RF>Anti-MCV; the specificity is Anti-MCV>Anti-CCP>RF.

[0006] Commonly used detection methods for RF include chemiluminescent immunoassay, enzyme-linked immunosorbent assay, or immunoturbidimetry. Anti-CCP is currently primarily detected using chemiluminescent immunoassay and enzyme-linked immunosorbent assay, while anti-MCV is primarily detected using enzyme-linked immunosorbent assay. These methodologies offer advantages such as rapidity, automation, quantitative detection, and low coefficients of variation. However, they are complex and time-consuming to perform, making them less suitable for rapid and convenient detection.

[0007] Immunochromatography is a rapid diagnostic technique with advantages such as accuracy, speed, and ease of use. Commonly used labeling materials include colloidal gold and fluorescent materials. Colloidal gold primarily uses its color to visually detect test results. However, for samples with extremely low antigen or antibody content, the color of colloidal gold is very light and difficult to visually interpret, resulting in low sensitivity and difficulty in achieving highly sensitive quantitative detection. Fluorescent materials, on the other hand, offer high fluorescence intensity, enabling highly sensitive quantitative detection.

[0008] Summary of the Invention

[0009] One of the purposes of the present application is to provide a fluorescent immunochromatographic test strip for the simultaneous detection of anti-mutant citrullinated vimentin antibodies (Anti-MCV), anti-cyclic citrullinated peptide antibodies (Anti-CCP) and rheumatoid factor (RF), providing a kit with high sensitivity, good specificity, strong stability and convenience for clinical diagnostic analysis, which can greatly improve the detection and diagnosis efficiency and solve the problems in the existing technology.

[0010] In the present application, the selection of the citrulline mutation site of MCV is different from that of CCP, so anti-CCP antibodies and anti-MCV antibodies can be well distinguished without cross-reaction, thus solving the interference problem between CCP and MCV.

[0011] The first aspect of the present application provides a fluorescent immunochromatographic test strip for detecting rheumatoid arthritis, the test strip comprising a sample loading area, a detection area, and a water absorption area; the detection area comprises a first sub-detection area immobilized with a mutant citrullinated vimentin, a second sub-detection area immobilized with a cyclic citrullinated peptide, and a third sub-detection area immobilized with a denatured human IgG; wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG are independently 0.15-0.75 μg; the amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1; the amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2; the sample loading area contains fluorescent material-labeled goat anti-human IgG, fluorescent material-labeled denatured human IgG, and fluorescent material-labeled goat anti-rabbit IgG in a mass ratio of 20:10:1.

[0012] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0013] In some embodiments, the loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

[0014] In some embodiments, the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0015] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first detection area, the second detection area, or the third detection area according to the flow direction of the sample on the test strip.

[0016] In some embodiments, the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

[0017] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0018] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL.

[0019] The second aspect of the present application provides a kit, which includes the fluorescent immunochromatographic test strip and instructions described in the first aspect.

[0020] The third aspect of the present application provides use of the fluorescent immunochromatographic test strip described in the first aspect in preparing a kit for detecting rheumatoid arthritis.

[0021] The fourth aspect of the present application provides a method for assessing whether a subject suffers from rheumatoid arthritis or the extent of the disease, comprising the following steps:

[0022] a. Adding a blood sample from the subject to the sample area of ​​the fluorescent immunochromatographic test strip;

[0023] The fluorescent immunochromatographic test strip comprises a sample loading area, a detection area and a water absorption area;

[0024] The detection area includes a first sub-detection area fixed with mutant citrullinated vimentin, a second sub-detection area fixed with cyclic citrullinated peptide, and a third sub-detection area fixed with denatured human IgG;

[0025] wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG are independently 0.15-0.75 μg;

[0026] The amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1;

[0027] The amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2;

[0028] The sample loading area contains fluorescent material-labeled goat anti-human IgG, fluorescent material-labeled denatured human IgG, and fluorescent material-labeled goat anti-rabbit IgG in a mass ratio of 20:10:1; and

[0029] b. Determine the qualitative or quantitative results of the fluorescence produced by the first sub-detection area, the second sub-detection area, and the third sub-detection area, and assess whether the subject has rheumatoid arthritis or the extent of the disease based on the qualitative or quantitative results.

[0030] In some embodiments, in step (b), when the first sub-detection zone, the second sub-detection zone, and the third sub-detection zone show positive fluorescence results, the subject is diagnosed with rheumatoid arthritis.

[0031] In some embodiments, the blood sample is whole blood, plasma, or serum.

[0032] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0033] In some embodiments, the loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

[0034] In some embodiments, the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0035] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first sub-detection area, the second sub-detection area, and the third sub-detection area according to the flow direction of the sample on the test strip.

[0036] In some embodiments, the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

[0037] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0038] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows a schematic diagram of a fluorescent immunochromatographic test strip, wherein 1 is the sample loading area, 2 is the detection area, 3 to 5 are sub-detection areas, 6 is the quality control area, 7 is the water absorption area, and 8 is the bottom plate.

[0040] Figure 2 shows a schematic diagram of another fluorescent immunochromatographic test strip, wherein 1 is the base plate, 2 is the sample area, 3 is the labeling area, 4 is the detection area, 5 to 7 are sub-detection areas, 8 is the quality control area, and 9 is the water absorption area.

[0041] FIG3 shows a comparison of the sensitivity of the fluorescent immunochromatographic method and the colloidal gold method under the condition of a coating concentration of 2 mg / mL.

[0042] Sequence Description

[0043] SEQ ID NO: 1 is the amino acid sequence of mutant citrullinated vimentin, wherein X is citrulline (Citrulline, Cit).

[0044] SEQ ID NO: 2 is the amino acid sequence of a cyclic citrullinated peptide, wherein a disulfide bond exists between C3 and C16, and X is citrulline (Citrulline). DETAILED DESCRIPTION

[0045] One of the purposes of the present application is to simultaneously detect anti-mutant citrullinated vimentin antibodies (Anti-MCV), anti-cyclic citrullinated peptide antibodies (Anti-CCP) and rheumatoid factor (RF) by the principle of fluorescent immunochromatography.

[0046] Anti-MCV uses an indirect detection principle. The Anti-MCV contained in the sample to be tested first binds to the fluorescent material coupled with goat anti-human IgG on the sample loading area and is captured by the recombinant MCV (T line, which can be T1 line, T2 line or T3 line) fixed on the detection area (for example, nitrocellulose membrane) through chromatography. The T line fluorescence signal can be quantitatively detected by the supporting instrument. The fluorescence intensity is positively correlated with the Anti-MCV concentration in the sample. The concentration of Anti-MCV in the sample can be quantitatively displayed by instrument calculation.

[0047] Anti-CCP uses an indirect detection principle. The Anti-CCP contained in the sample to be tested first binds to the fluorescent material conjugated with goat anti-human IgG on the sample loading area. It is then captured by chromatography by the recombinant CCP (T line, which can be T1 line, T2 line or T3 line) fixed on the detection area (e.g., nitrocellulose membrane). The T line fluorescence signal can be quantitatively detected by the supporting instrument. The fluorescence intensity is positively correlated with the Anti-CCP concentration in the sample. The instrument can quantitatively display the concentration of Anti-CCP in the sample.

[0048] RF adopts the double antigen sandwich detection principle. The RF contained in the sample to be tested first combines with the fluorescent material coupled with denatured human IgG on the loading area and is captured by the denatured human IgG (T line, which can be T1 line, T2 line or T3 line) fixed on the detection area (for example, nitrocellulose membrane) through chromatography. The T line fluorescence signal can be quantitatively detected by the supporting instrument. The fluorescence intensity is positively correlated with the RF concentration in the sample. The concentration of RF in the sample can be quantitatively displayed by instrument calculation.

[0049] The first aspect of the present application provides a fluorescent immunochromatographic test strip for detecting rheumatoid arthritis, the test strip comprising a sample loading area, a detection area, and a water absorption area; the detection area comprises a first sub-detection area immobilized with a mutant citrullinated vimentin, a second sub-detection area immobilized with a cyclic citrullinated peptide, and a third sub-detection area immobilized with a denatured human IgG; wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG are independently 0.15-0.75 μg; the amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1; the amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2; the sample loading area contains fluorescent material-labeled goat anti-human IgG, fluorescent material-labeled denatured human IgG, and fluorescent material-labeled goat anti-rabbit IgG in a mass ratio of 20:10:1.

[0050] In some embodiments, goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material are mixed and sprayed onto the pad at 4 μL / cm.

[0051] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0052] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL, and the coating concentration is 1.0 μL / cm.

[0053] In some embodiments, according to the flow direction of the sample on the test strip, the first sub-detection area, the second sub-detection area, and the third sub-detection area are arranged in sequence from upstream to downstream.

[0054] In some embodiments, according to the flow direction of the sample on the test strip, the first sub-detection area, the third sub-detection area, and the second sub-detection area are arranged in sequence from upstream to downstream.

[0055] In some embodiments, according to the flow direction of the sample on the test strip, the second sub-detection area, the first sub-detection area, and the third sub-detection area are arranged in sequence from upstream to downstream.

[0056] In some embodiments, according to the flow direction of the sample on the test strip, the second sub-detection area, the third sub-detection area, and the first sub-detection area are arranged in sequence from upstream to downstream.

[0057] In some embodiments, according to the flow direction of the sample on the test strip, the third sub-detection area, the first sub-detection area, and the second sub-detection area are arranged in sequence from upstream to downstream.

[0058] In some embodiments, according to the flow direction of the sample on the test strip, the third sub-detection area, the second sub-detection area, and the first sub-detection area are arranged in sequence from upstream to downstream.

[0059] In some embodiments, the coating amount of the mutant citrullinated vimentin is 0.15 μg, 0.155 μg, 0.16 μg, 0.165 μg, 0.17 μg, 0.175 μg, 0.18 μg, 0.185 μg, 0.19 μg, 0.195 μg, 0.2 μg, 0.205 μg, 0.21 μg, 0.215 μg, 0.22 μg, 0.225 μg, 0.23 μg, 0.235 μg, 0.24 μg, 0.245 μg, 0.25 μg, 0.255 μg, 0.26 μg, 0.265 μg, 0.27 μg, 0.275 μg, 0.285 μg, 0.295 μg, 0.305 μg, 0.315 μg, 0.325 μg, 0.335 μg, 0.345 μg, 0.35 .28μg, 0.285μg, 0.29μg, 0.295μg, 0.3μg, 0.305μg, 0.31μg, 0.315μg, 0.32μg, 0.325μg, 0.33μg, 0.335μg, 0.34μg, 0.345μg, 0.35μg, 0 .355μg, 0.36μg, 0.365μg, 0.37μg, 0.375μg, 0.38μg, 0.385μg, 0.39μg, 0.395μg, 0.4μg, 0.405μg, 0.41μg, 0.415μg, 0.42μg, 0.425μg, 0.43μg, 0.435μg, 0.44μg, 0.445μg, 0.45μg, 0.455μg, 0.46μg, 0.465μg, 0. 47μg, 0.475μg, 0.48μg, 0.485μg, 0.49μg, 0.495μg, 0.5μg, 0.505μg, 0.51μ g, 0.515μg, 0.52μg, 0.525μg, 0.53μg, 0.535μg, 0.54μg, 0.545μg, 0.55μg, 0.555μg, 0.56μg, 0.565μg, 0.57μg, 0.575μg, 0.58μg, 0.585μg, 0.59μg, 0. .g, 0.71 μg, 0.715 μg, 0.72 μg, 0.725 μg, 0.73 μg, 0.735 μg, 0.74 μg, 0.745 μg, 0.75 μg or a range between any of the above values.

[0060] In some embodiments, the coating concentration of the mutant citrullinated vimentin is 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or a range therebetween.

[0061] In some embodiments, the coating amount of the cyclic citrullinated peptide is 0.15 μg, 0.155 μg, 0.16 μg, 0.165 μg, 0.17 μg, 0.175 μg, 0.18 μg, 0.185 μg, 0.19 μg, 0.195 μg, 0.2 μg, 0.205 μg, 0.21 μg, 0.215 μg, 0.22 μg, 0.225 μg, 0.23 μg, 0.235 μg, 0.24 μg, 0.245 μg, 0.25 μg, 0.255 μg, 0.26 μg, 0.265 μg, 0.27 μg, 0.275 μg, 0.28 μg, 0.295 μg, 0.2 9μg, 0.295μg, 0.3μg, 0.305μg, 0.31μg, 0.315μg, 0.32μg, 0.325μg, 0. 33μg, 0.335μg, 0.34μg, 0.345μg, 0.35μg, 0.355μg, 0.36μg, 0.365μg, 0 .37μg, 0.375μg, 0.38μg, 0.385μg, 0.39μg, 0.395μg, 0.4μg, 0.405μg, 0.41μg, 0.415μg, 0.42μg, 0.425μg, 0.43μg, 0.435μg, 0.44μg, 0.445μg ,0.45μg, 0.455μg, 0.46μg, 0.465μg, 0.47μg, 0.475μg, 0.48μg, 0.485 μg, 0.49μg, 0.495μg, 0.5μg, 0.505μg, 0.51μg, 0.515μg, 0.52μg, 0.52 5μg, 0.53μg, 0.535μg, 0.54μg, 0.545μg, 0.55μg, 0.555μg, 0.56μg, 0. 565μg, 0.57μg, 0.575μg, 0.58μg, 0.585μg, 0.59μg, 0.595μg, 0.6μg, 0. 605μg, 0.61μg, 0.615μg, 0.62μg, 0.625μg, 0.63μg, 0.635μg, 0.64μg, 0.645μg, 0.65μg, 0.655μg, 0.66μg, 0.665μg, 0.67μg, 0.675μg, 0.68μg, 0.685μg, 0.69μg, 0.695μg, 0.7μg, 0.705μg, 0.71μg, 0.715μg, 0.72μg, 0.725μg, 0.73μg, 0.735μg, 0.74μg, 0.745μg, 0.75μg or a range between any of the above values.In some embodiments, the coating concentration of the cyclic citrullinated peptide is 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or a range therebetween.

[0062] In some embodiments, the modified human IgG package is 0.15μg、0.155μg、0.16μg、0.165μg、0.17μg、0.175μg、0.18μg、0.185μg 、0.19μg、0.195μg、0.2μg、0.205μg、0.21μg、0.215μg、0.22μg、0.225μg、0.23μg、0.235μg、0.24μg ,0.245μg,0.25μg,0.255μg,0.26μg,0.265μg,0.27μg,0.275μg,0.28μg,0.285μg,0.29μg,0.295μg,0.3μg,0.305μg,0.31μg,0.315μg,0.32μg,0.325μg,0.33μg,0.335μg,0.34μg,0.345μg,0.35 μg、0.355μg、0.36μg、0.365μg、0.37μg、0.375μg、0.38μg、0.385μg、0.39μg、0.395μg、0.4μg、0.40 5μg、0.41μg、0.415μg、0.42μg、0.425μg、0.43μg、0.435μg、0.44μg、0.445μg、0.45μg、0.455μg、0. 46μg, 0.465μg, 0.47μg, 0.475μg, 0.48μg, 0.485μg, 0.49μg, 0.495μg, 0.5μg, 0.505μg, 0.51μg, 0.515μg, 0.52μg, 0.525μg, 0.53μg, 0.535μg, 0.54μg, 0.545μg, 0.55μg, 0.555μg, 0.56μg, 0.565μg, 0.57μg, 0.575μg, 0.58μg, 0.585μg, 0.59μg, 0.595μg, 0.6μg, 0.605μg, 0.61μg, 0.615μg, 0.62μg, 0.625μg, 0.63μg, 0.635μg, 0.64μg, 0.645μg, 0.65μg, 0.655μg, 0.66μg, 0. 665μg、0.67μg、0.675μg、0.68μg、0.685μg、0.69μg、0.695μg、0.7μg、0.705μg、0.71μg、 0.715μg、0.72μg、0.725μg、0.73μg、0.735μg、0.74μg、0.745μg、0.75μg or the range between the above arbitrary values。

[0063] In some embodiments, the coating concentration of the denatured human IgG is 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or a range between any of the above values.

[0064] In some embodiments, the denaturation condition of the denatured human IgG is 60° C. water bath for 30 minutes.

[0065] In some embodiments, fluorescent materials include, but are not limited to, fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0066] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0067] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first sub-detection area, the second sub-detection area, or the third sub-detection area according to the flow direction of the sample on the test strip.

[0068] In some embodiments, the quality control area is coated with rabbit IgG.

[0069] In some embodiments, the coating concentration of the rabbit IgG is 0.5-1.5 mg / mL.

[0070] In some embodiments, the coating amount of the rabbit IgG is 0.15 μg, 0.155 μg, 0.16 μg, 0.165 μg, 0.17 μg, 0.175 μg, 0.18 μg, 0.185 μg, 0.19 μg, 0.195 μg, 0.2 μg, 0.205 μg, 0.21 μg, 0.215 μg, 0.22 μg, 0.225 μg, 0.23 μg, 0.235 μg, 0.24 μg, 0.245 μg, 0.25 μg, 0.255 μg, 0.26 μg, 0.265 μg, 0.27 μg, 0.275 μg, 0.28 μg, 0.285 μg, 0.29 μg, 0.295 μg, 0.3 μg, 0.305 μg, 0.31 μg, 0.315 μg, 0.32 μg, 0.325 μg, 0.33 μg, 0.335 μg, 0.34 μg, 0.345 μg, 0.35 μg, 0.355 μg, 0.36 μg, 0.365 μg, 0.37 μg, 0.375 μg, 0.38 μg, 0.385 μg, 0.39 μg, 0.395 μg, 0.4 μg, 0.405 μg, 0.41 μg, 0.415 μg, 0.42 μg, 0.425 μg, 0.43 μg, 0.435 μg, 0.44 μg, 0.445 μg, 0.45 μg, 0.455 μg, 0.46 μg, 0.465 μg, 0.47 μg, 0.475 μg, 0.48 μg, 0.485 μg, 0.49 μg, 0.495 μg, 0.5 μg, 0.505 μg, 0.51 μg, 0.515 μg, 0.52 μg, 0.525 μg, 0.53 μg, 0.535 μg, 0.54 μg, 0.545 μg, 0.55 μg, 0.555 μg, 0.56 μg, 0.565 μg, 0.57 μg, 0.575 μg, 0.58 μg, 0.585 μg, 0.59 μg, 0.595 μg, 0.6 μg, 0.605 μg, 0.61 μg, 0.615 μg, 0.62 μg, 0.625 μg, 0.63 μg, 0.635 μg, 0.64 μg, 0.645 μg, 0.65 μg, 0.655 μg, 0.66 μg, 0.665 μg, 0.67 μg, 0.675 μg, 0.68 μg, 0.685 μg, 0.69 μg, 0.695 μg, 0.7 μg, 0.705 μg, 0.71 μg, 0.715 μg, 0.72 μg, 0.725 μg, 0.73 μg, 0.735 μg, 0.74 μg, 0.745 μg, 0.75 μg or a range between any of the above values.

[0071] In some embodiments, the coating concentration of the rabbit IgG is 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or a range between any of the above values.

[0072] The fluorescent material of goat anti-rabbit IgG coated on the sample loading area can be captured by the rabbit IgG (C line, quality control line) fixed on the detection area (e.g., nitrocellulose membrane) through chromatography. The C line fluorescence signal can be quantitatively detected by the supporting instrument, thus achieving quality control.

[0073] The second aspect of the present application provides a kit, which includes the fluorescent immunochromatographic test strip and instructions described in the first aspect.

[0074] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0075] In some embodiments, the loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

[0076] In some embodiments, the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0077] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first detection area, the second detection area, or the third detection area according to the flow direction of the sample on the test strip.

[0078] In some embodiments, the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

[0079] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0080] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL.

[0081] The third aspect of the present application provides use of the fluorescent immunochromatographic test strip described in the first aspect in preparing a kit for detecting rheumatoid arthritis.

[0082] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0083] In some embodiments, the loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

[0084] In some embodiments, the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0085] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first detection area, the second detection area, or the third detection area according to the flow direction of the sample on the test strip.

[0086] In some embodiments, the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

[0087] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0088] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL.

[0089] The fourth aspect of the present application provides a method for diagnosing, assisting in diagnosis, detecting or evaluating whether a subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis or the risk of developing rheumatoid arthritis, comprising the following steps:

[0090] a. Adding a blood sample from the subject to the sample area of ​​the fluorescent immunochromatographic test strip;

[0091] The fluorescent immunochromatographic test strip comprises a sample loading area, a detection area and a water absorption area;

[0092] The detection area includes a first sub-detection area fixed with mutant citrullinated vimentin, a second sub-detection area fixed with cyclic citrullinated peptide, and a third sub-detection area fixed with denatured human IgG;

[0093] wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG are independently 0.15-0.75 μg;

[0094] The amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1;

[0095] The amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2;

[0096] The sample loading area contains fluorescent material-labeled goat anti-human IgG, fluorescent material-labeled denatured human IgG, and fluorescent material-labeled goat anti-rabbit IgG in a mass ratio of 20:10:1; and

[0097] b. Determine the qualitative or quantitative results produced by the fluorescence of the first sub-detection area, the second sub-detection area, and the third sub-detection area, and evaluate whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis (degree of disease), the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis based on the qualitative or quantitative results.

[0098] In some embodiments, in step (b), when the first sub-detection zone, the second sub-detection zone, and the third sub-detection zone show positive fluorescence results, the subject is diagnosed with rheumatoid arthritis.

[0099] In some embodiments, the qualitative or quantitative results generated by the fluorescence of the first sub-detection zone and the second sub-detection zone are used to assess whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0100] In some embodiments, the qualitative or quantitative results generated by the fluorescence of the first sub-detection zone and the third sub-detection zone are used to assess whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0101] In some embodiments, the qualitative or quantitative results generated by the fluorescence of the second sub-detection zone and the third sub-detection zone are used to assess whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0102] In some embodiments, the qualitative or quantitative result generated by the fluorescence of the first sub-detection zone is used to assess whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0103] In some embodiments, the qualitative or quantitative result generated by the fluorescence of the second sub-detection zone is used to assess whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0104] In some embodiments, the qualitative or quantitative result generated by the fluorescence of the third sub-detection zone is used to assess whether the subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0105] In some embodiments, if the first sub-detection area, the second sub-detection area, and the third sub-detection area show a fluorescence positive result, it is determined that the donor providing the blood sample has a 99.2% probability of suffering from rheumatoid arthritis.

[0106] In some embodiments, if the first sub-detection area and the second sub-detection area show a fluorescence positive result, it is determined that the donor providing the blood sample has a 94.6% probability of suffering from rheumatoid arthritis.

[0107] In some embodiments, if the first sub-detection zone and the third sub-detection zone show a fluorescence positive result, it is determined that the donor providing the blood sample has a 91.7% probability of suffering from rheumatoid arthritis.

[0108] In some embodiments, if the second sub-detection area and the third sub-detection area show a fluorescence positive result, it is determined that the donor providing the blood sample has a 93.6% probability of suffering from rheumatoid arthritis.

[0109] In some embodiments, if the first sub-detection zone shows a fluorescence positive result (or a value greater than 20 U / mL), it is determined that the donor providing the blood sample has an 88.5% probability of suffering from rheumatoid arthritis.

[0110] In some embodiments, if the second sub-detection zone shows a fluorescence positive result (or a value greater than 25 U / mL), it is determined that the donor providing the blood sample has a 92.3% probability of suffering from rheumatoid arthritis.

[0111] In some embodiments, if the third sub-detection zone shows a fluorescence positive result (or a value greater than 20 IU / mL), it is determined that the donor providing the blood sample has a 78.8% probability of suffering from rheumatoid arthritis.

[0112] RA patients were divided into non-bone erosion group (stage I) and bone erosion group (stage II-IV) according to X-ray staging criteria.

[0113] Stage I: There are no destructive changes in the joints or osteoporosis at the joint ends.

[0114] Stage II: Osteoporosis at the joint end, with mild subchondral cystic destruction or bone erosion; limited joint movement, no joint deformity; atrophy of adjacent joint muscles; and lesions in the extra-articular soft tissue.

[0115] Stage III: Obvious osteoporosis accompanied by destruction of articular cartilage or bone; narrowing of the joint space, joint subluxation and other deformities; extensive muscle atrophy; and lesions of extra-articular soft tissues.

[0116] Stage IV: In addition to the changes in stages II and III, there is fibrous or bony ankylosis.

[0117] In some embodiments, if the first test line value is within the range of 108.1-334.62 U / mL and the second test line value is within the range of 54.22-239.08 U / mL, the donor providing the blood sample is judged to be in stage I according to X-ray staging standards.

[0118] In some embodiments, if the first test line value is within the range of 209.69-466.65 U / mL and the second test line value is within the range of 192.78-449.86 U / mL, the donor providing the blood sample is judged to be in stage II according to X-ray staging standards.

[0119] In some embodiments, if the first test line value is within the range of 215.81-480.71 U / mL and the second test line value is within the range of 262.96-601.94 U / mL, the donor providing the blood sample is judged to be in stage III according to X-ray staging standards.

[0120] In some embodiments, if the first test line value is within the range of 129.97-333.55 U / mL and the second test line value is within the range of 572.11-1111.47 U / mL, the donor providing the blood sample is judged to be in stage IV according to X-ray staging standards.

[0121] In some embodiments, the first test line measurement, the second test line measurement, or the third test line measurement is calibrated with a standard curve.

[0122] In some embodiments, the first detection line value, the second detection line value, or the third detection line value is a quantitative result obtained by calculating the detected fluorescence numerical result using a standard curve.

[0123] In some embodiments, the blood sample is whole blood, plasma, or serum.

[0124] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0125] In some embodiments, the loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

[0126] In some embodiments, the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0127] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first detection area, the second detection area, or the third detection area according to the flow direction of the sample on the test strip.

[0128] In some embodiments, the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

[0129] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0130] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL.

[0131] In some embodiments, the fluorescent immunochromatographic test strip is used to diagnose, assist in diagnosis, detect, or evaluate whether a subject has rheumatoid arthritis, the severity of rheumatoid arthritis, the stage of rheumatoid arthritis, or the risk of developing rheumatoid arthritis.

[0132] In some embodiments, the fluorescence qualitative result of the fluorescent immunochromatographic test strip is used to assess whether a subject has rheumatoid arthritis.

[0133] In some embodiments, the fluorescence quantitative results of the fluorescent immunochromatographic test strip are used to assess the severity of rheumatoid arthritis in a subject.

[0134] The fifth aspect of the present application provides a method for detecting the content of anti-mutant citrullinated vimentin antibodies (Anti-MCV), anti-cyclic citrullinated peptide antibodies (Anti-CCP), and rheumatoid factor (RF) in a biological sample from a subject, comprising the following steps:

[0135] a. Adding a biological sample from a subject to the sample loading area of ​​a fluorescent immunochromatographic test strip;

[0136] The fluorescent immunochromatographic test strip comprises a sample loading area, a detection area and a water absorption area;

[0137] The detection area includes a first sub-detection area fixed with mutant citrullinated vimentin, a second sub-detection area fixed with cyclic citrullinated peptide, and a third sub-detection area fixed with denatured human IgG;

[0138] wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG are independently 0.15-0.75 μg;

[0139] The amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1;

[0140] The amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2;

[0141] The sample loading area contains fluorescent material-labeled goat anti-human IgG, fluorescent material-labeled denatured human IgG, and fluorescent material-labeled goat anti-rabbit IgG in a mass ratio of 20:10:1; and

[0142] b. Determine the quantitative results of the fluorescence generated by the first sub-detection area, the second sub-detection area, and the third sub-detection area, and determine the content of anti-mutant citrullinated vimentin antibodies (Anti-MCV), anti-cyclic citrullinated peptide antibodies (Anti-CCP), and rheumatoid factor (RF) in the biological sample based on the quantitative results.

[0143] In some embodiments, the biological sample is whole blood, plasma, or serum.

[0144] In some embodiments, the fluorescent immunochromatographic test strip further comprises a bottom plate.

[0145] In some embodiments, the loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

[0146] In some embodiments, the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

[0147] In some embodiments, the fluorescent immunochromatographic test strip further includes a quality control area, which is located downstream of the first detection area, the second detection area, or the third detection area according to the flow direction of the sample on the test strip.

[0148] In some embodiments, the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

[0149] In some embodiments, according to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

[0150] In some embodiments, the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL.

[0151] Example

[0152] The application will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the application in any way. Those skilled in the art will readily recognize various noncritical parameters that can be changed or modified to produce substantially the same result.

[0153] Example 1: Preparation of Fluorescent Immunochromatographic Test Strips for Quantitative Joint Detection of Anti-MCV Antibodies, Anti-CCP Antibodies, and RF

[0154] (a) Treatment of fluorescent materials:

[0155] The fluorescent material was dissolved in a phosphate (PB) buffer solution at pH 8.0. After the fluorescent material was fully resuspended, an activator, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), was added and mixed evenly. Then, an amount of a stabilizer, N-hydroxysuccinimide (NHS), equal to that of EDC, was added and mixed evenly. The mixture was incubated at room temperature for 2 h, followed by high-speed centrifugation at 14,000 rpm for 30 min. The supernatant was discarded, and the precipitate was washed with a phosphate (PB) buffer solution at pH 7.0 and redissolved. The concentrations of EDC and NHS were both 10 mg / mL.

[0156] (b) Antibody labeling:

[0157] Add goat anti-human IgG to the fluorescent material obtained in step (a), mix well, stir continuously, and react overnight at 4°C. After the reaction is completed, centrifuge at 14,000 rpm for 30 minutes, discard the supernatant, and wash the precipitate with phosphate buffer (PB) at pH 7.0 and redissolve it to obtain goat anti-human IgG labeled with the fluorescent material;

[0158] Add denatured human IgG to the fluorescent material solution obtained in step (a), mix well, stir continuously, and react overnight at 4°C. After the reaction, centrifuge at 14,000 rpm for 30 minutes, discard the supernatant, and wash the precipitate with a phosphate buffer solution (pH 7.0) and redissolve it to obtain denatured human IgG labeled with the fluorescent material;

[0159] Add goat anti-rabbit IgG to the fluorescent material solution obtained in step (a), mix well, stir continuously, and react at 4°C overnight. After the reaction, centrifuge at 14,000 rpm for 30 minutes, discard the supernatant, and wash the precipitate with a phosphate buffer solution (pH 7.0) and redissolve it to obtain fluorescent material-labeled goat anti-rabbit IgG;

[0160] (c) Preparation of the sample loading area: The fluorescent material-labeled goat anti-human IgG, fluorescent material-labeled denatured human IgG, and fluorescent material-labeled goat anti-rabbit IgG obtained in step (b) were coated on the sample loading area at a ratio of 20:10:1, and then dried at 37°C for 24 hours; wherein the coating concentration of the fluorescent material-labeled goat anti-human IgG was 2% (volume percentage, i.e., the ratio of the volume of the fluorescent material-labeled goat anti-human IgG to the total volume of the preparation solution), and after mixing with the other two markers, the coating concentration was 4 μL / cm2; the coating concentration of the fluorescent material-labeled denatured human IgG was 1%, and after mixing with the other two markers, the coating concentration was 4 μL / cm2; the coating concentration of the fluorescent material-labeled goat anti-rabbit IgG was 0.1%, and after mixing with the other two markers, the coating concentration was 4 μL / cm2

[0161] (d) Preparation of the detection area (2) (taking FIG1 as an example):

[0162] Preparation of test lines: Recombinant MCV (as shown in SEQ ID NO: 1), recombinant CCP (as shown in SEQ ID NO: 2), and denatured human IgG were diluted to a concentration of 1 mg / ml, and the first sub-test line (3), the second sub-test line (4), and the third sub-test line (5) on the test area were drawn at 1.0 μL / cm;

[0163] The amount of streaking is 1.0 μL / cm, the solution concentration is 1 mg / ml, the width of the cut is 0.4 cm, and the amount of protein coated on the test line is about 0.4 μg.

[0164] Preparation of quality control line (6): Rabbit IgG with a concentration of 1 mg / mL was streaked at the quality control line (6) on the test area at 1 μL / cm;

[0165] The coated detection area (nitrocellulose membrane) was dried at 37°C for 24 hours. After drying, the nitrocellulose membrane was removed and sealed for dry storage.

[0166] (e) Assembly:

[0167] The chromatographic membrane (2) obtained in step (d) is first pasted on the bottom plate (8), and then the sample loading area (1) is pasted on the end of the chromatographic membrane (2) close to the first detection line (3). The absorbent paper (7) is pasted on the end of the chromatographic membrane (2) close to the quality control line (6). The pasted bottom plate (8) is cut into 3.5 mm test strips, which are then placed in a plastic card shell and pressed to form a test kit. The test kit is assembled in an environment with a humidity of less than 30%.

[0168] Example 2: Another preparation of a fluorescent immunochromatographic test strip for quantitative combined detection of anti-MCV antibodies, anti-CCP antibodies, and RF (using FIG. 2 as an example)

[0169] 1) PVC board (1): The surface is coated with adhesive to support and fix the board.

[0170] 2) Sample area (2): Made of glass fiber, it is sprayed with a solution containing buffer, anti-erythrocyte antibodies, surfactants, sugars and other ingredients to buffer and separate interfering substances such as red blood cells.

[0171] 3) Labeling area (3): The composition is glass fiber polyester, etc., and the solution containing sheep antibody IgG fluorescent microsphere marker, denatured human IgG fluorescent microsphere marker, and sheep anti-rabbit IgG fluorescent microsphere marker is sprayed.

[0172] 4) Detection area (4): nitrocellulose membrane, a carrier for chromatography reaction, coated with the first detection line, the second detection line, the third detection line, and the quality control line in sequence.

[0173] 5) The first detection line (5, 6 or 7): coated on the NC membrane, contains recombinant MCV protein (as shown in SEQ ID NO: 1), and is used to detect anti-MCV antibodies.

[0174] 6) Second detection line (5, 6 or 7): coated on NC membrane, containing recombinant CCP protein (as shown in SEQ ID NO: 2), used for detecting anti-CCP antibodies.

[0175] 7) The third detection line (5, 6 or 7): coated on the NC membrane, contains denatured human IgG, and is used to detect RF.

[0176] 8) Quality control line (8): coated on the NC membrane, containing rabbit IgG, used to indicate the effectiveness of the product.

[0177] 9) Absorption pad (9): Made of plant fiber or cotton wool, it is used to absorb excess liquid and provide power for the chromatography reaction.

[0178] Example 3: Preparation of fluorescent immunochromatographic test strips (quantum dot immunochromatographic test strips) of anti-MCV antibodies, anti-CCP antibodies and RF.

[0179] By embedding quantum dots within inorganic materials, quantum dot microspheres can make them less susceptible to environmental influences, significantly improving their stability and detection sensitivity. The surface of quantum dot microspheres is hydrophilically coated, resulting in good biocompatibility and low nonspecific adsorption, making them excellent materials for high-sensitivity biological detection. As a type of fluorescent microsphere, quantum dot microspheres are assembled from nano- to micron-sized microspheres to protect the quantum dots. Their surfaces are easily modified and can be enriched with functional groups such as carboxyl (-COOH), amino (-NH2), p-toluenesulfonyl (-Ts), and streptavidin (-SA).

[0180] The preparation method of quantum dot immunochromatographic test strips is as follows:

[0181] (a) Reagents

[0182] 1) Carboxyl-modified quantum dot microspheres

[0183] 2) Goat anti-human IgG, denatured human IgG, goat anti-rabbit IgG

[0184] 3) Activators EDC, Sulfo-NHS

[0185] 4) Activation solution Buffer A (0.05M / L MES pH 6.0)

[0186] 5) Reaction solution Buffer B (0.05M / L MES pH 6.5)

[0187] 6) Blocking Buffer C (0.05 M / L TRIS-HCl + 1% W / V BSA pH 8.0)

[0188] 7) Storage solution Buffer D (0.05M / L TRIS-HCl + 1% W / V BSA + 10% W / V trehalose pH 8.0)

[0189] (b) Marking process

[0190] 1% quantum dot microspheres can be diluted 10 times, 5 times or the original times to mark different concentrations, that is, the concentration of microspheres during the reaction process can be 1 mg / mL, 2 mg / mL or 10 mg / mL; the sensitivity difference under the three concentrations is not large, but the original times mark is easy to agglomerate; if it is greater than 2 mg / mL, the microsphere concentration is high and the dispersion of the reaction process is difficult to observe. Therefore, it is preferred to dilute 10 times and the microsphere concentration is 1 mg / mL.

[0191] 1) Cleaning microspheres

[0192] Take 50 μL of 1% quantum dot microspheres and add them to a 1.5 mL centrifuge tube. Then add 450 μL of activation buffer A, shake at 1000 rpm for 1 min, and then centrifuge at 14800 rpm for 30 min. Remove the supernatant, add 400 μL of activation buffer A, redissolve, and wash once.

[0193] 2) Activated microspheres

[0194] Prepare a 10 mg / mL solution of EDC and Sulfo-NHS using activation buffer A. For example, weigh 1 mg of EDC and dissolve it in 100 μL of Buffer A. Add 50 μL of Sulfo-NHS solution and then 50 μL of EDC solution to the washed microspheres, resulting in a 1 mg / mL concentration of EDC and NHS. Activate the microspheres by shaking on a vortex shaker at 1000 rpm for 30 minutes.

[0195] 3) Cleaning after activation

[0196] After activation and washing, the solution is replaced with reaction solution Buffer B:

[0197] (1) After activation, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of reaction buffer B, reconstitute, and wash once.

[0198] (2) After washing, centrifuge at 14800 rpm for 20 min, remove the supernatant, and add 500 μL of reaction solution Buffer B to re-dissolve.

[0199] 4) Conjugated Antibodies

[0200] Add the antibody to the reconstituted microspheres and rotate to mix for 2 hours. The antibody labeling concentration range is 0.02-0.1 mg / mg, that is, 0.02-0.1 mg of antibody is added per 1 mg of microspheres.

[0201] Antibody volume = antibody labeling concentration x microsphere mass / antibody concentration.

[0202] 5) Closed

[0203] After 2.0 h of coupling reaction, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of blocking solution Buffer C, reconstitute, place on a rotating incubator, rotate and mix, and block for 60 min.

[0204] 6) Clean and store

[0205] After blocking, centrifuge at 14,800 rpm for 20 minutes, remove the supernatant, add 250 μL of Buffer D (final concentration of microspheres 2 mg / mL), and re-dissolve. After resuspending, store at 2-8°C until use.

[0206] (c) Preparation of the labeling area:

[0207] The fluorescent material labeled goat anti-human IgG, fluorescent material labeled denatured human IgG and fluorescent material labeled goat anti-rabbit IgG obtained in the above steps were coated on the sample loading area at a ratio of 20:10:1, and then dried at 37°C for 24 hours.

[0208] (d) Sample area preparation:

[0209] It is made of glass fiber and is sprayed with a solution containing buffers, anti-erythrocyte antibodies, surfactants, sugars and other ingredients to play a role in buffering and separating interfering substances such as red blood cells.

[0210] (e) Preparation of the detection area:

[0211] Detection area: nitrocellulose membrane, the carrier of the chromatography reaction, which is coated with the first detection line, the second detection line, the third detection line, and the quality control line in sequence.

[0212] 1) First test line (5, 6 or 7): Coated on the NC membrane, containing recombinant MCV protein (as shown in SEQ ID NO: 1), used to detect anti-MCV antibodies. Recombinant MCV was diluted to a concentration of 1 mg / ml and drawn at the first test line on the test area at 1.0 μL / cm

[0213] 2) Second detection line (5, 6 or 7): Coated on the NC membrane, containing recombinant CCP protein (as shown in SEQ ID NO: 2), used to detect anti-CCP antibodies. Recombinant CCP is streaked at the second sub-detection line on the detection zone.

[0214] 3) Third test line (5, 6, or 7): Coated on the NC membrane, containing denatured human IgG, used to detect RF. Denatured human IgG was diluted to a concentration of 1 mg / ml and streaked at the third sub-test line on the test area at 1.0 μL / cm.

[0215] 4) Quality control line (8): Coated on the NC membrane, containing rabbit IgG, used to indicate the effectiveness of the product. Rabbit IgG with a concentration of 1 mg / mL was streaked at the quality control line on the test area at 1 μL / cm.

[0216] The coated detection area (nitrocellulose membrane) was dried at 37°C for 24 hours. After drying, the nitrocellulose membrane was removed and sealed for dry storage.

[0217] (f) Absorbent pad:

[0218] The ingredients are plant fibers or cotton wool, etc., which are used to absorb excess liquid and provide power for the chromatography reaction.

[0219] (g) Assembly:

[0220] The chromatographic membrane (2) obtained in step (d) is first pasted on the bottom plate (8), and then the sample loading area (1) is pasted on the end of the chromatographic membrane (2) close to the first detection line (3). The absorbent paper (7) is pasted on the end of the chromatographic membrane (2) close to the quality control line (6). The pasted bottom plate (8) is cut into 3.5 mm test strips, which are then placed in a plastic card shell and pressed to form a test kit. The test kit is assembled in an environment with a humidity of less than 30%.

[0221] Example 4: Detection of rheumatoid arthritis using quantum dot immunochromatographic test strips.

[0222] A fluorescent immunochromatographic test strip was prepared according to the steps shown in Example 3, and rheumatoid arthritis was detected according to the following steps.

[0223] 1. Take 10 μL of human blood sample and add it to the sample diluent. Mix well. Then take 70 μL and add it to the sample area of ​​the fluorescent immunochromatographic test strip. Let it stand for 15 minutes.

[0224] 2. Use quantum dot immunoassay analyzer to read the results;

[0225] If the first sub-test area, the second sub-test area, and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 99.2% probability of suffering from rheumatoid arthritis;

[0226] If the first sub-detection area and the second sub-detection area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 94.6% probability of suffering from rheumatoid arthritis;

[0227] If the first sub-test area and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 91.7% probability of suffering from rheumatoid arthritis;

[0228] If the second sub-test area and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 93.6% probability of suffering from rheumatoid arthritis;

[0229] If the first sub-detection area shows a positive fluorescence result (or a value greater than 20 U / mL), it is judged that the donor providing the blood sample has an 88.5% probability of suffering from rheumatoid arthritis;

[0230] If the second sub-detection area shows a positive fluorescence result (or a value greater than 25 U / mL), it is judged that the donor providing the blood sample has a 92.3% probability of suffering from rheumatoid arthritis;

[0231] If the third sub-detection area shows a positive fluorescence result (or a value greater than 20 IU / mL), it is judged that the donor providing the blood sample has a 78.8% probability of suffering from rheumatoid arthritis;

[0232] According to the X-ray staging criteria, RA patients were divided into non-bone erosion group (stage Ⅰ) and bone erosion group (stage Ⅱ-Ⅳ).

[0233] Stage I: There are no destructive changes in the joints or osteoporosis at the joint ends.

[0234] Stage II: Osteoporosis at the joint end, with mild subchondral cystic destruction or bone erosion; limited joint movement, no joint deformity; atrophy of adjacent joint muscles; and lesions in the extra-articular soft tissue.

[0235] Stage III: Obvious osteoporosis accompanied by destruction of articular cartilage or bone; narrowing of the joint space, joint subluxation and other deformities; extensive muscle atrophy; and lesions of extra-articular soft tissues.

[0236] Stage IV: In addition to the changes in stages II and III, there is fibrous or bony ankylosis.

[0237] If the first test line value is within the range of 108.1 to 334.62 U / mL and the second test line value is within the range of 54.22 to 239.08 U / mL, it is determined that the donor providing the blood sample is in stage I according to the X-ray staging standard.

[0238] If the first test line value is within the range of 209.69 to 466.65 U / mL and the second test line value is within the range of 192.78 to 449.86 U / mL, it is determined that the donor providing the blood sample is in stage II according to the X-ray staging standard.

[0239] If the first test line value is within the range of 215.81 to 480.71 U / mL and the second test line value is within the range of 262.96 to 601.94 U / mL, it is determined that the donor providing the blood sample is in stage III according to the X-ray staging standard.

[0240] If the first test line value is within the range of 129.97 to 333.55 U / mL and the second test line value is within the range of 572.11 to 1111.47 U / mL, it is determined that the donor providing the blood sample is in stage IV according to the X-ray staging standard.

[0241] The first detection line measured value, the second detection line measured value, or the third detection line measured value can be calibrated using a standard curve.

[0242] The first detection line measurement value, the second detection line measurement value, or the third detection line measurement value may be a quantitative result obtained by calculating the fluorescence numerical result detected through a standard curve.

[0243] Example 5: Preparation of fluorescent immunochromatographic test strips (time-resolved fluorescent immunochromatographic test strips) for anti-MCV antibodies, anti-CCP antibodies, and RF.

[0244] The methodological principle of time-resolved fluorescent microspheres (TRFMs) is to use rare earth ions with long fluorescence half-lives as fluorescent materials. Because these fluorescent materials have a large Stokes shift (>150nm) and a fluorescence lifetime 5 to 6 orders of magnitude longer than that of the background substance, the interference of various nonspecific fluorescence can be effectively eliminated by simply delaying the measurement time and waiting for the background substance's fluorescence to fully decay before measuring the signal of this fluorescent material, achieving high sensitivity. As special functional microspheres, each microsphere can be encapsulated with thousands of fluorescent molecules, greatly improving the fluorescence labeling efficiency and effectively enhancing analytical sensitivity. The fluorescent microspheres are also modified with an appropriate density of carboxyl groups or other functional groups on the surface for covalent coupling with proteins or antibodies, thereby improving the stability of the label.

[0245] Preparation method of time-resolved fluorescence immunochromatographic test strips:

[0246] (a) Reagents

[0247] 1) Carboxyl-modified time-resolved fluorescent microspheres

[0248] 2) Goat anti-human IgG, denatured human IgG, goat anti-rabbit IgG

[0249] 3) Activators EDC, Sulfo-NHS

[0250] 4) Activation solution Buffer A (0.05M / L MES pH 6.0)

[0251] 5) Reaction solution Buffer B (0.05M / L MES pH 6.5)

[0252] 6) Blocking Buffer C (0.05 M / L TRIS-HCl + 1% W / V BSA pH 8.0)

[0253] 7) Storage solution Buffer D (0.05M / L TRIS-HCl + 1% W / V BSA + 10% W / V trehalose pH 8.0)

[0254] (b) Marking process

[0255] 1% time-resolved fluorescent microspheres can be diluted 10 times, 5 times, or the original times to mark different concentrations, that is, the microsphere concentration during the reaction process can be 1 mg / mL, 2 mg / mL, or 10 mg / mL; the sensitivity difference under the three concentrations is not large, but the original times markers are prone to aggregation; if it is greater than 2 mg / mL, the microsphere concentration is high and the dispersion of the reaction process is difficult to observe. Therefore, a 10-fold dilution is preferred, and the microsphere concentration used is 1 mg / mL.

[0256] 1) Cleaning microspheres

[0257] Take 50 μL of 1% time-resolved fluorescent microspheres and add them to a 1.5 mL centrifuge tube. Then add 450 μL of activation buffer A. Shake at 1000 rpm for 1 min, then centrifuge at 14800 rpm for 30 min. Remove the supernatant, add 400 μL of activation buffer A, re-dissolve, and wash once.

[0258] 2) Activated microspheres

[0259] Use activation buffer A to prepare a 10 mg / mL solution of EDC and Sulfo-NHS. For example, weigh 1 mg of EDC and dissolve it in 100 μL of Buffer A. Add 50 μL of Sulfo-NHS solution and then 50 μL of EDC solution to the washed microspheres, resulting in a 1 mg / mL concentration of EDC and NHS. Activate the microspheres by shaking on a vortex shaker at 1000 rpm for 30 minutes.

[0260] 3) Cleaning after activation

[0261] After activation and washing, the solution is replaced with reaction solution Buffer B:

[0262] (1) After activation, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of reaction buffer B, re-dissolve, and wash once.

[0263] (2) After washing, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of reaction solution Buffer B, and re-dissolve.

[0264] 4) Conjugated Antibodies

[0265] Add the antibody to the reconstituted microspheres and rotate to mix for 2 hours. The antibody labeling concentration range is 0.02-0.1 mg / mg, that is, 0.02-0.1 mg of antibody is added per 1 mg of microspheres.

[0266] Antibody volume = antibody labeling concentration x microsphere mass / antibody concentration.

[0267] 5) Closed

[0268] After 2.0 h of coupling reaction, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of blocking solution Buffer C, reconstitute, place on a rotating incubator, rotate and mix, and block for 60 min.

[0269] 6) Clean and store

[0270] After blocking, centrifuge at 14,800 rpm for 20 minutes, remove the supernatant, add 250 μL of Buffer D (final concentration of microspheres 2 mg / mL), and re-dissolve. After resuspending, store at 2-8°C until use.

[0271] (c) Preparation of the labeling area:

[0272] The fluorescent material labeled goat anti-human IgG, fluorescent material labeled denatured human IgG and fluorescent material labeled goat anti-rabbit IgG obtained in the above steps were coated on the sample loading area at a ratio of 20:10:1, and then dried at 37°C for 24 hours.

[0273] (d) Sample area preparation:

[0274] It is made of glass fiber and is sprayed with a solution containing buffers, anti-erythrocyte antibodies, surfactants, sugars and other ingredients to play a role in buffering and separating interfering substances such as red blood cells.

[0275] (e) Preparation of the detection area:

[0276] Detection area: nitrocellulose membrane, the carrier of the chromatography reaction, which is coated with the first detection line, the second detection line, the third detection line, and the quality control line in sequence.

[0277] 1) First test line (5, 6 or 7): Coated on the NC membrane, containing recombinant MCV protein (as shown in SEQ ID NO: 1), used to detect anti-MCV antibodies. Recombinant MCV was diluted to a concentration of 1 mg / ml and drawn at the first test line on the test area at 1.0 μL / cm

[0278] 2) Second detection line (5, 6 or 7): Coated on the NC membrane, containing recombinant CCP protein (as shown in SEQ ID NO: 2), used to detect anti-CCP antibodies. Recombinant CCP is streaked at the second sub-detection line on the detection zone.

[0279] 3) Third test line (5, 6, or 7): Coated on the NC membrane, containing denatured human IgG, used to detect RF. Denatured human IgG was diluted to a concentration of 1 mg / ml and streaked at the third sub-test line on the test area at 1.0 μL / cm.

[0280] 4) Quality control line (8): Coated on the NC membrane, containing rabbit IgG, used to indicate the effectiveness of the product. Rabbit IgG with a concentration of 1 mg / mL was streaked at the quality control line on the test area at 1 μL / cm.

[0281] The coated detection area (nitrocellulose membrane) was dried at 37°C for 24 hours. After drying, the nitrocellulose membrane was removed and sealed for dry storage.

[0282] (f) Absorbent pad:

[0283] The ingredients are plant fibers or cotton wool, etc., which are used to absorb excess liquid and provide power for the chromatography reaction.

[0284] (g) Assembly:

[0285] The chromatographic membrane (2) obtained in step (d) is first pasted on the bottom plate (8), and then the sample loading area (1) is pasted on the end of the chromatographic membrane (2) close to the first detection line (3). The absorbent paper (7) is pasted on the end of the chromatographic membrane (2) close to the quality control line (6). The pasted bottom plate (8) is cut into 3.5 mm test strips, which are then placed in a plastic card shell and pressed to form a test kit. The test kit is assembled in an environment with a humidity of less than 30%.

[0286] Example 6: Detection of rheumatoid arthritis using a time-resolved fluorescent immunochromatographic test strip.

[0287] Fluorescent immunochromatographic test strips were prepared according to the steps shown in Example 5, and rheumatoid arthritis was detected according to the following steps.

[0288] 1. Take 10 μL of human blood sample and add it to the sample diluent. Mix well. Then take 70 μL and add it to the sample area of ​​the fluorescent immunochromatographic test strip. Let it stand for 15 minutes.

[0289] 2. Read the results using a time-resolved fluorescence immunoassay analyzer;

[0290] If the first sub-test area, the second sub-test area, and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 99.2% probability of suffering from rheumatoid arthritis;

[0291] If the first sub-detection area and the second sub-detection area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 94.6% probability of suffering from rheumatoid arthritis;

[0292] If the first sub-test area and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 91.7% probability of suffering from rheumatoid arthritis;

[0293] If the second sub-test area and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 93.6% probability of suffering from rheumatoid arthritis;

[0294] If the first sub-detection area shows a positive fluorescence result (or a value greater than 20 U / mL), it is judged that the donor providing the blood sample has an 88.5% probability of suffering from rheumatoid arthritis;

[0295] If the second sub-detection area shows a positive fluorescence result (or a value greater than 25 U / mL), it is judged that the donor providing the blood sample has a 92.3% probability of suffering from rheumatoid arthritis;

[0296] If the third sub-detection area shows a positive fluorescence result (or a value greater than 20 IU / mL), it is judged that the donor providing the blood sample has a 78.8% probability of suffering from rheumatoid arthritis;

[0297] According to the X-ray staging criteria, RA patients were divided into non-bone erosion group (stage Ⅰ) and bone erosion group (stage Ⅱ-Ⅳ).

[0298] Stage I: There are no destructive changes in the joints or osteoporosis at the joint ends.

[0299] Stage II: Osteoporosis at the joint end, with mild subchondral cystic destruction or bone erosion; limited joint movement, no joint deformity; atrophy of adjacent joint muscles; and lesions in the extra-articular soft tissue.

[0300] Stage III: Obvious osteoporosis accompanied by destruction of articular cartilage or bone; narrowing of the joint space, joint subluxation and other deformities; extensive muscle atrophy; and lesions of extra-articular soft tissues.

[0301] Stage IV: In addition to the changes in stages II and III, there is fibrous or bony ankylosis.

[0302] If the first test line value is within the range of 108.1 to 334.62 U / mL and the second test line value is within the range of 54.22 to 239.08 U / mL, it is determined that the donor providing the blood sample is in stage I according to the X-ray staging standard.

[0303] If the first test line value is within the range of 209.69 to 466.65 U / mL and the second test line value is within the range of 192.78 to 449.86 U / mL, it is determined that the donor providing the blood sample is in stage II according to the X-ray staging standard.

[0304] If the first test line value is within the range of 215.81 to 480.71 U / mL and the second test line value is within the range of 262.96 to 601.94 U / mL, it is determined that the donor providing the blood sample is in stage III according to the X-ray staging standard.

[0305] If the first test line value is within the range of 129.97 to 333.55 U / mL and the second test line value is within the range of 572.11 to 1111.47 U / mL, it is determined that the donor providing the blood sample is in stage IV according to the X-ray staging standard.

[0306] The first detection line measured value, the second detection line measured value, or the third detection line measured value can be calibrated using a standard curve.

[0307] The first detection line measurement value, the second detection line measurement value, or the third detection line measurement value may be a quantitative result obtained by calculating the fluorescence numerical result detected through a standard curve.

[0308] Example 7: Preparation of fluorescent immunochromatographic test strips (aggregation-induced luminescence immunochromatographic test strips) of anti-MCV antibodies, anti-CCP antibodies and RF.

[0309] Aggregation-induced emission (AIE) materials have high aggregated luminescence efficiency. The use of a rigid main chain polymer structure can effectively restrict the vibrational and rotational structures of the molecules, significantly improving the solid-state luminescence efficiency of the prepared microspheres. Moreover, the large Stokes shift of the AIE molecules can efficiently separate the excitation light source and the signal acquisition window, resulting in a significant increase in the sensitivity of its fluorescence signal output. This technology uses high-efficiency AIE molecules as signal elements. Through monomer selection and process optimization, AIE fluorescent microspheres are in situ polymerized. The particle size of the microspheres can be effectively controlled from 50nm to 1000nm and micron level (particle size CV value is less than 5%), and the surface of the microspheres can be quantitatively modified as needed (functional groups such as carboxyl, amino, hydroxyl, streptavidin, biomacromolecules, etc.). With the help of coupling technology, it can be further connected to a variety of antibodies and antigen proteins, expanding its application in immunoassays. The fluorescent microspheres prepared by this technology far surpass similar products in terms of light color, efficiency and stability, and achieve full coverage of emission wavelengths (blue, green, red, etc.). The functional modification of AIE fluorescent microspheres is highly tunable and has obvious advantages in lateral flow technology, cell imaging, microfluidics technology and fluorescent enzyme-linked immunosorbent assay.

[0310] Preparation method of aggregation-induced luminescence immunochromatographic test strip

[0311] (a) Reagents

[0312] 1) Carboxyl-modified aggregation-induced emission microspheres

[0313] 2) Goat anti-human IgG, denatured human IgG, goat anti-rabbit IgG

[0314] 3) Activators EDC, Sulfo-NHS

[0315] 4) Activation solution Buffer A (0.05M / L MES pH 6.0)

[0316] 5) Reaction solution Buffer B (0.05M / L MES pH 6.5)

[0317] 6) Blocking Buffer C (0.05 M / L TRIS-HCl + 1% W / V BSA pH 8.0)

[0318] 7) Storage solution Buffer D (0.05M / L TRIS-HCl + 1% W / V BSA + 10% W / V trehalose pH 8.0)

[0319] (b) Marking process

[0320] 1% aggregation-induced luminescence microspheres can be diluted 10 times, 5 times, or the original times to mark different concentrations, that is, the microsphere concentration during the reaction process can be 1 mg / mL, 2 mg / mL, or 10 mg / mL; the sensitivity difference under the three concentrations is not large, but the original times mark is prone to aggregation; if it is greater than 2 mg / mL, the microsphere concentration is high and the dispersion during the reaction process is difficult to observe. Therefore, a 10-fold dilution is preferred, and the microsphere concentration used is 1 mg / mL.

[0321] 1) Cleaning microspheres

[0322] Take 50 μL of 1% aggregation-induced luminescence microspheres and add them to a 1.5 mL centrifuge tube. Then add 450 μL of activation buffer A, shake at 1000 rpm for 1 min, and then centrifuge at 14800 rpm for 30 min. Remove the supernatant, add 400 μL of activation buffer A, re-dissolve (ultrasonic dispersion can be used if necessary), and wash once.

[0323] 2) Activated microspheres

[0324] Use activation buffer A to prepare a 10 mg / mL solution of EDC and Sulfo-NHS. For example, weigh 1 mg of EDC and dissolve it in 100 μL of Buffer A. Add 50 μL of Sulfo-NHS solution and then 50 μL of EDC solution to the washed microspheres, resulting in a 1 mg / mL concentration of EDC and NHS. Activate the microspheres by shaking on a vortex shaker at 1000 rpm for 30 minutes.

[0325] 3) Cleaning after activation

[0326] After activation and washing, the solution is replaced with reaction solution Buffer B:

[0327] (1) After activation, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of reaction buffer B, re-dissolve, and wash once.

[0328] (2) After washing, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of reaction solution Buffer B, and re-dissolve.

[0329] 4) Conjugated Antibodies

[0330] Add the antibody to the reconstituted microspheres and rotate to mix for 2 hours. The antibody labeling concentration range is 0.02-0.1 mg / mg, that is, 0.02-0.1 mg of antibody is added per 1 mg of microspheres.

[0331] Antibody volume = antibody labeling concentration x microsphere mass / antibody concentration.

[0332] 5) Closed

[0333] After 2.0 h of coupling reaction, centrifuge at 14800 rpm for 20 min, remove the supernatant, add 500 μL of blocking solution Buffer C, reconstitute, place on a rotating incubator, rotate and mix, and block for 60 min.

[0334] 6) Clean and store

[0335] After blocking, centrifuge at 14,800 rpm for 20 minutes, remove the supernatant, add 250 μL of Buffer D (final concentration of microspheres 2 mg / mL), and re-dissolve. After resuspending, store at 2-8°C until use.

[0336] (c) Preparation of the labeling area:

[0337] The fluorescent material labeled goat anti-human IgG, fluorescent material labeled denatured human IgG and fluorescent material labeled goat anti-rabbit IgG obtained in the above steps were coated on the sample loading area at a ratio of 20:10:1, and then dried at 37°C for 24 hours.

[0338] (d) Sample area preparation:

[0339] It is made of glass fiber and is sprayed with a solution containing buffers, anti-erythrocyte antibodies, surfactants, sugars and other ingredients to play a role in buffering and separating interfering substances such as red blood cells.

[0340] (e) Preparation of the detection area:

[0341] Detection area: nitrocellulose membrane, the carrier of the chromatography reaction, which is coated with the first detection line, the second detection line, the third detection line, and the quality control line in sequence.

[0342] 1) First test line (5, 6 or 7): Coated on the NC membrane, containing recombinant MCV protein (as shown in SEQ ID NO: 1), used to detect anti-MCV antibodies. Recombinant MCV was diluted to a concentration of 1 mg / ml and drawn at the first test line on the test area at 1.0 μL / cm

[0343] 2) Second detection line (5, 6 or 7): Coated on the NC membrane, containing recombinant CCP protein (as shown in SEQ ID NO: 2), used to detect anti-CCP antibodies. Recombinant CCP is streaked at the second sub-detection line on the detection zone.

[0344] 3) Third test line (5, 6, or 7): Coated on the NC membrane, containing denatured human IgG, used to detect RF. Denatured human IgG was diluted to a concentration of 1 mg / ml and streaked at the third sub-test line on the test area at 1.0 μL / cm.

[0345] 4) Quality control line (8): Coated on the NC membrane, containing rabbit IgG, used to indicate the effectiveness of the product. Rabbit IgG with a concentration of 1 mg / mL was streaked at the quality control line on the test area at 1 μL / cm.

[0346] The coated detection area (nitrocellulose membrane) was dried at 37°C for 24 hours. After drying, the nitrocellulose membrane was removed and sealed for dry storage.

[0347] (f) Absorbent pad:

[0348] The ingredients are plant fibers or cotton wool, etc., which are used to absorb excess liquid and provide power for the chromatography reaction.

[0349] (g) Assembly:

[0350] The chromatographic membrane (2) obtained in step (d) is first pasted on the bottom plate (8), and then the sample loading area (1) is pasted on the end of the chromatographic membrane (2) close to the first detection line (3). The absorbent paper (7) is pasted on the end of the chromatographic membrane (2) close to the quality control line (6). The pasted bottom plate (8) is cut into 3.5 mm test strips, which are then placed in a plastic card shell and pressed to form a test kit. The test kit is assembled in an environment with a humidity of less than 30%.

[0351] Example 8: Detection of rheumatoid arthritis using aggregation-induced luminescence immunochromatographic test strips

[0352] Fluorescent immunochromatographic test strips were prepared according to the steps shown in Example 7, and rheumatoid arthritis was detected according to the following steps.

[0353] 1. Take 10 μL of human blood sample and add it to the sample diluent. Mix well. Then take 70 μL and add it to the sample area of ​​the fluorescent immunochromatographic test strip. Let it stand for 15 minutes.

[0354] 2. Read the results using an aggregation-induced luminescence immunoassay analyzer;

[0355] If the first sub-test area, the second sub-test area, and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 99.2% probability of suffering from rheumatoid arthritis;

[0356] If the first sub-detection area and the second sub-detection area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 94.6% probability of suffering from rheumatoid arthritis;

[0357] If the first sub-test area and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 91.7% probability of suffering from rheumatoid arthritis;

[0358] If the second sub-test area and the third sub-test area show a fluorescence positive result, it is judged that the donor providing the blood sample has a 93.6% probability of suffering from rheumatoid arthritis;

[0359] If the first sub-detection area shows a positive fluorescence result (or a value greater than 20 U / mL), it is judged that the donor providing the blood sample has an 88.5% probability of suffering from rheumatoid arthritis;

[0360] If the second sub-detection area shows a positive fluorescence result (or a value greater than 25 U / mL), it is judged that the donor providing the blood sample has a 92.3% probability of suffering from rheumatoid arthritis;

[0361] If the third sub-detection area shows a positive fluorescence result (or a value greater than 20 IU / mL), it is judged that the donor providing the blood sample has a 78.8% probability of suffering from rheumatoid arthritis;

[0362] According to the X-ray staging criteria, RA patients were divided into non-bone erosion group (stage Ⅰ) and bone erosion group (stage Ⅱ-Ⅳ).

[0363] Stage I: There are no destructive changes in the joints or osteoporosis at the joint ends.

[0364] Stage II: Osteoporosis at the joint end, with mild subchondral cystic destruction or bone erosion; limited joint movement, no joint deformity; atrophy of adjacent joint muscles; and lesions in the extra-articular soft tissue.

[0365] Stage III: Obvious osteoporosis accompanied by destruction of articular cartilage or bone; narrowing of the joint space, joint subluxation and other deformities; extensive muscle atrophy; and lesions of extra-articular soft tissues.

[0366] Stage IV: In addition to the changes in stages II and III, there is fibrous or bony ankylosis.

[0367] If the first test line value is within the range of 108.1 to 334.62 U / mL and the second test line value is within the range of 54.22 to 239.08 U / mL, it is determined that the donor providing the blood sample is in stage I according to the X-ray staging standard.

[0368] If the first test line value is within the range of 209.69 to 466.65 U / mL and the second test line value is within the range of 192.78 to 449.86 U / mL, it is determined that the donor providing the blood sample is in stage II according to the X-ray staging standard.

[0369] If the first test line value is within the range of 215.81 to 480.71 U / mL and the second test line value is within the range of 262.96 to 601.94 U / mL, it is determined that the donor providing the blood sample is in stage III according to the X-ray staging standard.

[0370] If the first test line value is within the range of 129.97 to 333.55 U / mL and the second test line value is within the range of 572.11 to 1111.47 U / mL, it is determined that the donor providing the blood sample is in stage IV according to the X-ray staging standard.

[0371] The first detection line measured value, the second detection line measured value, or the third detection line measured value can be calibrated using a standard curve.

[0372] The first detection line measurement value, the second detection line measurement value, or the third detection line measurement value may be a quantitative result obtained by calculating the fluorescence numerical result detected through a standard curve.

[0373] Example 9: Precision Testing

[0374] The T / C value is the ratio of the signal value of the test line (T line) to the signal value of the control line (C line) as read by the instrument. This calculation method can effectively improve the precision of test results. For example, when calculating the signal value of the test line T alone, the coefficient of variation is 10.81%, while when using the T / C calculation, the coefficient of variation drops to 4.11%. The lower the coefficient of variation, the better the precision of the test result and the more reliable the reagent.

[0375] The time-resolved fluorescent immunochromatographic test strip prepared in Example 5 was used for precision testing (coating concentration was 1 mg / mL). The results are shown in Table 1.

[0376] Table 1: Calculation of coefficient of variation CV (CV = SD / AV) using T / C values

[0377] The fluorescent immunochromatographic test strips disclosed herein for quantitatively detecting mutant citrullinated vimentin antibodies (Anti-MCV), anti-cyclic citrullinated peptide antibodies (Anti-CCP), and rheumatoid factor (RF) are equipped with detection lines for Anti-MCV, Anti-CCP, and RF. These strips can simultaneously detect Anti-MCV, Anti-CCP, and RF in human whole blood, plasma, and serum through a single sample addition. The strips are simple to operate and can reduce the cost of reagents and consumables. Furthermore, the strips can address the shortcomings of single-indicator diagnosing RA, helping to improve the accuracy of diagnosing RA and reduce misdiagnoses and missed diagnoses.

[0378] Example 10: Comparison of Fluorescence Immunochromatography and Colloidal Gold Method

[0379] Since the colloidal gold method performs grayscale analysis, while the fluorescent immunochromatography method performs fluorescence intensity analysis, the sensitivity and accuracy of colloidal gold are inferior to those of fluorescent immunochromatography in quantitative analysis.

[0380] The time-resolved fluorescence immunochromatographic test strip prepared in Example 5 was compared with the colloidal gold test strip under different conditions of coating concentration of 1 or 2 mg / mL. The experimental data are shown in Table 2 and Figure 3.

[0381] The T / C value is the ratio of the signal value of the test line (T line) read by the instrument to the signal value of the quality control line (C line).

[0382] Table 2: Comparison of Fluorescence Immunochromatography and Colloidal Gold Method

Claims

1. A fluorescent immunochromatographic test strip for detecting rheumatoid arthritis, the test strip comprising a sample loading area, a detection area and a water absorption area; The detection area includes a first sub-detection area fixed with mutant citrullinated vimentin, a second sub-detection area fixed with cyclic citrullinated peptide, and a third sub-detection area fixed with denatured human IgG; wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide and the denatured human IgG are independently 0.15-0.75 μg; The amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1; The amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2; The sample loading area contains goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material and goat anti-rabbit IgG labeled with fluorescent material in a mass ratio of 20:10:

1.

2. The fluorescent immunochromatographic test strip as claimed in claim 1, further comprising a base plate.

3. The fluorescent immunochromatographic test strip as claimed in claim 1, wherein the sample loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with a fluorescent material, denatured human IgG labeled with a fluorescent material, and goat anti-rabbit IgG labeled with a fluorescent material.

4. The fluorescent immunochromatographic test strip as claimed in claim 1, wherein the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres and aggregation-induced luminescence microspheres.

5. The fluorescent immunochromatographic test strip as described in claim 1, further comprising a quality control area, wherein the quality control area is located downstream of the first sub-detection area, the second sub-detection area and the third sub-detection area according to the flow direction of the sample on the test strip. 6 . The fluorescent immunochromatographic test strip according to claim 5 , wherein the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

7. The fluorescent immunochromatographic test strip according to claim 1, wherein: According to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

8. The fluorescent immunochromatographic test strip of claim 1, wherein the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide and the denatured human IgG is 0.5-1.5 mg / mL.

9. A kit comprising the fluorescent immunochromatographic test strip according to any one of claims 1 to 8 and instructions.

10. Use of the fluorescent immunochromatographic test strip according to any one of claims 1 to 8 in preparing a kit for detecting rheumatoid arthritis.

11. A method for assessing whether a subject has rheumatoid arthritis or the extent of the disease, comprising the following steps: a. Adding a blood sample from a subject to the sample loading area of ​​a fluorescent immunochromatographic test strip; The fluorescent immunochromatographic test strip comprises a sample loading area, a detection area and a water absorption area; The detection area includes a first sub-detection area fixed with mutant citrullinated vimentin, a second sub-detection area fixed with cyclic citrullinated peptide, and a third sub-detection area fixed with denatured human IgG; wherein the coating amounts of the mutant citrullinated vimentin, the cyclic citrullinated peptide and the denatured human IgG are independently 0.15-0.75 μg; The amino acid sequence of the mutant citrullinated vimentin is SEQ ID NO: 1; The amino acid sequence of the cyclic citrullinated peptide is SEQ ID NO: 2; The sample loading area contains goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material in a mass ratio of 20:10:1; and b. Determine the qualitative or quantitative results of the fluorescence produced by the first sub-detection area, the second sub-detection area, and the third sub-detection area, and evaluate whether the subject suffers from rheumatoid arthritis or the degree of the disease based on the qualitative or quantitative results.

12. The method of claim 11, wherein the blood sample is whole blood, plasma or serum.

13. The method according to claim 11, wherein the fluorescent immunochromatographic test strip further comprises a bottom plate.

14. The method of claim 11, wherein the sample loading area is divided into a sample area and a labeling area, the sample area is used to receive the sample, and the labeling area is used to coat goat anti-human IgG labeled with fluorescent material, denatured human IgG labeled with fluorescent material, and goat anti-rabbit IgG labeled with fluorescent material.

15. The method of claim 11, wherein the fluorescent material is selected from fluorescein isothiocyanate and its derivatives, rhodamine and its derivatives, phycoerythrin, fluorescent polystyrene microspheres, time-resolved microspheres, upconversion luminescent particles, quantum dot microspheres, and aggregation-induced emission microspheres.

16. The method of claim 11, wherein the fluorescent immunochromatographic test strip further comprises a quality control area, and the quality control area is located downstream of the first sub-detection area, the second sub-detection area, and the third sub-detection area according to the flow direction of the sample on the test strip.

17. The method of claim 16, wherein the quality control area is coated with rabbit IgG, and the coating amount of the rabbit IgG is 0.15-0.75 μg.

18. The method of claim 11, wherein: According to the flow direction of the sample on the test strip, the sample loading area, the detection area and the water absorption area are arranged from upstream to downstream.

19. The method of claim 11, wherein the coating concentration of the mutant citrullinated vimentin, the cyclic citrullinated peptide, and the denatured human IgG is 0.5-1.5 mg / mL.

Images