CN111308096A - Colloidal gold test strip for detecting rheumatoid arthritis - Google Patents
Colloidal gold test strip for detecting rheumatoid arthritis Download PDFInfo
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- CN111308096A CN111308096A CN202010146693.0A CN202010146693A CN111308096A CN 111308096 A CN111308096 A CN 111308096A CN 202010146693 A CN202010146693 A CN 202010146693A CN 111308096 A CN111308096 A CN 111308096A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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Abstract
The application relates to a colloidal gold test strip for rheumatoid arthritis detection, which comprises a detection area for rheumatoid arthritis related factors, wherein the detection area comprises a first sub-detection area coated with protein antigens of citrullinated vimentin and mutants thereof, a second sub-detection area coated with cyclic citrullinated short peptide antigens and a third sub-detection area coated with human IgG Fc fragments.
Description
Technical Field
The application relates to the field of biological detection, in particular to the detection of rheumatoid arthritis.
Background
Rheumatoid Arthritis (RA), a common chronic disabling autoimmune disease with a major manifestation of symmetrical, polyarticular synovitis, is ubiquitous worldwide, affecting approximately 1.0% of the world population. Currently, there are approximately 4500 million patients with rheumatoid arthritis worldwide, and approximately 140 million will be newly increased each year. In 2016, the global RA detection market has a total sales of about $ 3.47 billion, with a steady growth rate of 2% per year, while the major emerging markets for RA, brazil, canada, china, and mexico, will exhibit a rapid growth prospect. Meanwhile, with the aging of the population becoming serious, the need for diagnosis of rheumatoid arthritis will also increase. The incidence rate of rheumatoid arthritis in China is about 0.3-0.4%, and about 400-500 ten thousand patients exist, and about 15-20 ten thousand patients are newly increased every year.
At present, the markers selected for early diagnosis of rheumatoid arthritis are anti-cyclic citrullinated polypeptide antibodies and rheumatoid factors. Wherein the diagnostic effect of the rheumatoid factor is lower than that of the anti-cyclic citrullinated polypeptide antibody. Related clinical researches show that the sensitivity and specificity of the anti-mutant citrullinated vimentin antibody for diagnosing rheumatoid arthritis are higher than those of an anti-cyclic citrullinated peptide antibody.
Therefore, there is a need in the art for a new product and method for detecting rheumatoid arthritis.
Summary of The Invention
In a first aspect, the present application provides a colloidal gold test strip for rheumatoid arthritis detection, comprising a detection zone for rheumatoid arthritis-associated factors, wherein the detection zone comprises a first sub-detection zone coated with protein antigens of citrullinated vimentin and mutants thereof, a second sub-detection zone coated with cyclic citrullinated short peptide antigen, and a third sub-detection zone coated with human IgG Fc fragments.
In some embodiments, the amount of protein antigen coating of citrullinated vimentin and mutants thereof in the first sub-detection zone is 0.5-2.0mg/mL, and/or
In some embodiments, the amount of cyclic citrullinated short peptide antigen coated in the second sub-detection zone is 0.5-2.0mg/mL, and/or
In some embodiments, the amount of coating of human IgG-Fc fragment in the third sub-detection zone is between 0.5 and 1.5 mg/mL.
In some embodiments, the colloidal gold test strip further comprises a base plate and one or more of the following regions immobilized on the base plate: a sample loading area; the colloidal gold labeling area is used for attaching a colloidal gold label to an antibody to be detected in a detection sample; a water absorption area.
In some embodiments, the loading zone, the colloidal gold labeled zone, the detection zone, and the water absorption zone are arranged from upstream to downstream, in the direction of flow of the test sample on the strip.
In some embodiments, there is an overlap between adjacent regions.
In some embodiments, the overlap between the loading region and the colloidal gold labeled region is between 1 and 3 mm.
In some embodiments, the overlap between the colloidal gold labeled region and the detection region is between 1 and 3 mm.
In some embodiments, the overlap between the detection zone and the absorbent zone is between 1 and 3 mm.
In some embodiments, the loading zone is prepared from a sample pad.
In some embodiments, the sample pad is a polyester fiber film or a glass fiber film carrier.
In some embodiments, the colloidal gold labeled region is a first membrane loaded with a colloidal gold labeled first antibody.
In some embodiments, the first antibody is an antibody that recognizes IgG of human origin.
In some embodiments, the first antibody is a rabbit anti-human IgG antibody, a mouse anti-human IgG antibody, or a goat anti-human IgG antibody.
In some embodiments, the first antibody is loaded at 30-60 μ g.
In some embodiments, the first film is a fiberglass film or a polyester film.
In some embodiments, the colloidal gold has a particle average diameter of 40 to 60 nm.
In some embodiments, the absorbent region is prepared from an absorbent material.
In some embodiments, the absorbent region comprises an absorbent film and/or an absorbent paper.
In some embodiments, the water-absorbing capacity of the water-absorbing region is not less than 200 μ L.
In some embodiments, the detection zone further comprises a fourth sub-detection zone located downstream of the first to third sub-detection zones in the flow direction of the detection sample on the test strip for ensuring reliability of the detection result, and the fourth sub-detection zone is loaded with a second antibody capable of binding to the first antibody.
In some embodiments, the second antibody is a goat anti-rabbit IgG antibody and/or a mouse anti-rabbit IgG antibody.
In some embodiments, the second antibody is a rabbit anti-mouse IgG antibody and/or a goat anti-mouse IgG antibody.
In some embodiments, the second antibody is a murine anti-sheep IgG antibody and/or a rabbit anti-sheep IgG antibody.
In some embodiments, the spacing between each sub-detection zone is 1-1.5 cm.
In some embodiments, the detection zone comprises a second membrane, and each sub-detection zone is disposed on the second membrane.
In some embodiments, the second film is a polyester film or a glass fiber film.
In some embodiments, the colloidal gold test strip further comprises a hard shell, and a window for exposing the loading area and each sub-detection area is formed on the hard shell.
In a second aspect, the present application provides a kit for detecting rheumatoid arthritis, which comprises the colloidal gold test strip of the first aspect.
In some embodiments, the kit further comprises instructions.
In a third aspect, the application provides a use of the colloidal gold test strip of the first aspect in the preparation of a kit for detecting rheumatoid arthritis.
Drawings
Fig. 1 is a schematic diagram of a colloidal gold test strip of the present application. The detection area is (1) a sample loading area, (2) a colloidal gold labeling area, and (3) a detection area, wherein (31) is a first sub detection area, (32) a second sub detection area, (33) a third sub detection area, (34) a fourth sub detection area, (41) and (42) are water absorption areas, wherein (41) is a water absorption film, (42) is water absorption paper, and (5) is a bottom plate.
Fig. 2 is a color development schematic diagram of the colloidal gold test strip of the present application.
Wherein A in FIG. 2 and C in FIG. 2 are schematic diagrams of the colloidal gold test strip when the test strip is judged to be invalid; b in FIG. 2 is a schematic diagram showing a negative result; d in FIG. 2 is a schematic diagram showing the positive result of rheumatoid factor; e in FIG. 2 is a schematic diagram showing the positive result of the anti-mutant citrullinated vimentin antibody; f in FIG. 2 is a diagram showing the positive result of the anti-cyclic citrullinated peptide antibody; g in FIG. 2 is a schematic diagram showing the positive results of the rheumatoid factor and the anti-mutant citrullinated vimentin antibody; h in FIG. 2 is a schematic diagram of positive results of an anti-mutant citrullinated vimentin antibody and an anti-cyclic citrullinated peptide antibody; i in FIG. 2 is a schematic diagram showing the positive results of the rheumatoid factor and the cyclic citrullinated peptide; j in figure 2 is a schematic diagram showing positive results of the rheumatoid factor, the anti-mutant citrullinated vimentin antibody and the anti-cyclic citrullinated peptide antibody.
Detailed Description
In the application, 3 diagnostic markers are selected as joint inspection at the same time. Meanwhile, the content of the 3 markers in the human body is detected, whether the testee suffers from rheumatoid arthritis is comprehensively determined, and the accuracy of the detection result is more facilitated.
The colloidal gold test strip can simultaneously detect anti-mutant citrullinated vimentin antibodies, anti-cyclic citrullinated peptide antibodies and rheumatoid factors. By using the colloidal gold test strip, whether a subject suffers from rheumatoid arthritis can be judged by detecting the existence of an anti-mutant citrullinated vimentin antibody, an anti-cyclic citrullinated peptide antibody and a rheumatoid factor in human serum.
While this application contains many specifics, these should not be construed as limitations on the scope of the invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in the context of separate embodiments in this application can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.
Unless otherwise indicated, the terms herein have the same meaning as commonly understood by one of skill in the art, e.g., referring to colloidal gold test strips, kits, and units of value.
As used herein, the terms "comprises" and "comprising" mean either open or closed. For example, the term "comprises" or "comprising" may mean that other elements or steps or other elements not listed may also be included or included, or that only the listed elements or steps or other elements may be included or included.
Herein, the term "about" (e.g., in component amounts and reaction parameters) is to be interpreted in a sense that is generally understood by those skilled in the art. In general, the term "about" may be understood as any value within plus or minus 5% of a given value, for example, about X may represent any value in the range of 95% X to 105% X.
In a first aspect, the present application provides a colloidal gold test strip for rheumatoid arthritis detection, comprising a detection zone for rheumatoid arthritis-associated factors, wherein the detection zone comprises a first sub-detection zone coated with protein antigens of citrullinated vimentin and mutants thereof, a second sub-detection zone coated with cyclic citrullinated short peptide antigen, and a third sub-detection zone coated with human IgG Fc fragments.
In some embodiments, citrullinated vimentin and mutants thereof include fragments of citrullinated vimentin and mutants of the fragments.
In some embodiments, the amount of protein antigen coating of citrullinated vimentin and mutants thereof in the first sub-detection zone is 0.5-2.0mg/mL, and/or
In some embodiments, the amount of protein antigen coating of citrullinated vimentin and mutants thereof in the first sub-detection zone is 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, 1.6mg/mL, 1.7mg/mL, 1.8mg/mL, 1.9mg/mL, 2.0mg/mL, or any range therebetween.
In some embodiments, the cyclic citrullinated short peptide comprises a fragment of cyclic citrullinated short peptide.
In some embodiments, the amount of the cyclic citrullinated short peptide antigen coated in the second sub-detection zone is 0.5-2.0 mg/mL.
In some embodiments, the amount of the cyclic citrullinated short peptide antigen coated in the second sub-detection zone is 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, 1.6mg/mL, 1.7mg/mL, 1.8mg/mL, 1.9mg/mL, 2.0mg/mL, or any range therebetween.
In some embodiments, the amount of coating of human IgG Fc fragment in the third sub-detection zone is between 0.5 and 1.5 mg/mL.
In some embodiments, the amount of coating of human IgG Fc fragment in the third sub-detection zone is 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1.0mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, or any range therebetween.
In some embodiments, the colloidal gold test strip further comprises a base plate and one or more of the following regions immobilized on the base plate: a sample loading area; the colloidal gold labeling area is used for attaching a colloidal gold label to an antibody to be detected in a detection sample; a water absorption area.
In some embodiments, the loading zone, the colloidal gold labeled zone, the detection zone, and the water absorption zone are arranged from upstream to downstream, in the direction of flow of the test sample on the strip.
In some embodiments, there is an overlap between adjacent regions.
In some embodiments, there is an overlap interval between the loading region and the colloidal gold labeled region; in some embodiments, there is an overlap between the colloidal gold labeled region and the detection region; in some embodiments, there is an overlap interval between the detection zone and the bibulous zone.
In some embodiments, the overlap between the loading region and the colloidal gold labeled region is between 1 and 3 mm.
In some embodiments, the overlap between the loading zone and the colloidal gold labeling zone is 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm or any range therebetween.
In some embodiments, the overlap between the colloidal gold labeled region and the detection region is between 1 and 3 mm.
In some embodiments, the overlap between the colloidal gold labeled zone and the detection zone is 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm or any range therebetween.
In some embodiments, the overlap between the detection zone and the absorbent zone is between 1 and 3 mm.
In some embodiments, the overlap between the detection zone and the absorbent zone is 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm or any range therebetween.
In some embodiments, the sample loading zone is prepared from a sample pad.
In some embodiments, the sample pad is a polyester fiber film or a glass fiber film carrier.
In some embodiments, the sample pad is a polyester fiber film.
In some embodiments, the colloidal gold labeled region is a first membrane loaded with a colloidal gold labeled first antibody.
In some embodiments, the first antibody is an antibody capable of binding to an anti-mutant citrullinated vimentin antibody, an anti-cyclic citrullinated peptide antibody, a rheumatoid factor.
In some embodiments, the first antibody is a non-human mammalian anti-human IgG antibody.
In some embodiments, the first antibody is a rabbit anti-human IgG antibody, a mouse anti-human IgG antibody, or a goat anti-human IgG antibody.
In some embodiments, the first antibody is a rabbit anti-human IgG.
In some embodiments, the first antibody is loaded at 30-60 μ g.
In some embodiments, the first antibody is loaded at 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 μ g or any range therebetween.
In some embodiments, the first film is a fiberglass film or a polyester film.
In some embodiments, the first membrane is a fiberglass membrane.
In some embodiments, the colloidal gold has a particle average diameter of 40 to 60 nm.
In some embodiments, the colloidal gold has a particle average diameter of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60nm or any range therebetween.
In some embodiments, the absorbent region is prepared from an absorbent material.
In some embodiments, the absorbent region comprises an absorbent film and/or an absorbent paper.
In some embodiments, the water-absorbing capacity of the water-absorbing region is not less than 200 μ L.
In some embodiments, the absorbent paper absorbs no less than 200 μ L, 250 μ L, 300 μ L, 350 μ L, 400 μ L, 450 μ L, 500 μ L, 550 μ L, 600 μ L, 650 μ L, 700 μ L, 750 μ L, 800 μ L, 850 μ L, 900 μ L, 950 μ L, 1000 μ L of liquid.
In some embodiments, the detection zone further comprises a fourth sub-detection zone located downstream of the first to third sub-detection zones in the flow direction of the detection sample on the test strip for ensuring reliability of the detection result, and the fourth sub-detection zone is loaded with a second antibody capable of binding to the first antibody.
In some embodiments, the first sub-detection zone, the second sub-detection zone, the third sub-detection zone, and the fourth sub-detection zone are arranged in a flowing direction of the detection sample on the test strip.
In some embodiments, the first sub-detection zone, the third sub-detection zone, the second sub-detection zone, and the fourth sub-detection zone are arranged in a flowing direction of the test sample on the test strip.
In some embodiments, the second sub-detection zone, the first sub-detection zone, the third sub-detection zone, and the fourth sub-detection zone are arranged in a flowing direction of the detection sample on the test strip.
In some embodiments, the second sub-detection zone, the third sub-detection zone, the first sub-detection zone, and the fourth sub-detection zone are arranged in a flowing direction of the test sample on the test strip.
In some embodiments, the third sub-detection zone, the second sub-detection zone, the first sub-detection zone, and the fourth sub-detection zone are arranged in a flowing direction of the detection sample on the test strip.
In some embodiments, the third sub-detection zone, the first sub-detection zone, the second sub-detection zone, and the fourth sub-detection zone are arranged in a flowing direction of the detection sample on the test strip.
In some embodiments, the second antibody is a goat anti-rabbit IgG antibody and/or a mouse anti-rabbit IgG antibody.
In some embodiments, the second antibody is a rabbit anti-mouse IgG antibody and/or a goat anti-mouse IgG antibody.
In some embodiments, the second antibody is a murine anti-sheep IgG antibody and/or a rabbit anti-sheep IgG antibody.
In some embodiments, the second antibody is capable of binding to the first antibody and is selected from one or more of the following: goat anti-rabbit IgG, mouse anti-rabbit IgG, rabbit anti-mouse IgG, goat anti-mouse IgG, mouse anti-sheep IgG, and rabbit anti-sheep IgG.
In some embodiments, the first antibody is a rabbit anti-human IgG antibody; the second antibody is a goat anti-rabbit IgG antibody and/or a mouse anti-rabbit IgG antibody.
In some embodiments, the first antibody is a murine anti-human IgG antibody; the second antibody is a rabbit anti-mouse IgG antibody and/or a goat anti-mouse IgG antibody.
In some embodiments, the first antibody is a goat anti-human IgG antibody; the second antibody is a mouse anti-sheep IgG antibody and/or a rabbit anti-sheep IgG antibody.
In some embodiments, the first antibody is a rabbit anti-human IgG antibody and the second antibody is a goat anti-rabbit IgG.
In some embodiments, the spacing between each sub-detection zone is 1-1.5 cm.
In some embodiments, the distance between the first sub-detection zone coated with the protein antigen of citrullinated vimentin and mutants thereof and the second sub-detection zone coated with the cyclic citrullinated short peptide antigen is 1cm, 1.1cm, 1.2cm, 1.3cm, 1.4cm, 1.5cm or any range therebetween.
In some embodiments, the separation between the second sub-detection zone coated with the cyclic citrullinated short peptide antigen and the third sub-detection zone coated with the human IgG Fc fragment is 1cm, 1.1cm, 1.2cm, 1.3cm, 1.4cm, 1.5cm, or any range therebetween.
In some embodiments, the separation between the third sub-detection zone and the fourth sub-detection zone coated with human IgG Fc fragment is 1cm, 1.1cm, 1.2cm, 1.3cm, 1.4cm, 1.5cm, or any range therebetween.
In some embodiments, the detection zone comprises a second membrane, and each sub-detection zone is disposed on the second membrane.
In some embodiments, the sub-detection zones are distributed in stripes across the detection zone.
In some embodiments, the second film is a polyester film or a glass fiber film.
In some embodiments, the second membrane is a nitrocellulose membrane.
In some embodiments, the colloidal gold test strip further comprises a hard shell, and a window for exposing the loading area and each sub-detection area is formed on the hard shell.
In some embodiments, the colloidal gold test strip further comprises a hard shell, wherein a sample loading port and an observation window are arranged on the hard shell, the sample loading port exposes the sample loading area to the air, and the observation window obtains results of the detection line and the quality detection line.
In a second aspect, the present application provides a kit for detecting rheumatoid arthritis, which comprises the colloidal gold test strip of the first aspect.
In some embodiments, the kit further comprises instructions.
In a third aspect, the application provides a use of the colloidal gold test strip of the first aspect in the preparation of a kit for detecting rheumatoid arthritis.
In some embodiments, as shown in fig. 1, the present application provides a colloidal gold test strip for rheumatoid arthritis detection, which comprises a substrate (5), wherein a sample loading area (1), a colloidal gold labeling area (2), a detection area (3), and a water absorption area are sequentially arranged on the substrate (5); wherein the water absorption area comprises a water absorption film (41) and water absorption paper (42); rabbit anti-human IgG labeled by colloidal gold is adsorbed on the colloidal gold labeling area (2); the detection area (3) comprises a first sub detection area (31), a second sub detection area (32), a third sub detection area (33) and a fourth sub detection area (34); the first sub-detection area (31) is coated with citrullinated vimentin and protein antigens of mutants thereof, the second sub-detection area (32) is coated with cyclic citrullinated short peptide antigens, the third sub-detection area (33) is coated with human IgG Fc fragments, and the fourth sub-detection area (34) is coated with goat anti-rabbit IgG; the sub-detection zones (31, 32, 33 and 34) are distributed in strips on the detection zone (3).
In some embodiments, the present application provides a colloidal gold test strip for detecting anti-mutant citrullinated vimentin antibodies, anti-cyclic citrullinated peptide antibodies and rheumatoid factors, which comprises a base plate (5), wherein a sample pad (1), a glass fiber membrane (2), a nitrocellulose membrane (3), a water absorbing membrane (41) and a water absorbing paper (42) are sequentially arranged on the base plate (5); overlapping intervals are arranged between the sample pad (1) and the glass fiber membrane (2), between the glass fiber membrane (2) and the nitrocellulose membrane (3), between the nitrocellulose membrane (3) and the water absorbing membrane (41) and between the water absorbing membrane (41) and the water absorbing paper (42); wherein, the glass fiber membrane (2) is adsorbed with rabbit anti-human IgG labeled with colloidal gold; the detection kit comprises a nitrocellulose membrane (3) and is characterized by comprising a first sub-detection zone (31), a second sub-detection zone (32), a third sub-detection zone (33) and a fourth sub-detection zone (34); the first sub-detection area (31) is coated with citrullinated vimentin and protein antigens of mutants thereof, the second sub-detection area (32) is coated with cyclic citrullinated short peptide antigens, the third sub-detection area (33) is coated with human IgG Fc fragments, and the fourth sub-detection area (34) is coated with goat anti-rabbit IgG; the sub-detection zones (31, 32, 33 and 34) are distributed in stripes on the nitrocellulose membrane (3).
In some embodiments, the colloidal gold test strip uses the principle of immunological binding of antigen to antibody, and an indirect immunoassay is used.
In some embodiments, the protein antigen of citrullinated vimentin and mutants thereof, cyclic citrullinated short peptide antigen, human IgG Fc fragment are pre-coated on a nitrocellulose membrane.
In some embodiments, when a colloidal gold test strip is used, if an anti-mutant citrullinated vimentin antibody is present in the sample, the antibody binds to citrullinated vimentin to form a double antigen sandwich complex, showing a purple band on the detection zone of the test strip; if a negative sample is present, there is no purple band on the test strip at the test area.
In some embodiments, when a colloidal gold test strip is used, if an anti-cyclic citrullinated peptide antibody is present in the sample, the antibody binds to cyclic citrullinated peptide to form a double antigen sandwich complex, which exhibits a purple band on the test area of the test strip; if a negative sample is present, there is no purple band on the test strip at the test area.
In some embodiments, when a colloidal gold strip is used, if the sample contains rheumatoid factor, the rheumatoid factor binds to the human IgG Fc fragment to form a double antigen sandwich complex, showing a purple band on the detection zone of the strip; if a negative sample is present, there is no purple band on the test strip at the test area.
In some embodiments, the subject is assessed for rheumatoid arthritis using the color development results of a colloidal gold test strip. If the sub-detection area coated with the protein antigens of citrullinated vimentin and mutants thereof, the sub-detection area coated with the cyclic citrullinated short peptide antigen and the sub-detection area coated with the human IgG Fc fragment are all in color, and the sub-detection area coated with goat anti-rabbit IgG is also in color, the test subject is judged to be suffering from rheumatoid arthritis.
In some embodiments, the liquid to be detected is applied to a sample pad, which sufficiently absorbs the liquid to be detected. And then, the liquid to be detected is chromatographed on a glass cellulose membrane and reacts with the rabbit anti-human IgG marked by the colloidal gold, and when the liquid is chromatographed on a nitrocellulose membrane, the liquid is respectively reacted with citrullinated vimentin, cyclic citrullinated short peptide and human IgG Fc fragment, and when the content of the anti-mutant citrullinated vimentin antibody, the anti-cyclic citrullinated peptide antibody and the rheumatoid factor in the sample is higher than the lowest detection limit of the test strip on the test strip, a purple strip is presented in the corresponding sub-detection area. If the content of the anti-mutant citrullinated vimentin antibody, the anti-cyclic citrullinated peptide antibody and the rheumatoid factor in the sample is lower than the lowest detection limit of the test strip on the anti-mutant citrullinated vimentin antibody and the anti-cyclic citrullinated peptide antibody, a purple strip does not exist in the corresponding sub-detection area.
In some embodiments, a negative result is considered to be obtained when a purple band appears at the sub-detection zone that is coated with only goat anti-rabbit IgG.
In some embodiments, a positive result is considered to be obtained when a purple band is present at each of the sub-detection zones coated with goat anti-rabbit IgG, the sub-detection zone coated with citrullinated vimentin, the sub-detection zone coated with cyclic citrullinated short peptide, and the sub-detection zone coated with human IgG Fc fragment.
In some embodiments, when there is no purple band at the sub-detection zone coated with goat anti-rabbit IgG, the result is considered to be an invalid result regardless of the presence of a purple band at the sub-detection zone coated with citrullinated vimentin, at the sub-detection zone coated with cyclic citrullinated short peptides, at the sub-detection zone coated with human IgG Fc fragment.
The colloidal gold test strip is simple in design, capable of effectively reducing errors, simple, convenient and fast to operate, visual and accurate in result display, low in cost and high in sensitivity.
Examples
The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. Experimental procedures without specific conditions noted in the examples below are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages are by mass and ratios between components are in molar ratios. Unless defined otherwise, all terms of art or science used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are exemplary only.
Example 1: preparation of colloidal gold test strip
According to the structure shown in figure 1, colloidal gold test paper for detecting rheumatoid factors, anti-mutant citrullinated vimentin antibodies and anti-cyclic citrullinated peptide antibodies is prepared. The colloidal gold test strip comprises a bottom plate 5, wherein a sample pad 1, a glass fiber membrane 2, a nitrocellulose membrane 3, a water absorption membrane 41 and water absorption filter paper 42 which are closely connected from left to right are attached to the bottom plate 5, the sample pad presses the glass fiber membrane 2 for about 1-3mm when the sample pad 1 is closely connected with the glass fiber membrane 2, the glass fiber membrane 2 presses the nitrocellulose membrane 3 for about 1-3mm when the glass fiber membrane 2 is closely connected with the nitrocellulose membrane 3, the water absorption membrane 41 presses the nitrocellulose membrane 3 for about 1-3mm when the nitrocellulose membrane 3 is closely connected with the water absorption membrane 41, and the water absorption filter paper 42 presses the water absorption membrane 41 for about 1-3mm when the water absorption membrane 41 is closely connected with the water absorption filter paper 42; the glass cellulose membrane 2 is adsorbed with colloidal gold labeled human IgG Fc segment (as antigen) 31, citrullinated vimentin and its mutant protein antigen 32, cyclic citrullinated short peptide antigen 33 and goat anti-rabbit IgG 34.
Example 2: use and result of colloidal gold test strip
When the detection liquid chromatography detection device is used, detection liquid is added to the sample pad 1, the sample pad 1 fully absorbs the detection liquid, the detection liquid firstly chromatographs to the glass fiber membrane 2 to be completely contacted with the gold-labeled antigen sprayed with gold and fully reacts, and then chromatography is carried out to the nitrocellulose membrane 3 to react. The specific results are judged as follows:
negative results: only in control region C (where the nitrocellulose membrane 34 is coated with goat anti-rabbit IgG) is a red-purple line present, as shown in FIG. 2B, where the red-purple color is shown in grey.
Positive results: in addition to the red purple line in the control region C (goat anti-rabbit IgG coating of the nitrocellulose membrane 34), one, two or three red purple lines were present in the detection region (human IgG Fc fragment (31), citrullinated vimentin (32), cyclic citrullinated short peptide antigen (33) coating of the nitrocellulose membrane).
For example, red purple lines appeared on both the goat anti-human IgG coating on nitrocellulose membrane (34) and the human IgG Fc fragment coating on nitrocellulose membrane (31) (see D in fig. 2, where red purple is shown in grey), indicating positive for rheumatoid factor.
For example, red purple lines appeared in the goat anti-human IgG coating (34) on nitrocellulose membranes, in the citrullinated vimentin coating (32) on nitrocellulose membranes and its mutants (see E in fig. 2, where red purple is shown in grey), indicating that mutant citrullinated vimentin is positive.
For example, red purple lines appeared in the goat anti-human IgG coating (34) on the nitrocellulose membrane and the short cyclic citrullinated peptide antigen coating (33) on the nitrocellulose membrane (see F in FIG. 2, where red purple is shown in grey), indicating that the cyclic citrullinated peptide is positive.
For example, red purple lines (see G in fig. 2, where red purple is shown in grey) appeared in goat anti-human IgG coating (34) on nitrocellulose membrane, human IgG Fc fragment (31) on nitrocellulose membrane, citrullinated vimentin on nitrocellulose membrane, and protein antigen coating (32) on mutants thereof, indicating that rheumatoid factor, mutant citrullinated vimentin, was positive.
For example, red purple lines appeared in goat anti-human IgG coating (34) on nitrocellulose membrane, citrullinated vimentin and its mutant protein antigen coating (32) on nitrocellulose membrane, and cycotropin short peptide antigen coating (33) on nitrocellulose membrane (see H in fig. 2, where red purple is shown in gray), indicating that mutant citrullinated vimentin, cycotropin peptide antibodies were positive.
For example, red purple lines appeared in the goat anti-human IgG coating (34) of the nitrocellulose membrane, the human IgG Fc fragment coating (31) of the nitrocellulose membrane, and the cyclic citrullinated short peptide antigen coating (33) of the nitrocellulose membrane (see I in FIG. 2, in which red purple is shown in gray), indicating that the rheumatoid factor, cyclic citrullinated peptide antibody, is positive.
For example, red purple lines appeared in goat anti-human IgG coating (34) on nitrocellulose membrane, human IgG Fc fragment coating (31) on nitrocellulose membrane, citrullinated vimentin and its mutant protein antigen coating (32) on nitrocellulose membrane, and cycotinamic acid short peptide antigen coating (33) on nitrocellulose membrane (see J in fig. 2, where red purple is shown in gray), indicating that the antibodies were positive for rheumatoid factor, mutant citrullinated vimentin, and cycotinamic acid peptide.
Invalid result: when the control line (goat anti-human IgG coated on nitrocellulose membrane) did not show a reddish purple band, the colloidal gold test strip was judged to be invalid whether the detection line showed a reddish purple band, as shown in a of fig. 2 or C of fig. 2, where the reddish purple is represented in gray.
Finally, it should be understood that while the various aspects of the present specification describe specific embodiments, those skilled in the art will readily appreciate that the disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it is to be understood that, unless explicitly stated otherwise, the disclosed subject matter is not limited to the particular compositions, methods, and/or formulations, etc., described herein. Moreover, those of ordinary skill in the art will recognize that certain changes, modifications, permutations, variations, additions, subtractions and sub-combinations may be made in accordance with the teachings herein without departing from the spirit of the present specification. It is therefore intended that the following appended claims be interpreted as including all such alterations, modifications, permutations, variations, additions, subtractions and sub-combinations as fall within the true spirit and scope thereof. The scope of protection of this patent is not only to triple joint inspection, but also to include quadruple joint inspection or quintuplet inspection as extended from this patent.
Claims (10)
1. The colloidal gold test strip for detecting the rheumatoid arthritis comprises a detection area aiming at a rheumatoid arthritis related factor, wherein the detection area comprises a first sub-detection area coated with protein antigens of citrullinated vimentin and mutants thereof, a second sub-detection area coated with cyclic citrullinated short peptide antigen and a third sub-detection area coated with human IgG Fc fragments; optionally, the step of (a) is carried out,
the coating amount of the protein antigen of citrullinated vimentin and mutants thereof in the first sub-detection area is 0.5-2.0mg/mL, and/or
The coating amount of the cyclic citrullinated short peptide antigen in the second sub-detection area is 0.5-2.0mg/mL, and/or
The coating amount of the human IgG-Fc fragment in the third sub-detection area is 0.5-1.5 mg/mL.
2. The colloidal gold test strip of claim 1, further comprising a base plate and one or more of the following zones immobilized on the base plate:
a sample loading area;
the colloidal gold labeling area is used for attaching a colloidal gold label to an antibody to be detected in a detection sample;
a water absorption zone;
optionally, the sample loading area, the colloidal gold labeling area, the detection area and the water absorption area are arranged from upstream to downstream according to the flowing direction of the detection sample on the test strip;
further optionally, there is an overlapping portion between adjacent regions, further optionally, the overlapping interval between the loading region and the colloidal gold labeled region is 1-3mm and/or the overlapping interval between the colloidal gold labeled region and the detection region is 1-3mm and/or the overlapping interval between the detection region and the water absorption region is 1-3 mm.
3. The colloidal gold test strip of claim 2, wherein the sample loading zone is prepared from a sample pad, optionally a polyester film or glass film carrier.
4. The colloidal gold test strip of claim 2, wherein the colloidal gold labeled region is a first membrane loaded with a first antibody labeled with colloidal gold;
optionally, the first antibody is an antibody capable of recognizing human IgG, such as a rabbit anti-human IgG antibody, a mouse anti-human IgG antibody, or a goat anti-human IgG antibody; and/or
The loading amount of the first antibody is 30-60 mug; and/or
The first film is a glass fiber film or a polyester fiber film; and/or
The average diameter of the particles of the colloidal gold is 40-60 nm.
5. The colloidal gold test strip of claim 2, wherein the water-absorbing region is made of a water-absorbing material; optionally, the step of (a) is carried out,
the water absorption area comprises a water absorption film and/or water absorption paper; and/or
The water absorption capacity of the water absorption area is not lower than 200 mu L.
6. The colloidal gold test strip of any one of claims 1-5, wherein the detection zone further comprises a fourth sub-detection zone downstream of the first to third sub-detection zones in the flow direction of the test sample on the test strip for ensuring the reliability of the detection result, the fourth sub-detection zone being loaded with a second antibody capable of binding to the first antibody, optionally but not limited to the following antibodies,
the second antibody is a goat anti-rabbit IgG antibody and/or a mouse anti-rabbit IgG antibody; or
The second antibody is a rabbit anti-mouse IgG antibody and/or a sheep anti-mouse IgG antibody; or
The second antibody is a mouse anti-sheep IgG antibody and/or a rabbit anti-sheep IgG antibody.
7. The colloidal gold test strip of any one of claims 1-6, wherein the spacing between each sub-detection zone is 1-1.5 cm.
8. The colloidal gold test strip of any one of claims 1-7, wherein the detection zone comprises a second membrane, each sub-detection zone disposed on the second membrane, optionally,
the second film is a nitrocellulose film or a polyester film.
9. A kit for rheumatoid arthritis detection comprising the colloidal gold test strip of any one of claims 1-8, optionally further comprising instructions.
10. Use of the colloidal gold test strip of any one of claims 1-8 in the manufacture of a kit for the detection of rheumatoid arthritis.
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| CN202010146693.0A CN111308096A (en) | 2020-03-05 | 2020-03-05 | Colloidal gold test strip for detecting rheumatoid arthritis |
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