Preparation method of kit with low background value

Abstract

The invention relates to a preparation method of a kit with a low background value. The preparation method comprises the following steps: preparing antibody-coated magnetic particles and an antibody marked with alkaline phosphatase; the preparation method of the antibody marked with alkaline phosphatase comprises the following steps: (1) activating alkaline phosphatase; (2) activating the antibody; and (3) coupling of the activated alkaline phosphatase and the activated antibody: mixing the activated alkaline phosphatase obtained in the step (1) and the activated antibody obtained in the step (2), performing incubation reaction, and after the reaction is completed, performing sealing and dialysis to obtain the antibody marked with the alkaline phosphatase. According to the kit prepared by the method, the coupling efficiency of the magnetic beads and the antibody is improved, and compared with an existing coupling process, the background value is lower.

Description

Technical Field

[0001] The invention relates to the technical field of kit preparation, and in particular to a method for preparing a kit with a low background value. Background Art

[0002] Enzyme chemiluminescence enzyme immunoassay (CLEIA) uses the catalytic action of a labeled enzyme to make the luminescent agent (substrate) emit light. This type of luminescent agent that requires enzyme catalysis to emit light is called an enzyme-catalyzed luminescent agent or an enzyme-catalyzed luminescent substrate. The luminescent substrate of an enzyme-catalyzed reaction refers to a type of luminescent substrate that emits light after being degraded by an enzyme. At present, the enzymes commonly used in chemiluminescent enzyme immunoassay technology are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Enzymes such as alkaline phosphatase (AP) are used to label antigens or antibodies. After an immune reaction with the corresponding antigen (antibody) in the sample to be tested, a solid-phase coated antibody-antigen to be tested-enzyme-labeled antibody complex is formed. After washing, a substrate (luminescent agent) is added. The AP enzyme catalyzes and decomposes the substrate to emit light, which is received by the photon reading system. The photomultiplier tube converts the light signal into an electrical signal and amplifies it. Then, they are transmitted to the computer data processing system to calculate the concentration of the measured object.

[0003] Chemiluminescent reagents are often composed of two key parts, namely, antibody-magnetic bead solid phase reagents and antibody-enzyme labeling reagents. In immune response experiments, there is often interference from endogenous substances such as heterophilic antibodies and rheumatoid factors. Such interference can be eliminated by diluting or adding appropriate blocking agents. However, for the reagents themselves, such as abnormal aggregation of magnetic beads, the presence of some free luminescent substances that are not labeled with antigens or antibodies, will cause the background value of the reagent to be too high, thereby affecting the accuracy of the measurement of low-concentration clinical specimens.

[0004] Patent CN112362864B discloses a method for preparing a treatment agent for reducing the background luminescence value of an immunodiagnostic reagent. The treatment agent is used as a cleaning solution for a chemiluminescent instrument or as a component in a reagent and applied to an experimental reaction system to effectively reduce the background luminescence value of the immunodiagnostic reagent. However, the treatment agent has a complex component and may affect other components in the immunoreaction experiment. Patent CN110702907A discloses an enzymatic chemiluminescent immunoassay method for reducing the background of the luminescent substrate itself. The method is applied to a chemiluminescent method in which the marker enzyme used is HRP, but the method is not applicable to a chemiluminescent method marked with alkaline phosphatase. Summary of the invention

[0005] Aiming at the problem that the abnormal aggregation of magnetic beads existing in the prior art enzymatic chemiluminescent immunoassay and the presence of some free luminescent substances not labeled with antigens or antibodies lead to too high a background value of the reagent, thereby affecting the accuracy of the measurement of clinical low-concentration specimens, the present invention provides a method for preparing a kit with a low background value, which can effectively solve the problem of too high background luminescence value in immunodiagnostic reagents, greatly improve the sensitivity and product quality of the product, meet clinical requirements, and reduce the occurrence of misdiagnosis.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: A method for preparing a kit with a low background value, comprising the preparation of antibody-coated magnetic particles and antibodies labeled with alkaline phosphatase; The method for preparing the antibody labeled with alkaline phosphatase comprises the following steps: (1) Activation of alkaline phosphatase: N-succinimidyl-S-acetylthioacetate is added to a buffer containing alkaline phosphatase to carry out a cross-linking reaction. After the reaction is completed, the activated alkaline phosphatase is obtained after quenching, dialysis, activation and re-dialysis; (2) Antibody activation: The antibody is pre-treated with sulfhydryl blocking and sulfosuccinimide-4-( N -maleimidomethyl)cyclohexane-1-carboxylate is cross-linked with the pretreated antibody, and after the reaction is completed, the activated antibody is obtained by quenching and dialysis; (3) Coupling of activated alkaline phosphatase and activated antibody: The activated alkaline phosphatase obtained in the above step (1) and the activated antibody obtained in step (2) are mixed and incubated for reaction. After the reaction is completed, the antibody labeled with alkaline phosphatase is obtained after blocking and dialysis.

[0007] Furthermore, the buffer containing alkaline phosphatase in step (1) is a non-amine buffer, which is a PBS or HEPES buffer with a pH of 7.2-8.0; the cross-linking reaction conditions of N-succinimidyl-S-acetylthioacetate and the buffer containing alkaline phosphatase are: reaction at room temperature for 0.5-3h or at 2-8°C for 12-18h, and the molar ratio of N-succinimidyl-S-acetylthioacetate to alkaline phosphatase is 10-100:1.

[0008] Furthermore, the quenching conditions in step (1) and step (2) are as follows: adding 10% (v / v, volume ratio) of a 1M glycine buffer having a pH of 7.2 to the reaction solution after the cross-linking reaction, and reacting at room temperature for 0.5 to 2 hours.

[0009] Furthermore, the method for pre-treating the antibody for thiol blocking in step (2) is: adding a 1M thiol blocking agent with a mass concentration of 0.1-1% to a pH 7.2-8.0, 0.01-0.1M non-amine buffer containing the antibody, and reacting at room temperature for 0.5-1h; The sulfhydryl blocking agent is chloroacetamide.

[0010] Furthermore, in step (2), the cross-linking reaction conditions of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate and the pretreated antibody are: reaction at room temperature for 0.5 to 3 h or reaction at 2 to 8 ° C for 12 to 18 h, and the molar ratio of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate to the antibody is 10 to 100:1.

[0011] Furthermore, the incubation reaction conditions in step (3) are: room temperature for 2-4 hours or 2-8°C for 18-24 hours. The molar ratio of alkaline phosphatase to antibody is 1:1-3, preferably 1:1.

[0012] Furthermore, the method for preparing antibody-coated magnetic particles comprises the following steps: (a) Activation of magnetic particles; (b) antibody-magnetic particle coupling; (c) Sealing and washing.

[0013] Furthermore, the antibody-magnetic particle coupling method is as follows: the antibody is diluted with 100mM MES buffer, and the activated carboxyl magnetic particles are slowly added to the diluted antibody, vortexed to mix, and incubated at room temperature for 3h; the antibody:magnetic particle mass ratio is 1:20~1:200, preferably 1:100.

[0014] Further, the method of sealing and washing is: The coupled antibody-magnetic particles were adsorbed on a magnetic plate and the supernatant was discarded. Then, the first blocking solution with a pH of 7.5 was added. The blocking solution contained 0.02-0.1 M Tris, 150 mM NaCl, 0.05% Tween20, 0.05% sodium azide, and 0.02-0.1 M amino acids. The blocking was performed at room temperature for 30 min. Take the above-mentioned blocked magnetic beads, adsorb them again with a magnetic plate, discard the supernatant, and then add the second blocking solution with pH 7.5, which contains 0.02-0.1M Tris, 150mM NaCl, 0.05% Tween20, 0.05% sodium azide, and 1-5% protein blocking agent, and block at room temperature for 2-4 hours; Take the above-mentioned blocked magnetic beads, adsorb them on a magnetic plate, discard the supernatant, and wash them three times with TBST buffer at pH 7.4. After each wash, adsorb them on a magnetic plate and discard the supernatant. The TBST buffer contains 0.1M Tris, 150mM NaCl, and 0.05% Tween20. The coupled antibody-magnetic particles are stored in a buffer, thereby preparing antibody-coated magnetic particles.

[0015] The kit with low background value prepared according to the above method.

[0016] The present invention has the following beneficial effects: 1. The present invention provides a method for preparing a kit with a low background value. The antibody enzyme labeling technology uses an advanced heterobifunctional cross-linking method. Compared with the previous glutaraldehyde method and sodium periodate method, the cross-linking efficiency is significantly improved. A thiol blocking agent is added to reduce non-specific reactions that may occur during the detection process. Before activating the antibody, the free thiol groups on the antibody are blocked to avoid self-linking of the activated antibody, thereby improving the cross-linking efficiency. Compared with the existing coupling process, the background value is lower.

[0017] 2. The present invention provides a method for preparing a kit with a low background value. After activating the enzyme or antibody, a quencher is added to quench the excess cross-linking agent to prevent the residual cross-linking agent from reacting with irrelevant proteins.

[0018] 3. The present invention provides a method for preparing a low background value kit, which uses 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as an activator to couple sulfo-NHS with a carboxyl group to form an NHS ester that is much more stable than an O-acylisourea intermediate. The non-amine buffer used does not participate in or interfere with biochemical reactions, and can better couple the activated carboxyl magnetic beads with antibodies. DETAILED DESCRIPTION

[0019] The specific method for preparing the low background value kit of the present invention comprises the following steps: The preparation method of the kit mainly includes the preparation of antibody-coated magnetic particles and antibodies labeled with alkaline phosphatase.

[0020] The method for preparing antibody-coated magnetic particles comprises the following steps: (a) Activation of magnetic particles Take 10 mg of magnetic microparticles, wash with 1 mL of 100 mM MES (pH 5.0) buffer, incubate for 2 min, adsorb on magnetic plate, discard supernatant, and repeat this step once; Add 100 mM MES (pH 5.0) buffer and resuspend; Take 100mM MES (pH5.0) buffer to dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to 10-100mg / mL, take 100mM MES (pH5.0) to dissolve N-hydroxysulfosuccinimide (sulfo-NHS) to 10-100mg / mL, take appropriate amount of diluted EDC and sulfo-NHS and add them to the resuspended magnetic particles (the mass ratio of EDC to magnetic particles is 1:1-1:100, preferably 1:10; the mass ratio of sulfo-NHS to EDC is 1:1-3:1, preferably 2:1), vortex to mix, incubate at room temperature for 30min, after the incubation, adsorb on magnetic plate, discard supernatant, wash once with 100mM MES (pH5.0), adsorb on magnetic plate, discard supernatant; Activated magnetic particles are prepared by adding 10-100 mM Good's buffer at pH 5.0-7.5; the Good's buffer is preferably MES, PIPES, MOPSO, MOPS, HEPES, HEPPSO, EPPS, more preferably MES; the preferred concentration is 100 mM, and the preferred pH is 7.3.

[0021] (b) Antibody-magnetic particle coupling The antibody is diluted with 100 mM MES (pH 5.0-7.5, preferably pH 7.3) buffer, and the activated magnetic particles are slowly added to the diluted antibody (the mass ratio of antibody to magnetic particles is 1:20-1:200, preferably 1:100), vortexed to mix, and incubated at room temperature for 3 hours.

[0022] (c) Sealing and washing Take the coupled antibody-magnetic particles, adsorb them on the magnetic plate, discard the supernatant, add 1 mL of blocking solution containing 0.02~0.1M Tris (pH7.5), 150mM NaCl, 0.05% Tween20 (v / v), 0.05% sodium azide (w / v), 0.02~0.1M amino acids or protein-free compounds (alanine, glycine, lysine, arginine, ethanolamine, PEG or gelatin), and block at room temperature for 30 minutes; Take the above blocking antibody-magnetic particles, adsorb them on a magnetic plate, discard the supernatant, add 1 ml of blocking solution containing 0.02~0.1M Tris (pH7.5), 150mM NaCl, 0.05% Tween20 (v / v), 0.05% sodium azide (w / v), 1~5% protein blocking agent (w / v) (BSA, casein or sheep / horse serum), and block at room temperature for 2~4 hours; Take the above blocking antibody-magnetic particles, adsorb them on the magnetic plate, and discard the supernatant; The cells were washed three times with TBST buffer (0.1 M Tris, 150 mM NaCl, 0.05% Tween20 (v / v), pH 7.4), adsorbed on a magnetic plate, and the supernatant was discarded; The coupled antibody-magnetic particles are stored in a storage buffer to prepare antibody-coated magnetic particles. The storage buffer composition is: 0.1M PBS buffer (pH 6), 1% BSA, 0.05% Tween-20, 0.05% ProClin 300.

[0023] The preparation method of antibody-coated magnetic microparticles uses 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as an activator and introduces sulfo-NHS activated ester. After EDC activates the carboxyl group, it can be coupled with sulfo-NHS to form NHS esters that are much more stable than O-acylisourea intermediates, thereby improving the pH of the buffer during antibody coupling, maintaining the activity of the activated intermediate to a great extent, and greatly improving the coupling efficiency. The mild reaction conditions not only maintain the biological activity of the antibody, but also effectively reduce the hydrolysis of the carboxyl magnetic bead activation group, thereby greatly improving the efficiency of carboxyl magnetic bead coupling antibodies.

[0024] The method for preparing the antibody labeled with alkaline phosphatase of the present invention comprises the following steps: (1) Activation of alkaline phosphatase Add N-succinimidyl S-acetylthioacetate (SATA) to 0.01-0.1 M non-amine buffer (preferably PBS, HEPES) at pH 7.2-8.0 containing alkaline phosphatase (AP), vortex mix, and react at room temperature for 0.5h-3h, or at 2-8°C for 12-18h. The molar ratio of SATA to AP is 10-100:1.

[0025] Add 10% (v / v) glycine quencher to the reaction solution to remove unreacted SATA and react at room temperature for 0.5-2h.

[0026] The alkaline phosphatase after reaction was dialyzed in 10mM non-amine buffer (pH 7.0) at 4℃ for 12±1.2 hours to remove excess quencher and reactants; non-amine buffer is a buffer that does not contain primary amines, such as PBS, HEPES, etc. TRIS buffer is an amine-containing buffer.

[0027] Collect the dialyzed alkaline phosphatase, add 0.5M hydroxylamine·HCl, 25mM EDTA, 0.1M PBS solution, pH7.2-7.5, and react at room temperature for 2-3h; The alkaline phosphatase after reaction was dialyzed in a 10mM non-amine buffer (pH 7.0) containing 2~5mM metal chelator (such as ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, etc.) at 4℃ for 12±1.2 hours; Collect the dialyzed alkaline phosphatase and store it at 2-8°C for future use.

[0028] (2) Activating antibodies Add 0.1~1% of 1M thiol blocking agent (chloroacetamide) to 0.01~0.1M non-amine buffer (preferably PBS, HEPES) at pH 7.2~8.0 containing the antibody, and react at room temperature for 0.5~1h to block the free thiol groups on the antibody.

[0029] Sulfosuccinimide-4-( N -maleimidomethyl) cyclohexane-1-carboxylate (abbreviated as Sulfo-SMCC) is added to the treated antibody reaction solution and reacted at room temperature for 0.5-3 hours, or at 2-8°C for 12-18 hours. The molar ratio of Sulfo-SMCC to antibody is 10-100:1.

[0030] Add 10% (v / v) glycine quencher to the reaction solution to remove unreacted Sulfo-SMCC and react at room temperature for 0.5-2h.

[0031] The antibody was dialyzed in 10 mM non-amine buffer (pH 7.0) at 4°C for 12 ± 1.2 hours to remove excess glycine and reactants; Collect the dialyzed antibodies and store them at 2-8°C for later use.

[0032] (3) Conjugation of activated alkaline phosphatase and activated antibody The activated alkaline phosphatase of step (1) is mixed with the antibody of step (2), vortexed and reacted at room temperature for 2-4 hours, or at 2-8°C for 18-24 hours. The molar ratio of alkaline phosphatase to antibody is 1:1-3 (preferably 1:1).

[0033] Add 0.1~1% of 1M thiol blocking agent (chloroacetamide) to the reaction solution of the enzyme-labeled antibody to block the free thiol groups on alkaline phosphatase to prevent the formation of disulfide bonds. React at room temperature for 0.5~1h, and dialyze in 10mM non-amine buffer (pH7.0).

[0034] Collect the dialyzed enzyme-labeled antibody, add appropriate amount of buffer to the required concentration, and store at 2~8℃.

[0035] The preparation method of the antibody labeled with alkaline phosphatase is to use N-succinimidyl-S-acetylthioacetate (SATA) as a cross-linking agent, cross-link with AP to form an enzyme marker (AP-SATA) and purify it; use sulfosuccinimide-4-( N -Maleimidomethyl)cyclohexane-1-carboxylate (abbreviated as Sulfo-SMCC) is used as a cross-linking agent to cross-link with antibodies to form antibody conjugates and purify them. The modified AP enzyme marker is purified by hydroxylamine reduction and then cross-linked with the activated antibody. After purification, the antibody-enzyme marker is obtained. Pre-treating the antibody before activating it can prevent the antibody from self-linking. After the enzyme and antibody are activated, a quencher is added in time to quench the residual cross-linking agent. A thiol blocking agent is added to the final product to block the free thiol groups in the enzyme marker to avoid connecting other non-specific proteins. The mild purification method can maintain the biological activity of the enzyme and antibody. The heterobifunctional method can prevent the protein from self-linking to produce protein aggregates, improve the cross-linking efficiency, and keep the alkaline phosphatase labeled antibody in a stable state.

[0036] The kit prepared by the method of the present invention has a low background value, and after being placed in a 37° C. environment for a period of time, there is no phenomenon of increased background value.

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0038] The chemical reagents described in the following embodiments of the present invention can all be purchased directly from the market.

[0039] Example 1 Taking the human carbohydrate antigen 19-9 assay kit (chemiluminescence method) as an example, the preparation method of the antibody-coated magnetic particles and the antibody labeled with alkaline phosphatase in the kit is as follows.

[0040] The steps for preparing antibody-coated magnetic microparticles are as follows: (a) Activation of carboxyl magnetic beads 1. Take 10 mg of carboxyl magnetic beads, wash them with 1 mL of 100 mM MES (pH 5.0) buffer, then adsorb them on a magnetic plate, discard the supernatant, and repeat this step once; 2. Add 100mM MES (pH5.0) buffer and resuspend; 3. Dissolve EDC to 10 mg / mL in 100 mM MES (pH 5.0) buffer, dissolve sulfo-NHS to 10 mg / mL in 100 mM MES (pH 5.0), add appropriate amount of diluted EDC and sulfo-NHS to the carboxyl magnetic beads in step 2 (the mass ratio of EDC to carboxyl magnetic beads is 1:10; the mass ratio of sulfo-NHS to EDC is 2:1), vortex to mix, and incubate at room temperature for 30 min; 4. After the incubation, adsorb on the magnetic plate and discard the supernatant; 5. Wash once with 100mM MES (pH5.0) buffer, adsorb on magnetic plate, and discard the supernatant; 6. Add 100 mM MES (pH 7.3) buffer to prepare activated carboxyl magnetic beads.

[0041] (b) Antibody-magnetic bead coupling First, dilute the CA19-9 antibody with 100 mM MES (pH 7.3) buffer, then slowly add the carboxyl magnetic beads activated in step (a) to the diluted antibody (the mass ratio of antibody to magnetic beads is 1:100), vortex to mix, and incubate at room temperature for 3 hours.

[0042] (c) Sealing and washing 1. Take the antibody-magnetic beads coupled to step (b), adsorb on a magnetic plate, discard the supernatant, add 1 mL of blocking solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% sodium azide (w / v), and 100 mM glycine, and block at room temperature for 0.5 h; 2. Take the above-mentioned blocked magnetic beads, adsorb them on a magnetic plate, discard the supernatant, add 1 ml of blocking solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% sodium azide (w / v), and 1% BSA (w / v), and block at room temperature for 2 hours; 3. Take the above-mentioned blocked magnetic beads, adsorb them on the magnetic plate, and discard the supernatant; 4. Wash three times with TBST buffer (0.1 M Tris, 150 mM NaCl, 0.05% Tween-20 (v / v), pH 7.4), adsorb on magnetic plate, and discard the supernatant; The coupled magnetic beads were stored in 1 mL storage buffer to prepare antibody-coated magnetic particles. The storage buffer composition is: 0.1 M PBS buffer (pH 6), 1% BSA, 0.05% Tween-20, 0.05% ProClin 300.

[0043] The steps for preparing the antibody labeled with alkaline phosphatase are as follows: (1) Activation of alkaline phosphatase 1. Prepare SATA before use, using DMSO as solvent; 2. Add SATA to 0.1M PBS (pH 7.2) buffer containing alkaline phosphatase and react at room temperature for 0.5h. The molar ratio of SATA to AP is 100:1.

[0044] 3. Add 10% (v / v) 1M glycine buffer (pH 7.2) to the reaction solution and react at room temperature for 0.5 h; 4. The alkaline phosphatase after reaction was dialyzed in 10 mM PBS (pH 7.0) buffer at 4°C for 12 hours; 5. Collect the dialyzed alkaline phosphatase, add 0.5 M hydroxylamine·HCl, 25 mM EDTA, 0.1 M PBS solution, pH 7.2, and react at room temperature for 2 h; 6. Take out the alkaline phosphatase after the reaction and dialyze it in 5mM EDTA, 10mM PBS (pH7.0) at 4℃ for 12 hours; 7. Collect the dialyzed alkaline phosphatase and store it at 2-8°C for future use.

[0045] (2) Activated CA19-9 antibody 1. Add 1% (v / v) 1M chloroacetamide solution to 0.1M PBS (pH 7.2) buffer containing CA19-9 antibody and react at room temperature for 0.5h; 2. Prepare Sulfo-SMCC before use, using 0.1M PBS (pH 7.2) buffer as solvent; 3. Add Sulfo-SMCC to the CA19-9 antibody buffer after blocking the thiol groups, react at room temperature for 0.5 h, and the molar ratio of Sulfo-SMCC to antibody is 100:1; 4. Add 10% (v / v) 1M glycine buffer (pH 7.2) to the reaction solution and react at room temperature for 0.5h; 5. Take out the reacted CA19-9 antibody and dialyze it in 10mM PBS (pH7.0) at 4℃ for 12 hours; 6. Collect the dialyzed CA19-9 antibody and store it at 2-8°C for future use.

[0046] (3) Conjugation of alkaline phosphatase and CA19-9 antibody 1. Mix alkaline phosphatase and CA19-9 antibody and react at room temperature for 2 hours. The molar ratio of alkaline phosphatase to CA19-9 antibody is 1:1.

[0047] 2. Add 0.5% (v / v) 1M chloroacetamide solution to the enzyme-labeled antibody, react at room temperature for 0.5 h, and dialyze in 10 mM PBS (pH 7.0).

[0048] 3. Collect the dialyzed enzyme-labeled antibody, add appropriate amount of buffer to the required concentration, and store at 2-8℃.

[0049] Example 2 The difference from Example 1 lies in the preparation of antibody-coated magnetic microparticles. When activating carboxyl magnetic beads, the mass ratio of sulfo-NHS to EDC is 1:1.

[0050] Example 3 The difference from Example 1 lies in the preparation of antibody-coated magnetic microparticles. The formula of the first blocking solution in the blocking and washing operations is 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% sodium azide (w / v), 10% ethanolamine.

[0051] Example 4 The difference from Example 1 lies in the preparation of antibody-coated magnetic microparticles. The formula of the second blocking solution in the blocking and washing operations is 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% sodium azide (w / v), 1% casein.

[0052] Example 5 The difference from Example 1 lies in the preparation of the antibody labeled with alkaline phosphatase. The molar ratio of alkaline phosphatase to antibody during conjugation is 1:2.

[0053] Example 6 The difference from Example 1 lies in the preparation of the antibody labeled with alkaline phosphatase. The molar ratio of SATA to antibody is 20:1, and the molar ratio of Sulfo-SMCC to antibody is 20:1.

[0054] Comparative Example 1 The difference from Example 1 lies in the preparation of antibody-coated magnetic microparticles. Only EDC is used to activate carboxyl magnetic beads, and sulfo-NHS is not added.

[0055] Comparative Example 2 The difference from Example 1 lies in the preparation of antibody-coated magnetic microparticles. Only the blocking solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% sodium azide (w / v), and 100 mM glycine is used, and blocking is carried out at room temperature for 2 hours. In the blocking and washing operations, there is only the first blocking operation, and there is no second blocking operation.

[0056] Comparative Example 3 The difference from Example 1 is that the antibody-coated magnetic particles are prepared by using only a blocking solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (v / v), 0.05% sodium azide (w / v), and 5% BSA (w / v), and blocking for 2 hours at room temperature. In the blocking and washing operations, only the second blocking operation is performed, and the first blocking operation is not performed.

[0057] Comparative Example 4 The difference from Example 1 is that in the preparation of the antibody labeled with alkaline phosphatase, after the activation of alkaline phosphatase and the antibody, the quencher 1M glycine buffer was not used for treatment, but dialyzed directly.

[0058] Comparative Example 5 The difference from Example 1 is that in the preparation of the antibody labeled with alkaline phosphatase, after the alkaline phosphatase and the antibody are coupled, the thiol blocking agent 0.5% (v / v) 1M chloroacetamide solution is not used for thiol blocking, and the volume is directly fixed.

[0059] Comparative Example 6 The difference from Example 1 is that in the preparation of the antibody labeled with alkaline phosphatase, the antibody is not pre-treated with 1% (v / v) 1M chloroacetylaminothiol blocking in the operation of activating CA19-9 antibody.

[0060] Test results 1. Effect of different antibody-coated magnetic microparticle preparation schemes on background values The CA19-9 antibody-coated magnetic microparticles prepared in Examples 1-4 and Comparative Examples 1-3 were combined with the CA19-9 alkaline phosphatase-labeled antibody reagent of Example 1 to detect background values ​​on a fully automatic chemiluminescence immunoassay and record their RLU values. The relevant test results are shown in Table 1.

[0061] Table 1 Analysis of the influence of different antibody-coated magnetic microparticle preparation schemes on background values

[0062] As can be seen from Table 1 above, the background value (i.e., the value shown in the mean value row in Table 1 above) of the magnetic particles coated with CA19-9 antibody obtained in Examples 1-4 is significantly lower than that of Comparative Examples 1-3. Moreover, the background value after being placed in a 37°C biochemical incubator for 7 days did not show a significant increase. The background value of Comparative Examples 1-3 after being placed in a 37°C biochemical incubator for 7 days showed an increasing trend.

[0063] 2. Effect of different preparation schemes of antibodies labeled with alkaline phosphatase on background value The CA19-9 antibody labeled with alkaline phosphatase prepared in Examples 1, 5-6 and Comparative Examples 4-6 was used in combination with the CA19-9 antibody-magnetic particle composition reagent of Example 1 to detect background values ​​on a fully automatic chemiluminescence immunoassay and record their RLU values. The relevant test results are shown in Table 2.

[0064] Table 2 Analysis of the influence of different preparation schemes of antibodies labeled with alkaline phosphatase on background value

[0065] As can be seen from Table 2 above, the background values ​​of the CA19-9 antibody-enzyme marker obtained by coupling in Examples 1, 5, 6, and Comparative Example 4 are all low, but the signal-to-noise ratio of Example 1 is significantly higher than that of Examples 5, 6, and Comparative Example 4. After being placed in a 37°C biochemical incubator for 7 days, the background value of Comparative Example 4 increased. The background values ​​of Comparative Examples 5 and 6 are high, and the signal-to-noise ratio is lower than that of Example 1. After being placed in a 37°C biochemical incubator for 7 days, the background value also increased.

Claims

1. A method for preparing a kit with low background value, characterized in that: The invention comprises the preparation of antibody-coated magnetic particles and antibodies labeled with alkaline phosphatase; The method for preparing the antibody labeled with alkaline phosphatase comprises the following steps: (1) Activation of alkaline phosphatase: N-succinimidyl-S-acetylthioacetate is added to a buffer containing alkaline phosphatase to carry out a cross-linking reaction. After the reaction is completed, the activated alkaline phosphatase is obtained after quenching, dialysis, activation and re-dialysis; (2) Antibody activation: The antibody is pre-treated with sulfhydryl blocking and sulfosuccinimide-4-( N -maleimidomethyl)cyclohexane-1-carboxylate is cross-linked with the pretreated antibody, and after the reaction is completed, the activated antibody is obtained by quenching and dialysis; (3) Coupling of activated alkaline phosphatase and activated antibody: The activated alkaline phosphatase obtained in the above step (1) and the activated antibody obtained in step (2) are mixed and incubated for reaction. After the reaction is completed, the antibody labeled with alkaline phosphatase is obtained after blocking and dialysis.

2. The method for preparing the low background value kit according to claim 1, characterized in that: The buffer containing alkaline phosphatase in step (1) is a non-amine buffer; The cross-linking reaction conditions of N-succinimidyl-S-acetylthioacetate and the buffer containing alkaline phosphatase are: reaction at room temperature for 0.5-3 hours or at 2-8°C for 12-18 hours, and the molar ratio of N-succinimidyl-S-acetylthioacetate to alkaline phosphatase is 10-100:

1.

3. The method for preparing the low background value kit according to claim 1, characterized in that: The quenching conditions in step (1) and step (2) are as follows: adding a glycine buffer solution with a pH of 7.2 and a concentration of 1 M to the reaction solution after the cross-linking reaction, and reacting at room temperature for 0.5 to 2 hours.

4. The method for preparing the low background value kit according to claim 1, characterized in that: The method for pre-treating the antibody for thiol blocking in step (2) is: adding a 1M thiol blocking agent with a mass concentration of 0.1-1% to a pH 7.2-8.0, 0.01-0.1M non-amine buffer containing the antibody, and reacting at room temperature for 0.5-1h; The sulfhydryl blocking agent is chloroacetamide.

5. The method for preparing the kit with low background value according to claim 1, characterized in that: In step (2), the cross-linking reaction conditions of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate and the pretreated antibody are: reaction at room temperature for 0.5 to 3 h or reaction at 2 to 8 °C for 12 to 18 h, and the molar ratio of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate to antibody is 10 to 100:

1.

6. The method for preparing the kit with low background value according to claim 1, characterized in that: The incubation reaction conditions in step (3) are: room temperature for 2-4 hours or 2-8°C for 18-24 hours. The molar ratio of alkaline phosphatase to antibody is 1:1-3, preferably 1:

1.

7. The method for preparing the low background value kit according to claim 1, characterized in that: The method for preparing antibody-coated magnetic particles comprises the following steps: (a) Activation of magnetic particles; (b) antibody-magnetic particle coupling; (c) Sealing and washing.

8. The method for preparing the kit with low background value according to claim 7, characterized in that: The antibody-magnetic particle coupling method is as follows: the antibody is diluted with 100mM MES buffer, and the activated carboxyl magnetic particles are slowly added to the diluted antibody, vortexed to mix, and incubated at room temperature for 3h; the mass ratio of the antibody to the magnetic particles is 1:20~1:200, preferably 1:

100.

9. The method for preparing the kit with low background value according to claim 1, characterized in that: The method of blocking and washing is: The coupled antibody-magnetic particles were adsorbed on a magnetic plate and the supernatant was discarded. Then, the first blocking solution with a pH of 7.5 was added. The blocking solution contained 0.02-0.1 M Tris, 150 mM NaCl, 0.05% Tween20, 0.05% sodium azide, and 0.02-0.1 M amino acids. The blocking was performed at room temperature for 30 min. Take the above-mentioned blocked magnetic beads, adsorb them again with a magnetic plate, discard the supernatant, and then add the second blocking solution with pH 7.5, which contains 0.02-0.1M Tris, 150mM NaCl, 0.05% Tween20, 0.05% sodium azide, and 1-5% protein blocking agent, and block at room temperature for 2-4 hours; Take the above-mentioned blocked magnetic beads, adsorb them on a magnetic plate, discard the supernatant, and wash them three times with TBST buffer at pH 7.

4. After each wash, adsorb them on a magnetic plate and discard the supernatant. The TBST buffer contains 0.1M Tris, 150mM NaCl, and 0.05% Tween20. The coupled antibody-magnetic particles are stored in a buffer, thereby preparing antibody-coated magnetic particles.

10. A kit prepared according to the preparation method according to any one of claims 1 to 9.

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