WO2011012053A1

WO2011012053A1 - Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and method of preparing the same

Info

Publication number: WO2011012053A1
Application number: PCT/CN2010/075228
Authority: WO
Inventors: 张玥; 韩永俊; 高成秀
Original assignee: 上海科新生物技术股份有限公司
Priority date: 2009-07-31
Filing date: 2010-07-19
Publication date: 2011-02-03
Also published as: CN101988924B; CN101988924A

Abstract

A test strip for detecting anti-cyclic citrullinated peptide antibody based on colloidal gold immunochromatography and a method of preparing the same are provided. The test strip includes: a cellulose nitrate film (3), a conjugate pad (2), a sample pad (1) and a wicking pad (6), which are stuck on a substrate (7) and overlapping one another in sequence. A detection zone (4) and a control zone (5) are provided on the cellulose nitrate film. Cyclic citrullinated peptide antigen is coated on the detection zone, and antibody as a control is coated on the control zone. Two different antibodies labeled by colloidal gold are coated on the conjugate pad. Branched cyclic citrullinated peptide is used in the test strip. Pretreatment for sample pad and conjugate pad is improved in the preparing method. The test strip for detecting anti-cyclic citrullinated peptide antibody can be applied for diagnosing rheumatoid arthritis (RA).

Description

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;), 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. However, 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. To date, RF has been the only serological marker for RA in clinical use. However, 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.

In recent years, several new antibodies have been discovered in patients with RA, and their clinical features and possible pathological roles have also emerged. In particular, new anti-citrullin protein autoantibodies such as filaggrin and its cyclized form (ie, cyclic citrullinated peptide: CCP) have received much attention in the sensitivity and high specificity of RA patients, and Markers of early diagnosis and precursors to joint damage. Recent studies have confirmed that RA patients not only produce RF factors but also a variety of different antibodies. Although most antibodies are not specific for RA, citrullin autoantibodies (APF, AKA, anti-filaggrin, anti-CCP, and anti-Sa antibodies) are only recognized in RA, and anti-CCP antibodies are highly sensitive. Sex and specificity are of particular concern. 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. 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 (GICA) 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]. When using, it is only necessary to load the sample onto the sample pad of the test strip. In a few minutes, the positive result is judged based on the presence or absence of the purple-red band on the test line. Compared with other testing methods, 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.

At present, there is no 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.

Compared with the above advantages, the existing methods for detecting anti-cyclic citrullinated peptide antibodies have the following disadvantages:

1. The detection procedure is cumbersome and the detection time is long;

2. Detection reagents need to be refrigerated for transport and storage;

3. Detection requires special equipment. For example, the ELISA method requires a microplate reader;

4. Not suitable for single-person operation. For ELISA, both standard and negative-positive controls should be used at the same time. Multi-part simultaneous testing can save the cost of testing. Summary of the invention

In order to overcome the deficiencies of the above methodologies, 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).

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);

Wherein 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.

In a preferred embodiment, the test strip further comprises a sample pad and/or an absorbent pad.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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. Source, rabbit source, horse source, sheep source or guinea pig source, wherein antibody (a) may also be Staphylococcus A protein or Streptococcus G protein.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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:

1. 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. 2. Among 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. 3. 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. In addition, 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. DRAWINGS

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. detailed description

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.

In a preferred embodiment, the 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:

In a preferred embodiment, 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. 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, and the surfactant is preferably Triton X-100.

In a preferred embodiment, the bond pad is treated with a treatment liquid: 1% PVA in ultrapure water,

0.71% Na ₃ PO ₄ 1% BSA, 0.05% NaN ₃ 0.1% Triton X-100.

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.

In a preferred embodiment, the sample pad is treated with the following treatment solution: 0.05 MBorax in ultrapure water,

0.1% Sodium Casein 1% PEG 20000, 2% BSA, 0.05% Tween-20 0.05% NaN ₃ .

In the test strip of the present invention, in the horizontal direction, 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.

By "lapping" as used herein, it is meant that 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.

Various suitable antibodies are commercially available from the commercially available route and used as the above-mentioned antibodies of the present invention.

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. The unbound colloidal gold protein binding site is blocked, the non-specific reaction detected is reduced, and the label is then separated and purified by centrifugation.

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.

Since the cyclic citrulline peptide is a small molecule polypeptide, there is a need to increase the efficiency of the cyclic citrulline peptide coated on the membrane and to reduce the steric hindrance of the immune reaction, and the cyclic citrullinated peptide and the carrier protein are used. 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. In a preferred embodiment, 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 μl / η per antibody, for example, the amount It may be 1-3μ1/η, 1-2.5μ1/η, 1-2μ1/η, 0.5~2.5μ1/η, and the like. The amount of the detection antigen coated onto the detection line may be 1 to Ομΐ/cm, for example, 2 to 8 μl/η, 3 to 7 μl/η, 4 to 6 μl/η, 1 to 3 μl/η, and the like. The amount of the antibody (c) coated on the quality control line may be 1 to 10 μl / η, for example, 2 to 8 μl / η, 3 to 7 μl / η, 4 to 6 μl / η, 1 to 3 μl / η, 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.

Therefore, in a preferred embodiment of the present invention, 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). Membrane (3), bond pad (2), sample pad (1) and absorbent pad (6). 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). Wherein 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.

In another preferred embodiment of the present invention, the detection line on the nitrocellulose membrane detects a BSA-MAP conjugate having an antigen of 1.0 to 2.5 μl/η, 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 μl/η.

In another preferred embodiment of the present invention, 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 μl / η per antibody, for example, the amount may be 1-3 μl / η, 1-2.5 μl / η, 1-2 μl / η, etc.; The amount of detection antigen on the line is 1~10μ1/η, for example 2~8μ1/η, 3~7μ1/η, 4~6μ1/η, 1.0~2.5μ1/η, etc.; coated onto the quality control line The amount of the antibody (c) may be 1 to 10 μl / η, for example, 2 to 8 μl / η, 3 to 7 μl / η, 4 to 6 μl / η, 1.0 to 3.0 μl / η, and the like. A second aspect of the invention provides a method of preparing a test strip of the invention, the method comprising the steps of:

1) providing a bottom plate;

2) providing a binding pad and coating the binding pad with the gold standard antibody (a) and the gold standard antibody (b);

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 the quality control antibody is used. (c) coating the test line; and

4) The nitrocellulose membrane and the bonding pad are sequentially bonded to each other on the bottom plate to obtain the test strip.

In a preferred embodiment, the method of the invention further comprises the step of providing a sample pad and/or an absorbent pad.

In one embodiment, 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. In a preferred embodiment, 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.

In one embodiment, 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, and the surfactant is preferably Triton X-100. In a preferred embodiment, 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. In one embodiment, the binding pad is selected from the group consisting of a polyester film or a glass fiber membrane carrier.

In a preferred embodiment, 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 μl/η 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.

In one embodiment, 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 4~25μ _§ 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 μm membrane, and placed at 4 ° C until use.

Colloidal gold can be prepared in a conventional manner. In one embodiment, 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 μm) 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.

In one embodiment, 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 1~10μ1/η spray, and placed in an oven at 37 °C. Inside, dry and spare. In one embodiment, 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. in. In one embodiment, the detection antigen can be synthesized using prior art techniques or can be purchased commercially.

In one embodiment, after completing the above steps, 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.

In one embodiment, 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.

Accordingly, the present invention also provides a kit comprising the test strip of the present invention, a sampler, and instructions. In a preferred embodiment of the invention, 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. Dilute the avidin-biotinylated MAP conjugate to a concentration of 1.0 to 1.5 mg/mL, or dilute the streptavidin-biotinylated MAP conjugate with a coating buffer to a concentration of 1.2 to 1.5 mg/ The mL, BIO-Dot membrane tester is sprayed on the nitrocellulose membrane test line at a spray rate of 1.0 to 2.5 μl/η. In this embodiment, 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 3.0μ1/η was sprayed on the nitrocellulose membrane quality control line, and dried in an oven at 37 ° C for use.

In a specific embodiment, the method of the invention for preparing a test strip comprises:

1) providing a bottom plate;

2) Provide a sample pad;

3) providing a binding pad, wherein the binding pad is coated with a gold standard antibody (a) and a gold standard antibody (b);

4) providing a 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);

5) providing an absorbent pad;

6) sequentially bonding the nitrocellulose membrane, the bonding pad, the sample pad and the absorbent pad to each other on the bottom plate to obtain the test strip;

7) Providing an antigen: Synthetic cyclic citrullinated peptide or MAP-carrier protein conjugate, or commercially available;

8) Preparation of coating film: The antigen (preferably MAP-carrier protein conjugate) is diluted with a coating buffer to a concentration of 0.2 to 2.5 mg/mL, and 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 1~10μ1/η, and then placed in an oven at 37 ° C, and dried for use;

9) Preparation of gold standard antibody: Adjust the pH of the colloidal gold solution to 6.0~9.0 with 0.1M potassium carbonate, slowly add 4~25 g of the antibody to be labeled in each ml of colloidal gold solution, stir for 10~30 minutes, then add 10% BSA solution to a final concentration of 0.5 to 5%, continue to stir for 10 to 30 minutes, centrifuge, discard the supernatant, wash the precipitate with the washing solution 2 to 3 times, with 1/10 of the initial colloidal gold solution volume of the preservation solution Resuspend the pellet, mix and store at 4 °C, and set aside;

10) 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 μl/η, 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;

11) Paste the nitrocellulose membrane, bonding pad, sample pad and absorbent pad on the PVC rubber sheet, cut into test strips of different widths according to requirements, add appropriate amount of desiccant, and put into the aluminum foil bag for vacuum. Packaging; and

12) 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. At the time of detection, 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. If there is an anti-cyclic citrullinated peptide antibody in the serum sample, 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. , gradually enriched on the quality control line to form a deep purple-red band, and the excess unbound material continues to be chromatographed onto the absorbent pad, so that the band is judged to be positive on both the detection line and the quality control line; The serum sample does not contain anti-cyclic citrullinated peptide antibody. When 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. And 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

Different cyclic citrullinated peptides (CCPs) have subtle differences and exhibit different epitopes. One of the amino acid sequences of the cyclic citrullin peptide is:

HQC/HQE/STX/GRS/RCR/CGR/SGS

Wherein "S--S" denotes a disulfide bond, X denotes citrulline, and 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, according to the latest research progress, 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 MAP shown in the above structure was synthesized by Invitrogen, and the CCP structure was as described above and used in the following examples of the present invention. Example 2: Antigen coupling

1. 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:

_ , _ R-NH-CN-R'

Protein-COOH + NH ₂ -MAP ^ MAP-CO-NH-protein + R-NH- CO-NH-R'

2. 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.

Streptavidin

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.

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

1. Quality control antibody (c) and detection of antigen coating (detection antigen is MAP-BSA conjugate)

Dilute the MAP-BSA conjugate to 0.2~2.5mg/mL in the coating buffer, adjust the BIO-Dot instrument, and spray the amount of 2.2~2.5μ1/η on the nitrocellulose membrane with the end of the binding pad about 12mm as the detection line. (T); 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 μl/η. 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.

2. Debugging method for determining the ratio of two gold scales

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 Ιμΐ/cm.

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 μl.

Subsequently, 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 sprayed at 3μ1/η 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. Judgment basis: Critical reference value serum test, band can appear at the detection line (Τ), when the negative serum is detected, the detection line (Τ) does not appear the OD value of the band, that is, the application amount of the gold standard goat anti-human IgG. Conclusions obtained through this test OD60 is more in line with the requirements. Example 5: Scheme 2 for determining a gold-labeled antibody mixture

1. 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 μl/η 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 μl/η. 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.

2. Debugging method for determining the ratio of two gold scales

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 Ιμΐ/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 1μ1.

Subsequently, 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

3μ1/η was 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, and the positive serum, the critical reference serum and the negative serum were used as the debugging object. Judgment basis: Critical reference value serum test, band can appear at the detection line (Τ), when the negative serum is detected, the detection line (Τ) does not appear the OD value of the band, that is, the application amount of the gold standard goat anti-human IgG. Conclusions obtained through this test OD80 is more in line with the requirements. Example 6: Scheme 3 for determining a gold-labeled antibody mixture

1. 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 1.5~2.0μ1/η 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 1.0~3.0μ1/ η 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.

2. Debugging method for determining the ratio of two gold scales

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 Ιμΐ/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 1μ1.

3μ1/η was 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, and the positive serum, the critical reference serum and the negative serum were used as the debugging object. Judgment basis: critical parameter In the test serum test, a band can appear at the detection line (T), and when the negative serum is detected, the OD value of the band (T) does not appear to be the application amount of the gold-labeled goat anti-human IgG. The conclusion reached by this test is that OD60 is more in line with the requirements. Example 7: Preparation method of test strip

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

1. Preparation of coating film

Dilute the detection antigen to 0.2~2.5mg/mL with the coating buffer, adjust the BIO-Dot instrument, spray the detection line (T) 0.8~2.5μ1, about 12 mm from the end of the binding pad; dilute the goat anti-rabbit with the coating buffer IgG to 1.0~1.5mg/mL, spray the goat anti-rabbit IgG on the quality control line (C line) with the BIO-Dot instrument about 9 mm from the absorbent pad. 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.

2. Preparation of colloidal gold solution

Heat 0.01% of the HAuCl ₄ solution to boiling, add 1mL~2ml 1% trisodium citrate solution per 100mL HAuCl ₄ solution, start some blue, then light blue, black, red, boil 7~10min, transparent orange red . It is then filtered through an ultrafiltration or microfiltration membrane (0.45 μm) 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.

3. Capture antibody (a) marker

Adjust the pH of the colloidal gold to 0.1~9.0 with 0.1M potassium carbonate, add 8~25 g antibody (a) per ml of colloidal gold solution, magnetically stir for 10~30 minutes, then add 10% BSA to the final concentration 0.5~5 %, 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, and resuspend it with a solution of one tenth of the initial colloidal gold volume, and set it at 4 °C for later use.

4. Labeling of rabbit IgG (antibody (b))

Adjust the pH of the colloidal gold to 0.1~8.0 with 0.1M potassium carbonate, add 10~12μ _§ rabbit IgG per ml of colloidal gold solution, magnetically stir for 10~30 minutes, then add 10% BSA to the final concentration of 0.2~1%, Continue to stir for 10 to 30 minutes. 6000~10000g/min Centrifuge at 4°C for 20~40min, discard the supernatant, wash the pellet twice with the label washing solution, and resuspend it with the mark preservation solution of one tenth of the initial colloidal gold volume, and set it at 4°C for use.

5. Combination pad processing

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.

6. Processing of the sample pad

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

1) Cutting of the glass fiber membrane: The glass fiber membrane was cut into a length of 28 cm and a width of 2.4 cm by a cutter, and placed in a dry room for use.

2) 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. 3) 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.

4) 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%.

8. Cutting strips

Use a slitting machine to cut the large plate into single servings. The width of each person is cut into 2.5~4mm width according to certain requirements. Random sampling, sensitivity can detect indoor quality control samples (ie, critical value serum samples), strip display The color result is shown in Fig. 2b, and there is no non-specific band. The negative sample test result is shown in Fig. 2c, then the product passes the other quality control requirements and is qualified product.

9. Assembly, packaging

The cut test strips of one person are assembled in the prepared test paper card, so that the sample loading window corresponds to the sample pad of the test strip, the result display window corresponds to the detection area and the control area, and the assembly workshop temperature should be controlled at 25°. C ~ 37 ° C, humidity 20% ~ 30%. Then, it is packaged in an outer bag with a desiccant, instructions, and sampler, and stored at 4~25°C in the dark. Example 8: Capture antibody to mouse 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:

1. In step 3, the capture antibody is murine anti-human IgG. The specific labeling method is:

Adjust the pH of the colloidal gold solution to 6.5~7.5 with 0.1M potassium carbonate, add 8~10μ _§ mouse anti-human IgG per ml of colloidal gold solution, stir and mix for 10~30 minutes with magnetic stirrer, then add 10% BSA to The final concentration is 0.2~2%, and the magnetic stirrer is stirred and mixed for 10 to 30 minutes. 6000~10000g /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 it at 4 °C for use.

2. In 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. Specifically: 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 μl/η, dried at 37 ° C, and packaged in an aluminum foil pouch for use. Example 9: The capture antibody is Staphylococcal Protein A (SPA)

1. Capture the antibody to staphylococcal protein A in step 3. Adjust the pH of the colloidal gold solution to 5.0~6.5 with 0.1M potassium carbonate, add 8~15μ _§ 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.

2. In step 5, the corresponding gold standard antibody (a) is Staphylococcus aureus A protein, gold standard antibody (b) is gold standard rabbit

IgG, 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 μl/η, dried at 37 ° C, and packaged in an aluminum foil pouch for use. Example 10: Capture antibody is Streptococcus G protein (Protein G)

1. Capture the antibody to Streptococcus G protein in step 3.

Adjust the pH of colloidal gold to 6.5~7.5 with 0.1M potassium carbonate, add 9~14μ _§ Streptococcus G protein per ml of colloidal gold solution, magnetically stir for 10~30 minutes, then add 10% BSA to final concentration 0.2~ 1%, continue to stir for 10 to 30 minutes. 6000~10000g/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 it at 4°C for use.

2. In 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 μl/η, dried at 37 ° C, and packaged in an aluminum foil pouch for use. Example 11: Capture antibody is goat anti-human IgG

1. Capture the antibody in step 3 as goat anti-human IgG

Adjust the pH of the colloidal gold solution to 8.0~9.0 with 0.1M potassium carbonate, add 8~12μ _§ goat anti-human IgG per ml of colloidal gold solution, magnetically stir for 10~30 minutes, then add 10% BSA to final concentration 0.2~ 2.5%, continue to stir for 10 to 30 minutes. 6000~10000g/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 it at 4°C for use.

2. In 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 μl/η, 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):

1) Preparation of coating buffer: 9 g NaCK 1.15 g Na ₂ HP0 ₄ 0.23 g NaH ₂ P0 ₄ lOg sucrose, 0.5 g EDTA was dissolved in 1 L of ultrapure water, and filtered at 4 ° C for use.

2) Preparation of 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.

3) Preparation of 1% trisodium citrate: Dissolve trisodium citrate in ultrapure water, prepare a 1% solution, filter through 0.22μιη membrane, and valid for 1 month.

4) Preparation of 0.1MK ₂ CO ₃ : Prepared with ultrapure water, filtered through 0.22μιη membrane, set at 4°C for use, and valid for one month.

Formulation of 1000mL 0.1MK ₂ CO ₃ solution: 13.8gK ₂ C0 ₃ ; to a volume of 1000 mL of ultrapure water.

5) Preparation of 10% BSA: Prepared with ultrapure water, filtered with 0.05% (NaN ₃ ), 0.22 μm, and placed at 4 ° C for 2 weeks. lOOOOmL 10% BSA solution formulation: 100g BSA, 0.5g NaN ₃ ; ultrapure water to a volume of 1000 mL.

6) Preparation of the washing solution, ie, the preservation solution: 2% BSA, 0.05% NaN ₃ Ο.ΟΙΜρΗ 7.2 PBS, prepared with ultrapure water, filtered through a 0.22 μιη membrane, placed at 4 ° C for use, valid for two weeks. 1000 mL labeled wash preservation solution formulation: 20g BSA,

0.5 g NaN ₃ 0.01 M pH 7.2 PBS to a volume of 1000 mL.

7) Preparation of the bonding pad treatment solution:

1% PVA 0.71% Na ₃ PO ₄ 1% BSA, 0.05% NaN ₃ 0.1% TritonX-100, prepared in ultrapure water, set at 4 °C for 2 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.

8) Preparation of gold standard dilution:

Preparation of lOOOOmL dilution: 2.423gtris, lOgBSA, 0.2gNaN ₃ dissolved in ultrapure water, adjusted to pH 8.0, to a volume of 1000mL. After diluting the gold standard to the working concentration with a diluent, 20% Sucrose and 5% trehalose were added.

9) Preparation of sample pad treatment solution:

0.05MBorax 0.1% Sodium Casein, 1% PEG20000, 2% BSA, 0.05% Tween-20

0.05% NaN ₃ , prepared with ultrapure water, placed at 4 ° C for use, valid for two weeks. The required reagents for lOOOOmL: 19g Bor _ax , lg Sodium Casein 10g PEG20000, 20g BSA, 0.5ml Tween-20, 0.5g NaN ₃ . 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 μ 1 of the sample solution to be tested.

A sample of 50~70 μl of the above serum was pipetted using a micropipette to the sample pad, and the sample was slowly applied. Or use a pipette to slowly add 3~5 drops of blood sample to the sample pad, observe the results for 5~10 minutes, and judge the yin and positive results according to the appearance of the band. Example 14: Kit detection and clinical performance evaluation

1 Stability test

1.1 37 ° C acceleration stability

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.

1.2 4 ° C stability test

The 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.

2. Diagnostic sensitivity

A total of 323 sera diagnosed as rheumatoid arthritis (RA) patients were collected from the clinic, and the serum of RA patients collected above was tested using a self-made colloidal gold test strip according to the procedure described in the instructions. After 5 minutes, the statistics are as follows:

Based on the above statistics, based on the detection of 323 confirmed RA patients' serum, the number of samples with positive anti-CCP antibody was 229, and the number of negative samples was 94. So by calculation:

Diagnostic sensitivity (%) = 229/ (229+94) χ 100% = 70.9%

3. Diagnostic specificity Blood samples from healthy blood donors and non-A patients (including other rheumatic diseases and bone and joint diseases) were collected from the clinic. 300% of the serum was collected from the self-made colloidal gold test strips according to the instructions in the instructions. Serum was tested with non-RA patients. After 5 minutes, the statistical results are as follows:

According to the statistical results of 300 sera tests for non-RA patients and healthy blood donors, the number of samples that were negative for anti-CCP antibodies in healthy blood donors was 298, and the number of anti-CCP antibodies that were negative for RA patients was negative. 296 cases. So by calculation:

(Healthy blood donors) Diagnostic specificity (%) = 298/300 χ 100% = 99.3%

(Non-RA patient serum) Diagnostic specificity (%) = 296/300 = 98.7%

600 control groups (including healthy blood donors and non-RA patients)

Diagnostic specificity (%) = 594/600 = 99.0%

4. Diagnostic accuracy

Based on the above statistics of diagnostic specificity and diagnostic sensitivity, the following summary table is obtained:

According to the above statistical table, we can see that for the patients confirmed as RA, the number of positive samples detected was 229, and the serum samples of 600 healthy donors and non-RA patients were 594.

Ie: Diagnostic accuracy = (229+594) /923=89.2%

5. Intra-assay inaccuracy

Select one of the 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.

Zhongyang serum 0 10

Weak yang serum 0 10

Negative serum 10 0

According to the above results, four different sera were tested with the same batch of anti-CCP antibody colloidal gold test strips, and each test was performed 10 times, which can accurately distinguish the yin positive and get consistent. Yin positive results.

From the above results, the following conclusions can be drawn: There is no intra-assay imprecision in the anti-CCP antibody colloidal gold test strip.

6. Inter-assay inaccuracy

Three different batches of anti-CCP antibody colloidal gold test strips were used to select test strips of four different concentrations (high, medium, low, negative), 10 replicates per serum, and observations after 5 minutes. .

According to the above results, using three batches of anti-CCP antibody colloidal gold test strips produced in different batches, four different sera were tested, and each test was performed 10 times, which can accurately distinguish the yin positive and get consistent. Yin-positive results.

From the above results, the following conclusions can be drawn: There is no inter-assay imprecision in the anti-CCP antibody colloidal gold test strip.

7. Comparative test of anti-CCP antibody test kit (ELISA method) produced by German Ou Meng Company, which is different from the similar test items.

200 samples of random patient samples were collected from the clinic and tested with the Ou Meng ELISA kit and the self-made anti-CCP antibody colloidal gold test strips. The following results were obtained: Ou Meng ELISA kit detection method and reagent

Positive negative total positive 137 1 138 homemade colloidal gold

Negative 2 60 62 test strip

Total 139 61 200 Positive coincidence rate: 137/139 X 100%=98.6%

Negative coincidence rate: 60/61 X 100%=98.4%

Overall coincidence rate: (137+60) /200=98.5%

According to the above statistics, the total compliance rate of the self-made anti-CCP antibody colloidal gold detection reagent and the Ermen ELISA kit can reach 98.5%.

references

1. Osikowicz G et al. One-step chromatographic immunoassay for qualitative determination of choricogonadotropin in urine. Clin Chem. 1990, 36: 1583-1586.

2. Vallbracht I, Rieber J, Oppermann M. et al. Diagnostic and clinical value of anti-cyclic citmllinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis. Ann Rheum Dis. 2004 (63): 1079-1084.

3. Rantapaa-Dahlqvist S, de Jong BA, Berglin E. et al. Antibodies against cyclic citmllinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. 2003(48):2741-2749 _o

4. Forslind K, Ahlmen M, Eberhardt K. et al. Prediction of radiological outcome in early rheumatoid arthritis in clinical practice: role of antibodies to citmllinated peptides (anti-CCP). Ann Rheum Dis. 2004(63): 1090-1095 .

5. Meyer 0, Nicaise-Roland P, Santos MD. Serial determination of cyclic citmllinated peptide autoantibodies predicted five-year radiological outcomes in a prospective cohort of patients with early rheumatoid arthritis. Arthritis Res Ther. 2006, 8(2): R40.

6. Kohler G, Mil stein C. Continuous cultures of fUsed cells secreting antibody of predefined specificity. Nature. 1975(8): 495-497.

7. Clackson T, Hoogenboom HR, Griffiths AD. et al. Making antibody fragments using phage display libraries. Nature. 1991 (15): 624-628.

8. Marks JD, Hoogenboom HR, Bonnert TP. et al. By-passing immunization Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991(5): 581-597. 9. Zhu Liping, Chen Xueqing. Common experimental methods of immunology. People's Military Medical Press. 2000, 15-26.

10. J. Sambrook, D.W. Lace bait, Guide to Molecular Cloning, Third Edition, 2002: 1228-1232.

Claims

Claim

A test strip for detecting an anti-cyclic citrullinated peptide antibody, characterized in that the test strip comprises a bonding pad and a nitrocellulose membrane which are sequentially attached to each other in a horizontal direction and adhered to each other 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. Detection of antigen, quality control line coated with 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) does not bind to the anti-cyclic citrullinated peptide antibody and the detection antigen.

2. The test strip of claim 1 wherein the test strip further comprises a sample pad and/or an absorbent pad.

3. The test strip according to claim 1, wherein the test strip comprises a bottom plate and a sample pad, a bonding pad, and a nitrocellulose bonded to the bottom plate in a horizontal direction as viewed from each other. Membrane and absorbent pad.

The test strip according to any one of claims 1 to 3, wherein 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.

The test strip according to any one of claims 1 to 3, wherein the antibodies (a), (b) and (c) are selected from monoclonal antibodies or polyclonal antibodies, the single sheet The cloned antibody is of murine or rabbit origin, and the polyclonal antibody is a mouse, rabbit, horse, sheep or guinea pig source, wherein the antibody (a) may also be a staphylococcal protein A or a streptococcus G protein.

The test strip according to claim 5, wherein 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 test strip according to claim 5, wherein 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 mouse IgG and Mouse IgG.

The test strip according to any one of claims 1 to 3, wherein the detection antigen is a conjugate of a cyclic citrulline peptide and a carrier protein, and the carrier protein is selected from the group consisting of bovine serum white. Protein, avidin, streptavidin, thyroglobulin, keyhole limpet hemocyanin, egg protein and synthetic polylysine.

The test strip according to any one of claims 1 to 3, wherein the antigen comprises 2 to 8 cyclic citrullinated peptides and a polylysine core matrix to which the peptide is attached, Wherein each cyclic citrulline peptide may be the same or different antigenic epitope.

10. A method of preparing a test strip according to claim 1, characterized in that the method comprises the steps of: 1) providing a bottom plate; 2) providing a bonding pad, and using a gold standard antibody (a) and a gold standard The antibody (b) is coated with the binding pad; 3) providing a nitrocellulose membrane, wherein the nitrocellulose membrane has a detection line and a quality control line, and a detection antigen capable of specifically binding to the anti-cyclic citrullinated peptide antibody Coating the test line, 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 .