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WO2011012053A1 - Bande d’essai pour la détection d’un anticorps anti-peptide cyclique citrulliné dans le sang et procédé de préparation associé - Google Patents

Bande d’essai pour la détection d’un anticorps anti-peptide cyclique citrulliné dans le sang et procédé de préparation associé Download PDF

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Publication number
WO2011012053A1
WO2011012053A1 PCT/CN2010/075228 CN2010075228W WO2011012053A1 WO 2011012053 A1 WO2011012053 A1 WO 2011012053A1 CN 2010075228 W CN2010075228 W CN 2010075228W WO 2011012053 A1 WO2011012053 A1 WO 2011012053A1
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Prior art keywords
antibody
test strip
pad
detection
protein
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PCT/CN2010/075228
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English (en)
Chinese (zh)
Inventor
张玥
韩永俊
高成秀
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Shanghai Kexin Biotech Co Ltd
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Shanghai Kexin Biotech Co Ltd
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Publication of WO2011012053A1 publication Critical patent/WO2011012053A1/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/18—Post-translational modifications [PTMs] in chemical analysis of biological material citrullination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • 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

Definitions

  • Test strip for detecting blood anti-cyclic citrullinated peptide antibody and preparation method thereof
  • the invention belongs to the field of medical immunology, and particularly relates to a test strip for detecting an anti-cyclic citrullinated peptide antibody by colloidal gold immunochromatography and a preparation method thereof. Background technique
  • Rheumatoid arthritis also known as rheumatoid arthritis (RA;)
  • RA rheumatoid arthritis
  • RA is a systemic inflammatory disease characterized by chronic polyarticular lesions caused by abnormal growth of joint tissues or glenoids, resulting in random joint loss.
  • Recent studies have shown that joint injury in RA patients occurs two years after the onset of the disease, and early effective treatment can prevent subsequent progression of the disease. Therefore, early diagnosis and early treatment have become the focus.
  • patients with RA do not always show typical symptoms and symptoms early in the disease because they may not fully comply with the RA diagnostic criteria, which makes diagnosis difficult.
  • RA is classified as a systemic autoimmune disease due to the autoantibody of the IgG Fc segment corresponding to RF, and the presence of other antibodies.
  • RF has been the only serological marker for RA in clinical use.
  • the sensitivity of RF in RA is between 60-80%, and its specificity is also quite low, because RF can be widely and frequently recognized in other diseases, including various connective tissue diseases, chronic liver diseases and Infectious diseases, even among a few healthy people. Therefore, RF is applied to the diagnostic criteria of RA, but its early diagnostic value is not satisfactory.
  • Anti-CCP antibody is the first choice for the diagnosis of RA, especially early RA. It is suitable for the diagnosis of RA and the diagnosis of RA and other inflammatory arthritis and other forms of polyarthritis. It can also confirm the serological RF negative. RA. Specifically, it is manifested in the following four aspects: 1. The accuracy of anti-CCP antibody for clinical diagnosis of RA: specificity 95%, sensitivity 80% [1]; 2. Anti-CCP antibody can be detected in most patients with RA for the first time. Occurred in patients with early RA [2]; 3. Anti-CCP antibody titer associated with disease activity is a predictive outcome [3]; 4. Continuous monitoring of anti-CCP antibodies as an indicator of early RA aggressive progression [4].
  • the current detection method for anti-cyclic citrullinated peptide (CCP) antibody is enzyme-linked immunosorbent assay (ELISA), currently on the market.
  • ELISA enzyme-linked immunosorbent assay
  • the commercial anti-CCP antibody detection kit is mainly an ELISA kit.
  • the ELISA method is cumbersome. It takes about three hours to complete the whole experiment. It also requires professional immunology technicians to carry out the experiment in the laboratory, and the test results should be read by the microplate reader. This is the experiment in the primary medical institution. It is difficult to achieve the experimental test of the project in the room and small clinics. At the same time, the ELISA method is susceptible to various environmental factors such as temperature and incubation time, which brings many inconveniences to the test.
  • the gold-immunochromatography assay is a colloidal gold labeling technique.
  • the colloidal gold is used as the tracer, the strip fiber chromatography material is used as the solid phase, and the sample solution is applied to the chromatographic strip by the capillary effect.
  • the upper movement causes the analyte in the sample to immunoreact with the colloidal gold label on the binding pad, and is immunoreactive with the antigen (or antibody) on the strip fiber chromatography material to be trapped, thereby forming a visible purple Red strips, with intuitive experimental results, achieve rapid detection [5].
  • the positive result is judged based on the presence or absence of the purple-red band on the test line.
  • the stability is good, the operator does not need training, the operation is simple and fast, no cryopreservation is required, and storage and transportation are convenient.
  • test strip for detecting anti-CCP antibody colloidal gold immunochromatography.
  • the present invention introduces a cyclic citrulline peptide into a test strip to achieve high specificity, high sensitivity, and high accuracy of detection performance.
  • the interpretation results are simple and only take a few minutes; provide conditions for rapid screening of RA for early diagnosis and early treatment; meet the needs of grassroots laboratories, instant detection, and bedside detection.
  • the detection procedure is cumbersome and the detection time is long;
  • Detection reagents need to be refrigerated for transport and storage
  • Detection requires special equipment.
  • the ELISA method requires a microplate reader
  • the present invention applies colloidal gold chromatography to the detection of anti-cyclic citrullinated peptide antibodies, and at the same time applies the cyclic citrulline peptide to colloidal gold chromatography for the first time, using an indirect method.
  • realize the detection of anti-cyclic citrullinated peptide antibodies in plasma and serum samples greatly reduce the economic cost, achieve high specificity, high sensitivity, high accuracy of detection performance, and quickly and conveniently assist in the diagnosis of early rheumatoid arthritis (RA).
  • RA early rheumatoid arthritis
  • a first aspect of the present invention provides a test strip for detecting an anti-cyclic citrullinated peptide antibody, wherein the test strip comprises a bonding pad and a nitrocellulose membrane which are sequentially adhered to each other in a horizontal direction and adhered to a bottom plate, wherein The binding pad is coated with a gold standard antibody (a) and a gold standard antibody (b).
  • the nitrocellulose membrane has a detection line and a quality control line, and the detection line coating can specifically bind to the anti-cyclic citrullinated peptide antibody. Detection of antigen, quality control line coated with quality control antibody (c);
  • the antibody (a) binds to the detection antigen only in combination with the anti-cyclic citrullinated peptide antibody; Among them, the antibody (b) does not bind to the anti-cyclic citrullinated peptide antibody and the detection antigen.
  • test strip further comprises a sample pad and/or an absorbent pad.
  • the test strip comprises a bottom plate and a sample pad, a bonding pad, a nitrocellulose membrane and an absorbent pad which are sequentially bonded to each other on the bottom plate as viewed in the horizontal direction.
  • the OD ratio of the gold label antibody (a) and the gold label antibody (b) on the binding pad is 5:1 to 1 :1, preferably OD50 to 100/OD 10 to 40.
  • the antibodies (a), (b) and (c) are selected from a monoclonal antibody or a polyclonal antibody, the monoclonal antibody is a mouse or a rabbit source, and the polyclonal antibody is a mouse.
  • the antibody (a) is selected from the group consisting of murine anti-human IgG, staphylococcal protein A, streptococcal G protein, and goat anti-human IgG.
  • the antibodies (b) and (c) are selected from the group consisting of sheep IgG and anti-goat IgG, rabbit IgG and anti-rabbit IgG, and murine IgG and anti-mouse IgG.
  • the detection antigen is a conjugate of a cyclic citrulline peptide and a carrier protein selected from the group consisting of bovine serum albumin, avidin, streptavidin, thyroglobulin, Keyhole limpet hemocyanin, egg protein and synthetic polylysine.
  • the antigen comprises 2 to 8 cyclic citrullinated peptides and a polylysine core matrix (MAP) linked to the peptide, and the MAP can be linked to a carrier protein, wherein each of the cyclic melons
  • the acid peptides can be the same or different antigenic epitopes.
  • Preferred is a MAP-carrier protein conjugate.
  • a second aspect of the present invention provides a method of preparing a test strip of the present invention, the method comprising the steps of: 1) providing a bottom plate; 2) providing a binding pad, and wrapping the gold standard antibody (a) and the gold standard antibody (b) By the bonding pad; 3) providing a nitrocellulose membrane, wherein the nitrocellulose membrane has a detection line and a quality control line, and the detection line is coated with a detection antigen that specifically binds to the anti-cyclic citrullinated peptide antibody And coating the test line with the quality control antibody (c); and 4) sequentially affixing the nitrocellulose membrane and the bonding pad to each other on the bottom plate to obtain the test strip.
  • the invention also provides a kit comprising the test strip of the invention, a sampler, and instructions.
  • Advantages of the present invention over existing test strips and detection methods include:
  • the present invention introduces the cyclic citrulline peptide into the colloidal gold chromatography test strip for the first time, and realizes the rapid and simple auxiliary diagnosis of early rheumatoid arthritis.
  • the detection antigens used in the present invention the antigenic determinants of the branched cyclic citrulline peptides may be different, and the antigen epitopes are detected to be diversified, and the detection specificity is improved.
  • the cyclic citrulline peptide antigen of the present invention mimics the natural epitope conformation well, thereby improving its sensitivity to detection of rheumatoid arthritis. 4.
  • the present invention couples a cyclic citrulline peptide to a carrier protein to form a macromolecular protein, so that it can be well coated on the membrane.
  • the introduction of the carrier protein greatly increases the epitope space of citrulline, reduces the steric hindrance of the immune response, and improves the detection sensitivity. 5.
  • Samples directly loaded onto the test strip compared to the published detection method for anti-cyclic citrullinated peptide antibody detection After the pad, the results can be observed within 5 ⁇ 10min; no special instruments are needed, and bedside detection and outpatient clinic detection can be realized; the operation is simple, only one step is needed, the operator does not need training, so the detection cost is low; there is no special requirement for temperature. , no need to freeze, easy to store and transport, can be stored for 12 months at room temperature.
  • Figure 1 is a schematic view showing the side structure of the present invention.
  • the test strip is attached to the support rubber sheet (7) in sequence with the sample pad (1), the bonding pad (2), the nitrocellulose film (3), and the absorbent pad (6).
  • the nitrocellulose membrane (3) has a detection line (4) and a quality control line (5), the detection line (4) is coated with a detection antigen, and the quality control line (5) is coated with an antibody (c).
  • Figure 2 shows a plan view of the test strip of the present invention.
  • the numbers are the same as those in Figure 1.
  • Figure 3 is a schematic diagram of the detection results of the present invention. After the addition of the reaction, after 3 to 5 minutes of reaction, as shown in Fig. 2a and 2b, the result was positive, indicating that the serum contained the anti-cyclic citrullinated peptide antibody; as shown in Fig. 2c, the result was negative, indicating that the serum contained no anti- Cyclic citrullinated peptide antibody; as shown in Fig. 2d, 2e, the result is that the test strip fails, that is, there is no purple-red strip at the quality control line, no matter whether there is a strip at the detection line, it indicates that the test strip fails.
  • a first aspect of the invention relates to a test strip for detecting an anti-cyclic citrullinated peptide antibody, wherein the test strip comprises a bonding pad and a nitrocellulose membrane which are successively lapped to each other in a horizontal direction on a bottom plate, wherein The binding pad is coated with a gold standard antibody (a) and a gold standard antibody (b).
  • the nitrocellulose membrane has a detection line and a quality control line, and the detection line coating can specifically bind to the anti-cyclic citrullinated peptide antibody.
  • the detection antigen, the quality control line is coated with the quality control antibody (c); wherein, the antibody (a) binds to the detection antigen only in combination with the anti-cyclic citrullinated peptide antibody; wherein, the antibody (b) is not resistant Cyclic citrullinated peptide antibodies and detection of antigen binding.
  • test strip of the present invention further comprises a sample pad and/or an absorbent pad.
  • the substrate, sample pad, bond pad, nitrocellulose membrane, and absorbent pad used in the present invention can be prepared using various materials known in the art. Products of the following specifications are commercially available for the preparation of test strips of the present invention:
  • the binding pad of the present invention is selected from the group consisting of a polyester film or a glass fiber membrane carrier.
  • the bonding pad of the present invention may be a bonding pad impregnated with a treatment liquid (for example, selected from a polyester film or a glass fiber membrane carrier), and the treatment liquid contains 0.5% to 10% of one or several macromolecular inert proteins.
  • a treatment liquid for example, selected from a polyester film or a glass fiber membrane carrier
  • the treatment liquid contains 0.5% to 10% of one or several macromolecular inert proteins.
  • 0.1% ⁇ 3% ion salt slow Flush and 0.1% to 1% - one or several surfactants.
  • the surfactant can be an acidic, neutral, basic or amphoteric active agent. According to the process optimization experiment, select the appropriate reagent components, soak for 20 ⁇ 60 minutes, and dry at 37 °C.
  • the macromolecular inert protein is preferably PVA or BSA
  • the ionic salt buffer is preferably sodium phosphate
  • the surfactant is preferably Triton X-100.
  • the bond pad is treated with a treatment liquid: 1% PVA in ultrapure water,
  • the sample pad of the present invention can be treated by a sample pad treatment solution containing 0.5% to 10% - one or several macromolecular inert proteins, 0.1% to 3% ion salt buffer solution and 0.01% to 0.05% - kind or several surfactants.
  • the surfactant can be an acidic, neutral, basic or amphoteric active agent.
  • the appropriate reagent component can be selected according to the process optimization experiment, and the sample pad is evenly sprinkled on the sample pad by 30 ⁇ 50 mL/piece of the treatment liquid, and dried at 37 ° C as a spare sample pad.
  • the macromolecular inert protein is preferably BSA, PEG20000, sodium cholate, the ionic salt buffer is preferably Borax buffer, and the surfactant is preferably TWEEN-20.
  • the sample pad is treated with the following treatment solution: 0.05 MBorax in ultrapure water,
  • the sample pad (if present), the bonding pad, the nitrocellulose film, and the absorbent pad (if present) are sequentially bonded to each other on the substrate.
  • the "horizontal direction” or the like described herein refers to the test strip shown in FIG. 1 as an example, viewed from left to right, or from right to left, the order of overlapping of each mat or film is a sample mat. , a bonding pad, a nitrocellulose membrane and an absorbent pad. That is, the overlapping order of these films and mats does not change regardless of the direction from which they are viewed.
  • the pad or film has at least one end in contact with at least one end of the other pad or film, including at least a portion of at least one end of each pad or film being pressed against at least one end of the other pad or film.
  • At least a portion, or a contact that is pressed under the at least a portion, as shown in FIG. Figure 1 shows a preferred embodiment of the invention. However, they are in contact with each other in other ways, for example, from a vertical direction, a portion of the sample pad is pressed under the bonding pad, etc., and is also included in the range of "lapping" as long as the film and the pad are between Contact can effectively pass the sample through the membranes and pads in sequence based on capillary action.
  • the length of the sample pad is about 2.0 to 2.5 cm, and the overlapping portion with the bonding pad is about 4 to 8 mm long.
  • the length of the bonding pad is about 5 to 10 mm, and the overlap with the nitrocellulose film is about 1 to 3 mm.
  • the length of the nitrocellulose membrane is about 1.5 to 2.5 cm.
  • the length of the absorbent pad is about 1.5 to 3.0 cm, and the overlap with the nitrocellulose membrane is about 1 to 3 mm.
  • the width of the assembled test strip of the bottom plate, the sample pad, the bond pad, the nitrocellulose membrane, and the absorbent pad may be determined according to the actual situation, and is usually in the range of 2 mm to 5 cm, preferably 3 mm, 4 mm, or the like. Their thickness is also a conventional thickness in the art.
  • the width of the quality control line and the detection line on the bonding pad is usually about l-3 mm.
  • the distance between the detection line and the overlapping bonding pad end is about 8-13 mm, and the distance between the detecting line and the quality control line is usually 5-8 mm, and the quality control line and the overlapping absorbent pad end The distance is approximately 5-8mm.
  • the antibodies (a), (b) and (c) used in the present invention may be selected from monoclonal antibodies or polyclonal antibodies, which are murine or rabbit sources, and the polyclonal antibodies are murine and rabbit.
  • Source horse source, sheep source or guinea pig source, wherein the antibody (a) may also be a staphylococcal protein A or a streptococcus G protein.
  • the antibodies in their broadest sense include monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies, and antibody fragments so long as they have the specificity of the desired binding.
  • Monoclonal antibodies for use in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al. [6] or by recombinant DNA methods (see U.S. Patent 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as Clackson et al. [7] and Marks et al. [8].
  • the polyclonal antibody to be used according to the present invention can be produced by the animal immunization method described by Chen Xueqing et al. [9].
  • the protein A protein (Protein A) and the protein G protein (Protein G) to be used in the present invention are prepared by a prokaryotic expression cloning gene method described by J. Sambrook et al. [10].
  • Antibody (b) and antibody (c) are a pair of antigen-antibodies that can undergo a specific binding reaction, such as sheep IgG and anti-goat IgG, rabbit IgG and anti-rabbit IgG, mouse IgG and anti-mouse IgG, etc., For ease of explanation, in the specification and corresponding specific examples, antibody (b) and antibody (c) are correspondingly rabbit IgG and goat anti-rabbit IgG.
  • the binding pad coated tracer marker is a labeled mixture of colloidal gold particles and an antibody, specifically a colloidal gold tracer labeled capture antibody, and then a macromolecular inert protein such as bovine serum albumin, casein, etc.
  • a macromolecular inert protein such as bovine serum albumin, casein, etc.
  • the detection antigen of the present invention may be a cyclic citrullinated peptide or a branched antigenic peptide (MAP) structure.
  • MAP uses poly-lysine (Poly-Lys, PL) as the core matrix to chemically synthesize multiple cyclic citrullinated peptides on the branches, while the branched-chain citrullinated peptides can be found in the latest research.
  • the antigenic determinant with the scientific discovery of the corresponding update of the antigenic determinant on the branched chain, the cyclic citrullinated peptide, greatly improves the detection specificity.
  • This MAP structure is a dendritic structure, which increases the molecular weight and can well mimic the natural epitope conformation, thereby improving its sensitivity to RA detection.
  • the number of epitopes of MAP may be 2-10, preferably 2-8, more preferably 3-7.
  • the cyclic citrulline peptide-carrier protein conjugate is coupled by chemical synthesis, and does not affect the immunoreactivity of the antigenic determinant of the cyclic citrullinated peptide.
  • the experiment shows that the cyclic citrulline peptide-carrier is indeed added. The coating rate of the protein conjugate, the positive signal is greatly enhanced. This conjugate was used as the detection antigen of the detection system.
  • Suitable carrier proteins are bovine serum albumin, avidin, streptavidin, thyroglobulin, keyhole limpet hemocyanin (KLH), egg protein and synthetic polylysine.
  • the carrier is bovine serum albumin, avidin or streptavidin.
  • the amount of the antibodies (a) and (b) coated onto the binding pad may be 0.5 to 4 ⁇ l / ⁇ per antibody, for example, the amount It may be 1-3 ⁇ 1/ ⁇ , 1-2.5 ⁇ 1/ ⁇ , 1-2 ⁇ 1/ ⁇ , 0.5 ⁇ 2.5 ⁇ 1/ ⁇ , and the like.
  • the amount of the detection antigen coated onto the detection line may be 1 to ⁇ /cm, for example, 2 to 8 ⁇ l/ ⁇ , 3 to 7 ⁇ l/ ⁇ , 4 to 6 ⁇ l/ ⁇ , 1 to 3 ⁇ l/ ⁇ , and the like.
  • the amount of the antibody (c) coated on the quality control line may be 1 to 10 ⁇ l / ⁇ , for example, 2 to 8 ⁇ l / ⁇ , 3 to 7 ⁇ l / ⁇ , 4 to 6 ⁇ l / ⁇ , 1 to 3 ⁇ l / ⁇ , and the like.
  • the antibodies (a) and (b) of the present invention are colloidal gold-labeled.
  • Colloidal gold labeling technology is a novel immunolabeling technique that uses colloidal gold as a tracer and antigen-antibody reaction.
  • Colloidal gold is polymerized into gold particles of a specific size by chloroauric acid (HAuCl 4 ) under the action of reducing agents such as white phosphorus, tannic acid/sodium citrate and trisodium citrate (preferably trisodium citrate reducing agent). Since the electrostatic action becomes a stable colloidal state, it is called colloidal gold.
  • the colloidal gold mark is essentially a coating process in which the protein polymer is adsorbed onto the surface of the colloidal gold particles.
  • the adsorption mechanism is a negative charge on the surface of the colloidal gold particles, and forms a strong bond with the positive charge groups of the protein molecules due to electrostatic interaction.
  • This spherical colloidal gold particle has a high electron density and can be used in a non-covalent manner for a variety of biopolymer materials such as staphylococcus, immunoglobulins, toxins, glycoproteins, enzymes, antibiotics, hormones, bovine serum albumin, etc. The combination makes it an excellent marker for immune response and is therefore widely used for the detection of various substances.
  • the antibodies (a) and (b) of the present invention can be labeled using known colloidal gold labeling techniques.
  • the test strip of the present invention is as shown in Fig. 1, wherein nitrocellulose is sequentially adhered to each other in the vertical direction on the support rubber sheet PVC (7).
  • the binding pad (2) is coated with a colloidal gold-labeled antibody (a) and a colloidal gold-labeled antibody (b).
  • the nitrocellulose membrane (3) has a detection line (4) and a quality control line (5), the detection line (T) is coated with a detection antigen, and the quality control line (C) is coated with an antibody (c).
  • the detection antigen is a conjugate of MAP and a carrier protein.
  • Vertical direction refers to the direction from bottom to top in Figure 1. It is of course also possible to change the position of each pad or film in the vertical direction as long as it does not affect the sample's movement along the pads and membranes based on capillary action. These are included in the scope of the "overlay" in this article.
  • the detection line on the nitrocellulose membrane detects a BSA-MAP conjugate having an antigen of 1.0 to 2.5 ⁇ l/ ⁇ , an avidin-biotinylated MAP conjugate or a chain member.
  • the avidin-biotinylated MAP conjugate, the antibody (c) on the quality control line is a goat anti-rabbit IgG of 1.0 to 3.0 ⁇ l/ ⁇ .
  • the antibody (a) is selected from the group consisting of a mouse anti-human IgG, a grapevine A protein (SPA), a Streptococcus G protein or a goat anti-human IgG, and the antibody (b) is a rabbit IgG, an antibody (c) is a goat anti-rabbit IgG, the antigen is a BSA-MAP conjugate, an avidin-biotinylated MAP conjugate or a streptavidin-biotinylated MAP conjugate; an antibody coated onto a binding pad (a)
  • the amount of P (b) is 0.5 to 4 ⁇ l / ⁇ per antibody, for example, the amount may be 1-3 ⁇ l / ⁇ , 1-2.5 ⁇ l / ⁇ , 1-2 ⁇ l / ⁇ , etc.;
  • the amount of detection antigen on the line is 1 ⁇ 10 ⁇ 1/ ⁇ , for example 2 ⁇ 8 ⁇ 1/ ⁇ , 3 ⁇ 7 ⁇ 1/ ⁇ , 4 ⁇ 6 ⁇ 1/ ⁇ , 1.0 ⁇
  • nitrocellulose membrane wherein the nitrocellulose membrane has a detection line and a quality control line, and the detection line is coated with a detection antigen that specifically binds to the anti-cyclic citrullinated peptide antibody, and the quality control antibody is used. (c) coating the test line; and
  • the method of the invention further comprises the step of providing a sample pad and/or an absorbent pad.
  • the step of providing a sample pad of the present invention further comprises: treating the sample pad with a sample pad treatment solution, the treatment solution containing 0.5% to 10% - one or several macromolecular inert proteins, 0.1% to 3% Ionic salt buffer and 0.01% to 0.05% - one or several surfactants.
  • the surfactant can be an acidic, neutral, basic or amphoteric active agent.
  • the appropriate reagent component can be selected according to the process optimization experiment, and the sample pad is evenly sprinkled on the sample pad by 30 ⁇ 50 mL/piece of the treatment liquid, and dried at 37 ° C to be a spare sample pad.
  • the macromolecular inert protein is preferably BSA, PEG20000, sodium cholate, the ionic salt buffer is preferably Borax buffer, and the surfactant is preferably TWEEN-20.
  • the sample pad is treated with the following treatment solution: 0.05 MB orax, 0.1% Sodium Casein 1% PEG 20000, 2% BSA, 0.05% Tween-20 0.05% NaN 3 in ultrapure water.
  • the step of providing a bonding pad according to the present invention further comprises: treating the bonding pad with a treatment liquid, wherein the treatment liquid contains 0.5% to 10% of one or more macromolecular inert proteins, 0.1% to 3% of ions. Salt buffer and 0.1% ⁇ 1% one or several surfactants.
  • the surfactant can be an acidic, neutral, basic or amphoteric active agent. According to the process optimization experiment, select the appropriate reagent components, soak for 20 ⁇ 60 minutes, and dry at 37 °C.
  • the macromolecular inert protein is preferably PVA or BSA
  • the ionic salt buffer is preferably sodium phosphate
  • the surfactant is preferably Triton X-100.
  • the bond pad is treated with a treatment solution of 1% PVA, 0.71% Na 3 PO 4 , 1% BSA, 0.05% NaN 3 0.1% Triton X-100 o in ultrapure water in a preferred embodiment.
  • the binding pad is selected from the group consisting of a polyester film or a glass fiber membrane carrier.
  • the binding pad treated with the binding pad treatment solution is sprayed with a mixture of the gold standard antibody (a) and the gold standard antibody (b) at 0.5 to 4 ⁇ l/ ⁇ using a BIO-Dot instrument, 25 ° C Dry at ⁇ 30 °C, after drying, seal the bag and set at 2 °C ⁇ 8 °C for use.
  • the gold standard antibody (a) and the gold standard antibody (b) mixture can be diluted with the following dilutions: about 2.423-2.500 g tris dissolved in ultrapure water, 10-12 g BSA, 0.2-0.4 g NaN 3 , adjusted to pH 8.0, Make up to 1000mL. After diluting the gold standard to the working concentration with a diluent, 20% Sucrose and 5% trehalose were added.
  • the gold standard antibodies (a) and (b) are prepared by adjusting the pH of the colloidal gold solution to 6.0 to 9.0 with 0.05-0.2 M, preferably 0.1-0.15 potassium carbonate, in each ml of colloid. Slowly add 4 ⁇ 25 ⁇ ⁇ to be labeled antibody in the gold solution, stir for 10 ⁇ 30 minutes, then add 10% macromolecular substance solution to the final concentration of 0.5 ⁇ 5%, continue stirring for 10 ⁇ 30 minutes, centrifuge and discard the supernatant. The precipitate is washed 2 ⁇ 3 times with the washing solution, and 1/10 of the initial colloidal gold solution is used. The sediment is resuspended in the preservation solution, mixed and stored at 4 °C for use.
  • the macromolecular substance is selected from the group consisting of Staphylococcus, immunoglobulins, toxins, glycoproteins, enzymes, antibiotics, hormones, bovine serum albumin (BSA), preferably BSA.
  • the washing solution was prepared as follows: 2-3% BSA, 0.05-0.08% NaN 3 0.01-0.2 M pH 7.2 PBS, prepared with ultrapure water, filtered through a 0.22 ⁇ m membrane, and placed at 4 ° C until use.
  • Colloidal gold can be prepared in a conventional manner.
  • 0.01-0.05% of the HAuCl 4 solution is heated to boiling, and 1 mL to 2 ml of 1-3% trisodium citrate solution is added per 100 mL of HAuCl 4 solution, starting with some blue color, then light blue, black, red , boil 7 ⁇ 10min appears transparent orange red. It is then filtered through an ultrafiltration or microfiltration membrane (0.45 ⁇ m) to remove the polymer and other impurities that may be mixed.
  • the good quality colloidal gold should be pure, translucent, free of sediment and floating matter, and discarded when there is oil and a large amount of black granular precipitate on the liquid surface.
  • the nitrocellulose membrane is coated with a quality control antibody (c) and a detection antigen, including diluting the antigen, ie, the MAP conjugate, with a coating membrane buffer to a concentration of 0.2 to 2.5 mg/mL, and the antibody (c) Diluted to 0.3 ⁇ 3.0 mg/mL, sprayed the antigen and QC antibody (c) onto the nitrocellulose membrane with a BIO-Dot membrane meter at 1 ⁇ 10 ⁇ 1/ ⁇ spray, and placed in an oven at 37 °C. Inside, dry and spare.
  • the coating buffer comprises 9-13 g NaCl, 1.15-1.3 g Na 2 HP0 4 0.23-0.25 g NaH 2 P0 4 10-12 g sucrose, 0.5-0.7 g EDTA dissolved in 1 L ultrapure water.
  • the detection antigen can be synthesized using prior art techniques or can be purchased commercially.
  • the nitrocellulose membrane, the bonding pad, the sample pad and the absorbent pad are sequentially attached to each other on the PVC rubber sheet, and the test strips of different widths are cut as required, and the appropriate amount is added.
  • the desiccant is placed in an aluminum foil bag for vacuum packaging.
  • the method of the present invention further comprises providing a sample applicator and/or instructions and loading the foil pouch, the applicator and/or the instructions into the kit outer package.
  • the present invention also provides a kit comprising the test strip of the present invention, a sampler, and instructions.
  • the method of coating the detection line on the nitrocellulose membrane comprises: diluting the BSA-MAP conjugate with a coating buffer to a concentration of 0.2 to 2.5 mg/mL, or buffering with a coating membrane.
  • the antibody (c) on the quality control line is goat anti-rabbit IgG, and the antibody is diluted to a concentration of 1.0 to 1.5 mg/mL with a coating buffer, and a BIO-Dot membrane meter is used to 1.0 ⁇
  • the spray volume of 3.0 ⁇ 1/ ⁇ was sprayed on the nitrocellulose membrane quality control line, and dried in an oven at 37 ° C for use.
  • the method of the invention for preparing a test strip comprises:
  • nitrocellulose membrane wherein the nitrocellulose membrane has a detection line and a quality control line, and the detection line is coated
  • An antigen that specifically binds to an anti-cyclic citrullinated peptide antibody, and a quality control line coats a quality control antibody that specifically binds to a gold-labeled antibody (c);
  • the antigen preferably MAP-carrier protein conjugate
  • a coating buffer to a concentration of 0.2 to 2.5 mg/mL
  • the antibody (c) is diluted to 0.3 to 3.0 mg/mL.
  • the BIO-Dot membrane tester sprayed the antigen and the quality control antibody (c) on the nitrocellulose membrane at a spray rate of 1 ⁇ 10 ⁇ 1/ ⁇ , and then placed in an oven at 37 ° C, and dried for use;
  • the prepared gold standard antibody solution is sprayed on the pretreated polyester film by a BIO-Dot membrane meter at a spray rate of 0.5 to 4 ⁇ l/ ⁇ , and dried in an oven at 37 ° C until the drying is completed. Packed in a sealed bag, stored at 2 ⁇ 8 °C, ready for assembly of sticky sheets;
  • the aluminum foil bag, the sampler and the instructions are placed in the outer box of the kit to be the anti-cyclic citrullinated peptide antibody test strip.
  • the present invention introduces a quality control line, that is, a colloidal gold-labeled antibody (b) is chromatographed with a capillary effect, and is immunologically reacted with an antibody (c) immobilized on the quality control line (C) to form an immune complex. Interception, gradually enriched to form a deeper purple band.
  • the antibody (b) and the antibody (c) do not immunoreact with the capture antibody, the colloidal gold antibody (a), and the detection antigen, the cyclic citrullin peptide, and do not cause an immunological cross-reactivity.
  • the purple-red band (C) presented by the introduced quality control line is a criterion for determining whether there are enough specimens, whether the chromatographic process is normal, and also as an internal control standard for the reagent.
  • the detection principle of the invention is that the cyclophosphamide peptide detection antigen is sprayed on the detection line, the colloidal gold label mixture of the gold standard antibody (a) and the gold standard antibody (b) is sprayed on the binding pad, and the indirect method is used to detect whether the serum sample is used. Contains an anti-cyclic citrullinated peptide antibody.
  • the sample migrates to the binding pad along the chromatogram, infiltrating the gold standard mixture covering the binding pad, wherein the human IgG and the gold standard antibody (a) combine to form a human IgG-gold standard antibody (a) complex due to capillary Effect, the mixture moves forward along the nitrocellulose membrane.
  • the mixture specifically reacts with the detection antigen coated on the nitrocellulose membrane to form a triplet complex. And being trapped on the test line, Enrichment forms a deeper purplish red band; as the capillary effect continues to move forward, at the same time, the gold-labeled antibody (b) is trapped with a specific immune response to the antibody (C) coated on the quality control line.
  • the serum sample does not contain anti-cyclic citrullinated peptide antibody.
  • the gold-labeled antibody (a) reaches the detection line, it does not immunoreact with the detection antigen coated on the detection line, so no purple-red band appears at the detection line.
  • the gold-labeled antibody (b) continues to move forward and is specifically trapped in the antibody (c) at the quality control line and is trapped, and gradually enriched to form a purple-red band on the quality control line, so only in the quality The judgement of the occurrence of the band was negative.
  • the test is to qualitatively determine anti-cyclic citrullinated peptide antibodies in serum samples to aid in the diagnosis of rheumatoid arthritis.
  • the sample refers to from a mammal, more preferably from a human, including serum, plasma, and whole blood. It should be understood that the features described in the first aspect of the invention are also applicable to the technical solution of the second aspect, and the technical features described in the second aspect are also applicable to the technical solution of the first aspect. Features described in both aspects and any combination of the various ranges are within the scope of the invention.
  • the invention will be described below in the form of specific embodiments. It is to be understood that these embodiments are illustrative and not restrictive.
  • the preparations used in the examples are all conventional preparations, and the amounts and units of calculation are also conventionally used. Further, Example 12 describes various solutions used in the specific embodiments of the present application, such as coating buffers and the like.
  • Example 1 Cyclic citrulline peptide structure
  • CCPs cyclic citrullinated peptides
  • S--S denotes a disulfide bond
  • X denotes citrulline
  • the other letters are single-letter symbols of common amino acids, that is, H represents histidine, Q represents glutamine, C represents cysteine, E It means glutamic acid, S means serine, T means threonine, G means glycine, and R means arginine.
  • the branched antigen peptide (MAP) structure of the cyclic citrullinated peptide is based on poly-lysine (PL), which generally takes 2 to 8 branches, and these branches may be different.
  • the epitope of the cyclic citrullinated peptide increases the new branched antigen epitope and improves the detection performance. Taking four branches as an example, the structure is as follows:
  • the CCP CCP diagram shows the expanded epitope map of the same four CCPs.
  • the above synthetic MAP structure, purity requirements HPLC method is greater than 95%.
  • the carrier is bovine serum albumin (BSA)
  • the bovine serum albumin is coupled to MAP using a carbodiimide method, which is a very chemically active bifunctional reagent that condenses with both the carboxyl group on the hapten and the amino group on the hapten. .
  • the method is to mix the amino group of the hapten polypeptide with the carrier protein bovine serum albumin in a certain molecular ratio (the ratio of 1:1 in this study) in a suitable solution, then add carbodiimide, stir for 1 ⁇ 2h, and set at room temperature. After reacting for 24 hours, the unreacted hapten and the carrier protein were finally dialyzed to obtain a MAP conjugate. The concentration is then determined by ultraviolet spectroscopy.
  • the response mode is as follows:
  • the carrier is avidin
  • the avidin is first diluted with ultrapure water to 5 ⁇ 10mg/ml, diluted with 0.01M PBS to l ⁇ 3mg/ml, and then mixed with the commercial biotinylated MAP conjugate at a molar ratio of 1:4. Incubation overnight at 4 ° C became an avidin-biotinylated MAP conjugate.
  • the avidin is first diluted with ultrapure water to 5 ⁇ 10mg/ml, diluted with 0.01M PBS to l ⁇ 3mg/ml, and then mixed with the commercial biotinylated MAP conjugate at a molar ratio of 1:4. Streptavidin-biotinylation by overnight incubation at 4 °C MAP conjugate.
  • Example 3 Antibody preparation
  • the antibody (a) is an immunogen prepared by purifying human IgG, and is prepared by immunizing a mammal with an anti-human IgG polyclonal antibody or a monoclonal antibody, and can also express a grapevine A protein (SPA) or a streptococcus G protein by prokaryotic expression of the cloned gene. Protein G) Or purchase commercial reagents (Shanghai Yueke Biotechnology Co., Ltd.) to achieve test strip preparation.
  • SPA grapevine A protein
  • streptococcus G protein a streptococcus G protein
  • Antibodies (b) and antibodies (c) can immunoreact to form immune complexes, which can be prepared by routine experimentation. Or purchase commercial reagents (Shanghai Yueke Biotechnology Co., Ltd.).
  • Example 4 Scheme 1 for determining a gold-labeled antibody mixture
  • Quality control antibody (c) and detection of antigen coating detection antigen is MAP-BSA conjugate
  • the goat anti-rabbit IgG was diluted to 1.3 to 1.5 mg/mL with a coating buffer, and the spray amount was 1.0 to 3.0 ⁇ l/ ⁇ .
  • the spray pad was about 9 mm as a quality control line (C). The distance between the two lines is about 5 ⁇ 8 mm, and the spray line should be uniform in thickness. Dry at 37 ° C, packaged for use.
  • the MAP-BSA conjugate was used as the detection antigen, and the gold standard goat anti-human IgG and rabbit IgG were used as examples to illustrate the determination of the ratio.
  • Other antibodies such as mouse anti-human IgG and rabbit IgG, grapevine A protein and rabbit IgG, streptococcus G Protein and rabbit IgG can also be determined according to this method.
  • the OD20 of the gold-labeled rabbit IgG was basically determined by preliminary experiments, and sprayed on the bonding pad with BIO-Dot spray volume ⁇ /cm.
  • MPBS 0.01 MPBS is the loading buffer, which is the band that can obtain the expected color intensity, and the application OD 20 spray of rabbit IgG is determined to be 1 ⁇ l.
  • the gold-labeled goat anti-human IgG was serially diluted to a final concentration of OD 100, 80, 60, 40, 20, and then the gold-labeled rabbit IgG was diluted to a final concentration of OD 20, and the above gold-labeled goat anti-human IgG was used with BIO-Dot.
  • the gold-labeled rabbit IgG mixture sprayed at 3 ⁇ 1/ ⁇ sprayed on the treated polyester film, and the prepared nitrocellulose membrane, bonding pad, sample pad and absorbent pad were sequentially attached to the plastic substrate, using positive serum, critical reference Value serum and negative serum are the target of debugging.
  • Quality control antibody (c) and detection of antigen coating detection antigen is avidin-biotinylated MAP
  • the coating buffer was used to dilute the avidin-biotinylated MAP to 1.0 to 1.5 mg/mL, and the BIO-Dot instrument was adjusted to have a spray amount of 1.5 to 2.0 ⁇ l/ ⁇ on the nitrocellulose membrane of the binding pad end of about 12 mm.
  • Detection line (T); with coating buffer The goat anti-rabbit IgG was diluted to 1.0 to 1.5 mg/mL, and the spray amount was 1.0 to 3.0 ⁇ l/ ⁇ .
  • the spray pad was about 9 mm as the quality control line (C). The distance between the two lines is about 5 ⁇ 8 mm, and the spray line should be uniform in thickness. Dry at 37 ° C, packaged for use.
  • the avidin-biotinylated MAP was used as the detection antigen, and the gold standard goat anti-human IgG and rabbit IgG were used as examples to illustrate the determination of the ratio.
  • Other antibodies such as mouse anti-human IgG and rabbit IgG, grapevine A protein and rabbit IgG, Streptococcal G protein and rabbit IgG can also be determined according to this method.
  • the OD20 of the gold-labeled rabbit IgG was basically determined by preliminary experiments, and sprayed on the binding pad with BIO-Dot spray volume ⁇ /cm, and O.OIMPBS was used as the loading buffer to obtain the band of the expected color intensity, and the rabbit IgG was determined.
  • the application OD 20 spray volume is 1 ⁇ 1.
  • the gold-labeled goat anti-human IgG was serially diluted to a final concentration of OD 100, 80, 60, 40, 20, and then the gold-labeled rabbit IgG was diluted to a final concentration of OD 20, and the above gold-labeled goat anti-human IgG was used with BIO-Dot. And the gold-labeled rabbit IgG mixture spray volume is
  • Quality control antibody (c) and detection of antigen coating detection antigen is streptavidin-biotinylated MAP) coating buffer diluted streptavidin-biotinylated MAP to 1.2 ⁇ 1.5mg /mL, adjust the BIO-Dot instrument, spray the amount of 1.5 ⁇ 2.0 ⁇ 1/ ⁇ on the nitrocellulose membrane with the end of the binding pad about 12mm as the detection line (T); dilute the goat anti-rabbit IgG to 1.0 with the coating buffer ⁇ 1.5mg/mL, spray volume is 1.0 ⁇ 3.0 ⁇ 1/ ⁇ spray distance absorbent pad is about 9mm for quality control line (C). The distance between the two lines is about 5 ⁇ 8 mm, and the spray line should be uniform in thickness. Dry at 37 ° C, packaged for use.
  • Streptavidin-biotinylated MAP was used as the detection antigen, and gold standard goat anti-human IgG and rabbit IgG were used as examples to illustrate the determination of ratios.
  • Other antibodies such as mouse anti-human IgG and rabbit IgG, grape globulin A protein and rabbit IgG, Streptococcal G protein and rabbit IgG can also be determined according to this method.
  • the OD20 of the gold-labeled rabbit IgG was determined by preliminary experiments, and the spray volume was ⁇ /cm. BIO-Dot was sprayed on the binding pad, and 0.01 M PBS was used as the loading buffer to obtain the band of the expected color intensity, and the rabbit IgG was determined. The application OD 20 spray volume is 1 ⁇ 1.
  • the gold-labeled goat anti-human IgG was serially diluted to a final concentration of OD 100, 80, 60, 40, 20, and then the gold-labeled rabbit IgG was diluted to a final concentration of OD 20, and the above gold-labeled goat anti-human IgG was used with BIO-Dot. And the gold-labeled rabbit IgG mixture spray volume is
  • the preparation method of the test strip for detecting anti-cyclic citrullinated peptide antibody in the sample by the colloidal gold chromatography method of the present invention is as follows
  • the distance between the two lines is about 5 ⁇ 8 mm, and the spray line should be uniform in thickness. Dry at 37 ° C, packaged for use.
  • the bonding pad was immersed in the bonding pad treatment solution for 30 minutes and dried at 37 °C.
  • the application OD and the spray amount of the gold standard antibody mixture were determined by referring to Example 4, Example 5, and Example 6, and sprayed on the pretreated polyester film with BIO-Dot, dried at 37 ° C, and packaged in an aluminum foil pouch. spare.
  • the sample pad was evenly sprinkled on the sample pad with a sample pad treatment solution of 45 mL/piece. After drying at 37 ° C, it is packaged in aluminum foil bag and used. 7. Pre-cutting of raw materials
  • Cutting of absorbent paper Use a paper cutter to cut the absorbent paper to a length of 28 cm and a width of 3 cm.
  • Cutting of the polyester film Cut according to the length of the spray film of 28 cm in length and 1 cm in width, and place it in a dry room for later use.
  • the nitrocellulose membrane, the polyester film, the glass fiber membrane, and the absorbent paper are sequentially laminated on the PVC base plate as shown in Fig. 1 to form a large plate.
  • the temperature of the assembly workshop should be controlled at 25 ° C ⁇ 37 ° C, humidity 20% ⁇ 30%.
  • the method for preparing a test strip for detecting a blood anti-cyclic citrullinated peptide antibody by the colloidal gold chromatography described in this embodiment differs from the embodiment 7 in that:
  • step 3 the capture antibody is murine anti-human IgG.
  • the specific labeling method is:
  • step 5 the corresponding gold standard antibody (a) is gold-labeled mouse anti-human IgG, gold standard antibody (b) is gold-labeled rabbit IgG, and the experimental method is the same as the step.
  • binding pad pretreatment is in combination The pad treatment solution was immersed for 30 minutes and dried at 37 °C. The gold standard mixture was sprayed on the pretreated polyester film with BIO-Dot at 2-4 ⁇ l/ ⁇ , dried at 37 ° C, and packaged in an aluminum foil pouch for use.
  • the capture antibody is Staphylococcal Protein A (SPA)
  • the method for preparing a test strip for detecting a blood anti-cyclic citrullinated peptide antibody by the colloidal gold chromatography described in this embodiment differs from the embodiment 7 in that:
  • step 3 Adjust the pH of the colloidal gold solution to 5.0 ⁇ 6.5 with 0.1M potassium carbonate, add 8 ⁇ 15 ⁇ ⁇ staphylococcal prion protein per ml of colloidal gold solution, magnetically stir for 10 ⁇ 30 minutes, then add 10% BSA to final concentration 0.2 ⁇ 3%, continue to stir for 10 to 30 minutes. 6000 ⁇ 10000 g/min Centrifuge at 4 °C for 20 ⁇ 40min, discard the supernatant, wash the precipitate twice with the washing solution, resuspend with the preservation solution of one tenth of the initial colloidal gold volume, and set at 4 °C for use.
  • step 5 the corresponding gold standard antibody (a) is Staphylococcus aureus A protein, gold standard antibody (b) is gold standard rabbit
  • the experimental method is the same as step 5: the bonding pad pretreatment is immersed in the bonding pad treatment solution for 30 minutes, and dried at 37 ° C.
  • the gold standard mixture was sprayed on the pretreated polyester film with BIO-Dot at 2-4 ⁇ l/ ⁇ , dried at 37 ° C, and packaged in an aluminum foil pouch for use.
  • Capture antibody is Streptococcus G protein (Protein G)
  • the method for preparing a test strip for detecting a blood anti-cyclic citrullinated peptide antibody by the colloidal gold chromatography described in this embodiment differs from the embodiment 7 in that:
  • step 5 the corresponding gold standard antibody (a) is the gold-stranded streptococcus G protein, and the gold-labeled antibody (b) is the gold-labeled rabbit IgG.
  • the experimental method is the same as step 5: the binding pad pretreatment is treated in the bonding pad. The solution was immersed for 30 minutes and dried at 37 °C. The gold standard mixture was sprayed on the pretreated polyester film with BIO-Dot at 2-4 ⁇ l/ ⁇ , dried at 37 ° C, and packaged in an aluminum foil pouch for use.
  • Example 11 Capture antibody is goat anti-human IgG
  • the method for preparing a test strip for detecting a blood anti-cyclic citrullinated peptide antibody by the colloidal gold chromatography described in this embodiment differs from the embodiment 7 in that:
  • step 5 the corresponding gold standard antibody (a) is gold-labeled goat anti-human IgG, and the gold-labeled antibody (b) is gold-labeled rabbit IgG.
  • the experimental method is the same as step 5: binding pad pretreatment is combined The pad treatment solution was immersed for 30 minutes and dried at 37 °C. The gold standard mixture was sprayed on the pretreated polyester film with BIO-Dot at 2-4 ⁇ l/ ⁇ , dried at 37 ° C, and packaged in an aluminum foil pouch for use.
  • Example 12 Test strip composition and reagent preparation
  • the invention provides a colloidal gold chromatography method for detecting a composition of test strips of anti-cyclic citrullinated peptide antibodies in a sample: the substrates are coated on the support rubber sheet PVC (7), and the coated antigens are sequentially attached to each other. And the quality control antibody of nitrocellulose membrane (3), binding pad (2), sample pad (1), and absorbent pad (6).
  • the binding pad (2) is coated with a gold standard antibody (a) and a gold standard antibody (b) rabbit IgG.
  • the nitrocellulose membrane (3) has a detection line and a quality control line, the detection line (T) is coated with a detection antigen, and the quality control line (C) is coated with a goat anti-rabbit IgG.
  • the colloidal gold immunochromatographic assay of the present invention detects test strips of anti-cyclic citrullinated peptide antibodies in a sample, and the reagents used are prepared as follows (the ingredients used are commercially available):
  • HAuCl 4 Dissolve HAuCl 4 in ultrapure water, prepare a 1% solution, and set at 4 °C for a period of four months.
  • lOOOOmL 1%HAUC1 4 solution formulation 10gHAuCl 4 ; ultrapure water to a volume of 1000 mL.
  • washing solution ie, the preservation solution: 2% BSA, 0.05% NaN 3 ⁇ . ⁇ 7.2 PBS, prepared with ultrapure water, filtered through a 0.22 ⁇ membrane, placed at 4 ° C for use, valid for two weeks.
  • the required reagents for 1000 ml are: 10 g PVA, 7.1 g Na 3 PO 4 , 10 g BSA, 0.5 g NaN 3 , 1 ml Triton X-100.
  • Example 13 Sample processing and sample loading method
  • Intravenous blood collection l-5ml serum is naturally precipitated, centrifuged at 3000g / min 5 ⁇ 10 minutes, take the supernatant to get the sample solution to be tested, at least 100 ⁇ 1 of the sample solution to be tested.
  • the test strip was placed at 37 ° C for accelerated test, and the indoor control product was taken out for 1 day, 3 days, 7 days, 21 days, January, February, March, and April.
  • the intra-assay precision of the negative-positive rate of the product (10 parts each) was used to judge the stability of the test strip. After 4 months, the results showed that the quality control products were in line with expectations, and the positive-positive rate of each positive-positive reference product was 100%.
  • the negative-positive reference was 10 anti-CCP antibody-positive sera and 10 anti-CCP antibody-negative sera.
  • test strips were placed at 4 ° C for routine stability testing, and the indoor quality control products were taken out monthly.
  • the test papers were also judged by the intra-assay precision of the negative-positive coincidence rate of the negative-positive reference products (10 each).
  • the stability of the strip After 12 months, the results showed that the quality control product test results were in line with expectations, and the positive-yellow coincidence rate of each negative-positive reference product was 100%. After 14 months, the results showed that the positive-yellow coincidence rate of each negative-positive reference product was still 100%. After 18 months, the test results showed that there was a false negative. Based on the above results, the test strips are stored at 2 ⁇ 8 °C and are stable for at least one year.
  • control groups including healthy blood donors and non-RA patients.
  • test strips produced, and select one of four different concentrations of serum (high, medium, low, negative), and make 10 replicates for each serum. Observe the results after 5 minutes. The number of positive results of different serum negative results is strong.
  • the total compliance rate of the self-made anti-CCP antibody colloidal gold detection reagent and the Ermen ELISA kit can reach 98.5%.

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Abstract

La présente invention concerne une bande d’essai pour la détection d’un anticorps anti-peptides cycliques citrullinés basée sur l’immunochromatographie avec particules d’or colloïdal et un procédé de préparation associé. La bande de test comprend : un film en nitrate de cellulose (3), un tampon de conjugué (2), un tampon témoin (1) et un tampon d’aspiration (6), qui sont collés sur un substrat (7) et qui se chevauchent à la suite. Une zone de détection (4) et une zone témoin (5) sont prévues sur le film en nitrate de cellulose. Un antigène peptide cyclique citrulliné est enduit sur la zone de détection, et un anticorps comme témoin est enduit sur la zone témoin. Deux anticorps différents marqués à l’or colloïdal sont enduits sur le tampon de conjugué. Un peptide cyclique citrulliné ramifié est utilisé dans la bande d’essai. Le prétraitement du tampon témoin et du tampon de conjugué est amélioré dans le procédé de préparation. La bande d’essai permettant la détection d’un anticorps anti-peptide cyclique citrulliné peut être appliquée pour diagnostiquer la polyarthrite rhumatoïde.
PCT/CN2010/075228 2009-07-31 2010-07-19 Bande d’essai pour la détection d’un anticorps anti-peptide cyclique citrulliné dans le sang et procédé de préparation associé Ceased WO2011012053A1 (fr)

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CN200910055724.5A CN101988924B (zh) 2009-07-31 2009-07-31 检测血液抗环瓜氨酸多肽抗体的试纸条及制备方法

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CN113189331A (zh) * 2021-04-15 2021-07-30 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) 一种烟草青枯病菌快速免疫检测试纸条及其制备方法和应用
CN113189331B (zh) * 2021-04-15 2023-07-18 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) 一种烟草青枯病菌快速免疫检测试纸条及其制备方法和应用
CN114113615A (zh) * 2021-12-03 2022-03-01 河南省农业科学院 一种通用型单克隆抗体筛选的免疫层析检测试纸条及检测方法
CN114113615B (zh) * 2021-12-03 2024-03-05 河南省农业科学院 一种通用型单克隆抗体筛选的免疫层析检测试纸条及检测方法
CN114397458A (zh) * 2022-01-25 2022-04-26 山东康华生物医疗科技股份有限公司 一种用于肺炎支原体抗原检测的检测盒
CN116027035A (zh) * 2023-03-30 2023-04-28 济南玖方生物科技有限公司 一种用于提高hiv1/2尿液胶体金免疫层析检测准确率的试剂盒及制备方法
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