Anti-MDA5 antibody detection kit and its preparation method

Abstract

This invention relates to a detection kit for anti-MDA5 antibodies and a method for preparing the same. The kit includes reagents A1, A2, and A3. Reagent A1 includes MDA5 antigen coated with magnetic beads, labeling buffer, blocking buffer, washing solution, and magnetic bead dilution solution. Reagent A2 includes anti-human IgG conjugate labeled with a chemiluminescent marker and labeling buffer. Reagent A3 includes calibrators.

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology, specifically to a kit for quantitative detection of anti-MDA5 antibodies using magnetic beads chemiluminescence, and its preparation and detection methods. Background Technology

[0002] Idiopathic inflammatory myopathy (IIM) is a systemic disease primarily affecting skeletal muscles, clinically characterized by skeletal muscle weakness, skin lesions, systemic organ damage, and the production of specific antibodies. Polymyositis (PM) and dermatomyositis (DM) are the two most common types. Dermatomyositis is a group of autoimmune diseases mainly affecting the skin and muscles, possibly related to autoimmune factors, genetics, and infection. Melanoma differentiation-associated gene 5 (MDA5) has been identified as an autoantigen highly associated with a specific subtype of dermatomyositis (DM). MDA5+DM is a rare autoimmune disease prevalent in East Asia, a group of multisystemic inflammatory diseases characterized by interstitial pneumonia and skin rashes, belonging to a specific subtype of dermatomyositis. Lung imaging suggests interstitial pneumonia, and serum antibody testing reveals positive anti-MDA5 antibodies.

[0003] MDA5, encoded by the helicase C-induced interferon domain protein 1 (IFIH1) gene, is a key protein in the physiological recognition of viral double-stranded RNA (dsRNA). When an individual is exposed to certain environmental factors, such as hepatitis A virus, Coxsackievirus B, enteroviruses, and rhinoviruses, these microviruses can activate MDA5, producing type I IFN (IFNα and IFNβ) and other inflammatory cytokines involved in antiviral responses. Overactivation of the type I interferon pathway may lead to autoinflammatory diseases.

[0004] Anti-MDA5 antibody levels are associated with disease prognosis. A study by SATO et al. (SATO et al. RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen inpatients with clinically amyopathic dermatomyositis: association with rapidly progressive interstitial lung disease[J]. Arthritis Rheum, 2009, 60(7): 2193-2200.) showed that anti-MDA5 antibody levels in patients with effective treatment were significantly lower than in those without, and anti-MDA5 antibody levels in patients with poor treatment outcomes remained persistently high. Therefore, dynamic monitoring of anti-MDA5 antibody levels can help assess disease prognosis. Detection of serum anti-MDA5 antibody titers can not only preliminarily assess whether DM patients have concurrent interstitial lung diseases (ILD), but also serve as a serological marker for the potential development of acute progressive interstitial pneumonia (RPILD) in DM patients. Furthermore, recent studies have also suggested that serum anti-MDA5 antibody titers are closely related to disease activity in DM patients. Therefore, quantitative detection of anti-MDA5 antibodies is not only helpful for the early assessment of ILD progression, but also significant for monitoring disease activity and evaluating treatment efficacy.

[0005] Currently, immunological detection methods for anti-MDA5 antibodies include qualitative methods such as Western blotting, immunostrip assays, and ELISA. However, these methods suffer from poor consistency, low sensitivity, narrow linear range, low reproducibility, complex operation (low automation), low throughput, and the inability to quantitatively detect anti-MDA5 antibody levels in patient serum, thus failing to adequately meet clinical needs. Therefore, rapidly and accurately diagnosing this type of disease and quantitatively detecting anti-MDA5 antibody levels in patient serum is a crucial problem that urgently needs to be solved.

[0006] On the other hand, the MDA5 antigen has a large molecular weight, making expression difficult. Existing research reagents all use truncated protein fragments for research applications, resulting in lower detection rates compared to the full-length fragment. Moreover, the full-length protein is expensive, and the ELISA method has low sensitivity and high cost. Even if the full-length protein is successfully expressed, due to its unique sequence structure, it cannot be dissolved in conventional buffer solutions, requiring the introduction of reagents such as urea to increase its solubility. However, these reagents severely affect the labeling and coupling efficiency of magnetic beads and antigens, which may also be one of the methodological gaps in this indicator.

[0007] Magnetic bead chemiluminescence immunoassay offers significant improvements over previous membrane strip immunoassays and enzyme-linked immunosorbent assays in terms of detection sensitivity, detection range, detection time, and automation, while also being pollution-free and widely applicable in clinical settings. However, the application of magnetic bead chemiluminescence immunoassay in the immunoassay of anti-MDA5 antibody IgG products is currently unseen. Summary of the Invention

[0008] Through extensive experimental research, the inventors unexpectedly discovered that in the quantitative detection of anti-MDA5 antibodies, the use of a specific reagent composition can reduce the non-specific adsorption of biological substances in the sample by magnetic beads, thereby significantly improving the coupling efficiency, stability, and sensitivity. Based on this discovery, the inventors developed a chemiluminescent immunoassay kit for the quantitative detection of anti-MDA5 antibodies.

[0009] In one aspect, the present invention provides a chemiluminescent immunoassay kit for the quantitative detection of anti-MDA5 antibodies, comprising reagent A1, reagent A2, and reagent A3; wherein reagent A1 comprises magnetic beads coated with MDA5; reagent A2 comprises a labeled anti-human IgG conjugate; and reagent A3 comprises a calibrator.

[0010] In some embodiments, the A1 reagent is prepared from magnetic beads, MDA5 antigen, labeling buffer, blocking buffer, washing solution, and magnetic bead dilution solution.

[0011] In some embodiments, the A2 reagent is prepared from a chemiluminescent label, anti-human IgG, and a labeling buffer.

[0012] In some embodiments, the magnetic bead particles have a particle size of 1-3 μm.

[0013] In some preferred embodiments, the magnetic bead particles have a particle size of 1 μm.

[0014] In some embodiments, the labeling buffer comprises 0.015 mol / L morpholine ethanesulfonic acid (MES) at a pH of 6.0–7.0.

[0015] In some embodiments, the blocking buffer is selected from either a first blocking buffer or a second blocking buffer, wherein the first blocking buffer comprises 0.05 mol / L Tris and 0.5% gelatin; and the second blocking buffer comprises 5% BSA and MPC polymer.

[0016] In some preferred embodiments, the MPC polymer is Biolipidure 206.

[0017] In some embodiments, the cleaning solution includes arginine and Brij35.

[0018] In some embodiments, the magnetic bead diluent comprises 0.05 mol / L Tris, 0.5% casein, 0.2% Proclin 300, and polysaccharides.

[0019] In some preferred embodiments, the polysaccharide is pranolose and sulfated dextran.

[0020] In some embodiments, the amount of MDA5 antigen in the A1 reagent is 10-30 μg antigen / mg magnetic beads.

[0021] In some preferred embodiments, the amount of MDA5 antigen in the A1 reagent is 20 μg antigen / mg magnetic beads.

[0022] In some embodiments, the concentration of the anti-human IgG conjugate labeled in the A2 reagent is 0.05-0.2 ng / ml.

[0023] In some preferred embodiments, the concentration of the anti-human IgG conjugate labeled in the A2 reagent is 0.1 ng / ml.

[0024] In some embodiments, the calibrator in the A3 reagent is a dilution of positive serum for anti-MDA5 antibody detection in CAMD patients using Western blot assay.

[0025] In some embodiments, the concentration of the calibrator in the A3 reagent is 5-400 Ru / ml.

[0026] In some preferred embodiments, the concentration of the calibrator in the A3 reagent is 15-200 Ru / ml.

[0027] In some embodiments, the chemiluminescent label is luminol, isoluminol, terpyridine ruthenium, or acridine ester.

[0028] In some embodiments, the kit further includes a sample buffer comprising 0.05 M Tris, 1% gelatin, and 0.2% Tween 20.

[0029] In some embodiments, the method of using the A1 reagent includes the following steps:

[0030] 1) Place the magnetic bead stock solution into a centrifuge tube and remove the supernatant; add labeling buffer, remove the supernatant, and resuspend; add EDC and mix by rolling at room temperature for 30 min; add labeling buffer and remove the supernatant; add labeling buffer and add MDA5 antigen, and mix by rolling at room temperature for 2 h.

[0031] 2) Discard the supernatant from step 1), add blocking buffer, and discard the supernatant; add blocking buffer and mix well, then block at 37 degrees Celsius and 1500 rpm for 5-18 hours.

[0032] 3) Discard the supernatant from step 2), add the cleaning solution, and discard the supernatant; add the cleaning solution and mix well, then wash overnight at room temperature and 1500 rpm.

[0033] 4) Add magnetic bead diluent and remove the supernatant; add magnetic bead diluent to obtain A1 reagent with a final concentration of 0.1-0.2 mg / ml.

[0034] In some embodiments, the method of using the A1 reagent includes the following steps:

[0035] 1) Place the magnetic bead stock solution into a centrifuge tube and remove the supernatant; add labeling buffer, mix thoroughly, adsorb, remove supernatant, repeat 3 times; add labeling buffer and resuspend; add EDC, mix by rolling at room temperature for 30 min; add labeling buffer, mix, adsorb, remove supernatant, repeat once; add labeling buffer, add MDA5 antigen, mix by rolling at room temperature for 2 h.

[0036] 2) Discard the supernatant from step 1), add blocking buffer and mix well, adsorb, remove supernatant, repeat once, for a total of 2 times; discard the supernatant, add blocking buffer and mix well, block at 37 degrees and 1500 rpm for 5-6 hours.

[0037] 3) Discard the supernatant from step 2), add the cleaning solution and mix well, adsorb, remove the supernatant, repeat once, for a total of 2 times; discard the supernatant, add the cleaning solution and mix well, wash overnight at room temperature and 1500 rpm.

[0038] 4) Add magnetic bead diluent, mix well, adsorb, remove supernatant, repeat once, for a total of 2 times; add magnetic bead diluent to obtain A1 reagent with a final concentration of 0.1-0.2 mg / ml.

[0039] In some implementations, the method of using the A2 reagent includes the following steps:

[0040] 1) Take anti-human IgG into an EP tube, add labeling buffer, add chemiluminescent label, wrap the centrifuge tube with a layer of aluminum foil to protect it from light, place it on a rotary mixer, and mix and react at room temperature for a period of time.

[0041] 2) Add 1% Lys, place in a light-protected environment on a rotary mixer, and mix at room temperature for 30 min.

[0042] 3) Purify the chemiluminescent label-antibody conjugate, add glycerol, and store at -20 degrees Celsius for later use;

[0043] 4) Take the chemiluminescent label-antibody conjugate, add it to the A2 reagent diluent, mix thoroughly, and obtain the A2 reagent with a final concentration of 0.05 ng / ml-0.2 ng / ml.

[0044] In some implementations, the method of using the A2 reagent includes the following steps:

[0045] 1) Take anti-human IgG into an EP tube, add labeling buffer to 0.3 ml, take acridinium ester working solution, add it to the above protein solution, mix immediately, wrap the centrifuge tube with a layer of aluminum foil to protect it from light, place it on a rotary mixer, and mix and react for a period of time at room temperature.

[0046] 2) Take 1% Lys, add it to the centrifuge tube above, keep it in the dark, place it on a rotary mixer, and mix and react at room temperature for 30 min.

[0047] 3) Purify the acridinium ester-antibody conjugate, add glycerol, and store at -20 degrees Celsius for later use;

[0048] 4) Take the chemiluminescent label-antibody conjugate, add it to the A2 reagent diluent, mix thoroughly, and obtain the A2 reagent with a final concentration of 0.05 ng / ml-0.2 ng / ml.

[0049] In some implementations, the method of using the A3 reagent includes the following steps:

[0050] Take the calibrator stock solution and add the calibrator diluent to obtain A3 reagents with final concentrations of 15 Ru / ml and 200 Ru / ml, respectively. The calibrator diluent is selected from either the first calibrator diluent or the second calibrator diluent, wherein the first calibrator diluent includes 1% casein and 0.1% Tween 20; and the second calibrator diluent includes 5% BSA.

[0051] In a second aspect, the present invention provides a method of using the above-mentioned kit, which includes using reagents A1, A2, and A3 to determine the concentration of anti-MDA5-IgG antibody in the sample to be tested.

[0052] The superior technical effects of the reagent kit described in this invention are mainly in the following aspects:

[0053] 1. After extensive raw material screening, debugging, pairing and other combination of conditions, the kit of the present invention uses small-diameter magnetic beads, high labeling pH and activation ratio, and the addition of biolipidure206 or gelatin and biolipidure206 to the blocking solution can reduce the non-specific adsorption of biological substances in the sample by the magnetic beads.

[0054] 2. After the magnetic beads are labeled, they are cleaned with a combination of arginine and brij35 to increase their stability.

[0055] 3. Polysaccharides were added to the magnetic bead diluent, which greatly improved its coupling efficiency, stability and sensitivity.

[0056] 4. Conventional detection systems have poor P / N ratios. By introducing reagents such as polysaccharides and casein into the reaction system, as well as the pairing of secondary antibodies, the P / N ratio can be significantly improved. Attached Figure Description

[0057] Figure 1 The preparation process of a chemiluminescent immunoassay kit for the quantitative detection of anti-MDA5 antibody;

[0058] Figure 2 The reaction calibration curve is for the chemiluminescent immunoassay kit for the quantitative detection of anti-MDA5 antibody. Detailed Implementation

[0059] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are merely for illustrating the features and advantages of the invention and are not intended to limit the scope of the claims. Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those skilled in the art.

[0060] definition

[0061] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0062] As used in this article, “detection” refers to the qualitative analysis of the presence or absence of a substance.

[0063] As used herein, “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0064] As used herein, the conjunction "composed of..." excludes any unspecified element, step, or component. If used in a claim, this phrase makes the claim closed, excluding any material other than those described, except for conventional impurities associated with them. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the element described in that clause; other elements are not excluded from the claim as a whole.

[0065] As used herein, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0066] As used herein, “and / or” is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0067] As used in this article, "multiple" refers to two or more.

[0068] As used herein, the term "buffer solution" refers to a solution that prevents significant pH changes through the action of its acid-base paired components. Such substances are well known to those skilled in the art; see, for example, Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffer solutions include MES, MOPS, MOPSO, Tris, HEPES, phosphates, acetates, citrates, succinates, ammonium salts, casein, BSA, Biolipidure 206, etc.

[0069] As used in this article, the term "Brij35" refers to a chemical detergent, a nonionic detergent, namely dodecyl polyethylene glycol ether, also known as polyoxyethylene lauryl ether.

[0070] As used in this article, the term "Proclin 300" refers to a highly effective preservative composed of MIT / CMIT.

[0071] As used herein, the term "MPC polymer" refers to a chemical substance, 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0072] As used in this article, the term "bovine serum albumin (BSA)" refers to a globulin found in bovine serum containing 607 amino acid residues, with a molecular weight of 66.446 kDa and an isoelectric point of 4.7. BSA is generally used as a stabilizer in protein preservation solutions and reaction solutions. The addition of BSA may act as a "protector" or "carrier".

[0073] As used in this article, the term "casein" refers to casein, a phosphoprotein found in animal milk. Its structural formula is NH₂RCOO. It is generally used as a stabilizer in protein preservation solutions and reaction solutions; its addition may provide protection.

[0074] As used in this article, the term "EDC" refers to an activator, namely 1-ethyl-(3-dimethylaminopropyl)carbodiimide, which is often used as a carboxyl activator in the preparation of amides from primary amines.

[0075] As used herein, the term "immunological assay" refers to a determination that utilizes the specific interaction / binding affinity between an antigen and antibody, and is generally used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, etc. In some embodiments, the immunological assay is an enzyme immunoassay (EIA), such as an ELISA, Elispot assay, or CLEIA assay. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch. 7, Paul W., ed., 2nd edition, Raven Press, NY (1989).

[0076] As used herein, the term "detectable reaction" refers to any composition that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. In this invention, it is particularly preferred that such labels be applicable to immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence immunoassays, chemiluminescence immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750), acrid esters, magnetic beads (e.g., This includes, but is not limited to, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of such markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0077] Example

[0078] Since the antigen preservation solution of MDA5 contains urea, and the antigen will precipitate when urea is removed by dialysis or other methods, and different magnetic beads have different tolerances to urea, it is necessary to screen and optimize the types of magnetic beads and labeling conditions.

[0079] Example 1: Magnetic Bead Screening

[0080] 1.1 MDA5 antigen was labeled with JSR-Tsoyl, JSR-MS160-carboxyl, Thermo-Myone-carboxyl, and JSR-MS300 magnetic beads, respectively. After labeling, the mixture was diluted to the working concentration with magnetic bead diluent.

[0081] 1.2 Dilute the chemiluminescently labeled anti-human IgG antibody to the working concentration;

[0082] 1.3 The magnetic beads labeled in 1.1 were combined with the secondary antibody working solution and sample buffer in 1.2 to perform calibrator detection, and the calibrator detection results and the state of the magnetic beads when the antigen was labeled were compared.

[0083] The results are shown in Table 1 below. Myone magnetic bead labeling showed the best magnetic bead state, with only slight agglutination. This can be optimized by adding the labeling buffer, blocking buffer, and washing solution provided in this application to completely improve the magnetic bead agglutination problem. MS160 and other magnetic bead-labeled antigens all showed more severe agglutination.

[0084] Table 1: Magnetic Bead Screening Results

[0085]

[0086] Example 2: Screening with magnetic bead-labeled buffer solution

[0087] 2.1 Using the screened Myone magnetic beads, MDA5 antigen was labeled with 0.015 mol / L MES at pH 5.5, pH 6.0, and pH 6.5, respectively. After labeling, the solution was diluted to the working concentration with magnetic bead dilution buffer.

[0088] 2.2 Dilute the chemiluminescently labeled anti-human IgG antibody to the working concentration;

[0089] 2.3 The magnetic beads labeled in 2.1 were combined with the secondary antibody working solution and sample buffer in 2.2 to perform calibrator detection, and the calibrator detection results were compared with the state of the magnetic beads when the antigen was labeled.

[0090] The results are shown in Table 2 below. As the pH value of the labeling buffer increases, the labeling effect is enhanced, the luminescence value increases and the P / N ratio increases. The magnetic beads are in the best condition at pH 6.5, with no aggregation and only slight adhesion to the wall.

[0091] Table 2: Screening Results of Magnetic Bead Labeling Buffer

[0092]

[0093] Example 3: Screening of blocking solution buffer

[0094] 3.1 Using the screened Myone magnetic beads, MDA5 antigen was labeled with 0.015 mol / L MES at pH 6.5 as the labeling buffer. Then, the magnetic beads were blocked with 5% BSA, 5% BSA + MPC polymer, 0.05 mol / L Tris + 0.5% gelatin, and IBS002 blocking buffer, respectively. After labeling, the magnetic beads were diluted to the working concentration with magnetic bead diluent.

[0095] 3.2 Dilute the chemiluminescently labeled anti-human IgG antibody to the working concentration;

[0096] 3.3 The magnetic beads labeled in 3.1 were combined with the secondary antibody working solution and sample buffer solution in 3.2 to perform sample detection, and the sample detection results were compared.

[0097] The results are shown in Table 3 below. Blocking with 5% BSA+MPC polymer is as effective as blocking with gelatin only, both of which can reduce false positives and false negatives.

[0098] Table 3: Screening Results of Blocking Buffer Solution

[0099]

[0100]

[0101] Despite the above screening and labeling processes using magnetic beads, false negatives and missed detections still exist. Further screening of magnetic bead diluents and sample diluents is necessary.

[0102] Example 4: Screening of magnetic bead dilution solution

[0103] 4.1 Using the screened Myone magnetic beads, MDA5 antigen was labeled with 0.015 mol / L MES at pH 6.5 as the labeling buffer. Then, the magnetic beads were blocked with 5% BSA + MPC polymer or 0.5% gelatin blocking buffer. After labeling, the magnetic beads were diluted to the working concentration with magnetic bead dilution buffer containing 1% BSA, magnetic bead dilution buffer containing casein + polysaccharide, and magnetic bead dilution buffer containing 1% casein, respectively.

[0104] 4.2 Dilute the chemiluminescently labeled anti-human IgG antibody to the working concentration;

[0105] 4.3 The magnetic beads labeled in 4.1 were combined with the secondary antibody working solution and sample buffer solution in 4.2 to perform sample detection, and the sample detection results were compared.

[0106] The results are shown in Table 4 below. Comparing the positive and negative concordance rates, it can be seen that the magnetic bead diluent containing casein is more effective than the magnetic bead diluent containing BSA.

[0107] Table 4: Screening Results of Magnetic Bead Diluent

[0108]

[0109]

[0110]

[0111] Furthermore, the accelerated stability of different magnetic bead diluents was analyzed, and the results are shown in Table 5 below. Adding polysaccharides to the magnetic bead diluent can improve the stability of the magnetic beads, but the stability is still not ideal and further optimization is needed.

[0112] Table 5: Acceleration stability results for different magnetized systems

[0113]

[0114] Example 5: Screening of Cleaning Fluid

[0115] 5.1 Using the screened Myone magnetic beads, MDA5 antigen was labeled with 0.015 mol / L MES labeling buffer at pH 6.5, and then the magnetic beads were blocked with 5% BSA + MPC polymer or 0.5% gelatin blocking buffer. After blocking, the magnetic beads were washed overnight with arginine + Brij35 washing solution. After washing, the magnetic beads were diluted to the working concentration with magnetic bead diluent containing casein + polysaccharide.

[0116] 5.2 Dilute the chemiluminescently labeled anti-human IgG antibody to the working concentration;

[0117] 5.3 The magnetic beads labeled in 5.1 were combined with the secondary antibody working solution and sample buffer solution in 5.2 to perform sample detection, and the sample detection results were compared.

[0118] The results are shown in Table 6 below. After sealing, washing with arginine and Brij35 can improve the stability of the magnetic beads.

[0119] Table 6: Results of Cleaning Fluid Screening

[0120]

[0121] Example 6: Preparation of a chemiluminescent immunoassay kit for the quantitative detection of anti-MDA5 antibodies

[0122] Step 1: Preparation of A1 reagent

[0123] 1) Place the magnetic bead stock solution into a centrifuge tube and remove the supernatant; add labeling buffer, mix thoroughly, adsorb, remove supernatant, repeat 3 times; add labeling buffer and resuspend; add EDC, mix by rolling at room temperature for 30 min; add labeling buffer, mix, adsorb, remove supernatant, repeat once; add labeling buffer, add MDA5 antigen, mix by rolling at room temperature for 2 h.

[0124] 2) Discard the supernatant from step 1), add blocking buffer and mix well, adsorb, remove supernatant, repeat once, for a total of 2 times; discard supernatant, add blocking buffer and mix well, block at 37 degrees and 1500 rpm for 5 hours.

[0125] 3) Discard the supernatant from step 2), add the washing solution (arginine + brij35), mix well, adsorb, remove the supernatant, repeat once, for a total of 2 times; discard the supernatant, add the washing solution, mix well, and wash overnight at room temperature and 1500 rpm.

[0126] 4) Add magnetic bead diluent, mix well, adsorb, remove supernatant, repeat once, for a total of 2 times; add magnetic bead diluent to obtain reagent A1 with a final concentration of 0.1 mg / ml;

[0127] Step 2: Preparation of A2 reagent

[0128] 1) Take anti-human IgG into an EP tube, add labeling buffer to 0.3 ml, take acridinium ester working solution, add it to the above protein solution, mix immediately, wrap the centrifuge tube with a layer of aluminum foil to protect it from light, place it on a rotary mixer, and mix and react for a period of time at room temperature.

[0129] 2) Take 1% Lys, add it to the centrifuge tube above, keep it in the dark, place it on a rotary mixer, and mix and react at room temperature for 30 min.

[0130] 3) Purify the acridinium ester-antibody conjugate, add glycerol, and store at -20 degrees Celsius for later use;

[0131] 4) Take the chemiluminescent label-antibody conjugate, add it to the A2 reagent diluent, mix thoroughly, and obtain the A2 reagent with a final concentration of 0.05 ng / ml-0.2 ng / ml;

[0132] Step 3: Preparation of A3 reagent

[0133] Take the calibrator stock solution and add calibrator diluent (1% casein + 0.1% Tween 20 or 5% BSA) to obtain A3 reagents with final concentrations of 15 Ru / ml and 200 Ru / ml, respectively.

[0134] Step 4: Design of Standard Curve

[0135] Using the prepared A1 and A2 reagents, calibrators with concentrations of 0 RU / ml, 5 RU / ml, 15 RU / ml, 50 RU / ml, 200 RU / ml, and 500 RU / ml were tested. The luminescence values ​​and concentrations were written into the fitting software to obtain the calibration curves.

[0136] Example 7: Performance Evaluation of the Reagent Kit

[0137] The accuracy (recovery test) results are shown in Table 7 below: the recovery rate was 104.5%, which is within the range of 90%-110%, indicating good accuracy.

[0138] Table 7: Accuracy (Recovery Test)

[0139]

[0140]

[0141] The interference test results are shown in Table 8 below: 2000 IU / ml RF, 15 mg / ml hemoglobin, 20 mg / ml triglycerides, 0.4 mg / ml bilirubin, and 600 ng / ml HAMA had no effect on the detection results.

[0142] Table 8: Interference Tests

[0143]

[0144] The repeatability results are shown in Table 9 below: 10 tests were conducted on each of the two samples with different concentrations, and the repeatability was less than 10% for both.

[0145] Table 9: Repeatability Tests

[0146] Sample number Luminous value Concentration value Sample number Luminous value Concentration value M52 186086 10.9 M63 2195963 97.49 M52 196910 11.46 M63 2006147 89.86 M52 172216 10.18 M63 2134600 95.03 M52 179592 10.56 M63 2140662 95.27 M52 175572 10.35 M63 2110027 94.04 M52 173518 10.24 M63 2034493 91.01 M52 190079 11.11 M63 2038843 91.18 M52 184877 10.84 M63 2203415 97.79 M52 188557 11.03 M63 2168384 96.38 M52 189271 11.06 M63 2106354 93.89 average value 183668 10.8 2113889 94.2 CV 4.4％ 3.9％ 3.3％ 2.9％

[0147] The detection limits are shown in Table 10 below: ≤2RU / ml

[0148] Table 10: Limit of Detection Test

[0149]

[0150] The linear results are shown in Table 11 below: the linear correlation is good in the range of 2-400 RU / ml.

[0151] Table 11: Linearity Test

[0152]

[0153] Example 8: Instructions for use of the chemiluminescent immunoassay kit for anti-MDA5 antibodies :

[0154] 8.1 Test calibrators and calibration curves;

[0155] 8.2 Add the diluted sample and magnetic beads; the anti-MDA5 antibody in the sample reacts with the antigen on the magnetic beads.

[0156] 8.3 Under the influence of a magnetic field, the magnetic beads are adsorbed onto the wall of the reaction tube, and the unbound substances are washed away by the washing liquid;

[0157] 8.4 Add acridil ester-labeled anti-human IgG, and the magnetic bead-labeled MDA5 antigen-anti-MDA5 antibody-IgG complex in the sample reacts with the acridil-labeled anti-human IgG antibody;

[0158] 8.5 After washing the precipitated complex, an excitation solution is added; under alkaline conditions, hydrogen peroxide in the excitation solution reacts with acridine ester to form an excited-state acridine ketone, which releases photons during its return to the ground state, forming a luminescence reaction. The luminescence intensity of the reaction can be detected using a luminometer.

[0159] 8.6 Within the detection range, the MDA5-IgG concentration of the sample to be tested is calculated and read by fitting the curve.

[0160] It should be understood that although the present invention has been described by way of example according to its preferred embodiments, it should not be limited to the above embodiments. Various modifications and variations can be made to the present invention by those skilled in the art. The reaction reagents, reaction conditions, etc., involved in the preparation of the chemiluminescent immunoassay kit for anti-MDA5 antibody can be adjusted and changed according to specific needs. Therefore, those skilled in the art can make several simple substitutions without departing from the concept and principles of the present invention, and these should all be included within the scope of protection of the present invention.

Claims

1. A chemiluminescent immunoassay kit for the quantitative detection of anti-MDA5 antibodies, comprising reagents A1, A2, and A3; The A1 reagent comprises magnetic beads coated with MDA5, prepared from magnetic beads, MDA5 antigen, labeling buffer, blocking buffer, washing buffer, and magnetic bead diluent; wherein the magnetic bead particles have a particle size of 1 μm and the groups modified by the magnetic beads are carboxyl groups; the washing buffer comprises arginine and Brij35; the magnetic bead diluent comprises 0.05 mol / L Tris, 0.5% casein, 0.2% Proclin 300, and polysaccharide; the labeling buffer comprises 0.015 mol / L morpholine ethanesulfonic acid with a pH of 6.5; the blocking buffer is selected from either a first blocking buffer or a second blocking buffer, wherein the first blocking buffer comprises 0.05 mol / L Tris and 0.5% gelatin; the second blocking buffer comprises 5% BSA and MPC polymer, wherein the MPC polymer is Biolipidure 206; in, The preparation method of the A1 reagent includes the following steps: 1) Place the magnetic bead stock solution into a centrifuge tube and remove the supernatant; add labeling buffer, remove the supernatant, and resuspend; add EDC and mix by rolling at room temperature; add labeling buffer and remove the supernatant; add labeling buffer and add MDA5 antigen, and mix by rolling at room temperature. 2) Discard the supernatant from step 1), add blocking buffer, discard the supernatant; add blocking buffer, mix well, and block. 3) Discard the supernatant from step 2), add the cleaning solution, discard the supernatant; add the cleaning solution and mix well, wash overnight at room temperature; 4) Add magnetic bead diluent and discard the supernatant; add magnetic bead diluent to obtain reagent A1; The A2 reagent comprises a labeled anti-human IgG conjugate, which is prepared from a chemiluminescent label, anti-human IgG, and a labeling buffer. The A3 reagent includes calibrators.

2. The kit according to claim 1, wherein, The polysaccharide is pranolose and sulfated dextran.

3. The kit according to claim 1, wherein, The amount of MDA5 antigen used in the A1 reagent is 10-30 μg antigen / mg magnetic beads.

4. The kit according to claim 3, wherein, The amount of MDA5 antigen used in the A1 reagent is 20 μg antigen / mg magnetic bead.

5. The kit according to claim 1, wherein, The concentration of the labeled anti-human IgG conjugate in the A2 reagent is 0.05-0.2 ng / ml.

6. The kit according to claim 5, wherein, The concentration of the labeled anti-human IgG conjugate in the A2 reagent is 0.1 ng / ml.

7. The kit according to claim 1, wherein, The concentration of the calibrator in the A3 reagent is 5-400 Ru / ml.

8. The kit according to claim 7, wherein, The concentration of the calibrator in the A3 reagent is 15-200 Ru / ml.

9. The kit according to claim 1, wherein, The chemiluminescent label is luminol, isoluminol, terpyridine ruthenium, or acridine ester.

10. The kit according to claim 1, wherein, The kit also includes a sample buffer comprising 0.05 M Tris, 1% gelatin and 0.2% Tween 20.

11. The kit according to claim 1, wherein, The preparation method of the A1 reagent includes the following steps: 1) Place the magnetic bead stock solution into a centrifuge tube and remove the supernatant; add labeling buffer, mix thoroughly, adsorb, remove supernatant, repeat 3 times; add labeling buffer and resuspend; add EDC, mix by rolling at room temperature for 30 min; add labeling buffer, mix, adsorb, remove supernatant, repeat once; add labeling buffer, add MDA5 antigen, mix by rolling at room temperature for 2 h. 2) Discard the supernatant from step 1), add blocking buffer and mix well, adsorb, remove supernatant, repeat once, for a total of 2 times; discard the supernatant, add blocking buffer and mix well, block at 37 degrees and 1500 rpm for 5-6 hours. 3) Discard the supernatant from step 2), add the cleaning solution and mix well, adsorb, remove the supernatant, repeat once, for a total of 2 times; discard the supernatant, add the cleaning solution and mix well, wash overnight at room temperature and 1500 rpm. 4) Add magnetic bead diluent, mix well, adsorb, remove supernatant, repeat once, for a total of 2 times; add magnetic bead diluent to obtain A1 reagent with a final concentration of 0.1-0.2 mg / ml.

12. The kit according to claim 1, wherein, The preparation method of reagent A2 includes the following steps: 1) Take anti-human IgG into an EP tube, add labeling buffer, add chemiluminescent label, wrap the centrifuge tube with a layer of aluminum foil to protect it from light, place it on a rotary mixer, and mix and react at room temperature for a period of time. 2) Add 1% Lys, place in a light-protected environment on a rotary mixer, and mix at room temperature for 30 min. 3) Purify the chemiluminescent label-antibody conjugate, add glycerol, and store at -20 degrees Celsius for later use; 4) Take the chemiluminescent label-antibody conjugate, add it to the A2 reagent diluent, mix thoroughly, and obtain the A2 reagent with a final concentration of 0.05 ng / ml-0.2 ng / ml.

13. The kit according to claim 12, wherein, The preparation method of the A2 reagent includes the following steps: 1) Take anti-human IgG into an EP tube, add labeling buffer to 0.3 ml, take acridinium ester working solution, add it to the above protein solution, mix immediately, wrap the centrifuge tube with a layer of aluminum foil to protect it from light, place it on a rotary mixer, and mix and react for a period of time at room temperature. 2) Take 1% Lys and add it to the centrifuge tube above. Keep it in the dark and place it on a rotary mixer to mix and react at room temperature for 30 min. 3) Purify the acridinium ester-antibody conjugate, add glycerol, and store at -20 degrees Celsius for later use; 4) Take the chemiluminescent label-antibody conjugate, add it to the A2 reagent diluent, mix thoroughly, and obtain the A2 reagent with a final concentration of 0.05 ng / ml-0.2 ng / ml.

14. The kit according to claim 1, wherein, The preparation method of the A3 reagent includes the following steps: Take the calibrator stock solution and add the calibrator diluent to obtain A3 reagents with final concentrations of 15 Ru / ml and 200 Ru / ml, respectively. The calibrator diluent is selected from either the first calibrator diluent or the second calibrator diluent, wherein the first calibrator diluent includes 1% casein and 0.1% Tween 20; and the second calibrator diluent includes 5% BSA.

Images