Method for improving stability of immunodetection reagent and reducing interference of rheumatoid factors

Abstract

A method for improving immunodetection reagent stability and reducing rheumatoid factor interference comprises the following steps: (1) activation of magnetic particles: EDC and NHS are prepared into an activation solution for activation of the magnetic particles, and the temperature is controlled to be 2-8 DEG C in the preparation process of the activation solution; (2) antibody-magnetic particle coupling: diluting an antibody and an anti-interference protein with a buffer solution, then adding the magnetic particles activated in the step (1) into the diluted antibody and anti-interference protein, uniformly mixing, and incubating to obtain coupled magnetic particles; (3) terminating the reaction: cleaning the coupled magnetic particles by using a terminating reaction solution, removing supernatant, and continuously adding the terminating reaction solution for terminating the reaction; and (4) sealing and washing: sealing the coupling magnetic particles after the reaction is terminated in the step (3) by using a sealing liquid, washing after the sealing is completed, and diluting and storing the washed coupling magnetic particles by using a magnetic particle antibody working liquid to prepare the antibody-magnetic particle conjugate. When the antibody-magnetic particle conjugate prepared by the method is used in the preparation of a kit, the detection of the kit is more reliable and repeatable.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme - catalyzed chemiluminescence immunoassay, and particularly relates to a method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor. Background Art

[0002] The enzyme - catalyzed chemiluminescence kit mainly includes magnetic microparticles - antibody / antigen conjugates, enzyme - labeled antibodies (alkaline phosphatase - antibody / antigen conjugates) and washing solution, and performs detection by using the magnetic microparticle chemiluminescence immunoassay method and applying principles such as the double - antibody sandwich method / competition method. In addition, according to the specific kit type and application, other necessary components may also be included, such as substrates, auxiliary reagents, calibrators and quality control products, etc., to ensure the accuracy and reliability of the experiment.

[0003] The preparation process of the magnetic microparticle antibody working solution generally includes steps such as activation of carboxyl magnetic beads, coupling of antibody and magnetic beads, blocking, and washing. The activation method of carboxyl magnetic beads adopts a two - step method, which divides the reaction of activating carboxyl by EDC and the reaction of forming an amide bond with protein amino groups into two steps. This method usually requires the introduction of N - hydroxysuccinimide (NHS) in the first step of activating carboxyl. NHS is a hydrophilic small molecule that can react quickly with amino groups. After EDC activates carboxyl, a stable intermediate is formed with NHS activated ester. After removing the excess EDC, antibody protein is added, and the amino group on the protein reacts with the NHS intermediate to form an amide bond. The two - step method can effectively avoid the aggregation of antibody protein caused by EDC residue, and at the same time, the introduction of NHS activated ester can effectively improve the efficiency of amide bond formation. EDC and NHS activated ester are commonly used activators for forming covalent bonds between biomolecules. However, during the process of using EDC and NHS to activate magnetic beads, different activation conditions are very likely to cause changes in the physical properties of magnetic beads, such as size, shape or magnetism, including the control of reaction rate and other such changes may affect the efficiency of magnetic beads capturing specific molecules from the mixture.

[0004] The coupling of antibody and magnetic beads is to add a certain concentration of antibody solution to the magnetic bead suspension, and make the antibody covalently bind to the functional groups on the surface of the magnetic beads through EDC and NHS. Generally, parameters such as pH value, temperature and reaction time are adjusted to achieve the best coupling effect, usually carried out under mild conditions to avoid antibody denaturation. Rheumatoid factor (RF) is an autoantibody, mainly present in the serum and synovial fluid of patients with rheumatoid arthritis. RF can bind to the Fc segment of immunoglobulin (Ig) G, and may cause false positive and false negative results in immunoassays.

[0005] In immunology experiments, the role of blocking agents is to reduce nonspecific binding. Commonly used blocking agents include skimmed milk powder, BSA (bovine serum albumin), serum, and Fab fragment single-chain secondary antibodies. These blocking agents bind to the blank gaps on the membrane surface by adsorption, thereby avoiding nonspecific binding of the primary antibody. In the process of magnetic bead coupling antibodies, the use of proteins for blocking can improve the stability of signal values in subsequent use, greatly reduce nonspecific binding, reduce nonspecificity, and maintain good repeatability and stability.

[0006] The accuracy and stability of the enzymatic chemiluminescent reagent are closely related to the coupling process of the magnetic particle antibody working solution and the enzyme-labeled antibody working solution, and the selection of the reagent buffer is also crucial. Application No. CN200910239511.8 discloses a method for stabilizing alkaline phosphatase or its marker, which discloses a stabilizer containing protein, zinc ions, magnesium and calcium ions, and sodium ions mixed with alkaline phosphatase or its marker to dissolve into a solution to explore its 37°C accelerated stability. However, the patent does not involve the long-term stable storage of the enzymatic chemiluminescent reagent at 2-8°C and the reduction of RF interference. Summary of the invention

[0007] In view of the problems that in the magnetic particle activation process of the existing enzymatic chemiluminescent immunoassay kit, the activation process is too fast and the activation efficiency is uncontrollable, the antibody and magnetic particle coupling process is susceptible to RF interference and false positives or false negatives occur, and the long-term stability of the enzymatic chemiluminescent reagent needs to be improved, the present invention provides a method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factors.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: A method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factors, the method comprising the following steps: (1) Activation of magnetic particles: EDC and NH are prepared into an activation solution for activation of magnetic particles, wherein the temperature of the activation solution preparation process is controlled at 2 to 8° C.; (2) Antibody-magnetic particle coupling: dilute the antibody and anti-interference protein with a buffer, then add the magnetic particles activated in step (1) thereto, mix well, and incubate to obtain coupled magnetic particles; (3) Termination reaction: Wash the coupled magnetic microparticles with the termination reaction solution. After removing the supernatant, add the termination reaction solution again to terminate the reaction. If the magnetic beads activated by EDC and NHS are not promptly bound to the target molecule, the remaining activated groups may react with non-target substances, resulting in non-specific binding. Therefore, terminating the non-specific reaction is necessary. Glycine and ethanolamine are small molecules containing primary amines that competitively bind to the unreacted activated groups on the magnetic beads, thereby preventing subsequent non-specific binding. This can avoid the reaction of the magnetic beads with other proteins or molecules in the sample during subsequent use and improve the specificity of coupling. Additionally, the buffering capacity of ethanolamine and glycine may also contribute to maintaining the pH stability of the reaction.

[0009] (4) Blocking and washing: Block the coupled magnetic microparticles after the termination reaction in step (3) with the blocking solution. After completion of blocking, wash them. Dilute and store the washed coupled magnetic microparticles with the magnetic microparticle antibody working solution, and thus the antibody-magnetic microparticle conjugate is prepared. The magnetic microparticle antibody diluent includes one of 20 - 100 mM / L PBS, 10 - 200 mM / L NaCl, 0.1 - 8% of BSA, casein, or γ-globulin, 0.05 - 2% Tween 20, one or two of disodium ethylenediaminetetraacetate (EDTA·2Na) and hydroxyethylethylenediaminetriacetic acid, and one or two of 0.05% - 1% Proclin 300, Proclin 950, and sodium azide, with a pH of 3.0 - 7.0.

[0010] Furthermore, the total concentration of disodium ethylenediaminetetraacetate and / or hydroxyethylethylenediaminetriacetic acid in the magnetic microparticle antibody diluent is ≥5 mM / L.

[0011] Furthermore, the activation method of the magnetic microparticles is specifically as follows: Take the magnetic microparticles, wash them with the buffer solution and then remove the supernatant. Continue to resuspend the magnetic microparticles with the buffer solution, then add the activation solution to the magnetic microparticles to obtain a mixture, mix the mixture evenly and carry out the activation reaction. After the incubation ends, remove the supernatant, wash the magnetic microparticles with the buffer solution, and add the buffer solution to the washed magnetic microparticles to prepare the activated magnetic microparticles.

[0012] Furthermore, the concentration of the activation solution is 10 - 100 mg / ml, the activation reaction time is 30 min; the buffer solution is 100 mM / L MES pH 5.0; The mass ratio of the magnetic microparticles to EDC in the mixture is 1 - 20:1, and the mass ratio of the magnetic microparticles to NHS in the mixture is 1 - 20:1.

[0013] Furthermore, the mass ratio of the magnetic microparticles to EDC in the mixture is 10:1, and the mass ratio of the magnetic microparticles to NHS in the mixture is 10:1.

[0014] Further, in step (2), the mass ratio of the antibody to the anti-interference protein is 1:1 to 200, and the mass ratio of the antibody to the magnetic particles is 1:20 to 200; The anti-interference protein is at least one of protein A and protein G; The incubation time is 3 to 24 h.

[0015] Further, the mass ratio of the antibody to the anti-interference protein is 1:1, and the mass ratio of the antibody to the magnetic particles is 1:100; The anti-interference protein is protein A.

[0016] Further, the termination reaction solution includes 100 mM / L Tris pH 7.4, 150 mM / L NaCl, 0.05% Tween 20, 10 to 100 mM / L amino acid or small molecule peptide or both of the above, and one or both of 0.1% to 20% (v / v) alcohol compounds; The amino acid is glycine, lysine, alanine or arginine, the small molecule peptide is glycylglycine, carnosine, carnosine or tripeptide, and the alcohol compound is ethanolamine, diethanolamine, triethanolamine, cyclohexanol, isopropanol or benzyl alcohol; The termination reaction time is 0.5 to 2 h; Preferably, the amino acid is glycine; the glycine concentration is 50 mM / L, preferably the alcohol compound is ethanolamine; the ethanolamine is 10%.

[0017] A kit, the kit includes an antibody-magnetic particle conjugate, the antibody-magnetic particle conjugate is prepared by the method for improving the stability of the immunoassay reagent and reducing the interference of rheumatoid factor, the kit further includes an enzyme-labeled antibody diluent, and the enzyme-labeled antibody diluent includes: 100 mM / L PBS, 150 mM / L NaCl, 0.5% BSA, 0.05 to 2% Tween 20, 0.1 mM / L zinc ion, 2 mM / L magnesium ion, 3% trehalose, 0.05% Proclin 300, 0.09% sodium azide, and the pH is 6.8.

[0018] Further, the kit further includes an auxiliary reagent, and the auxiliary reagent components include: 100 mM / L PBS, 150 mM / L NaCl, 2% BSA, 0.05% Tween 20, 3% trehalose, 0.05% Proclin 300, 0.09% sodium azide, EDTA·2Na ≥ 2 mM / L, and the pH is 7.2.

[0019] The present invention has the following beneficial effects: 1. The present invention provides a method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor. During the activation process of magnetic microparticles, low-temperature operating conditions are maintained. Compared with the normal-temperature operation in the prior art, the low-temperature environment helps to maintain the stability of magnetic beads and their surface modifications, slows down the rate of chemical reactions, makes the activation process more controllable, and avoids the occurrence of side reactions that may be caused by the too-fast reaction rate in normal-temperature activation operations, thereby improving the activation efficiency.

[0020] 2. The present invention provides a method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor. During the coupling process of antibodies and magnetic microparticles, anti-interference proteins are introduced. By using proteins such as protein A / G, the interference caused by RF can be reduced, the specificity and purity of the experiment can be improved, and the addition of proteins such as protein A / G can increase the binding stability between magnetic beads and antibodies, preventing the antibodies from falling off the magnetic beads. These proteins can provide stronger binding force to ensure that the antibodies are firmly bound to the magnetic beads, thereby improving the reliability and repeatability of the experiment.

[0021] 3. The present invention provides a method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor. In order to further improve the stability of the reagents, a termination reaction step is added before blocking, that is, treated with small molecule substances and alkanolamines. After treatment with glycine and ethanolamine, protein is used for blocking, mainly to further improve specificity and stability to obtain a highly efficient and specific magnetic bead-antibody complex.

[0022] 4. The present invention provides a method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor. By selecting a suitable metal chelator, corresponding concentration, and addition method to ensure that it does not affect the normal function of the enzyme and does not interfere with the chemiluminescence reaction, while being helpful for the performance of in vitro diagnostic kits. The synergistic effect of metal ions and chelating agents in the enzyme-labeled antibody diluent is beneficial to the long-term stable preservation of enzyme-promoted reagents, thereby maintaining the long-term stability of the reagents, solving the problem of poor stability of enzyme-promoted chemiluminescence immunoassay kit reagents, and improving the diagnostic effect and accuracy of the kit. Specific Embodiments

[0023] During the enzyme-promoted chemiluminescence immunoassay process, the method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor includes the following operations: (1) Activation of magnetic microparticles (hereinafter carboxyl magnetic beads are taken as an example), The present invention uses a two-step method (EDC, NHS) to activate carboxyl magnetic beads and controls the low-temperature treatment during the preparation of the activation solution.

[0024] The specific activation steps are as follows: 1. Take 10 mg of magnetic beads, wash and incubate them with 1 ml of 100 mM / L MES (pH 5.0) for 2 min, adsorb them on a magnetic plate, and discard the supernatant. 2. Resuspend with 100 mM / L MES (pH 5.0) 1 - 3 times, and discard the supernatant. 3. Add 100 mM / L MES (pH 5.0) and mix. 4. Dissolve EDC and NHS in 100 mM / L MES (pH 5.0, temperature 2 - 8 °C) to 10 - 100 mg / mL respectively, and add appropriate amounts of the diluted EDC and NHS to the magnetic beads in step 3 (mass ratio of magnetic beads to EDC is 1 - 20:1, mass ratio of magnetic beads to NHS is 1 - 20:1, preferably mass ratio of magnetic beads to EDC is 10:1, mass ratio of magnetic beads to NHS is 10:1), vortex and mix well, incubate at room temperature for 30 min. 5. After incubation, adsorb on a magnetic plate and discard the supernatant. 6. Wash with 100 mM / L MES (pH 5.0) 1 - 3 times, adsorb on a magnetic plate, and discard the supernatant. 7. Add 100 mM / L MES (pH 5.0) buffer to obtain the activated carboxyl magnetic beads. (2) Antibody - magnetic particle coupling In the present invention, an anti - interference protein (protein A / G) is introduced into the coupling process of the activated magnetic beads and the antibody.

[0025] The specific coupling steps are as follows: Dilute the antibody and the anti - interference protein with 100 mM / L MES (pH 5.0) (preferably mass ratio of antibody to anti - interference protein is 1:1 - 200, most preferably mass ratio of antibody to anti - interference protein is 1:1; the anti - interference protein is preferably protein A / G, most preferably protein A), and add the activated carboxyl magnetic beads in step (1) to the diluted antibody and anti - interference protein (mass ratio of antibody to magnetic beads is 1:20 - 200, preferably 1:100), vortex and mix well, incubate at room temperature for 3 - 24 hours.

[0026] Protein A is a cell wall protein isolated from Staphylococcus aureus type A. It is also known as protein A and can bind to the Fc segment of most types of human IgG (except IgG3). Therefore, it can be used to capture RF in the sample, thereby reducing its interference with subsequent detections; protein G is a cell wall protein isolated from Streptococcus type G and can also specifically bind to the Fc segment of IgG, thus preventing RF from binding to the antibody - binding site in the detection system. Specifically, protein A and protein G reduce the chance of RF binding to the antibody in the detection reagent by competitively binding to the Fc fragment, thereby reducing the interference effect of RF. (3) Terminate the reaction 1. Take the antibody-conjugated magnetic beads from step (2), adsorb them on a magnetic plate, discard the supernatant, add 1 ml of a solution containing 0.1 M Tris (pH 7.4), 150 mM / L NaCl, 0.05% Tween 20, 10 - 100 mM / L amino acid or small molecule peptide or two of the above (preferably glycine, lysine, alanine, arginine, glycylglycine, lipeptide, carnosine, tripeptide, etc., most preferably glycine; the concentration is preferably 50 mM / L), 0.1% - 20% (v / v) of one or two of the following alcohol compounds (preferably ethanolamine, diethanolamine, triethanolamine, cyclohexanol, isopropanol, benzyl alcohol, etc., most preferably ethanolamine; the concentration is preferably 10%) to wash the magnetic beads 1 - 3 times, adsorb them on a magnetic plate, and discard the supernatant.

[0027] 2. After adding the above buffer again, incubate at room temperature for 0.5 - 2 hours; The use of glycine and ethanolamine in terminating the reaction can help block the unreacted active sites and reduce non-specific binding.

[0028] (4) Block and wash 1. Take the conjugated magnetic beads from step (3), adsorb them on a magnetic plate, discard the supernatant, add 1 ml of a solution containing 0.1 M Tris (pH 7.4), 150 mM / L NaCl, 0.05% Tween 20, and 5% BSA to block for 3 h - 20 h; 2. Wash 3 times with TBST (0.1 M Tris, 150 mM / L NaCl, 0.05% Tween 20 (v / v), pH 7.4), adsorb them on a magnetic plate, and discard the supernatant; Preserve the conjugated magnetic beads with 1 ml of magnetic particle antibody diluent, and the antibody-magnetic particle conjugate is thus prepared.

[0029] The magnetic particle antibody diluent includes: 20 - 101 mM / L PBS, 10 - 200 mM / L NaCl, one of 0.1 - 8% BSA, casein, γ-globulin, 0.05 - 2% Tween 20, one or two of EDTA·2Na, hydroxyethyl ethylenediaminetriacetic acid (HEDTA), one or two of 0.05% - 1% Proclin 300, Proclin 950, sodium azide, and the pH is 3.0 - 7.0. The total concentration of EDTA·2Na and / or hydroxyethyl ethylenediaminetriacetic acid (HEDTA) in the magnetic particle antibody diluent is ≥5 mM / L.

[0030] Metal chelators (EDTA·2Na, HEDTA) are added to the magnetic particle antibody diluent, and Zn is added to the dilution buffer for the enzyme-labeled antibody working solution 2+ 、Mg 2+。The concentration, type, and addition method of metal chelators in enzyme - catalyzed chemiluminescence immunoassay reagents have a great impact on the accuracy and stability of the reagents (37 °C accelerated stability, 2 - 8 °C long - term stability). If the metal chelator is directly added to the enzyme - labeled antibody diluent, it is easy to chelate with Zn 2+ and Mg 2+ . And Zn 2+ and Mg 2+ are important metal cofactors of alkaline phosphatase and are crucial for stabilizing the activity of alkaline phosphatase. In the research and development of enzyme - immuno - chemiluminescence immunoassay kits, it is found that the simultaneous addition of metal chelators (EDTA·2Na, HEDTA) and Mg 2+ , Zn 2+ can maintain the stability of enzyme activity for a long time, that is, maintain the long - term stability of the reagent. However, due to the differences in kits, the addition methods and concentrations are also different.

[0031] The kit also includes enzyme - labeled antibodies. The enzyme - labeled antibody diluent is used to store or dilute enzyme - labeled antibodies. Specifically, the components of the enzyme - labeled antibody diluent in the present invention include: 20 - 100 mM / L PBS, 10 - 200 mM / L NaCl, one of 0.1 - 8% BSA, casein, and γ - globulin, 0.05 - 2% Tween 20, 0.01 - 1 mM / L zinc ion, one or two of 0.01 - 10 mM / L magnesium ion, calcium ion, and manganese ion, one or two of 0.5% - 10% trehalose or sucrose, one or two of 0.05% - 1% Proclin300, Proclin950, and sodium azide, and the pH is 5.0 - 7.0.

[0032] Some kits also need to use auxiliary reagents. The components of the auxiliary reagents include: 100 mM / L PBS, 150 mM / L NaCl, 2% BSA, 0.05% Tween 20, 3% trehalose, 0.05% Proclin300, 0.09% sodium azide, the concentration of EDTA·2Na ≥ 2 mM / L, and the pH is 7.2.

[0033] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with specific embodiments.

[0034] The chemical reagents described in the following embodiments of the present invention can all be directly purchased from the market, and the implementation conditions not specified are conventional conditions in this industry.

[0035] Example 1: (1) Activation of carboxyl magnetic beads 1. Take 10 mg of magnetic beads, wash and incubate with 1 ml of 100 mM / L MES (pH 5.0) for 2 min, adsorb with a magnetic plate, and discard the supernatant; 2. Resuspend 1 - 3 times with 100 mM / L MES (pH 5.0), discard the supernatant; 3. Add 100 mM / L MES (pH 5.0) and mix; 4. Dissolve EDC and NHS in 100 mM / L MES (pH 5.0, temperature 2 - 8 °C) to 10 mg / ml respectively, and immediately take an appropriate amount of the diluted EDC and NHS and add them to the magnetic beads in step 3 (mass ratio of magnetic beads: EDC is 10:1, mass ratio of magnetic beads: NHS is 10:1), vortex and mix well, incubate at room temperature for 30 min; 5. After incubation, wash 3 times with 100 mM / L MES (pH 5.0), adsorb with a magnetic plate, discard the supernatant; 6. Add 100 mM / L MES (pH 5.0) to obtain activated carboxyl magnetic beads; (2) Antibody - magnetic bead coupling 1. Dilute the target antibody and protein A with 100 mM / L MES (pH 5.0) respectively (mass ratio of antibody: magnetic beads is 1:100, mass ratio of protein: magnetic beads is 1:100).

[0036] 2. Add the activated carboxyl magnetic beads in step (1) to the diluted antibody, vortex and mix well, incubate at room temperature for 24 hours.

[0037] (3) Terminate the reaction 1. Take the coupled magnetic beads in step (2), adsorb with a magnetic plate, discard the supernatant, add 1 ml of a termination solution containing 0.1 M Tris (pH 7.4), 150 mM / L NaCl, 0.05% Tween20, 50 mM / L glycine amino acid, 10% ethanolamine (v / v) and wash once; 2. Add 1 ml of a termination solution containing 0.1 M Tris (pH 7.4), 150 mM / L NaCl, 0.05% Tween20, 50 mM / L glycine amino acid, 10% ethanolamine (v / v) to the coupled magnetic beads, incubate at room temperature for 30 minutes.

[0038] (4) Block and wash 1. Take the coupled magnetic beads in step (3), adsorb with a magnetic plate, discard the supernatant, add 1 ml of a blocking solution containing 0.1 M Tris (pH 7.4), 150 mM / L NaCl, 5% BSA (w / v), 0.05% Tween20 (v / v), 0.05% sodium azide (w / v) and block overnight; 2. Adsorb with a magnetic plate, discard the supernatant; 3. Wash 3 times with TBST (0.1 M Tris, 150 mM / L NaCl, 0.05% Tween20 (v / v), pH 7.4), adsorb with a magnetic plate, discard the supernatant; 4. The conjugated magnetic beads are stored with 1 ml of magnetic particle antibody diluent to prepare the magnetic particle antibody conjugate.

[0039] The magnetic particle antibody diluent includes: 100 mM / L PBS, 150 mM / L NaCl, 0.5% BSA, 0.05% Tween 20, 5 mM / L HEDTA, 0.05% Proclin 300, 0.09% sodium azide, and the pH is 6.0.

[0040] The enzyme-labeled antibody diluent includes: 100 mM / L PBS, 150 mM / L NaCl, 0.5% BSA, 0.05 - 2% Tween 20, 0.1 mM / L zinc ion, 2 mM / L magnesium ion, 3% trehalose, 0.05% Proclin 300, 0.09% sodium azide, and the pH is 6.8.

[0041] Example 2 Different from Example 1, this example also includes auxiliary reagents.

[0042] The auxiliary reagents include: 100 mM / L PBS, 150 mM / L NaCl, 2% BSA, 0.05% Tween 20, 3% trehalose, 0.05% Proclin 300, 0.09% sodium azide, 2 mM / L EDTA·2Na, and the pH is 7.2. (The requirements vary depending on the type of kit).

[0043] Example 3 The difference between Example 3 and Example 2 is that in this example, HEDTA > 5 mM / L in the magnetic particle antibody diluent, and the HEDTA concentration in this example is 6 mM / L; the concentration of EDTA·2Na in the auxiliary reagents is > 2 mM / L, and the EDTA·2Na concentration in this example is 3 mM / L.

[0044] Example 4 The difference between Example 4 and Example 1 is that in this example, HEDTA > 5 mM / L in the magnetic particle antibody diluent, and the HEDTA concentration in this example is 6 mM / L.

[0045] Comparative Example 1 The difference from Example 1 is that Protein A was not introduced when the antibody was conjugated with carboxyl magnetic beads.

[0046] Comparative Example 2 The difference from Example 1 is that the magnetic particle antibody diluent does not contain HEDTA.

[0047] Comparative Example 3 The difference from Example 1 is that the enzyme-labeled antibody diluent contains HEDTA, and the concentration of HEDTA is 2 mM / L.

[0048] Comparative Example 4 The difference between this comparative example and Example 1 is that the concentration of HEDTA in the magnetic particle antibody diluent in this comparative example is < 5 mM / L, and the HEDTA concentration in this comparative example is 4 mM / L.

[0049] Comparative Example 5 The difference from Example 2 is that Protein A was not introduced when the antibody was conjugated with carboxyl magnetic beads.

[0050] Comparative Example 6 The difference from Example 2 is that the magnetic particle antibody diluent does not contain HEDTA.

[0051] Comparative Example 7 The difference from Example 2 is that the enzyme-labeled antibody diluent contains HEDTA, and the concentration of HEDTA is 2 mM / L.

[0052] Comparative Example 8 The difference between this comparative example and Example 2 is that the concentration of HEDTA in the magnetic particle antibody diluent in this comparative example is < 5 mM / L, and the HEDTA concentration in this comparative example is 4 mM / L); the concentration of EDTA·2Na in the auxiliary reagent is < 2 mM / L, and the HEDTA concentration in this comparative example is 1 mM / L).

[0053] Comparative Example 9 The difference between this comparative example and Example 2 is that the concentration of HEDTA in the magnetic particle antibody diluent in this comparative example is > 5 mM / L, and the HEDTA concentration in this comparative example is 6 mM / L; the concentration of EDTA·2Na in the auxiliary reagent is < 2 mM / L, and the concentration of EDTA·2Na in this comparative example is 1 mM / L.

[0054] Test Results The antibody-magnetic particle conjugate, enzyme-labeled antibody and auxiliary reagent prepared according to the methods of the above Examples 1 to 4 and Comparative Examples 1 to 9 were diluted with the corresponding magnetic particle antibody diluents of Examples 1 to 4 and Comparative Examples 1 to 9 as solid-phase reagents, and the enzyme-labeled antibody was diluted with the enzyme-labeled antibody diluents of Examples 1 to 4 and Comparative Examples 1 to 9 as labeling reagents. The auxiliary reagent was used alone in the enzyme-labeled plate for automatic chemiluminescence immunoassay analyzer to detect samples respectively. The relevant test results are shown in the following table.

[0055] 1. Detection of thyroid stimulating hormone (TSH), and the influence of the TSH antibody-magnetic particle conjugation scheme on reducing RF interference and stability (37 °C acceleration and 4 °C long-term stability).

[0056] Auxiliary reagents are required in the TSH detection process. Therefore, the TSH antibody-magnetic particle conjugates prepared in Examples 2-3 and Comparative Examples 5-9 were diluted with a magnetic particle antibody diluent at a volume ratio of 1:10 to prepare a solid-phase reagent, and the TSH antibody-AP (alkaline phosphatase) was diluted with the enzyme-labeled antibody diluent described in the present invention at a ratio of 1:40 to prepare a labeling reagent. The auxiliary reagents were placed separately in the detection well plate, and the samples were detected on an automatic chemiluminescence immunoassay analyzer. The relevant test results are shown in the following table.

[0057] Table 1 Analysis of the determination results of TSH reagents for RF samples Control group Example 2 Comparative Example 5 Interference rate of Example 2 Interference rate of Comparative Example 5 Serum sample 1 14.381 14.534 16.345 1.06％ 13.66％ Serum sample 2 26.586 25.921 29.583 -2.50％ 11.27％ Serum sample 3 34.714 33.682 37.261 -2.97％ 7.34％ The control group in Table 1 refers to the concentration of thyroid-stimulating hormone in the human serum sample tested by the imported reagent. It can be seen from the results in Table 1 that when protein A is introduced during the conjugation of the TSH antibody and magnetic beads in the TSH reagent obtained in Example 2, the influence of RF on the test results can be reduced, and the accuracy of the chemiluminescence reagent detection results can be improved.

[0058] Table 2 Analysis of the influence of the accelerated stability of TSH reagents at 37°C Table 3 Analysis of the influence of the accelerated stability of TSH reagents at 4°C It can be seen from Table 2 and Table 3 that the TSH reagents obtained in Examples 2-3 are more stable in terms of accelerated stability at 37°C compared with Comparative Examples 6-8. According to the results of Comparative Example 7, HEDTA cannot be directly added to the dilution buffer of the enzyme-labeled antibody working solution, which easily leads to the inactivation of the enzyme and thus poor stability of the reagent. It can be seen from Examples 2, 3, Comparative Example 8, and Comparative Example 9 that the metal chelators (EDTA·2Na, HEDTA), Zn 2+ , Mg 2+ coexist and HEDTA ≥ 5 mM / L in the magnetic particle antibody diluent, and at the same time, the concentration of EDTA·2Na in the auxiliary reagent is ≥ 2 mM / L, which is helpful for the accelerated stability and long-term stability of the reagent.

[0059] 2. Detection of different alpha-fetoprotein (AFP), the influence of the AFP antibody-magnetic bead conjugation scheme on reducing RF interference and stability (37°C acceleration and 4°C long-term stability) During the AFP detection process, no auxiliary reagents are required. The AFP antibody-magnetic particle conjugates prepared in Example 1, Example 4, and Comparative Examples 1-4 are diluted at a ratio of 1:10 with the magnetic particle antibody diluent of the present invention to prepare a solid-phase reagent, and the AFP antibody-AP is diluted at a ratio of 1:40 with the enzyme-labeled antibody diluent of the present invention to prepare a labeling reagent. Samples are detected on an automatic chemiluminescence immunoassay analyzer, and the relevant test results are shown in the following table.

[0060] Table 4 Analysis of the determination results of AFP reagents for RF samples Control group Example 1 Comparative Example 1 Interference rate of Example 1 Interference rate of Comparative Example 1 Serum sample 1 30.54 29.88 33.55 -2.16％ 9.86％ Serum sample 2 69.21 73.52 77.49 6.23％ 11.96％ Serum sample 3 121.04 117.24 133.97 -3.14％ 10.68％ The control group in Table 4 refers to the concentration of alpha-fetoprotein in human serum samples detected by imported reagents. As can be seen from the results in Table 4, when protein A is introduced during the coupling of the AFP antibody and magnetic beads in the AFP reagent obtained in Example 1, the influence of RF on the test results can be reduced, and the accuracy of the chemiluminescence reagent detection results can be improved.

[0061] Table 5 Analysis of the influence of the accelerated stability of AFP reagents at 37°C

[0062] Table 6 Analysis of the influence of the long-term stability of AFP reagents at 4°C As can be seen from Table 5 and Table 6, the AFP reagent obtained in Example 1 has better long-term stability at 4°C compared with Comparative Examples 2-4. According to the results of Comparative Example 2, the presence of HEDTA in the magnetic particle antibody diluent is beneficial to the long-term stability of the reagent. According to the results of Comparative Example 3, when HEDTA is directly added to the enzyme-labeled antibody diluent, although it does not have a significant destructive effect on the long-term stability of the AFP kit as in the case of TSH detection, its results are not as good as the long-term stability effects shown in Example 1 and Example 4. According to the results of Example 1, Example 4, and Comparative Example 4, the coexistence of metal chelators (HEDTA), Zn 2+ , Mg 2+ and a total amount of HEDTA in the magnetic particle antibody diluent ≥ 5 mM / L contribute to the accelerated stability and long-term stability of the reagent.

[0063] In summary, the stability of TSH reagents and AFP reagents and RF interference are all affected by the preparation of magnetic particle-antibody conjugates and the metal chelators in reagent diluents (magnetic particle antibody diluent, enzyme-labeled antibody diluent, auxiliary reagents), including the type, concentration, and addition method of metal chelators. Different enzyme-catalyzed chemiluminescence kits depend on the type of kit.

Claims

1. A method for improving the stability of immunoassay reagents and reducing the interference of rheumatoid factor, characterized in that, The method includes the following steps: (1) Activation of magnetic particles: Prepare an activation solution with EDC and NHS for the activation of magnetic particles, and control the temperature at 2-8°C during the preparation of the activation solution; (2) Conjugation of antibody-magnetic particles: Dilute the antibody and anti-interference protein with a buffer solution, then add the magnetic particles activated in step (1) thereto, mix well and incubate to obtain conjugated magnetic particles; (3) Termination of reaction: Wash the conjugated magnetic particles with a termination reaction solution, remove the supernatant, and then continue to add the termination reaction solution to terminate the reaction; (4) Blocking and washing: Block the conjugated magnetic particles after the termination reaction in step (3) with a blocking solution, wash them after blocking, and dilute and store the washed conjugated magnetic particles with a magnetic particle antibody diluent to prepare an antibody-magnetic particle conjugate; The magnetic particle antibody diluent includes one of 20-100 mM / L PBS, 10-200 mM / L NaCl, 0.1-8% BSA, casein, and γ-globulin, 0.05-2% Tween 20, one or two of disodium ethylenediaminetetraacetate and hydroxyethyl ethylenediaminetriacetic acid, and one or two of 0.05%-1% Proclin 300, Proclin 950, and sodium azide, with a pH of 3.0-7.

0.

2. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 1, characterized in that, The total concentration of disodium ethylenediaminetetraacetate and / or hydroxyethyl ethylenediaminetriacetic acid in the magnetic particle antibody diluent is ≥5 mM / L.

3. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 1, characterized in that The specific method for activating magnetic particles is as follows: Take magnetic particles, wash them with a buffer solution and then remove the supernatant; Resuspend the magnetic particles with the buffer solution again, then add the activation solution to the magnetic particles to obtain a mixed solution, mix the mixed solution well and carry out an activation reaction; After the incubation ends, remove the supernatant, wash the magnetic particles with a buffer solution, and add the buffer solution to the washed magnetic particles to prepare the activated magnetic particles.

4. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 3, characterized in that, The concentration of the activation solution is 10-100 mg / ml, the activation reaction time is 30 min; the buffer solution is 100 mM / L MES pH 5.0; The mass ratio of magnetic particles to EDC in the mixed solution is 1-20:1, and the mass ratio of magnetic particles to NHS is 1-20:

1.

5. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 4, characterized in that, The mass ratio of magnetic particles to EDC in the mixed solution is 10:1, and the mass ratio of magnetic particles to NHS is 10:

1.

6. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 1, characterized in that, In step (2), the mass ratio of the antibody to the anti-interference protein is 1:1-200, and the mass ratio of the antibody to the magnetic particles is 1:20-200; The anti-interference protein is at least one of protein A and protein G; The incubation time is 3-24 h.

7. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 6, wherein The mass ratio of the antibody to the anti-interference protein is 1:1, and the mass ratio of the antibody to the magnetic particles is 1:100; The anti-interference protein is protein A.

8. The method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to claim 1, characterized in that, The termination reaction solution includes 100 mM / L Tris pH 7.4, 150 mM / L NaCl, 0.05% Tween 20, 10-100 mM / L amino acid or small molecule peptide or two of them, and one or two of 0.1%-20% (v / v) alcohol compounds; The amino acid is glycine, lysine, alanine or arginine, the small molecule peptide is glycylglycine, carnosine, carnosine or tripeptide, and the alcohol compound is ethanolamine, diethanolamine, triethanolamine, cyclohexanol, isopropanol or benzyl alcohol; The termination reaction time is 0.5 to 2 h; Preferably, the amino acid is glycine; the glycine concentration is 50 mM / L, preferably the alcohol compound is ethanolamine; the ethanolamine is 10%.

9. A kit, the kit includes an antibody-magnetic particle conjugate, the antibody-magnetic particle conjugate is prepared by the method for improving the stability of an immunoassay reagent and reducing the interference of rheumatoid factor according to any one of claims 1 to 8, the kit further includes an enzyme-labeled antibody diluent, and the enzyme-labeled antibody diluent includes: 100 mM / L PBS, 150 mM / L NaCl, 0.5% BSA, 0.05 - 2% Tween 20, 0.1 mM / L zinc ion, 2 mM / L magnesium ion, 3% trehalose, 0.05% Proclin 300, 0.09% sodium azide, pH 6.

8.

10. The kit according to claim 9, wherein the kit further comprises an auxiliary reagent, and the components of the auxiliary reagent include: 100 mM / L PBS, 150 mM / L NaCl, 2% BSA, 0.05% Tween 20, 3% trehalose, 0.05% Proclin 300, 0.09% sodium azide, EDTA·2Na ≥ 2 mM / L, pH 7.2.

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