F-actin antigen composition, antigen coating substance and application thereof

Abstract

The invention provides an F-actin antigen composition, an antigen coating substance and application thereof, and relates to the technical field of in-vitro detection. The F-actin antigen composition comprises an F-actin natural antigen and an F-actin recombinant antigen in a mass ratio of (1-2): 1. The antigen coating substance is prepared by firstly mixing an F-actin natural antigen and an F-actin recombinant antigen and then coating the mixture on a solid-phase carrier; or the F-actin natural antigen and the F-actin recombinant antigen are respectively coated on a solid-phase carrier and then are mixed to prepare the F-actin recombinant antigen. The F-actin antigen composition and the antigen coating substance alleviate the defects of poor repeatability and low stability in detection of an anti-F-actin antibody in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection, and in particular to an F-actin antigen composition, an antigen coating and applications thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Autoimmune hepatitis (AIH) is a chronic, progressive liver disease caused by abnormal autoimmune reactions that lead to inflammatory damage to liver cells. In severe cases, it can progress to cirrhosis and liver failure. AIH can be divided into type I and type II based on different serum autoantibodies. Type I autoimmune hepatitis (AIH-1) accounts for about 90% of all AIH cases. Anti-smooth muscle autoantibodies (ASMA) positivity is one of the clinical criteria for diagnosing AIH-1. F-actin is the main specific target antigen of ASMA, and the detection of its antibodies has high sensitivity and specificity for the diagnosis of AIH-1.

[0004] At present, the detection kits of anti-F-actin antibody (AFA) on the market mainly use enzyme-linked immunosorbent assay and indirect immunofluorescence assay for detection. The main principle of the kit is that the anti-F-actin antibody in the sample is captured by the antigen coated on the carrier, and then combined with the secondary antibody labeled with enzyme or fluorescein. The enzyme-linked immunosorbent assay detects the color intensity of the liquid in the experimental group after adding the chromogenic substrate, and the indirect immunofluorescence assay uses a microscope to observe the fluorescence model, compares it with the reference substance, and then reflects the concentration of anti-F-actin antibody in the sample. The above methods all have problems such as low repeatability and stability.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide an F-actin antigen composition and an antigen coating and their application, so as to alleviate at least one of the defects of low repeatability and low stability in the prior art for detecting anti-F-actin antibodies.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In this article, F-actin refers to filamentous actin, which is one of the components of the cytoskeleton and is a polymer assembled from globular actin (G-actin). In this article, "F-actin" is also referred to as "F-actin", and the two are used interchangeably.

[0009] Herein, the natural F-actin antigen refers to the F-actin that exists in nature and has not been artificially modified or synthesized. In an optional embodiment, the natural F-actin antigen is the F-actin obtained by assembling monomers extracted from animals in vitro.

[0010] Herein, the F-actin recombinant antigen refers to F-actin synthesized and expressed in vitro using genetic engineering and protein engineering techniques. In an optional embodiment, the F-actin recombinant antigen is F-actin obtained by assembling monomers obtained by in vitro expression in vitro.

[0011] The term "signal substance" herein refers to a substance that can provide a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art; the signal substance can directly provide a signal, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation or luminescence; or it can indirectly provide a signal through a subsequent reaction in which the signal substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals. Examples of signal substances include, but are not limited to, one or more of enzymes, luminescent labels, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radiocontrast agents, paramagnetic ions, metals, and photosensitizers.

[0012] Herein, "anti-F-actin antibody-positive disease" refers to a disease that can cause the patient's anti-F-actin antibodies to be higher than the level when the patient is not suffering from the disease. Exemplary anti-F-actin antibody-positive diseases include but are not limited to autoimmune hepatitis.

[0013] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual such relationship, order, or importance between such entities or actions, such as numbers (I) and (II), and (i), (ii)...(v).

[0014] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0015] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0016] In a first aspect, a F-actin antigen composition is provided, which comprises a natural F-actin antigen and a recombinant F-actin antigen, wherein the mass ratio of the natural F-actin antigen to the recombinant F-actin antigen is 1 to 2:1, for example, it can be but is not limited to 1:1, 1.5:1 or 2:1.

[0017] In an optional embodiment, the species of origin of the F-actin natural antigen includes but is not limited to one or more of chicken, rabbit, mouse, pig and cattle, preferably cattle.

[0018] In an optional embodiment, the F-actin natural antigen is derived from heart tissue.

[0019] In an optional embodiment, the amino acid sequence of the monomer of the F-actin recombinant antigen is as shown in SEQ ID NO.1 or 2, preferably as shown in SEQ ID NO.1.

[0020] In a second aspect, there is provided use of the F-actin antigen composition described in the first aspect in preparing an antigen coating for detecting anti-F-actin antibodies.

[0021] In a third aspect, a method for preparing an F-actin antigen coating is provided, the preparation method comprising (I) or (II):

[0022] (I) First, the natural F-actin antigen and the recombinant F-actin antigen are mixed at a mass ratio of (1-1.5):1, and then coated on a solid phase carrier.

[0023] (II) coating the natural F-actin antigen on a solid phase carrier to obtain a first coating; coating the recombinant F-actin antigen on a solid phase carrier to obtain a second coating; and mixing the first coating and the second coating at a mass ratio of (1-2):1.

[0024] In an optional embodiment, in (I), the mass ratio of the natural F-actin antigen to the recombinant F-actin antigen is 1:1.

[0025] In an optional embodiment, in (II), the mass ratio of the first coating material to the second coating material is 1.5:1.

[0026] In an optional embodiment, the preparation method includes coating the antigen on a solid phase carrier in a coating buffer.

[0027] In an optional embodiment, the solid phase carrier is magnetic particles.

[0028] In an optional embodiment, the magnetic particles are tosyl magnetic particles.

[0029] In an optional embodiment, the magnetic particles are tosyl magnetic particles, and the coating buffer contains MgCl2, KCl and a buffer component, and the buffer component is selected from tris(hydroxymethylaminomethane), 4-hydroxyethylpiperazineethanesulfonic acid, phosphate, carbonate, borate or 2-morpholineethanesulfonic acid.

[0030] In an optional embodiment, the coating buffer contains 1.8-2.5mM MgCl2 (for example, but not limited to 1.8, 2.0, 2.2 or 2.5mM), 40-60mM KCl (for example, but not limited to 40, 45, 50, 55 or 60mM) and 8-12mM tris(hydroxymethyl)aminomethane at a pH of 7.0-8.0. This preferred coating buffer can maintain the stability of the F-actin antigen conformation during the coating process, improve the coating titer of the magnetic particles, and the obtained reagent has a low detection background, a high signal-to-noise ratio, and good repeatability. It has good differentiation of the reactivity of weak positive, positive and strong positive samples, ensuring the accuracy and repeatability of the test results.

[0031] In an alternative embodiment, the coating buffer contains 2 mM MgCl2, 50 mM KCl and 10 mM Tris at pH 7.5.

[0032] In an optional embodiment, the feed ratio of antigen to magnetic particles is 20 μg:1 mg.

[0033] In an optional embodiment, the preparation method includes coating the antigen on the magnetic particles according to the following method: adding the washed magnetic particles and antigen to the coating buffer, and coating at 37°C for 20 to 25 hours; after separating the magnetic particles, washing the magnetic particles with the washing buffer, and then adding the blocking buffer, blocking at 37°C for 4 to 6 hours; after separating the magnetic particles, washing and storing them in the magnetic particle diluent.

[0034] In a fourth aspect, an antigen coating for detecting anti-F-actin antibodies is provided, wherein the antigen coating comprises complex I prepared by (I) in the preparation method described in the third aspect, and / or complex II prepared by (II) in the preparation method described in the third aspect.

[0035] In a fifth aspect, there is provided the use of the F-actin antigen composition of the first aspect, or the antigen coating of the fourth aspect in any of the following:

[0036] (i) Detection of anti-F-actin antibodies for non-diagnostic and non-therapeutic purposes;

[0037] (ii) preparing a kit for detecting anti-F-actin antibodies;

[0038] (iii) Detection of anti-smooth muscle antibodies for non-diagnostic and non-therapeutic purposes;

[0039] (iv) preparing a kit for detecting anti-smooth muscle antibodies;

[0040] (v) Preparation of a kit for detecting anti-F-actin antibody positive diseases.

[0041] In an optional embodiment, the anti-F-actin antibody-positive disease includes autoimmune hepatitis.

[0042] In a sixth aspect, a reagent or a kit is provided, wherein the reagent or the kit is used for detecting anti-F-actin antibodies, anti-smooth muscle antibodies or anti-F-actin antibody positive diseases. The reagent or the kit comprises the F-actin antigen composition described in the first aspect, or the antigen coating described in the fourth aspect.

[0043] The kit may also optionally include reagents and / or consumables known to those skilled in the art for detection reactions, including but not limited to one or more of buffer reagents, salts, secondary antibodies, signal substances, blocking solutions, washing solutions, solvents, eluents, negative controls, positive controls, standards, and quality control products.

[0044] In an optional embodiment, the antigen coating is obtained by coating the antigen on magnetic particles and stored in a magnetic particle diluent, wherein the magnetic particle diluent contains 8-12mM tris (for example but not limited to 8, 10 or 12mM), 1.8-2.5mM MgCl2 (for example but not limited to 1.8, 2.0, 2.2 or 2.5mM), 40-60mM KCl (for example but not limited to 40, 45, 50, 55 or 60mM), 0.03-0.10w / v% ProClin300 (for example but not limited to 0.03, 0.05, 0.08 or 0.10w / v%), 0.5-1.0w / v% BSA (for example but not limited to 0.5, 0.8 or 1.0w / v%) and 0.75-1.50mM ATP (for example, but not limited to, 0.75, 1.0, 1.25 or 1.50 mM), pH 7.0-7.5.

[0045] In an optional embodiment, the kit is used to detect autoimmune hepatitis and also includes a reagent for detecting at least one of the following autoantibodies: anti-hepatocyte cytoplasmic type I antibodies (anti-LC-1 antibodies), anti-liver and kidney microsomal antibodies (anti-LKM-1 antibodies), anti-nuclear antibodies (ANA), anti-soluble liver antigen / liver and pancreatic antigen antibodies (anti-SLA / LP) and atypical perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA).

[0046] In an optional embodiment, the kit is a chemiluminescent detection kit, and further includes conventional chemiluminescent detection reagent components, including but not limited to one or more of an F-actin detection antibody working solution coupled with an alkaline phosphatase marker, a sample diluent, and a luminescent substrate.

[0047] In the seventh aspect, a method for detecting anti-F-actin antibodies for non-diagnostic and therapeutic purposes is provided, the method comprising using the F-actin antigen composition described in the first aspect, or the antigen coating described in the fourth aspect, or the reagent or kit described in the sixth aspect.

[0048] In an optional embodiment, chemiluminescence is used to detect the anti-F-actin antibody captured in the test sample.

[0049] In an optional embodiment, the F-actin antigen composition or the antigen coating is used to capture anti-F-actin antibodies in the sample to be tested.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The F-actin antigen composition provided by the present invention comprises a natural F-actin antigen and a recombinant FF-actin antigen. The antigen coating for detecting anti-F-actin antibodies prepared by using the F-actin antigen composition has good long-term stability and precision. In the preferred embodiment, after the kit is placed at 2-8°C for 12 months, the signal retention rate can reach more than 95%; and the coefficient of variation CV can reach less than 3% when detecting samples of three different concentrations, low, medium and high. Compared with the magnetic particles coated with a single natural F-actin antigen or recombinant antigen as a capture reagent, the antigen coating has a significantly higher positive coincidence rate under the premise that the negative coincidence rate reaches 100%, and the positive and negative coincidence rates are all above 98%, wherein the positive and negative coincidence rate of the antigen coating prepared by coating the natural F-actin antigen and the recombinant F-actin antigen on a solid phase carrier respectively and then mixing them can reach 100%.

[0052] The reagent or kit based on the antigen composition and antigen coating provided by the present invention has the advantages of simple operation, good repeatability, stability and clinical sensitivity; and good specificity, signal-to-noise ratio, precision and long-term stability. In a preferred embodiment, the detection rate of the reagent or kit for anti-F-actin antibody positive samples can reach more than 96%, and in a more preferred embodiment, the clinical specificity and sensitivity can both reach 100%, enriching the diagnostic products for autoimmune hepatitis. DETAILED DESCRIPTION

[0053] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] In the following examples, the magnetic microparticles used are tosyl magnetic beads, and the tosyl groups on the surface of the magnetic beads react chemically with the amino groups in the target protein to form a covalent bond, and the coupling can be completed without activation.

[0055] In the following examples, all relevant experiments were performed using the fully automatic chemiluminescence immunoassay analyzer SMART 6500H produced by Chongqing Cosmay Technology Co., Ltd.

[0056] The implementation process and reagent performance of the present invention will be described in detail below in conjunction with specific examples. The reagents and materials used in the following examples, except for those with clear sources or manufacturers, are all sold by conventional manufacturers on the market, and the clinical samples used are provided by cooperative hospitals.

[0057] Example 1

[0058] 1. This embodiment provides a coating method for F-actin antigen, and the specific steps are as follows:

[0059] (1) Take the tosyl magnetic bead mother solution, discard the supernatant using a magnetic separation device, and then add the pre-coating washing solution (PB buffer, pH 7.0-8.0) for washing.

[0060] (2) Add appropriate amount of magnetic beads and antigen in coating buffer (10 mM Tris, 2 mM MgCl2, 50 mM KCl, pH 7.5) at a mass ratio of total antigen: magnetic beads = 20 μg: 1 mg, and rotate at 37°C for 20 to 25 h.

[0061] (3) After discarding the supernatant using a magnetic separation device, add post-coating washing solution (PB buffer, containing 0.1% w / v% BSA, 0.05 w / v% ProClin300, pH 7.0-8.0) for washing.

[0062] (4) Use a magnetic separation device to discard the supernatant, add blocking buffer (Tris buffer, containing 0.5% w / v% BSA, 0.05 w / v% ProClin300, pH 7.0-8.0), and rotate at 37°C for 4-6 hours.

[0063] (5) Use a magnetic separation device to discard the supernatant, and add a post-blocking washing solution (Tris buffer system, containing 0.5 w / v% Tween 20, pH 7.0-8.0) for washing.

[0064] (6) Use a magnetic separation device to discard the supernatant and store the coated product in magnetic bead diluent at 2-8°C.

[0065] 2. This embodiment involves the coating of natural F-actin and recombinant F-actin antigens, as follows:

[0066] Table 1

[0067] Group F-actin source Antigen type Experimental Group 1 chicken natural Experimental Group 2 rabbit natural Experimental Group 3 mouse natural Experimental Group 4 pig natural Experimental Group 5 ox natural Experimental Group 6 Type α Reorganization Test Group 7 Gamma Reorganization

[0068] In this embodiment, the natural or recombinant antigen raw material used is partially in monomeric form and needs to be polymerized in vitro into a polymeric form, namely F-actin. The in vitro polymerization steps of F-actin are as follows:

[0069] (1) The lyophilized powder of F-actin antigen was reconstituted to 10 mg / mL with G solution (0.2 mM CaCl2, 5 mM Tris-HCl, 0.2 mM ATP, 0.5 mM DTT, pH 8.0, prepared in D2O).

[0070] (2) Incubate on ice for 60 min.

[0071] (3) Centrifuge at 14,000 rpm for 15 min at 4°C and transfer the supernatant to a new centrifuge tube.

[0072] (4) Remove 30-200 μL of supernatant and transfer to a new centrifuge tube. Add 1 / 10 volume of 10×F solution (100 mM Tris-HCl, 20 mM MgCl2, 500 mM KCl, 10 mM ATP, pH 7.5, prepared in D2O) and incubate at room temperature for 1 h.

[0073] (5) Centrifuge at 25°C and 14,000 r / min for 1 h, discard 90% of the supernatant, and transfer the remainder to a new centrifuge tube to obtain the polymerized F-actin product, which can be used for coating.

[0074] Among them, F-actin from natural sources: including heart tissues of chicken, rabbit, mouse, pig and cow, was obtained by referring to the Purification of muscleactin method written by Pardee JD and Spudich JA in Methods in Cell Biology, with a purity greater than 90%.

[0075] The recombinant antigen was purchased from abbexa and the sequence is as follows:

[0076] Amino acid sequence of α-type recombinant F-actin antigen:

[0077]

[0078] Amino acid sequence of γ-type recombinant F-actin antigen:

[0079]

[0080] Example 2

[0081] In this embodiment, magnetic beads coated with different antigens were tested. After being diluted to the same concentration with magnetic bead diluent, they were matched with the same concentration of alkaline phosphatase-labeled IgG antibody working solution (produced by Zhuhai Lihe Medical Diagnostic Products Co., Ltd.) to detect 158 ​​positive clinical samples, and the positive rate of the test results was statistically analyzed.

[0082] In this example, the fully automatic chemiluminescence immunoassay analyzer SMART 6500H was used for detection:

[0083] The two-step reaction was adopted. After the instrument absorbed the sample (75 μL), it was diluted in the machine with the sample diluent, and then F-actin magnetic beads (0.5 mg / mL, 50 μL, prepared according to the method of Example 1) were added, incubated at 37°C for 10 min, and F-actin enzyme-labeled antibody (0.5 μg / mL, 100 μL) was added after washing, and incubated at 37°C for 10 min. After washing again, 200 μL of substrate solution (AMPPD solution, purchased from Zhuhai Lihe Medical Diagnostic Products Co., Ltd., item number: M1008) was added and incubated at 37°C for 5 min, and the luminescence value was read. The detection of the coating effect of F-actin from different sources is shown in Tables 2 and 3 below:

[0084] Table 2 Effect of natural F-actin coating

[0085]

[0086]

[0087] Table 3 Detection of the coating effect of recombinant F-actin

[0088]

[0089] The above results show that among the natural F-actin antigens, the bovine F-actin was selected as the coating antigen, and the positive detection rate was the best; among the recombinant antigens, the α-type F-actin was selected as the coating antigen, and the positive detection rate was the best. However, the results also show that the detection reagent obtained by coating a single antigen has a maximum positive detection rate of only about 90%, and the performance needs to be further improved.

[0090] Example 3

[0091] In this embodiment, two preferred coating materials in Example 2 were mixed for coating by two schemes to screen a suitable coating process. The specific process is as follows:

[0092] Process 1: Mix natural bovine species and recombinant α-type F-actin antigens at a natural antigen: recombinant antigen (natural: recombinant) ratio of 2:1, 1.5:1, 1:1, 1:1.5, and 1:2, and then coat them together according to the coating steps in Example 1 to obtain a mixed coated magnetic bead mother solution.

[0093] Process 2: The natural bovine species and recombinant α-type F-actin antigens are first coated according to the coating steps in Example 1 to obtain single antigen-coupled magnetic beads. The magnetic beads coated with the natural bovine antigen are recorded as magnetic beads 1, and the magnetic beads coated with the recombinant α-type antigen are recorded as magnetic beads 2. The beads 1: magnetic beads 2 are then mixed at a ratio of 2:1, 1.5:1, 1:1, 1:1.5, and 1:2 to obtain a mixed magnetic bead mother liquor after coating.

[0094] The magnetic bead mother solution obtained by the above two processes was diluted to 0.5 mg / mL with magnetic bead diluent to obtain magnetic bead working solution. 158 positive clinical samples were tested and the positive detection rate was statistically analyzed. The results are as follows:

[0095] Table 4 Process 1 test results

[0096]

[0097] Table 5 Process 2 test results

[0098]

[0099]

[0100] The above test results show that: in scheme 1, when the mixed coating ratio is natural antigen: recombinant antigen = 1:1, the positive detection rate is the highest, which is the optimal mixed coating ratio; in scheme 2, when the mixing ratio is magnetic beads 1 (natural antigen coupled magnetic microparticles): magnetic beads 2 (recombinant antigen coupled magnetic microparticles) = 1.5:1, the positive detection rate is the highest, which is the optimal magnetic bead coating post-mixing ratio. Among them, scheme 2, i.e. the preferred post-coating mixing process, has the best positive detection rate of the reagent, which can reach 100%.

[0101] This embodiment provides two feasible anti-F-actin magnetic bead coating processes, and the results show that the positive detection rate of the magnetic beads obtained by coating with a suitable ratio is significantly improved compared with the magnetic beads coated with a single antigen. The combined coating scheme is feasible, but different processes have different ratio requirements.

[0102] Example 4

[0103] In this embodiment, the post-coating mixing scheme of test group 14 was taken as an example for research. The coating buffer in the coating step (2) of Example 1, except for 2mM MgCl2 and 50mM KCl as common components, was used with 10mM tris(hydroxymethylaminomethane) (Tris, pH7.5), 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, pH7.5), 10mM phosphate (PB, pH7.5), 100mM carbonate (CB, pH9.2), 10mM borate (pH8.0), 50mM 2-morpholineethanesulfonic acid (MES, pH6.5) buffer system. After coating equal amounts of magnetic beads and antigens, the same concentration of alkaline phosphatase-labeled F-actin antibody working solution was used to detect the same mixed clinical samples, and the luminescence value, signal-to-noise ratio and repeatability (CV) were compared.

[0104] The test results are as follows:

[0105] Table 6

[0106]

[0107]

[0108] The above test results show that when Tris is used as a buffer system to coat F-actin, the resulting magnetic beads are matched with the enzyme-labeled working solution to detect weakly positive, positive and strongly positive samples, and the reactivity is higher than that of negative samples, and the background is lower, that is, the signal-to-noise ratio is higher. At the same time, the repeatability (CV) of the test results for the same sample is good, all within 4%. Therefore, compared with 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, pH7.5), 10mM phosphate (PB, pH7.5), 100mM carbonate (CB, pH9.2), 10mM borate (pH8.0) and 50mM 2-morpholineethanesulfonic acid (MES, pH6.5), 10mM Tris (pH7.5) with 2mMMgCl2 and 50mM KCl is the best F-actin coating buffer.

[0109] Example 5

[0110] In this embodiment, the post-coating mixing scheme of test group 14 was taken as an example for research. According to the coating method in Example 1, in step (2), the components of the F-actin coating buffer were: 10mM Tris, 2mM MgCl2, 50mM KCl, and the pH was adjusted to 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. After coating equal amounts of magnetic beads and antigens, the same concentration of alkaline phosphatase-labeled F-actin antibody working solution was used to detect the same mixed clinical samples, and the luminescence value, signal-to-noise ratio and repeatability (CV) were compared.

[0111] The test results are as follows:

[0112] Table 7

[0113]

[0114]

[0115] The above test results show that the pH of the coating buffer has a significant effect on the performance of the magnetic beads. 7.0-8.0 is selected as the optimal pH of the F-actin coating buffer. The obtained magnetic beads are matched with the enzyme-labeled working solution to detect negative, weakly positive, positive and strongly positive samples. The background is low, the reactivity and signal-to-noise ratio are high, and the CV is small. Among them, the coating effect of pH 7.5 is the best.

[0116] Example 6

[0117] The magnetic beads obtained from test groups 5, 6, 10, and 14 in Examples 2 and 4 were diluted to the same working concentration, matched with the same enzyme labeling working solution, loaded into reagent bottles, sealed and stored at 2-8° C. The day when the magnetic bead working solution was prepared was recorded as the 0th month, and the same samples were tested at 0th, 3rd, 6th, 9th, 12th, and 13th months, respectively, and compared with the signal value results detected at 0th month to obtain the signal retention rate at each time point, and then investigate its long-term stability.

[0118] The above signal retention rate calculation method is:

[0119] Signal retention rate at month N = (RLU at month N stored at 2-8°C / RLU at month 0 stored at 2-8°C)%, where N = 3, 6, 9, 12, 13.

[0120] The qualified standard for long-term stability is: the signal retention rate of the reagent is ≥95% when stored at 2-8°C for 12 months.

[0121] The experimental results are as follows:

[0122] Table 8

[0123]

[0124]

[0125] The above test results show that when the natural bovine antigen or recombinant α-type antigen is coated separately and matched with the same enzyme label, the signal retention rate is below 95% after being placed at 2-8°C for 12 months (experimental group 5 and experimental group 6), which does not meet the long-term stability standard; when the two antigens are mixed and coated or mixed separately, the obtained magnetic beads are matched with the same enzyme label, the signal retention rate after 12 months is above 95% (experimental group 10 and experimental group 14), among which the reagent system obtained by the post-coating mixing process (experimental group 14) has the best long-term stability.

[0126] Example 7

[0127] The magnetic beads obtained from test groups 10 and 14 in Example 4 were used to test clinical samples of three different concentrations, low, medium and high, for 10 times to examine the precision performance of the reagent.

[0128] The experimental results are as follows:

[0129] Table 9

[0130]

[0131] The above test results show that the CVs of the two mixed coating processes of the anti-F-actin antibody detection reagents in test groups 10 and 14 were both within 3%, and the reagent precision performance was good.

[0132] Example 8

[0133] The reagents obtained from test groups 5, 6, 10, and 14 in Examples 3 and 4 were used to detect 243 clinical samples at the same time as the control reagent EUROIMMUN anti-F-actin antibody IgG detection kit (indirect immunofluorescence method), and the positive and negative coincidence rates of the results were statistically analyzed to examine the clinical performance.

[0134] The experimental results are as follows:

[0135] Table 10

[0136]

[0137] The above test results show that when the negative compliance rate of the four groups of reagents reached 100%, the positive compliance rate of the reagents obtained by coating the natural bovine antigen or the recombinant α-type antigen alone (test groups 5 and 6) was about 90%, which was a low compliance rate; when the two antigens were mixed for coating or mixed after coating, the positive and negative compliance rates of the obtained reagents were all above 98% (test groups 10 and 14), among which the total positive and negative compliance rate of the reagents obtained by mixing after coating (group 14) was the highest.

[0138] That is, the anti-F-actin antibody detection kits obtained by the two F-actin antigen coating processes provided by test groups 10 and 14 have good clinical sensitivity and specificity and have good clinical application prospects.

[0139] Example 9

[0140] In this example, the magnetic bead mother solution obtained in test group 14 was used to further optimize the magnetic bead diluent formula. The performance of different diluent formulas was tested, and the test results were as follows:

[0141] Table 11

[0142]

[0143]

[0144] Each group can meet the better detection effect.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An F-actin antigen composition, characterized in that: The method comprises a natural F-actin antigen and a recombinant F-actin antigen, wherein the mass ratio of the natural F-actin antigen to the recombinant F-actin antigen is 1 to 2:

1.

2. The F-actin antigen composition according to claim 1, characterized in that The species of the natural F-actin antigen include one or more of chicken, rabbit, mouse, pig and cattle, preferably cattle; And / or, the amino acid sequence of the monomer of the F-actin recombinant antigen is shown as SEQ ID NO.1 or 2.

3. Use of the F-actin antigen composition according to claim 1 or 2 in preparing an antigen coating for detecting anti-F-actin antibodies.

4. A method for preparing a F-actin antigen coating, characterized in that: Includes (I) or (II): (I) firstly mix the natural F-actin antigen and the recombinant F-actin antigen in a mass ratio of (1-1.5):1, and then coat them on a solid phase carrier; (II) coating a natural F-actin antigen on a solid phase carrier to obtain a first coating; coating a recombinant F-actin antigen on a solid phase carrier to obtain a second coating; mixing the first coating and the second coating at a mass ratio of (1-2):1; Optionally, in (I), the mass ratio of the natural F-actin antigen to the recombinant F-actin antigen is 1:1; Optionally, in (II), the mass ratio of the first coating material to the second coating material is 1.5:

1.

5. The preparation method according to claim 4, characterized in that: Coating the antigen on the solid phase carrier in a coating buffer; Optionally, the solid phase carrier is a magnetic particle; Optionally, the magnetic particles are tosyl magnetic particles, the coating buffer contains MgCl2, KCl and a buffer component, and the buffer component is selected from tris(hydroxymethylaminomethane), 4-hydroxyethylpiperazineethanesulfonic acid, phosphate, carbonate, borate or 2-morpholineethanesulfonic acid; Optionally, the coating buffer contains 1.8-2.5 mM MgCl2, 40-60 mM KCl, and 8-12 mM tris(hydroxymethyl)aminomethane at a pH of 7.0-8.0; Optionally, the coating buffer contains 2 mM MgCl2, 50 mM KCl and 10 mM Tris at pH 7.5; Optionally, the feed ratio of antigen to magnetic particles is 20 μg:1 mg.

6. The preparation method according to any one of claims 4 to 5, characterized in that: Antigens were coated on magnetic particles as follows: washed magnetic particles and antigens were added to coating buffer and coated at 37°C for 20-25 hours; after separation of magnetic particles, the magnetic particles were washed with washing buffer, and then blocking buffer was added and blocked at 37°C for 4-6 hours; after separation of magnetic particles, the magnetic particles were washed and stored in magnetic particle diluent.

7. An antigen coating for detecting anti-F-actin antibodies, characterized in that: It comprises the complex I prepared by (I) in the preparation method according to any one of claims 4 to 6 and / or the complex II prepared by (II) in the preparation method according to any one of claims 4 to 6.

8. Use of the F-actin antigen composition according to any one of claims 1 to 2, or the antigen coating according to claim 7 in any of the following: (i) Detection of anti-F-actin antibodies for non-diagnostic and therapeutic purposes; (ii) preparing a kit for detecting anti-F-actin antibodies; (iii) Detection of anti-smooth muscle antibodies for non-diagnostic and non-therapeutic purposes; (iv) preparing a kit for detecting anti-smooth muscle antibodies; (v) preparing a kit for detecting anti-F-actin antibody positive diseases; Optionally, the anti-F-actin antibody-positive disease comprises autoimmune hepatitis.

9. A reagent or a kit, characterized in that Comprising the F-actin antigen composition according to any one of claims 1 to 2, or the antigen coating according to claim 7; Optionally, the antigen coating is obtained by coating the antigen on magnetic particles and stored in a magnetic particle diluent, wherein the magnetic particle diluent contains 8-12 mM tris(hydroxymethyl)aminomethane, 1.8-2.5 mM MgCl2, 40-60 mM KCl, 0.03-0.10 w / v% ProClin300, 0.5-1.0 w / v% BSA and 0.75-1.50 mM ATP, and the pH is 7.0-7.5; Optionally, the kit is used to detect autoimmune hepatitis and also includes a reagent for detecting at least one of the following autoantibodies: anti-hepatocyte cytoplasmic type I antibodies, anti-liver and kidney microsomal antibodies, anti-nuclear antibodies, anti-soluble liver antigen / liver and pancreatic antigen antibodies and atypical perinuclear anti-neutrophil cytoplasmic antibodies.

10. A method for detecting anti-F-actin antibodies for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises using the F-actin antigen composition according to any one of claims 1 to 2, or the antigen coating according to claim 7, or the reagent or kit according to claim 9.

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