Christmas liquefied carbohydrate chain antigen chemiluminescence detection kit and preparation method thereof

Abstract

The invention relates to the technical field of in-vitro diagnostic reagent chemiluminescence detection, in particular to a Christmas liquefied carbohydrate chain antigen chemiluminescence detection kit and a preparation method thereof. The chemiluminescence detection kit comprises the following components: a KL-6 calibrator, an amino magnetic bead coupled KL-6 monoclonal antibody, an alkaline phosphatase labeled KL-6 monoclonal antibody, an AMPPD rapid chemiluminescence substrate solution and a cleaning solution, and the chemiluminescence detection kit prepared from the components is simple in detection process, easy to operate, high in automation degree and high in detection sensitivity. The kit is high in sensitivity, good in specificity and good in stability, a result can be automatically obtained within 12.5 min, the requirement for clinical auxiliary diagnosis of interstitial lung diseases can be met, and the problem that in the prior art, a chemiluminescence detection kit prepared by adopting an acridinium ester magnetic particle chemiluminescence immunoassay method or a biotin-streptavidin detection system is poor in specificity and high in sensitivity is solved. The technical problems of low marking efficiency, high manufacturing cost and poor detection result reliability in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagent chemiluminescence detection, and in particular to a liquefied sugar chain antigen chemiluminescence detection kit and a preparation method thereof. Background Art

[0002] KL-6 was discovered and named by Japanese scholar KOHNO in 1985 when he was studying tumor markers of lung adenocarcinoma cells. It belongs to the 9th family of lung cell antigens and is a type of high-molecular-weight mucin glycoprotein encoded by the epithelial mucin 1 (MUC1) gene. MUC1 is a transmembrane high-molecular-weight mucin with a highly glycosylated extracellular domain and a sialylated sugar chain with a molecular weight of about 200 kd. The sugar chain is mainly composed of a core tandem repeat sequence of 20 amino acid residues and has a spatial structural epitope that can be identified by antibodies. KL-6 is one of the sialylated carbohydrate antigens on the N-terminal domain of MUC1, located on the cell surface, and is released into the blood due to the dissolution of its extracellular region by proteases, and exists in the blood in a soluble form.

[0003] KL-6 has a strong chemotactic effect on fibroblasts, promoting the proliferation and migration of fibroblasts, thereby causing the development of fibrosis in diseased tissues. KL-6 is mainly produced by type II alveolar epithelial cells. When epithelial cells are damaged, KL-6 will enter the blood circulation. Detecting its level can help determine alveolar damage, type II alveolar cell regeneration, and the activity of various lung diseases. KL-6 was initially used as a serum biomarker for tumors, but subsequent studies have found that it is also a biomarker for interstitial lung disease, and the degree of KL-6 elevation is closely related to the range of interstitial lung disease lesions. As an indicator of alveolar epithelial cell destruction and regeneration, the detection of KL-6 concentration is fast, simple, economical, repeatable, and non-invasive, which is superior to classic methods such as high-resolution lung CT, alveolar lavage, and lung biopsy. The determination of KL-6 levels in patients can effectively avoid the irreversible formation of pulmonary fibrosis and has a certain clinical preventive value.

[0004] Commonly used immunoassay methods in clinical practice include latex immunoturbidimetry and chemiluminescence immunoassay. Among them, latex-enhanced immunoturbidimetry is a commonly used immunoassay method used to detect the concentration of specific antigens or antibodies in serum or body fluids. This method combines immunoturbidimetry and latex enhancement technology to produce turbidity changes through the binding reaction of latex particles with antigens or antibodies, thereby indirectly determining the concentration of the target substance. Latex-enhanced immunoturbidimetry is a simple, cost-effective method that does not require complex instruments and equipment. However, latex-enhanced immunoturbidimetry has low sensitivity, and the agglutination reaction may be interfered by other non-specific factors, resulting in inaccurate agglutination reaction results. Secondly, the heterogeneity of latex particles can lead to errors in the measurement results. At the same time, latex particles are prone to agglutination or precipitation during storage, affecting the reliability of the experimental results. Chemiluminescence technology is an immunoassay that has developed very rapidly worldwide in the past decade. It combines highly sensitive chemiluminescence measurement technology with highly specific immune response and is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. It has the advantages of high sensitivity, wide detection range, simple and fast operation, good marker stability, no pollution, simple and economical instruments, etc. At present, this technology has become a routine detection method in clinical laboratory medicine.

[0005] Chinese patent applications CN 111856016A, CN 114137204A and CN 114544527A all disclose a KL-6 assay kit and its preparation and detection method. The three patents are all prepared and produced by latex immunoturbidimetry. Patent CN 112946255 A discloses a reagent or kit for chemiluminescent detection of sialylated sugar chain antigen magnetic microparticles and its application. The method adopts acridinium ester magnetic microparticle chemiluminescent immunoassay. The luminescence of acridinium ester is flash type and short in time. The substrate needs to be added in situ and the light is measured at the same time. The instrument requirements are high. Acridinium ester is highly hydrophobic and easily non-specifically adsorbed, which brings interference and affects the detection results. In addition, the stability of acridinium ester is poor. For small molecule projects, acridinium ester labeling is difficult and the labeling efficiency is low; Patent CN 117538538 A discloses a sialylated sugar chain antigen chemiluminescence detection kit and a preparation method thereof, the method adopts a biotin-streptavidin detection system, the nanomagnetic material used in the system has a high production cost, and the SA group of the streptavidin (SA)-labeled nanomagnetic material is easily detached during storage and use, which may cause stability problems, affecting the reliability of the detection results, and biotin interference is a common problem in the detection of the avidin-biotin detection system. When the avidin-biotin system is used for immunoassay, if there is a high concentration of free biotin in the sample to be tested, it will compete with the biotinylated antibody for binding to the binding site of the avidin, thereby affecting the detection results.

[0006] In view of this, the purpose of the present invention is to provide a KL-6 magnetic particle chemiluminescence immunoassay kit to solve the above problems, which has a simple detection process, is easy to operate, has a high degree of automation, high sensitivity, good specificity, good kit stability, and can automatically produce results in 12.5 minutes. KL-6 magnetic particle chemiluminescence detection kit and its preparation method can meet the clinical auxiliary diagnosis of interstitial lung disease. Summary of the invention

[0007] The object of the present invention is to provide a chemiluminescent detection kit for liquefied sugar chain antigens and a preparation method thereof, which solves the technical problems that the stability of acridinium ester is poor when the acridinium ester magnetic particle chemiluminescent immunoassay method is adopted in the existing chemiluminescent detection kit, acridinium ester labeling is difficult for small molecule projects, the labeling efficiency is low, and the production cost is high and the reliability of the detection result is poor when the biotin-streptavidin detection system is adopted.

[0008] To achieve the above-mentioned object, the present invention provides a chemiluminescent detection kit for liquefied sugar chain antigens, the components of the kit comprising: KL-6 calibrator, amino magnetic beads coupled KL-6 monoclonal antibody, alkaline phosphatase labeled KL-6 monoclonal antibody, AMPPD rapid chemiluminescent substrate solution and cleaning solution.

[0009] The specific preparation method of the KL-6 calibrator is as follows: first prepare a buffer solution of the KL-6 calibrator, and then dissolve the recombinant KL-6 antigen in the buffer solution.

[0010] The specific preparation method of the amino magnetic bead-coupled KL-6 monoclonal antibody prepared by coating process is as follows:

[0011] Magnetic bead pretreatment, resuspending amino magnetic beads by vortexing, transferring 100 μL of microspheres to a 1.5 mL centrifuge tube, and then placing the centrifuge tube on a magnetic rack for 1-3 minutes. After the solution becomes clear, the supernatant is removed, and the solution is washed three times with 300-500 μL of buffer, and then the supernatant is removed by adsorption by the magnetic rack, wherein the magnetic beads have a particle size of 1.5-3 μm, and the magnetic beads are amino-modified superparamagnetic microspheres;

[0012] Activation, add 100uL of freshly prepared glutaraldehyde solution to the centrifuge tube, vortex and oscillate to fully suspend the magnetic beads, then wrap them with tin foil, and react on a magnetic bead rotator at 25°C for 0.5-2h, wherein the concentration of the glutaraldehyde solution is 20-50%, and the activation process needs to be protected from light to prevent cross-linking polymerization of glutaraldehyde itself;

[0013] Washing after activation: after the magnetic beads are activated, the centrifuge tube is placed on the magnetic stand for 1-3 minutes. After the solution becomes clear, the supernatant is removed and the beads are washed three times with 500 μL of the buffer solution;

[0014] Magnetic bead coupling, adding 300-500uL coupling buffer to the centrifuge tube, coupling KL-6 monoclonal antibody, and then adding 0.05-0.1% surfactant, gently mixing, wrapping with tin foil to react at 25°C in the dark, and coupling on a magnetic bead rotator, wherein the coupling conditions are: coupling time 2-6h, or coupling time 24h at a coupling temperature of 2-8°C, and the ratio of antibody to magnetic beads is 1:50-1:150;

[0015] Blocking, placing the centrifuge tube on the magnetic rack for 1-3 minutes, after the solution becomes clear, removing the supernatant, adding 500-1000uL of blocking solution, blocking at 37°C for 1-3 hours, and coupling on a magnetic bead rotator, wherein the blocking solution is one of animal serum albumin, casein, gelatin, arginine, glycine, surfactant and PEG series;

[0016] Preservation: After sealing is completed, the centrifuge tube is placed on a magnetic separation rack for magnetic separation, and the supernatant is removed after separation. After washing 3 times with 500 μL PBS solution or preservation solution, it is resuspended in the preservation solution. The preservation temperature is 4°C. If necessary, 0.02% sodium azide should be added to the preservation solution to inhibit bacterial growth.

[0017] The specific preparation method of the alkaline phosphatase-labeled KL-6 monoclonal antibody is as follows:

[0018] Take the required labeled amount of antibody, add the diluted 20-50 mM SPDP, vortex to mix, centrifuge, and react at 20-37° C. in the dark for 0.5-1 h, wherein the molar ratio of the antibody to the SPDP is 1:10-1:80;

[0019] After the reaction is completed, the reaction solution is desalted 2-3 times using an ultrafiltration tube with a molecular cutoff of less than 40 kD;

[0020] First, equilibrate the DTT powder to room temperature, then use freshly prepared 0.02-0.05M acetate buffer to prepare a sufficient amount of 120mg / mL DTT solution, add DTT at a ratio of 10-30μL 120mg / mL DTT per 100μL antibody, mix with a pipette to avoid bubbles, and treat at room temperature away from light for 0.5-1h;

[0021] After the reaction is completed, the reaction solution is desalted 2-3 times using the ultrafiltration tube with a molecular weight cutoff of less than 40 kD;

[0022] Take out the alkaline phosphatase AP and balance it to room temperature, add the balanced AP to the centrifuge tube, then add 0.05M MOPS and 20mM hetero-bifunctional cross-linker to make the final reaction concentration of the AP 2mg / mL, vortex and mix, centrifuge, protect from light, and react at 37°C for 0.5-1h;

[0023] The activated antibody derivative and the ALP modified with thiol groups are reacted in a mass ratio of 1:0.5-1:2 at a temperature of 20-37°C for 1-3 hours in a light-proof manner;

[0024] After the reaction is completed, the reaction solution is desalted 2-3 times again using the ultrafiltration tube with a retention molecule of less than 40KD to obtain the alkaline phosphatase-labeled KL-6 monoclonal antibody. Finally, the antibody is stored at -20°C with a preservation solution and diluted to a suitable concentration with an enzyme-labeled diluent for use.

[0025] The specific preparation method of the enzyme-labeled diluent is as follows: 10-50 mM Tris buffer, 4.5-18 g / L sodium chloride, 5-20 g / L sugar, 0.1-1 g / L protein, 0.01-0.5 mM zinc ion, 0.1-10 mM magnesium ion, 5-30 g / L PEG2000, 300.5-3 g / L polyvinyl pyrrolidone, 10-30 mL / L alcohol, 2-10 g / L 6-aminoacetic acid, 0.01-0.1% surfactant and 1-5 g / L preservative are mixed to form the prepared diluent, the prepared diluent has a pH range of 7.0-8.0, is filtered through a 0.22 um filter membrane after preparation, and is stored at 2-8° C. for standby use.

[0026] The AMPPD rapid chemiluminescent substrate solution is mainly composed of AMPPD, a fluorescent agent and a buffer solution containing a surfactant, and the cleaning solution is a concentrated cleaning solution, which is composed of a TRIS buffer system, sodium chloride, Tween-20, Triton X-100, and proclin-300.

[0027] The present invention also provides a method for preparing a liquefied sugar chain antigen chemiluminescence detection kit, comprising the following steps:

[0028] 10 μL of sample, 50 μL of amino magnetic beads coated with KL-6, and 100 μL of KL-6 monoclonal antibody labeled with alkaline phosphatase were added to the reaction tube and incubated to form a solid-phase antibody-antigen-enzyme-labeled MMP-3 antibody complex;

[0029] Afterwards, a cleaning solution is added to the reaction tube, and the unbound enzyme-labeled antibodies and other substances are washed away by the cleaning solution;

[0030] Then, AMPPD fast chemiluminescent substrate solution is added. The luminescent substrate solution emits photons under the catalysis of alkaline phosphatase. The number of photons generated is proportional to the concentration of KL-6 in the sample, thereby realizing the detection of the sample.

[0031] The invention discloses a chemiluminescent detection kit for liquefied sugar chain antigen and a preparation method thereof. By providing a coating process of amino magnetic beads coupled to KL-6 monoclonal antibody with high sensitivity and good stability and providing a high titer alkaline phosphatase labeled KL-6 monoclonal antibody labeling process and enzyme labeling diluent, the prepared chemiluminescent detection kit has a simple detection process, is easy to operate, has a high degree of automation, high sensitivity, good specificity, good stability of the kit, and automatically produces results in 12.5 minutes. The kit can meet the needs of clinical auxiliary diagnosis of interstitial lung diseases and solve the technical problems that the stability of acridinium ester is poor when the acridinium ester magnetic particle chemiluminescent immunoassay method is adopted in the existing chemiluminescent detection kit, acridinium ester labeling is difficult for small molecule projects, the labeling efficiency is low, and the production cost is high and the reliability of the detection result is poor when the biotin-streptavidin detection system is adopted. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0033] Figure 1 It is a schematic diagram of the correlation between Example 11 of the present invention and Fuji clinical practice.

[0034] Figure 2 It is a schematic diagram of the correlation between Example 12 of the present invention and Fuji clinical practice.

[0035] Figure 3 It is a flow chart of the preparation method of the liquefied sugar chain antigen chemiluminescence detection kit of the present invention. DETAILED DESCRIPTION

[0036] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0037] Example 1

[0038] Preparation of the kit of the present invention

[0039] 1. Coating process of amino magnetic beads coupled with KL-6 monoclonal antibody:

[0040] (1) Magnetic bead pretreatment: resuspend the amino magnetic beads by vortexing, transfer 100 μL of the microspheres to a 1.5 mL centrifuge tube, and place the centrifuge tube on a magnetic rack for 1 min. After the solution becomes clear, remove the supernatant, wash three times with 500 μL of buffer, and then remove the supernatant by adsorption on the magnetic rack. The magnetic beads are amino-modified superparamagnetic microspheres with a particle size of 1 μm.

[0041] (2) Activation: Add 100uL of freshly prepared glutaraldehyde solution to a centrifuge tube with a glutaraldehyde concentration of 20%, vortex to fully suspend the magnetic beads, wrap them with tin foil, and react at 25°C for 0.5h (protect from light to prevent cross-linking and polymerization of glutaraldehyde itself), and react on a magnetic bead rotator to ensure that the magnetic beads are in a suspended state;

[0042] (3) Post-activation washing: After the magnetic beads were activated, the centrifuge tube was placed on the magnetic stand for 1 min. After the solution became clear, the supernatant was removed and the beads were washed three times with 500 ul of buffer.

[0043] (4) Magnetic bead coupling: add 500uL coupling buffer to the centrifuge tube, couple the KL-6 monoclonal antibody, add 0.05% surfactant, mix gently, wrap with tin foil and react at 25°C in the dark, couple for 2h, and couple on a magnetic bead rotator to ensure the suspension of the magnetic beads. In this coupling reaction, the ratio of antibody to magnetic beads is 1:50;

[0044] (5) Blocking (blocking is required after coupling to reduce nonspecific binding and improve the stability of the magnetic beads after coupling): place the centrifuge tube on the magnetic stand for 1 min. After the solution becomes clear, remove the supernatant and add 500 uL of blocking solution. Block at 37° C. for 1 h and couple on a magnetic bead rotator to ensure the suspension of the magnetic beads. The blocking solution is bovine serum albumin.

[0045] (6) Storage: After sealing, the centrifuge tube is placed on a magnetic separation rack for magnetic separation, and the supernatant is removed after separation. The tube is washed three times with 500 μL PBS solution (pH 7.2) or storage solution, and then resuspended in storage solution. The storage temperature is 4°C. If necessary, 0.02% (w / v) sodium azide (NaN3) should be added to the storage solution to inhibit bacterial growth.

[0046] 2. Preparation of alkaline phosphatase labeled KL-6 monoclonal antibody:

[0047] (1) Take the required labeled amount of antibody, add the diluted 20 mM SPDP, vortex to mix, centrifuge, and react at 37°C in the dark for 0.5 h, wherein the molar ratio of antibody to SPDP is 1:20;

[0048] (2) After the reaction is completed, the reaction solution is desalted 2-3 times using an ultrafiltration tube with a molecular weight cutoff of less than 40 kD;

[0049] (3) First, equilibrate the DTT powder to room temperature (about 30 minutes), then use freshly prepared 0.02 M acetate buffer (pH 4.5) to prepare a sufficient amount of 120 mg / mL DTT solution. Add DTT at a ratio of 10 μL 120 mg / mL DTT per 100 μL of antibody, mix with a pipette to avoid bubbles, and treat at room temperature in the dark for 0.5 h;

[0050] (4) After the reaction is completed, the reaction solution is desalted 2-3 times using the ultrafiltration tube with a molecular weight cutoff of less than 40 kD;

[0051] (5) Take out the alkaline phosphatase AP and equilibrate it to room temperature (about 30 minutes), add the equilibrated AP into the centrifuge tube, add 0.05M MOPS (pH 7.3) and 20mM SMCC to make the final reaction concentration of AP 2mg / mL, vortex and oscillate to mix, centrifuge, protect from light, and react at 37°C for 0.5h, wherein the molar ratio of AP to SMCC is 1:20;

[0052] (6) reacting the activated antibody derivative and the thiol-modified ALP at a mass ratio of 1:0.5 at 37° C. for 1 h in a light-proof manner;

[0053] (7) After the reaction is completed, the reaction solution is desalted 2-3 times again using the ultrafiltration tube with a molecular weight cutoff of less than 40KD to obtain the alkaline phosphatase-labeled KL-6 monoclonal antibody. Finally, the antibody is stored at -20°C with a preservation solution and diluted to a suitable concentration with an enzyme-labeled diluent for use.

[0054] 3. Preparation of enzyme-labeled diluent:

[0055] The enzyme label diluent is prepared by mixing Tris buffer 50mM, sodium chloride 4.5g / L, pullulan 5g / L, BSA 0.1g / L, zinc ion 0.05mM, magnesium ion 5mM, PEG200010g / L, polyvinylpyrrolidone 302g / L, glycerol 10mL / L, 6-aminoacetic acid 2g / L, Tween-20 0.01% and KroVin3001g / L, with a pH range of 7.4±0.05.

[0056] Example 2

[0057] Preparation of the kit of the present invention

[0058] 1. Preparation of amino magnetic beads coupled to KL-6 monoclonal antibody: The particle size of the magnetic beads used is preferably 1.8 μm, and the remaining steps are the same as in Example 1.

[0059] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: same as Example 1.

[0060] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0061] Example 3

[0062] Preparation of the kit of the present invention

[0063] 1. Preparation of KL-6 monoclonal antibody coupled with amino magnetic beads: During the magnetic bead coupling process, the ratio of antibody to magnetic beads was 1:100, and the other steps were the same as in Example 1.

[0064] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: same as Example 1.

[0065] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0066] Example 4

[0067] Preparation of the kit of the present invention

[0068] 1. Preparation of amino magnetic beads coupled to KL-6 monoclonal antibody: During the magnetic bead blocking process, the magnetic beads were blocked at 37° C. for 1 h and coupled on a magnetic bead rotator to ensure the suspension of the magnetic beads. Casein was used as the blocking solution. The other steps were the same as in Example 1.

[0069] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: same as Example 1.

[0070] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0071] Example 5

[0072] Preparation of the kit of the present invention

[0073] 1. Preparation of KL-6 monoclonal antibody coupled with amino magnetic beads: During the magnetic bead blocking process, the magnetic beads were blocked at 37° C. for 2 h, and coupled on a magnetic bead rotator to ensure that the magnetic beads were in a suspended state. Glycine was used as the blocking solution, and the other steps were the same as in Example 1.

[0074] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: same as Example 1.

[0075] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0076] Example 6

[0077] Preparation of the kit of the present invention

[0078] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0079] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: Take the required labeled amount of antibody, add diluted 50 mM SPDP, vortex to mix, centrifuge, react at 37° C. in the dark for 0.5 h, the molar ratio of antibody to SPDP is 1:20, and the other steps are the same as in Example 1.

[0080] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0081] Example 7

[0082] Preparation of the kit of the present invention

[0083] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0084] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: Take the required labeled amount of antibody, add diluted 20 mM SPDP, vortex to mix, centrifuge, react at 37° C. in the dark for 0.5 h, the molar ratio of antibody to SPDP is 1:60, and the other steps are the same as in Example 1.

[0085] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0086] Example 8

[0087] Preparation of the kit of the present invention

[0088] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0089] 2. Preparation of alkaline phosphatase labeled KL-6 monoclonal antibody: After taking out alkaline phosphatase AP and equilibrating it to room temperature (about 30 minutes), add the equilibrated AP into the centrifuge tube, and then add 0.05M MOPS (pH7.3) and 20mM SM (PEG) 6 (so that the final reaction concentration of AP is 2 mg / mL), wherein the molar ratio of AP to SM (PEG) 6 is 1:20, vortex to mix, centrifuge, protect from light, and react at 37°C for 0.5h.

[0090] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0091] Example 9

[0092] Preparation of the kit of the present invention

[0093] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0094] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: The activated antibody derivative and ALP modified with thiol groups were reacted in a mass ratio of 1:1 at 37° C. for 1 h under light-blocking conditions. The other steps were the same as in Example 1.

[0095] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0096] Example 10

[0097] Preparation of the kit of the present invention

[0098] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0099] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: The activated antibody derivative and ALP modified with thiol groups were reacted at a mass ratio of 1:1.5 at 37° C. for 1 h under light-blocking conditions. The other steps were the same as those in Example 1.

[0100] 3. Preparation of enzyme-labeled diluent: same as in Example 1.

[0101] Embodiment 11

[0102] Preparation of the kit of the present invention

[0103] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0104] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: same as Example 1.

[0105] 3. Preparation of enzyme-labeled diluent: The enzyme-labeled diluent consists of 10 mM Tris buffer, 18 g / L sodium chloride, 10 g / L pullulan, 1 g / L BSA, 0.05 mM zinc ion, 5 mM magnesium ion, 20 g / L PEG2000, 301 g / L polyvinylpyrrolidone, 20 mL / L glycerol, 2 g / L 6-aminoacetic acid, 0.05% Tween-20, and 1 g / L proclin-300, with a pH range of 7.4 ± 0.05.

[0106] Example 12

[0107] Preparation of the kit of the present invention

[0108] 1. Preparation of amino magnetic beads coupled KL-6 monoclonal antibody: same as Example 1.

[0109] 2. Preparation of alkaline phosphatase-labeled KL-6 monoclonal antibody: same as Example 1.

[0110] 3. Preparation of enzyme-labeled diluent: The enzyme-labeled diluent consists of Tris buffer 50mM, sodium chloride 18g / L, sucrose 10g / L, HSA 1g / L, zinc ion 0.05mM, magnesium ion 5mM, PEG200020g / L, polyvinylpyrrolidone 301g / L, glycerol 20mL / L, 6-aminoacetic acid 2g / L, Tween-80 0.05%, ProClin9501g / L, and the pH range is 7.4±0.05.

[0111] The KL-6 magnetic microparticle chemiluminescent immunoassay kit prepared in the above-mentioned embodiments can be used in conjunction with Guilin Ulite fully automatic chemiluminescent immunoassay analyzer IAC200 (detection range is 40U / mL-10000U / mL) to detect samples, and the results can be obtained in 12.5 minutes.

[0112] The steps for testing samples are as follows: Figure 3 As shown:

[0113] S101, add 10 μL of sample, 50 μL of amino magnetic beads coated with KL-6 and 100 μL of KL-6 monoclonal antibody labeled with alkaline phosphatase into the reaction tube, and incubate to form a solid phase antibody-antigen-enzyme-labeled MMP-3 antibody complex;

[0114] S102, then, adding a cleaning solution to the reaction tube, and washing with the cleaning solution to remove the unbound enzyme-labeled antibody and other substances;

[0115] S103, adding AMPPD fast chemiluminescent substrate solution, the luminescent substrate solution emits photons under the catalysis of alkaline phosphatase, the number of photons generated is proportional to the concentration of KL-6 in the sample, and the detection of the sample is achieved.

[0116] The KL-6 magnetic microparticle chemiluminescent immunoassay kit prepared in each of the above examples was used to perform performance tests:

[0117] 1. Potency test: Use the kits of Examples 1-5 to perform potency tests on blank samples and internal reference products on the Ulite fully automatic chemiluminescence immunoassay analyzer IAC200.

[0118] Table 1: Examples 1-5 titer test

[0119]

[0120] 2. Magnetic bead accelerated thermal stability test: The coated magnetic beads in Example 1-5 were divided into two, and placed at 2-8°C / 37°C for 7 days respectively. Internal reference products of different concentrations were taken for testing, and the signal retention rate of the magnetic beads after 7 days of acceleration at 37°C was calculated.

[0121] 2-1: Example 1 Magnetic Bead Acceleration Stability Test

[0122]

[0123] Table 2-2: Example 2 Magnetic Bead Accelerated Stability Test

[0124]

[0125]

[0126] Table 2-3: Example 3 Magnetic Bead Accelerated Stability Test

[0127]

[0128] Table 2-4: Example 4 Magnetic Bead Accelerated Stability Test

[0129]

[0130] Table 2-5: Example 5 Magnetic Bead Accelerated Stability Test

[0131]

[0132]

[0133] Repeatability test: Use Examples 6-10 to test 2 different concentrations of internal reference products of the company, repeat the test 10 times, and calculate the average value of the test results. and standard deviation (SD) and coefficient of variation CV.

[0135] Table 3: Repeatability test of Examples 6-10

[0136]

[0137]

[0138] Enzyme-labeled antibody accelerated thermal stability test: The alkaline phosphatase-labeled KL-6 monoclonal antibody in Example 11-12 was diluted to a working concentration with an enzyme-labeled diluent, divided into two, and placed at 2-8°C / 37°C for 7 days respectively. Internal reference products of different concentrations were tested on the Uni-Person fully automatic chemiluminescence immunoassay analyzer IAC200, and the signal retention rate after 7 days of acceleration at 37°C was calculated.

[0139] Table 4-1: Example 11 Enzyme-labeled Antibody Accelerated Stability Test

[0140]

[0141] Table 4-2: Example 12 Enzyme-labeled Antibody Accelerated Stability Test

[0142]

[0143]

[0144] Clinical comparison with benchmark companies: The KL-6 magnetic microparticle chemiluminescence detection kit prepared in Examples 11-12 was tested on 40 KL-6 clinical samples, and the clinical correlation coefficient r with the Japanese Fuji approved by the drug supervision and management department was calculated. The comparison results are as follows: Figure 1 and Figure 2 shown.

[0145] Table 4: Clinical test (unit: ng / ml)

[0146]

[0147]

[0148] Test results:

[0149] In the potency test, the signal-to-noise ratio S1 / S0 of Examples 1-5 is about 60, the low-end sensitivity is high, and the magnetic beads are accelerated for 7 days at 37°C with a signal retention rate of >90%, which has good stability and can meet the stability requirements of clinical diagnosis. It is explained that in the kit prepared by the present invention, the magnetic bead particle size used is preferably 1.5μm-3μm, and the ratio of antibody to magnetic beads during the coupling process of amino magnetic beads coupled to KL-6 monoclonal antibody magnetic beads is 1:50 and 1:100. After coupling, it is necessary to block at 37°C for 1-3h. The blocking solution can meet the requirements of sensitivity and accelerated stability with BSA and casein.

[0150] In the repeatability test, the coefficient of variation CV of Examples 6 to 10 was less than 3%, indicating that the preparation process of the alkaline phosphatase-labeled KL-6 monoclonal antibody provided by the kit of the present invention is simple and has good repeatability.

[0151] In the accelerated thermal stability test of the enzyme-labeled antibody, the signal retention rate of the enzyme-labeled antibody accelerated thermal stability test at 37°C for 7 days in Examples 11 and 12 was >90%, indicating that the kit of the present invention provides an alkaline phosphatase enzyme-labeled diluent with good stability.

[0152] In the clinical comparison with the benchmark enterprise, it can be seen from the data of Example 11 and Example 12 that the clinically relevant dilution r of the detection kit of the present invention and the Fuji detection kit is greater than 0.98, the detection results have good consistency, and there is almost no difference in the clinical comparison, indicating that the detection kit of the present invention has high detection accuracy.

[0153] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A chemiluminescent detection kit for liquefied sugar chain antigens, characterized in that: The components of the kit include: KL-6 calibrator, amino magnetic beads coupled KL-6 monoclonal antibody, alkaline phosphatase labeled KL-6 monoclonal antibody, AMPPD fast chemiluminescent substrate solution and cleaning solution.

2. The chemiluminescent detection kit for liquefied sugar chain antigen according to claim 1, characterized in that: The specific preparation method of the KL-6 calibrator is: first prepare a buffer solution of the KL-6 calibrator, and then dissolve the recombinant KL-6 antigen in the buffer solution.

3. The chemiluminescent detection kit for liquefied sugar chain antigen according to claim 1, characterized in that: The specific preparation method of the amino magnetic bead-coupled KL-6 monoclonal antibody prepared by coating process is as follows: Magnetic bead pretreatment, resuspending amino magnetic beads by vortexing, transferring 100 μL of microspheres to a 1.5 mL centrifuge tube, and then placing the centrifuge tube on a magnetic rack for 1-3 minutes. After the solution becomes clear, the supernatant is removed, and the solution is washed three times with 300-500 μL of buffer, and then the supernatant is removed by adsorption by the magnetic rack, wherein the magnetic beads have a particle size of 1.5-3 μm, and the magnetic beads are amino-modified superparamagnetic microspheres; Activation, add 100uL of freshly prepared glutaraldehyde solution to the centrifuge tube, vortex and oscillate to fully suspend the magnetic beads, then wrap them with tin foil, and react on a magnetic bead rotator at 25°C for 0.5-2h, wherein the concentration of the glutaraldehyde solution is 20-50%, and the activation process needs to be protected from light to prevent cross-linking polymerization of glutaraldehyde itself; Washing after activation: after the magnetic beads are activated, the centrifuge tube is placed on the magnetic stand for 1-3 minutes. After the solution becomes clear, the supernatant is removed and the beads are washed three times with 500 μL of the buffer solution; Magnetic bead coupling, adding 300-500uL coupling buffer to the centrifuge tube, coupling KL-6 monoclonal antibody, and then adding 0.05-0.1% surfactant, gently mixing, wrapping with tin foil to react at 25°C in the dark, and coupling on a magnetic bead rotator, wherein the coupling conditions are: coupling time 2-6h, or coupling time 24h at a coupling temperature of 2-8°C, and the ratio of antibody to magnetic beads is 1:50-1:150; Blocking, placing the centrifuge tube on the magnetic rack for 1-3 minutes, after the solution becomes clear, removing the supernatant, adding 500-1000uL of blocking solution, blocking at 37°C for 1-3 hours, and coupling on a magnetic bead rotator, wherein the blocking solution is one of animal serum albumin, casein, gelatin, arginine, glycine, surfactant and PEG series; Preservation: After sealing is completed, the centrifuge tube is placed on a magnetic separation rack for magnetic separation, and the supernatant is removed after separation. After washing 3 times with 500 μL PBS solution or preservation solution, it is resuspended in the preservation solution. The preservation temperature is 4°C. If necessary, 0.02% sodium azide should be added to the preservation solution to inhibit bacterial growth.

4. The chemiluminescent detection kit for liquefied sugar chain antigens according to claim 1, characterized in that: The specific preparation method of the alkaline phosphatase-labeled KL-6 monoclonal antibody is as follows: Take the required labeled amount of antibody, add the diluted 20-50 mM SPDP, vortex to mix, centrifuge, and react at 20-37° C. in the dark for 0.5-1 h, wherein the molar ratio of the antibody to the SPDP is 1:10-1:80; After the reaction is completed, the reaction solution is desalted 2-3 times using an ultrafiltration tube with a molecular weight cutoff of less than 40 kD; First, equilibrate the DTT powder to room temperature, then use freshly prepared 0.02-0.05M acetate buffer to prepare a sufficient amount of 120mg / mL DTT solution, add DTT at a ratio of 10-30μL 120mg / mL DTT per 100μL antibody, mix with a pipette to avoid bubbles, and treat at room temperature away from light for 0.5-1h; After the reaction is completed, the reaction solution is desalted 2-3 times using the ultrafiltration tube with a molecular weight cutoff of less than 40 kD; Take out the alkaline phosphatase AP and balance it to room temperature, add the balanced AP to the centrifuge tube, then add 0.05M MOPS and 20mM hetero-bifunctional cross-linker to make the final reaction concentration of the AP 2mg / mL, vortex and mix, centrifuge, protect from light, and react at 37°C for 0.5-1h; The activated antibody derivative and the ALP modified with thiol groups are reacted in a mass ratio of 1:0.5-1:2 at a temperature of 20-37°C for 1-3 hours in a light-proof manner; After the reaction is completed, the reaction solution is desalted 2-3 times again using the ultrafiltration tube with a retention molecule of less than 40KD to obtain the alkaline phosphatase-labeled KL-6 monoclonal antibody. Finally, the antibody is stored at -20°C with a preservation solution and diluted to a suitable concentration with an enzyme-labeled diluent for use.

5. The chemiluminescent detection kit for liquefied sugar chain antigens according to claim 4, characterized in that: The specific preparation method of the enzyme label diluent is: Tris buffer 10-50mM, sodium chloride 4.5-18g / L, sugar 5-20g / L, protein 0.1-1g / L, zinc ion 0.01-0.5mM, magnesium ion 0.1-10mM, PEG20005-30g / L, polyvinyl pyrrolidone 300.5-3g / L, alcohol 10-30mL / L, 6-aminoacetic acid 2-10g / L, surfactant 0.01-0.1% and preservative 1-5g / L are mixed to prepare the prepared diluent, the pH range of which is 7.0-8.0, and after preparation, the prepared diluent is filtered through a 0.22um filter membrane and stored at 2-8°C for standby use.

6. The chemiluminescent detection kit for liquefied sugar chain antigens according to claim 1, characterized in that: The AMPPD fast chemiluminescent substrate solution is mainly composed of AMPPD, a fluorescent agent and a buffer solution containing a surfactant. The cleaning solution is a concentrated cleaning solution, which is composed of a TRIS buffer system, sodium chloride, Tween-20, Triton X-100 and Proclin-300.

7. A method for preparing a chemiluminescent detection kit for liquefied sugar chain antigens, applied to the chemiluminescent detection kit for liquefied sugar chain antigens according to any one of claims 1 to 6, characterized in that: The following steps are involved: 10 μL of sample, 50 μL of amino magnetic beads coated with KL-6, and 100 μL of KL-6 monoclonal antibody labeled with alkaline phosphatase were added to the reaction tube and incubated to form a solid-phase antibody-antigen-enzyme-labeled MMP-3 antibody complex; Afterwards, a cleaning solution is added to the reaction tube, and the unbound enzyme-labeled antibodies and other substances are washed away by the cleaning solution; Then, AMPPD fast chemiluminescent substrate solution is added. The luminescent substrate solution emits photons under the catalysis of alkaline phosphatase. The number of photons generated is proportional to the concentration of KL-6 in the sample, thereby realizing the detection of the sample.

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