Preparation method of reagent for detecting β2-microglobulin

Abstract

The present invention relates to a method for preparing a reagent for detecting β2-microglobulin, comprising 1) diluting a first microsphere and a first antibody with a buffer to obtain a mixed solution of the first microsphere and the first antibody, adding a cross-linking agent for incubation, adding a blocking agent for blocking, and obtaining a labeled first microsphere; 2) diluting a second microsphere and a second antibody with a buffer to obtain a mixed solution of the second microsphere and the second antibody, adding a cross-linking agent for incubation, adding a blocking agent for blocking, and obtaining a labeled second microsphere; 3) mixing the labeled first microsphere with the labeled second microsphere; wherein the first microsphere and the second microsphere are carboxyl microspheres with different diameters; the first antibody is different from the second antibody; and the pH of the buffer is 6.3 to 9.5. The present invention also relates to a reagent prepared by the method and a kit containing the reagent.

Description

Technical Field

[0001] The invention belongs to the field of immunoassay, and in particular relates to a method for preparing a reagent for detecting beta 2-microglobulin. Background Art

[0002] β2-microglobulin is mainly used to evaluate renal function and as a non-specific tumor marker. Increased urine concentration and normal serum level is due to reduced tubular reabsorption, which is seen in renal tubular diseases such as Fanconi syndrome, Lowe syndrome, chronic chromium poisoning, acute tubular necrosis, cystinosis, kidney transplantation, acute or chronic pyelonephritis, while normal in cystitis; increased serum level and normal urine concentration is due to reduced glomerular filtration rate, which is seen in acute or chronic nephritis; increased serum and urine levels are seen in increased production that exceeds the tubular reabsorption capacity, which is seen in malignant tumors such as hepatocellular carcinoma, lung cancer, gastric and colon cancer, multiple myeloma, malignant lymphoma, and chronic lymphocytic leukemia.

[0003] β2-microglobulin is generally measured by RIA, ELISA, CLIA, immunoturbidimetry, etc. However, in clinical practice, diagnostic reagents prepared by immunological methods using latex as carrier microspheres can be used with automatic analysis instruments, and therefore have been widely used in clinical examinations in recent years.

[0004] At present, the main methods for connecting microspheres to the labeled substance are physical adsorption and chemical coupling. Among them, chemical coupling is to use an activated cross-linking agent to connect the labeled substance and the microspheres through a chemical reaction, and the stability after connection is better than physical adsorption (JL Ortega-Vinuesa et al., Journal of Colloid & Interface Science, 1995). The surface of the microspheres used for chemical coupling will be modified with some chemical groups, such as carboxyl, amino and hydroxyl (corresponding to carboxyl microspheres, amino microspheres and hydroxyl microspheres). Among these, carboxyl microspheres are more widely used.

[0005] The method of preparing carboxyl microspheres using an activator is well known. For example, CN106353507A discloses a method of coupling an antibody to a microsphere, in which EDC and sulfo-NHS are added to the microspheres for activation reaction for 15 minutes after the microspheres are resuspended in a buffer solution, and the reaction is centrifuged and washed and ultrasonically resuspended after the reaction; the dissolved antibody is then mixed with the microspheres for coupling reaction, and centrifugation and resuspending are required again after coupling. For another example, CN105606822A discloses a method of using antibody-sensitized polystyrene latex particles, specifically washing the latex three times with MES at pH 5.0 and ultrasonically dispersing it, adding EDAC for activation for 15 minutes, washing twice and ultrasonically dispersing it, and then adding antibodies in sequence to mix and seal to obtain sensitized particles.

[0006] However, the above method not only requires centrifugation to remove free activators and free unbound antibodies during the preparation process, but also requires ultrasonic resuspension of the microspheres to disperse them. Although these steps remove unnecessary components and solve the problem of easy agglomeration during the preparation of carboxyl microspheres, they make the operation process inefficient and time-consuming, which is not conducive to industrial preparation.

[0007] In response to this situation, a method that does not require purification has been proposed. For example, CN107167586A discloses the following method: first, latex is added to a buffer solution, and then EDC is added for activation for 15 minutes. Then, the pH is adjusted to 7.60, and then SLO is added for cross-linking reaction for 2 hours. Finally, two blocking agents are added to block to obtain the finished product. This method does not require centrifugation or ultrafiltration steps, thereby improving the preparation efficiency.

[0008] However, this method still requires the microspheres to be activated with a crosslinking agent for a period of time, and then the pH value needs to be adjusted before the crosslinking reaction can be carried out. This inevitably prolongs the preparation time and increases the complexity of the operation, making it inconvenient for application in industry.

[0009] Therefore, when detecting β2-microglobulin, there is still a need for a carboxyl microsphere labeling method that is simpler to operate and less time-consuming. Summary of the invention

[0010] To this end, the technical problem to be solved by the present invention is to provide a method for labeling carboxyl microspheres for detecting β2-microglobulin with simple operation. On the other hand, the problem of how to prevent microspheres from agglutinating during the labeling process is further solved. On the other hand, the problem of how to improve the labeling efficiency is also solved. On the other hand, the problem of improving the stability of the reagent after labeling is also solved.

[0011] To achieve the above objectives, the inventors first studied the general carboxyl microsphere labeling method, and unexpectedly found that by adjusting the pH of the microsphere dilution buffer and the antibody dilution buffer, the pH can be kept within the optimal range after the addition of the cross-linking agent (i.e., for the activation reaction), which ensures that the carboxyl microspheres have better stability after sensitization, thereby eliminating the need for pre-activation of the microspheres and the need for additional adjustment of the pH after the addition of the cross-linking agent. During the research process, the inventors also found that at least by reducing the step of pre-activation of the cross-linking agent (i.e., first contacting and mixing the microspheres with the substance to be labeled and then adding the cross-linking agent to cross-link the microspheres with the substance to be labeled), the sensitivity of the microspheres is reduced, making it less likely for aggregation to occur during the labeling process, thereby eliminating the need for centrifugation and / or dispersion steps during the labeling process.

[0012] That is to say, by adjusting the pH parameters and the labeling step at the same time, the inventors have established a universal carboxyl microsphere labeling method, which can be used to prepare labeled carboxyl microspheres and does not require purification and centrifugation during the preparation process, thereby simplifying the operation process and shortening the operation time. In addition, since large-scale centrifugation equipment is not required during production, the safety of production is also improved.

[0013] The labeled microspheres prepared by the above method can be used alone or in combination as detection reagents to detect specific substances. For example, based on the above improved labeling method, the inventors further prepared a reagent for detecting β2-microglobulin.

[0014] At present, there are reagents for measuring β2-microglobulin in blood and urine on the market. On the one hand, the concentration of β2-microglobulin in urine (reference range <300μg / L) is much lower than that in blood, which requires the reagent to have high low-end sensitivity; on the other hand, the linear range of most existing reagents on the market is between 0 and 18 mg / L, and a few reagents have a linear range of 0 to 50 mg / L. In clinical applications, there are certain limitations for measuring samples above 50 mg / L. Therefore, if you want to take into account both higher low-end sensitivity and wider linear range when detecting β2-microglobulin, you need two different labeled microspheres.

[0015] The inventors used the improved microsphere labeling method to prepare two labeled microspheres of different particle sizes, and mixed them to obtain a β2-microglobulin detection reagent, which has both low-end sensitivity and a wide linear range. In addition, the present invention further selects a microsphere particle size suitable for detecting β2-microglobulin. As confirmed below, the reagent prepared in this way can be used to detect β2-microglobulin.

[0016] Accordingly, the present invention provides a method for preparing a reagent for detecting β2-microglobulin, the method comprising the following steps:

[0017] 1) diluting the first microspheres and the first anti-β2-microglobulin antibody with a buffer to obtain a mixed solution of the first microspheres and the first anti-β2-microglobulin antibody, adding a cross-linking agent for incubation, and adding a blocking agent for blocking to obtain labeled first microspheres;

[0018] 2) Repeat step 1) using the second microsphere and the second anti-β2-microglobulin antibody to obtain a labeled second microsphere

[0019] 3) mixing the labeled first microspheres with the labeled second microspheres;

[0020] Wherein, the first microsphere and the second microsphere are carboxyl microspheres with different diameters; the first antibody is different from the second antibody; and the pH of the buffer is 6.3-9.5.

[0021] In some embodiments, the pH of the buffer in steps 1) and 2) is about 7.0 to 9.0.

[0022] In some embodiments, the dilution with buffer in steps 1) and 2) can be performed according to any conventional dilution method, for example, the microspheres and the anti-β2-microglobulin antibody can be separated and then the two can be mixed; or the microspheres can be first diluted with a buffer and then the anti-β2-microglobulin antibody can be added thereto. In some embodiments, the step of diluting the microspheres can also be included after the blocking step. Preferably, the microspheres can be diluted with a buffer commonly used in the art, including but not limited to Good's buffer, amino acid buffer, etc. Under the pH conditions involved in the method of the present invention, the inventors further found that the selection of the type of buffer and its ion buffer can help to further prevent the aggregation of the microspheres and improve the labeling efficiency.

[0023] Therefore, the method of the present invention also involves:

[0024] In one embodiment, the buffer in step 1) is a Good's buffer having an ionic strength of about 3-30 mM.

[0025] Preferably, the Good's buffer is one or more selected from HEPES, MPOS, MES, TES, TAPS and HEPPSO.

[0026] Preferably, the ionic strength of the Good's buffer is about 5 mmol / L to 25 mmol / L.

[0027] In another embodiment, the buffer in step 1) is an amino acid buffer with an ionic strength of about 10-500 mM.

[0028] Preferably, the amino acid buffer is a neutral aliphatic amino acid buffer and / or an alkaline amino acid buffer.

[0029] More preferably, the neutral aliphatic amino acid buffer is a glycine buffer and / or an alanine buffer.

[0030] Further preferably, the alkaline amino acid buffer is an arginine buffer or a lysine buffer.

[0031] In some embodiments, the cross-linking agent is EDC, DCC or DIC.

[0032] Preferably, the ionic strength of the amino acid buffer is about 15 mmol / L to 300 mmol / L.

[0033] In addition, for the method of the present invention, the inventors have also found that by adjusting the amount of cross-linking agent used, the number of activated microspheres can be maintained in a relatively appropriate range, thereby helping to avoid agglutination during the labeling process due to excessive microsphere activation and competition for the labeled substance.

[0034] Therefore, the method of the present invention also involves:

[0035] In one embodiment, the weight ratio of the crosslinking agent to the microspheres is about 0.5% to 4.0%.

[0036] Preferably, the weight ratio of the crosslinking agent to the microspheres is about 1.0% to 3.5%.

[0037] In one embodiment, the method of the present invention further comprises:

[0038] The step of adding a protective agent, preservative and / or surfactant after the blocking step.

[0039] In one embodiment, the method of the present invention does not comprise any purification step.

[0040] Preferably, the purification step is performed by ultrafiltration and / or centrifugation.

[0041] In one embodiment, the method of the present invention does not include any step for dispersing the microspheres.

[0042] Preferably, the step of dispersing the microspheres is performed using ultrasound.

[0043] In a specific embodiment, the diameter of the first microsphere is preferably 30 to 120 nm, more preferably 40 to 110 nm; the diameter of the second microsphere is preferably 140 to 400 nm, more preferably 160 to 300 nm.

[0044] In another aspect, the present invention also relates to a reagent for detecting β2-microglobulin, which is prepared by the method of the present invention.

[0045] In yet another aspect, the present invention also relates to a kit for detecting β2-microglobulin, the kit comprising the labeled microsphere solution of the present invention.

[0046] Therefore, the method of the present invention simplifies the operation process of the microsphere labeling method and reduces the operation time, improves the safety of the production process, and is more conducive to industrialization. Further, the method of the present invention also prevents the agglutination of the microspheres during the labeling process. Further, the method of the present invention also improves the labeling efficiency of the microspheres. Further, the method of the present invention improves the stability of the microsphere reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a picture of the appearance of the microspheres during the labeling process;

[0048] Figure 2 This is a picture of the appearance of the microspheres during the labeling process;

[0049] FIG3 shows a dose-response curve of the reagents prepared in Examples 1 to 2 during detection;

[0050] Figure 4 The metering response curve of the β2-microglobulin detection reagent prepared in Example 4 when testing β2-microglobulin in a sample is shown. DETAILED DESCRIPTION

[0051] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0052] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0053] Microspheres

[0054] In the present invention, "microspheres" refer to a collection of tiny entities that can be used as carriers of substances to be labeled for immunological detection, and their diameters are generally less than 5 microns.

[0055] As used in the present invention, "carboxyl microspheres" refer to microspheres with carboxyl groups modified on the surface for covalent binding with the amino termini of the labeled substance. There is a bridging chemical arm between the carboxyl microspheres and the antibody, which reduces the steric effect, not only improves the binding rate of the antibody, but also provides a suitable three-dimensional spatial structure for the antibody, effectively protecting the active area for the antibody to bind to the antigen. Many companies abroad have provided microsphere particle raw materials with carboxyl groups modified on the surface, for example, carboxyl microspheres produced by Sichuan New Materials Company, carboxyl microspheres produced by Bangs Laboratories, etc.

[0056] In the present invention, any suitable type of carboxyl microspheres can be used as long as they can be coupled to the amino-containing labeled substance. Common microspheres can be, for example: latex (e.g., polystyrene microspheres, chitosan microspheres, polylactic acid microspheres, polyacrylic acid microspheres and / or starch microspheres, etc.), fluorescent microspheres, magnetic beads. However, the present invention is not limited thereto.

[0057] The present invention has no particular limitation on the diameter of the carboxyl microspheres. For example, diameters in the range of 20 nm to 5000 nm can be used in the labeling method of the present invention.

[0058] In some embodiments, the microspheres of the present invention include a "first microsphere" and a "second microsphere".

[0059] As used in the present invention, "first microsphere" refers to a microsphere with a diameter of about 30 to 120 nm; the second microsphere refers to a microsphere with a diameter of about 140 to 400 nm, wherein the diameters of the first microsphere and the second microsphere are different.

[0060] Preferably, the diameter of the first microsphere may be about 40 to 110 nm, for example, about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm; preferably, the diameter of the second microsphere may be about 160 to 300 nm, for example, about 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm or 290 nm, etc.

[0061] Objects to be marked

[0062] As used in the present invention, "substance to be labeled" refers to a substance that can be coupled to carboxyl microspheres and can be used to specifically bind to a substance to be detected in a sample.

[0063] Substances that specifically bind to an analyte are well known in the art. For example, in a method for diagnosing hepatitis B, a hepatitis B surface antibody can be used as an analyte. In this case, a hepatitis B surface antigen can be used as a marker for the microspheres of the present invention. Similarly, among these well-known markers, those skilled in the art know which markers can be coupled to carboxyl microspheres.

[0064] The present invention has no particular limitation on the type of the substance to be labeled, as long as it can be coupled to the carboxyl microspheres. For example, the substance to be labeled can be an antibody or an antigen. For another example, the substance to be labeled can be a polypeptide or a fluorescent compound.

[0065] When the substance to be labeled is an antibody, it can be selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or an antibody fragment (preferably a fragment that binds to an antigen) and the like.

[0066] In some embodiments, the antibody can be derived from rabbit, mouse, goat, sheep, chicken and / or human.

[0067] When the substance to be labeled is an antigen, it can be selected from an antigen or a fragment thereof (preferably an antigen epitope) and the like.

[0068] The object to be detected of the present invention can be obtained by purification, expression and / or artificial synthesis.

[0069] Exemplary markers include: anti-serum amyloid A antibody, retinol binding protein antibody, β2 microglobulin polyclonal antibody, anti-human cystatin C polyclonal antibody, rabbit anti-human lipoprotein a antibody, anti-myoglobin polyclonal antibody or anti-human D-dimer monoclonal antibody, etc. However, the present invention is not limited thereto.

[0070] In some embodiments, the substance to be labeled may be modified.

[0071] In a specific embodiment, a primary antibody and a secondary antibody are used.

[0072] In the present invention, "first antibody" and "first anti-β2-microglobulin antibody" have the same meaning; similarly, "second antibody" and "second anti-β2-microglobulin antibody" have the same meaning.

[0073] In the present invention, the first antibody and the second antibody are different anti-β2-microglobulin antibodies.

[0074] As used in the present invention, "different anti-β2-microglobulin antibodies" refers to a combination of one or more of the following: antibodies from different species, antibodies are polyclonal antibodies and monoclonal antibodies, and antibodies are specific for different epitopes of the same antigen. Exemplary "different anti-β2-microglobulin antibodies" include: rabbit anti-β2-microglobulin antibody 1 and rabbit anti-β2-microglobulin antibody 2 (both for different epitopes of β2-microglobulin), mouse anti-β2-microglobulin antibody 1 and mouse anti-β2-microglobulin antibody 2 (both for different epitopes of β2-microglobulin), mouse anti-β2-microglobulin antibody and rabbit anti-β2-microglobulin antibody, mouse anti-β2-microglobulin monoclonal antibody and rabbit anti-β2-microglobulin polyclonal antibody, but the present invention is not limited thereto.

[0075] Buffer

[0076] The method of the present invention comprises a microsphere dilution buffer and an antibody dilution buffer.

[0077] The microsphere dilution buffer and the antibody dilution buffer can be selected from Good's buffer or amino acid buffer.

[0078] The pH of the buffer of the present invention may be 6.3 to 9.5, for example 6.3, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4 or 9.5.

[0079] The inventors further found that in the method of the present invention, Good's buffer with low ionic strength can further reduce the risk of latex agglutination.

[0080] Therefore, in a preferred embodiment, the microsphere dilution buffer and the antibody dilution buffer of the present invention are Good's buffers having an ionic strength of about 3 to 30 mM.

[0081] Good's buffer is also called zwitterionic buffer. They have common characteristics when used as buffer systems: 1) pKa is between 6-8; 2) high solubility in water; 3) not easy to penetrate biological membranes; 4) small salt effect; 5) ion concentration, solution composition and temperature have little effect on dissociation; 6) do not form complexes or precipitation with metal ions; 7) the buffer is chemically stable; 8) low light absorption in the ultraviolet and visible wavelength range; 9) easy to prepare high-purity salt. Commonly used Good's buffers include: MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, HEPES, TES, DIPSO, TAPSO, TRIZAMA, HEPPSO, POPSO, EPPS, TEA, Tricine, Bicine, TAPS, AMPSO, CHES, CAPSO or AMP, etc.

[0082] In a more preferred embodiment, the Good's buffer of the present invention may be one or more of MOPS, HPEPS, MES, TAPS and HEPPSO.

[0083] When the microsphere dilution buffer and the antibody dilution buffer are selected from Good's buffer, the ionic strength thereof can be 3 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM or 30 mM.

[0084] In addition, the inventors unexpectedly discovered that the use of an amino acid buffer in the method of the present invention can further reduce the risk of latex agglutination, and when the amino acid buffer is used, its ionic strength can be applied in a wider range. We speculate that this is because the amino acid buffer also participates in cross-linking to a certain extent, slowing down the cross-linking speed, thereby making it less likely for agglutination to occur during the labeling process.

[0085] Therefore, in another preferred embodiment, the microsphere dilution buffer and the antibody dilution buffer are amino acid buffers with an ionic strength of about 10 to 500 mM. Commonly used amino acid buffers include glycine buffer, alanine buffer, valine buffer, leucine buffer, isoleucine buffer, histidine buffer, arginine buffer or lysine buffer.

[0086] In a more preferred embodiment, the amino acid buffer of the present invention is a neutral aliphatic amino acid buffer, for example, a glycine buffer, an alanine buffer, a valine buffer, a leucine buffer or an isoleucine buffer.

[0087] In another more preferred embodiment, the amino acid buffer of the present invention is an alkaline amino acid buffer, such as arginine buffer, histidine buffer or lysine buffer.

[0088] When the microsphere dilution buffer and the antibody dilution buffer are selected from amino acid buffers, their ionic strength can be 10mM, 15mM, 40mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM or 500mM.

[0089] In the method of the present invention, the microsphere dilution buffer and the antibody dilution buffer may be the same or different. In a preferred embodiment, the microsphere dilution buffer and the antibody dilution buffer are the same.

[0090] Sealing agent

[0091] As used herein, "blocking agent" refers to an agent that can block unbound carboxyl groups on microspheres.

[0092] Those skilled in the art will appreciate that any reagent capable of blocking a carboxyl group can be used as a blocking agent of the present invention. Exemplary blocking agents can be selected from protein blocking agents, such as BSA, casein, skim milk powder or amino-containing substances, such as TRIS, amino acid blocking agents (such as alanine, arginine, lysine, etc.) One or more.

[0093] Regarding the dosage of the blocking agent, those skilled in the art can select it according to its type. For example, when BSA is used, a concentration of 1 to 5 g / L can be used.

[0094] Protective agent

[0095] In the present invention, the protective agent refers to an agent used to protect the activity of the label and improve the stability of the labeled microparticles.

[0096] The present invention has no particular limitation on the type of protective agent, and any common protective agent in the field of diagnostic reagents can be used. Exemplary protective agents can be selected from carbohydrate protective agents, such as sucrose, trehalose, dextran, glucose, and fructose; protein protective agents, such as BSA, casein, and skim milk powder; and alcohol protective agents, such as mannitol. For the dosage of the protective agent, those skilled in the art can select it according to the type of protective agent, for example, when using trehalose, a concentration of 5 to 100 g / L can be used.

[0097] preservative

[0098] In the present invention, a preservative refers to an agent used to prolong the shelf life of labeled microparticles.

[0099] The present invention has no particular limitation on the type of preservative, and any common preservative commonly used in the field of diagnostic reagents can be used. Exemplary preservatives can be selected from one or more of sodium azide, phenol, p-hydroxybenzoic acid, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium propionate, xylenol, parabens, permanganate, antibiotics (such as gentamicin, dichloroacetamide, ProClin series launched by SUPELCO, etc.), ethyl p-hydroxybenzoate and ethylmercuric sodium thiosulfate.

[0100] Regarding the dosage of the preservative, those skilled in the art can select it according to the type of the preservative. For example, when using ProClin300, a concentration of 0.1 to 10 g / L can be used.

[0101] Surfactants

[0102] In the present invention, surfactant refers to a substance that can significantly change the interface state of its solution system when added in a small amount; it has fixed hydrophilic and lipophilic groups and can be arranged in a directional manner on the surface of the solution.

[0103] For the type of surfactant, the present invention has no particular limitation, and any common surfactant commonly used in the field of diagnostic reagents can be used. Exemplary surfactants can be selected from one or more of Tween, SPAN, Triton, EMULGEN series surfactants (such as Tween 20, Tween 40 and Triton 100) and lauric acid macrogol glyceride, anionic alkyl polyglycosides, dodecyl dimethyl betaine, 3ARAMT1, SHO62C and AM PHITOL.

[0104] Regarding the dosage of the surfactant, those skilled in the art can select it according to the type of the surfactant. For example, when using Triton 100, a concentration of 1 to 20 g / L can be used.

[0105] Preparation method

[0106] The present invention provides a method for detecting a microsphere reagent of β2-microglobulin, the method comprising the following steps:

[0107] 1) diluting the first microspheres and the first antibody using a buffer;

[0108] 2) obtaining a mixed solution of the microspheres and the substance to be labeled;

[0109] 3) adding a cross-linking agent for incubation;

[0110] 4) Add sealing agent to seal;

[0111] 5) obtaining a labeled first microsphere;

[0112] 6) using the second microsphere and the second antibody to replace the first question and the first antibody respectively, repeating the above steps 1) to 5) to obtain a labeled second microsphere;

[0113] 7) mixing the labeled first microspheres with the labeled second microspheres;

[0114] Wherein, the first microsphere and the second microsphere are carboxyl microspheres with different diameters; the pH of the buffer in step 1) is 6.3-9.5.

[0115] In the method of the present invention, diluting the microspheres and antibodies with a buffer may be performed in the following manner:

[0116] Dilute the microspheres and the substance to be labeled using buffer A and buffer B respectively to obtain a microsphere solution and a substance to be labeled solution, wherein buffer A and buffer B may be the same or different; or

[0117] The microspheres are first diluted with a buffer to obtain a microsphere solution, and then the substance to be labeled is directly added to the microsphere solution.

[0118] When the microspheres and the substance to be labeled are diluted separately, the concentration of the microsphere solution can be, for example, 0.01-100 mg / ml, or 0.1-50 mg / ml; and the concentration of the substance to be labeled solution can be, for example, 0.1-100 mg / ml, or 0.1-30 mg / ml.

[0119] In some embodiments, the substance to be labeled can be used in a weight ratio of 0.1% to 30% relative to the microspheres, and the weight can also be 0.5% to 25%, or even 1% to 15%.

[0120] As described in the method of the present invention, the method does not include any step of activating the microspheres using an activating agent before step 4).

[0121] Any cross-linking agent for carboxyl microspheres in the art can be used in the method of the present invention. Exemplary cross-linking agents include, but are not limited to, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DCC (dicyclohexylcarbodiimide) and DIC (N,N'-diisopropylcarbodiimide), which are usually in the form of dry powders. In some preferred embodiments, the cross-linking agent is preferably used in a weight ratio of 0.5 to 4% relative to the microspheres, as demonstrated in the examples below, when the method of the present invention uses the cross-linking agent, it will further help to obtain carboxyl microspheres with good labeling effects. In addition, the crosslinking agent is more preferably used in a weight ratio of 1.0% to 3.5% relative to the microspheres, and the weight ratio can be 1.0%, 1.5%, 2%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9% or 4.0%.

[0122] As shown in the following examples, no purification or dispersion step is required in the method of the present invention. The purification step may include, for example, a purification step for removing free activators after activating the microspheres, and a purification step for removing free substances to be labeled after the labeling step; and the dispersion step may include a step for resuspending the microspheres after centrifugation or agglutination. Common purification steps include centrifugation (low temperature) or ultrafiltration, and common dispersion steps include ultrasound or oscillation. These methods will inevitably prolong the reaction time and increase the requirements for the reaction environment and equipment. The method of the present invention overcomes the above-mentioned defects.

[0123] The incubation process of step 3) in the method of the present invention actually synchronizes the activation of the carboxyl microspheres with the labeling (coupling) of the microspheres. During the incubation process, the incubation time can be greater than or equal to 10 minutes, for example, 10 minutes, 20 minutes, 1 hour, 2 hours, 4 hours, etc.; the incubation temperature can be 2 to 80° C., for example, room temperature (25° C.).

[0124] In step 4) of the method of the present invention, blocking can be performed, for example, for at least 10 minutes, for example, 10 minutes, 20 minutes, half an hour, 1 hour, and the like.

[0125] In some embodiments, the method of the present invention includes a step of adding one or more of a protective agent, a preservative, and a surfactant between the blocking step and the step of obtaining the labeled microparticles. When multiple agents are added, they can be added simultaneously, sequentially, or in combination thereof.

[0126] In an exemplary embodiment, the mixing ratio of the labeled first microspheres and the labeled second microspheres of the present invention is 1:1 to 20:1, or 1.5:1 to 15:1, or 5:1.

[0127] Reagent test kit

[0128] The labeled microspheres produced by the method of the present invention can be used to prepare an immunoturbidimetric kit for detecting β2-microglobulin.

[0129] For example, in one embodiment, the reagent produced by the method of the present invention can be used as a detection reagent (R2) in an immunoturbidimetric kit. In this case, the immunoturbidimetric kit may also include a reagent (R1) for incubating samples, a calibrator / calibrator for preparing a working curve, and a quality control product / quality control material for quality control.

[0130] Analytical methods

[0131] Instruments and Materials

[0132] Prepare R1 reagent for incubating samples

[0133]

[0134] The pH was adjusted to 7.4 ± 0.1.

[0135] R2 reagent for detection: prepared by the method of the present invention, see Examples 1, 2, and 4 for details.

[0136] Instrument: Hitachi 7180 fully automatic biochemical analyzer.

[0137] Cystatin C instrument parameter settings:

[0138] Methods: 2-point endpoint method;

[0139] Sample / reagent 1 / reagent 2 were 2 μl / 155 μl / 25 μl respectively;

[0140] Main wavelength: 546nm;

[0141] Sub-wavelength: 700nm;

[0142] Reaction time: 10min;

[0143] Metering points: 19 to 34 points;

[0144] Reaction temperature: 37°C.

[0145] β2-microglobulin instrument parameter settings:

[0146] Methods: 2-point endpoint method;

[0147] Sample / reagent 1 / reagent 2 were 2 μl / 180 μl / 80 μl respectively;

[0148] Main wavelength: 570nm;

[0149] Sub-wavelength: No

[0150] Reaction time: 10min;

[0151] Metering points: 19 to 34 points;

[0152] Reaction temperature: 37°C.

[0153] Calibrators (Cystatin C): Standard 1, 0.00 mg / L; Standard 2, 0.50 mg / L; Standard 3, 1.00 mg / L; Standard 4, 2.00 mg / L; Standard 5, 4.00 mg / L; Standard 6, 8.00 mg / L.

[0154] Calibrator (β2-microglobulin): Calibrator 1, 0 mg / L; Standard 2, 1.5 mg / L; Standard 3, 10 mg / L; Standard 4, 50 mg / L; Standard 5, 80 mg / L.

[0155] Quality control product (cystatin C): Quality control product level 1, labeled value 0.86 (0.68 ~ 1.04) mg / L; Quality control product level 2, labeled value 3.8 (3.04 ~ 4.56) mg / L.

[0156] Quality control product (β2-microglobulin): Quality control product level 1, labeled value 1.32 (1.05~1.58) mg / L; Quality control product level 2, labeled value 2.33 (1.86~2.80) mg / L.

[0157] High-value cystatin C sample: human serum sample, cystatin C concentration is about 36 mg / L.

[0158] β2-microglobulin high value sample: recombinant β2-microglobulin, β2-microglobulin concentration is about 1500mg / L.

[0159] Calibration curve signal determination

[0160] The prepared reagents were calibrated and the quality control products were measured and analyzed using a fully automatic biochemical analyzer. For specific results, please see Examples 1, 2, and 5 below.

[0161] Analytical sensitivity determination

[0162] The determination was repeated twice using CysC calibrator 6 and calibrator 5, and the average of the absorbance difference was calculated and converted to ΔA of 1.00 mg / L. The calculation formula is as follows:

[0163]

[0164] Where:

[0165] represents the average value of Cal-6 absorbance difference;

[0166] represents the average value of Cal-5 absorbance difference;

[0167] C6 indicates the labeled concentration of Cal-6;

[0168] C5 represents the labeled concentration of Cal-5.

[0169] According to the conversion results, if the ΔA of 1.00 mg / L is higher, it means that the analytical sensitivity is higher, and the reagent has a better reaction gradient when measuring high-end concentration calibrants. It also indirectly indicates that the labeling effect of the reagent is better. Please see the following Examples 1 and 2 for the results.

[0170] Intra-assay CV determination

[0171] The quality control product level 1 and quality control product level 2 were used to repeat the measurement 10 times respectively, and the intra-assay coefficient of variation (CV) was calculated according to the following formula:

[0172]

[0173] Where:

[0174] CV means coefficient of variation;

[0175] S represents standard deviation;

[0176] Indicates the measured mean.

[0177] The difference in reagent repeatability is judged based on the CV value. The smaller the CV, the better the reagent repeatability, and vice versa.

[0178] Equivalence band determination

[0179] CysC band effect:

[0180] The high-value sample of cystatin C was diluted with physiological saline to prepare a series of samples containing different concentrations of antigen (theoretical concentrations were 4.00 mg / L, 8.00 mg / L, 16.00 mg / L, 20.00 mg / L, 25.00 mg / L, 30.00 mg / L, 32.00 mg / L, 34.00 mg / L, and 36.00 mg / L); then the concentrations were measured from low concentration to high concentration, and the trend of the measurement results was first increased and then decreased. When the measured concentration was lower than the upper limit of the linear range (8.00 mg / L) for the second time, the experiment could be terminated, and then the dose-response curve was drawn with the theoretical concentration of the sample as the abscissa and the measured concentration as the ordinate. When the measured concentration was again equivalent to the upper limit of the linear range (8.00 mg / L), the concentration range of this interval was the equivalent band of the reagent. Please see the following Example 3, Table 14 and Figure 3 for the experimental results.

[0181] β2-Microglobulin Band Effect:

[0182] The β2-microglobulin antigen was diluted with physiological saline to prepare a series of samples containing different concentrations of antigen (theoretical concentrations are 80.00 mg / L, 120.00 mg / L, 160.00 mg / L, 200.00 mg / L, 240.00 mg / L, and 320.00 mg / L); then the concentrations were measured from low to high concentrations, and the trend of the measurement results was first increased and then decreased. When the measured concentration was lower than the upper limit of the linear range (80.00 mg / L) for the second time, the experiment was terminated, and then the dose-response curve was drawn with the theoretical concentration of the sample as the horizontal axis and the measured concentration as the vertical axis. When the measured concentration was again equivalent to the upper limit of the linear range (80.00 mg / L), the concentration range of this interval was the equivalent band of the reagent. For experimental results, please see the following Examples 5 and Figure 4 .

[0183] Long-term stability test at 2-8°C

[0184] Appearance observation: Mix the prepared reagent and take out about 3 ml into a test tube, seal and store at 2-8°C. Take out the reagent at 0 day, 6 months, and 12 months (or 24 months) and observe it under light with the naked eye. If it is a milky white suspension without precipitation, the appearance of the reagent is normal, and the stability of the reagent can be further determined by the test results of quality control products. On the contrary, if there is precipitation in the reagent, it means that the stability of the reagent is poor, and the stability of the reagent is acceptable can be further determined by the test results of quality control products.

[0185] Quality control product determination method: After using the series of calibrators to calibrate the reagents, the quality control product level 1 and level 2 (three replicates in each group) were measured at 0 days, 6 months, and 12 months, and the average values ​​were calculated respectively. The relative deviation (%) was then calculated using the following formula:

[0186] (The average value of the quality control level after storage for a certain period of time - the average value of the quality control level at 0 days) / the average value of the quality control level at 0 days × 100%.

[0187] Reagent stability is generally considered to be poor and unacceptable when the relative deviation differs by more than ±10% from the initial determination.

[0188] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples.

[0189] Example 1 Establishment of a microsphere labeling method based on Good's buffer

[0190] Conventional method A

[0191] A commonly used carboxyl microsphere labeling method is as follows:

[0192] 1) Anti-human cystatin C polyclonal antibody (Sichuan MacBio New Materials Technology Co., Ltd.) was dissolved in 50 mM acetic acid, pH 6.0, to an antibody concentration of 1 mg / mL;

[0193] 2) suspend the microspheres in 50 mM MES (pH 6.0) to a concentration of 1% w / v;

[0194] 3) Add one volume of antibody solution to 10 volumes of carboxyl microsphere (98 nm latex, Sichuan MyBio New Materials Technology Co., Ltd.) suspension while stirring, and continue stirring at room temperature for 20 minutes;

[0195] 4) adding EDC solution so that the weight ratio of EDC to microspheres is 5%;

[0196] 5) Immediately adjust the pH to 7.4 at room temperature, mix and react for 30 minutes;

[0197] 6) Remove the supernatant by centrifugation and resuspend in storage buffer (2 g / L Tris, 10 g / L sucrose and 5 g / L sorbic acid) by ultrasonication.

[0198] As can be seen from the above content, this method requires a step of adjusting the pH value after adding the cross-linking agent; at the same time, in order to prevent the microspheres from aggregating, the method requires centrifugation after cross-linking to remove the free activator and free unbound antibodies in the supernatant, and also requires an ultrasonic resuspension step for the microspheres to disperse them. Therefore, there are problems such as a complicated and time-consuming operation process, the need for large-scale equipment, and being unfavorable for industrialization.

[0199] Based on this, the present invention conducted the following experiments.

[0200] Experiment 1

[0201] method:

[0202] The carboxyl microspheres and anti-human cystatin C polyclonal antibody were diluted to 20 mg / ml and 30 mg / ml respectively using 30 mM HEPES buffer at pH 6.3; the diluted antibody solution and the diluted microsphere solution were mixed to make the weight ratio of the antibody to the microspheres 14-16%.

[0203] The mixed solution was added with EDC stock solution to make the weight ratio of EDC to microspheres 5%, and labeled at 25°C for 30 minutes.

[0204] TRIS (final concentration 2 g / L) was then added for blocking for 30 minutes.

[0205] Sucrose (final concentration 10 g / L) and sorbic acid (5 g / L) were added, and the solution was allowed to stand for 30 minutes to obtain antibody-labeled microspheres.

[0206] Experiments 2 to 6 and Comparative Example A1

[0207] In comparison with Experiment 1, the operating parameters of Experiments 2 to 6 and Comparative Example A1 are given in Table 1 below, wherein the parameters not given are the same as those of Experiment 1.

[0208] Table 1

[0209] Buffer pH Buffer Type Ionic strength mM Experiment 1 6.3 HEPES 30 Experiment 2 7.0 MES 5 Experiment 3 8.5 MOPS 20 Experiment 4 7.8 HEPES 10 Experiment 5 9.0 MES 25 Experiment 6 9.5 MOPS 3 Comparative Example A1 6.0 HEPES 20

[0210] result:

[0211] In order to verify the dispersibility, reactivity and labeling efficiency of the reagents prepared in Experiments 1 to 6, Comparative Example A1 and Conventional Method A mentioned above, the appearance of the reagents during labeling was observed. The results are as follows: Figure 1As shown in the figure, the experimental group of Experiment 4 remained clear during the labeling process, while both Conventional Method A and Comparative Example A1 showed severe agglutination during the labeling process. Further, according to the description in the above "Analysis Method" section, the calibration curve signal and analysis sensitivity were measured. The results are shown in Table 2 below.

[0212] Table 2

[0213]

[0214] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0215] As shown in Table 2, first, when the method of the present invention is used for labeling, experiments 1 to 6 using Good's buffer at pH 6.3 to 9.5 remain clear during the labeling process; conventional method A shows agglutination during the preparation process, and centrifugation and ultrasonic resuspension operations are performed in the subsequent experimental process; and comparative example A1 with a buffer pH of 6.0 also shows severe agglutination during the labeling process, and centrifugation and ultrasonic resuspension have to be performed in the subsequent experimental process. Secondly, it can be seen from the calibration curve signal results that the blank absorbance at the reaction end point of experiments 1 to 6 is much smaller than that of conventional method A and comparative example A1, indicating that the reagents obtained in experiments 1 to 6 have better dispersibility; in addition, at the calibration points of 0.50, 1.00, 2.00, 4.00 and 8.00, the absorbance of experiments 1 to 6 is significantly higher than that of conventional method A and comparative example A1, which proves that the reaction of experiments 1 to 6 is more intense and the labeling effect is better. Finally, from the analytical sensitivity results, it can be seen that the analytical sensitivity of Experiments 1 to 6 is significantly higher than that of Conventional Method A and Comparative Example A1, which means that the reaction reagents prepared in Experiments 1 to 6 have better labeling efficiency.

[0216] Furthermore, by comparing the calibration curves of experiments 1 to 6 and the results of analytical sensitivity experiments, it can be seen that: compared with the buffer solutions of pH 6.3 and pH 9.5, the reagents prepared at pH 7.0 to 9.0 have better dispersibility and labeling efficiency; and at pH 7.8 to 8.5, the dispersibility and labeling efficiency of the reagents are optimal.

[0217] In addition, by comparing the results of experiments 1 to 6, it can be seen that: compared with Good's buffer with an ionic strength of 3mM or 30mM, the reagent prepared with an ionic strength of 5mM to 25mM has better dispersibility and labeling efficiency; and at an ionic strength of 10mM to 20mM, the dispersibility and labeling efficiency of the reagent are optimal.

[0218] Comparative Example A2

[0219] method:

[0220] In order to verify that the method of the present invention can achieve better results when using 3-30 mM Good's buffer, Comparative Example A2 was conducted. The ionic strength of the buffer used in this comparative example was 50 mM, and the other experimental conditions were the same as those of Experiment 3.

[0221] result:

[0222] In order to verify the dispersibility, reactivity and labeling effect of the reagent prepared in Comparative Example A2, the appearance of the reagent during labeling was observed. Figure 1 It can be seen that the microspheres in Comparative Example A2 were severely agglutinated during the labeling process; at the same time, according to the description in the above "Analysis Method" section, the calibration curve signal and analysis sensitivity were measured. The results are shown in Table 3 below.

[0223] Table 3

[0224]

[0225] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0226] As shown in Table 3, when labeling is performed using the method of the present invention, using a Good's buffer with an ionic strength of 3 mM to 30 mM can achieve better dispersibility, reactivity and labeling effect than using a Good's buffer with an ionic strength of 50 mM.

[0227] Experiments 7-10

[0228] method:

[0229] Experiment 3 among Experiments 1 to 6 was selected, and Experiments 7 to 10 were carried out according to this method. The changed parameters are shown in Table 4 below, and the other experimental conditions are the same as those of Experiment 3.

[0230] Table 4

[0231] Weight ratio of EDC to microspheres Experiment 3 5.0％ Experiment 7 4.0％ Experiment 8 3.0％ Experiment 9 1.0％ Experiment 10 0.5％

[0232] result:

[0233] In order to verify the dispersibility, reactivity and labeling effect of the reagents prepared in Experiments 7 to 10, the calibration curve signal and analytical sensitivity were measured according to the description in the above "Analysis Method" section. The results are shown in Table 5 below.

[0234] Table 5

[0235]

[0236] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0237] As shown in Table 5, by adjusting the weight ratio of EDC to microspheres from 5.0% to 0.5% to 4.0% based on the method and parameters of Experiment 3, the dispersibility, reactivity and labeling effect of the prepared reagent can be further optimized. Among them, the best effect is achieved when the weight ratio of EDC to microspheres is about 3.0%.

[0238] Experiments 11-13

[0239] method:

[0240] Experiment 8, which had the best effect among experiments 7 to 10, was selected, and experiments 11 to 13 were continued according to the method. The changed parameters were shown in Table 6 below, and the other experimental conditions were the same as those of experiment 8.

[0241] Table 6

[0242] Buffer Type Microsphere particle size nm Experiment 8 MOPS 98 Experiment 11 MOPS 300 Experiment 12 TAPS 98 Experiment 13 HEPPSO 98

[0243] result:

[0244] In order to verify the dispersibility, reactivity and labeling effect of the reagents prepared in Experiments 11 to 13, the calibration curve signal and analytical sensitivity were measured according to the description in the above "Analysis Method" section. The results are shown in Table 7 below.

[0245] Table 7

[0246]

[0247] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0248] It can be seen from Table 7 that after adjusting the particle size of the microspheres or replacing the buffer with other Good's buffers, there is no significant effect on the dispersibility, reactivity and labeling effect of the reagent prepared by the method of the present invention.

[0249] Example 2 Establishment of a microsphere labeling method based on amino acid buffer

[0250] Conventional method B

[0251] A commonly used carboxyl microsphere labeling method is as follows:

[0252] 1) Anti-human cystatin C polyclonal antibody (Sichuan MacBio New Materials Technology Co., Ltd.) was dissolved in 500 mM pH 6.0 glycine buffer to an antibody concentration of 1 mg / mL;

[0253] 2) Microspheres (126 nm latex, Sichuan MacBio New Materials Technology Co., Ltd.) were suspended in 500 mM glycine buffer at pH 6.0 to a concentration of 1% w / v;

[0254] 3) Add one volume of antibody solution to 10 volumes of microsphere suspension while stirring, and continue stirring at room temperature for 20 minutes;

[0255] 4) adding EDC solution so that the weight ratio of EDC to microspheres is 5%;

[0256] 5) Immediately adjust the pH to 7.4 at room temperature, mix and react for 30 minutes;

[0257] 6) Remove the supernatant by centrifugation and resuspend in storage buffer (2 g / L Tris, 10 g / L sucrose and 5 g / L sorbic acid) by ultrasonication.

[0258] Experiment 21

[0259] method:

[0260] Carboxyl microspheres (126 nm latex, Sichuan Mike Biotech New Material Technology Co., Ltd.) and anti-human cystatin C polyclonal antibodies were diluted to 15 mg / ml and 20 mg / ml respectively using 500 mM, pH 6.3 glycine buffer; the diluted antibody solution and the diluted microsphere solution were mixed so that the weight ratio of the antibody to the microspheres was 14-16%.

[0261] The mixed solution was added with EDC stock solution to make the weight ratio of EDC to microspheres 5%, and labeled at 25°C for 20 minutes.

[0262] Then, casein (final concentration 5 g / L) was added for blocking for 20 minutes.

[0263] Trehalose (final concentration 5 g / L) and phenol (1 g / L) were added, and the solution was allowed to stand for 20 minutes to obtain antibody-labeled microspheres.

[0264] Experiments 22 to 26 and Comparative Example B

[0265] In comparison with Experiment 21, the operating parameters of Experiments 22 to 26 and Comparative Example B are given in Table 8 below, wherein the parameters not given are the same as those of Experiment 21.

[0266] Table 8

[0267]

[0268]

[0269] result:

[0270] In order to verify the dispersibility, reactivity and labeling efficiency of the reagents prepared in Experiments 21 to 26 and Comparative Example B, the appearance of the reagents during labeling was observed. The results are as follows: Figure 2As shown in the figure, the experimental group of Experiment 24 remained clear during the labeling process, while Comparative Example B showed severe agglutination during the labeling process. At the same time, according to the description in the above "Analysis Method" section, the calibration curve signal and analysis sensitivity were measured. The results are shown in Table 9 below.

[0271] Table 9

[0272]

[0273] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0274] As shown in Table 9, first, when the method of the present invention is used for labeling, experiments 21 to 26 using amino acid buffers of pH 6.3 to 9.5 remain clear during the labeling process; conventional method B shows agglutination during the preparation process, and centrifugation and ultrasonic resuspension operations are performed during the subsequent experiments; comparative example B with a buffer pH of 6.0 also shows severe agglutination during the labeling process, and centrifugation and ultrasonic resuspension are also performed during the subsequent experiments. Secondly, it can be seen from the calibration curve signal results that the blank absorbance at the reaction end point of experiments 21 to 26 is much smaller than that of conventional method B and comparative example B, indicating that the reagents obtained in experiments 21 to 26 have better dispersibility; and at the calibration points of 0.50, 1.00, 2.00, 4.00 and 8.00, the absorbance of experiments 21 to 26 is significantly higher than that of conventional method B and comparative example B, which proves that the reaction of experiments 21 to 26 is more intense and the labeling effect is better. Finally, from the analytical sensitivity results, it can be seen that the analytical sensitivity of Experiments 21 to 26 is significantly higher than that of Conventional Method B and Comparative Example B, which indicates that the reaction reagents prepared in Experiments 21 to 26 have better labeling efficiency.

[0275] Furthermore, by comparing the calibration curves of Experiments 21 to 26 and the analytical sensitivity experimental results, it can be seen that: compared with the buffer solutions of pH 6.3 and pH 9.5, the reagents prepared at pH 7.0 to 9.0 have better dispersibility and labeling efficiency; and at pH 7.8 to 8.5, the dispersibility and labeling efficiency of the reagents are optimal.

[0276] In addition, by comparing the results of Experiments 21 to 26, it can be seen that: relative to amino acid buffers with an ionic strength of 10mM or 500mM, the reagents prepared with an ionic strength of 15mM to 300mM have better dispersibility and labeling efficiency; and at an ionic strength of 40mM to 250mM, the dispersibility and labeling efficiency of the reagents are optimal.

[0277] Experiments 27-30

[0278] method:

[0279] Experiment 24 among Experiments 21 to 26 was selected, and Experiments 27 to 30 were conducted according to this method. The changed parameters are shown in Table 10 below, and the other experimental conditions were the same as those of Experiment 24.

[0280] Table 10

[0281] Weight ratio of EDC to microspheres Experiment 24 5.0％ Experiment 27 4.0％ Experiment 28 3.0％ Experiment 29 1.0％ Experiment 30 0.5％

[0282] result:

[0283] In order to verify the dispersibility, reactivity and labeling effect of the reagents prepared in Experiments 27 to 30, the calibration curve signal and analytical sensitivity were measured according to the description in the above "Analysis Method" section. The results are shown in Table 11 below.

[0284] Table 11

[0285]

[0286]

[0287] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0288] As shown in Table 11, the dispersibility, reactivity and labeling effect of the prepared reagent can be further optimized by adjusting the weight ratio of EDC to microspheres from 5.0% to 0.5% to 4.0% based on the method and parameters of Experiment 24. Among them, the best effect is achieved when the weight ratio of EDC to microspheres is about 3.0%.

[0289] Experiments 31-33

[0290] method:

[0291] Experiment 28 among Experiments 27 to 30 was selected, and Experiments 31 to 33 were continued according to this method. The changed parameters are shown in Table 12 below, and the other experimental conditions were the same as those of Experiment 28.

[0292] Table 12

[0293] Buffer Type Microsphere particle size nm Experiment 28 Arginine 126 Experiment 31 Arginine 350 Experiment 32 Alanine 126 Experiment 33 Lysine 126

[0294] result:

[0295] In order to verify the dispersibility, reactivity and labeling effect of the reagents prepared in Experiments 31 to 33, the calibration curve signal and analytical sensitivity were measured according to the description in the above "Analysis Method" section. The results are shown in Table 13 below.

[0296] Table 13

[0297]

[0298] Note: The blank absorbance in brackets is the end point of the reagent reaction

[0299] It can be seen from Table 13 that the effect achieved by the method of the present invention can still be achieved after adjusting the particle size of the microspheres or replacing the buffer with other amino acid buffers.

[0300] Example 3 Performance Test

[0301] According to the description in the "Analysis Method" section above, the conventional method, the experiment of the present invention and the comparative example were subjected to intra-batch CV test, equivalence band and long-term stability at 2-8°C to verify the effect of the method of the present invention when used as a diagnostic reagent. The results are shown in Table 14 and Figure 3.

[0302] Table 14

[0303]

[0304]

[0305] Note: “Milky white” means milky white suspension

[0306] It can be seen from Table 14 and Figure 3 that, compared with the conventional method and the comparative example, the reagent prepared by the experimental method of the present invention has a lower intra-batch CV, and thus has better repeatability; at the same time, the reagent prepared by the experimental method of the present invention has a wider equivalence band, which proves that the method of the present invention further improves the labeling efficiency of the microspheres and the labeling efficiency is gradually improved with the gradual optimization of the parameters, thereby reducing the probability of false positives or false negatives in detection applications; in addition, from the stability results, the absolute values ​​of the relative deviations of the quality control of the reagents prepared by the method of the present invention are less than 10% at 6 months and 12 months, and the stability is good; while the conventional method used in the art for labeling microspheres has a small amount of precipitation at 6 months, and a large amount of precipitation at 12 months, and the stability is poor.

[0307] Thus, according to Examples 1 to 3, we have successfully established a simple and easy-to-operate carboxyl microsphere labeling method, and verified the advantages of the reagent prepared by this method in terms of dispersibility, reactivity, labeling efficiency and stability. Next, we will use this method to prepare a reagent for detecting β2-microglobulin and further study the preparation parameters.

[0308] Example 4 Preparation of a reagent for detecting β2-microglobulin

[0309] Preparation Example 1

[0310] method:

[0311] 1) 80 nm latex (Sichuan Mike Biotech New Materials Technology Co., Ltd.) and rabbit anti-β2-microglobulin polyclonal antibody 1 (Sichuan Mike Biotech New Materials Technology Co., Ltd.) were diluted to 20 mg / ml and 30 mg / ml respectively using 20 mM, pH 8.5 TES buffer;

[0312] Mixing the diluted antibody solution and the diluted microsphere solution so that the weight ratio of the antibody to the microsphere is 14-16%;

[0313] Add EDC mother solution to the mixed solution so that the weight ratio of EDC to microspheres is 5%, and label at 25°C for 3 to 4 hours;

[0314] TRIS (final concentration 2 g / L) was then added for blocking for 2 h;

[0315] Trehalose (final concentration 10 g / L) and ProClin300 (1 g / L) were added, and the solution was allowed to stand for 2 hours to obtain the first labeled microsphere solution;

[0316] 2) Repeat step 1) using latex with a diameter of 210 nm (Sichuan Mike Biotech New Material Technology Co., Ltd.) and rabbit anti-β2-microglobulin polyclonal antibody 2 (Sichuan Mike Biotech New Material Technology Co., Ltd.) to obtain a labeled second microsphere solution (Note: the above-mentioned rabbit anti-β2-microglobulin polyclonal antibody 1 and rabbit anti-β2-microglobulin polyclonal antibody 2 are two antibodies targeting different epitopes of β2-microglobulin, respectively);

[0317] 3) The first microsphere solution and the second microsphere solution are mixed at a volume ratio of 5:1 to obtain a reagent for detecting β2-microglobulin.

[0318] Preparation Examples 2 to 7 and Comparative Example C

[0319] method:

[0320] In comparison with Preparation Example 1, the operating parameters of Preparation Examples 2 to 7 and Comparative Example C are given in Table 15 below, wherein the parameters not given are the same as those of Preparation Example 1.

[0321] Table 15

[0322]

[0323]

[0324] Note: Rabbit monoclonal antibody and mouse monoclonal antibody are produced by Sichuan Mike Biotech New Materials Technology Co., Ltd.

[0325] In comparison with Preparation Example 1, the operating parameters of Comparative Examples C2 to C3 are given in Table 16 below, wherein the parameters not given are the same as those of Preparation Example 1.

[0326] Table 16

[0327] Buffer ionic strength (mM) Buffer pH Weight ratio of EDC to microspheres % Preparation Example 1 15 8.5 3.0 Comparative Example C2 50 8.5 5.0 Comparative Example C3 15 6.0 5.0

[0328] Conventional method C

[0329] 1) labeling the microspheres using conventional method A, wherein the microspheres are replaced with 80 nm latex and the antibody is replaced with rabbit anti-β2-microglobulin polyclonal antibody 1 to obtain a labeled first microsphere solution;

[0330] 2) labeling the microspheres using conventional method A, wherein the microspheres are replaced with 210 nm latex and the antibody is replaced with rabbit anti-β2-microglobulin polyclonal antibody 2 to obtain a labeled second microsphere solution;

[0331] 3) The first microsphere solution and the second microsphere solution are mixed at a volume ratio of 5:1 to obtain a reagent for detecting β2-microglobulin.

[0332] Preparation Example 21

[0333] method:

[0334] 1) Using 250 mM arginine buffer at pH 8.5, 80 nm latex and rabbit anti-β2-microglobulin polyclonal antibody 1 were diluted to 20 mg / ml and 30 mg / ml respectively;

[0335] Mixing the diluted antibody solution and the diluted microsphere solution so that the weight ratio of the antibody to the microsphere is 14-16%;

[0336] Add EDC mother solution to the mixed solution so that the weight ratio of EDC to microspheres is 5%, and label at 25°C for 3 hours;

[0337] TRIS (final concentration 2 g / L) was then added for blocking for 2 h;

[0338] Trehalose (final concentration 10 g / L) and ProClin300 (1.5 g / L) were added and allowed to stand for 2 hours to obtain a labeled first microsphere solution;

[0339] 2) Repeat step 1) using latex with a diameter of 210 nm and rabbit anti-β2-microglobulin polyclonal antibody 2 to obtain a labeled second microsphere solution;

[0340] 3) The first microsphere solution and the second microsphere solution are mixed at a volume ratio of 5:1 to obtain a reagent for detecting β2-microglobulin.

[0341] Preparation Examples 22 to 28 and Comparative Example D

[0342] method:

[0343] In comparison with Preparation Example 21, the operating parameters of Preparation Examples 22 to 28 and Comparative Example D are given in Table 17 below, wherein the parameters not given are the same as those of Preparation Example 21.

[0344] Table 17

[0345]

[0346] Note: Rabbit monoclonal antibody and mouse monoclonal antibody are produced by Sichuan Mike Biotech New Materials Technology Co., Ltd.

[0347] Example 5 Testing of reagents for detecting β2-microglobulin

[0348] Calibration curve signal determination:

[0349] The prepared reagents were calibrated using the calibrator β2-microglobulin mentioned in the “Analysis Method” and an automatic biochemical analyzer. The specific results are shown in Table 18 below.

[0350] Table 18

[0351]

[0352]

[0353] Judging from the results, Comparative Examples C2, C3 and D2 agglutinated severely during the preparation process, and could not be used later without ultrasound and centrifugation. Conventional method C also agglutinated, requiring ultrasound and centrifugation for dispersion. However, no agglutination occurred in the preparation examples and Comparative Examples C1 and D1. Among them, Comparative Examples C1 and D1 failed to pass the calibration because the diameter of the microspheres selected in the preparation examples was not suitable for specific β2-microglobulin detection compared to the comparative examples. For Preparation Examples 1 to 7, considering the high-end (80 mg / L, 50 mg / L) gradient and the low-end (1.5 mg / L) sensitivity, it can be known that the reagents of Preparation Examples 1, 5 and 6 are superior to other reagents including reagents obtained by methods conventionally used in the art in terms of calibration curve signals; similarly, in Preparation Examples 21 to 28, Preparation Examples 21 to 23, 26 and 27 are superior to other reagents.

[0354] Intra-assay CV determination

[0355] The calibrated reagents were tested using the quality control product (β2-microglobulin) mentioned in the "Analytical Methods" and the intra-batch CV determination method. The results are shown in Table 19.

[0356] Table 19

[0357]

[0358] As can be seen from Table 19, the deviations of all reagents are less than 5%, thus meeting the requirements of subsequent experiments. Overall, the CV values ​​of Preparation Examples 1, 5, 6, 21-23, 26 and 27 are smaller than those of Preparation Examples 7 and 25, and are more obviously smaller than other reagents including reagents obtained by methods conventionally used in the art.

[0359] Dose response curve

[0360] method:

[0361] The high-value sample of β2-microglobulin was diluted with physiological saline to prepare a series of samples containing different concentrations of antigen (theoretical concentrations are 80 mg / L, 120 mg / L, 160 mg / L, 200 mg / L, 240 mg / L, and 320 mg / L); then the concentrations were measured from low to high concentrations to obtain the measured values ​​of β2-microglobulin concentrations, and then the dose-response curve was drawn with the theoretical concentration as the horizontal axis and the measured concentration as the vertical axis. The results are shown in Figure 2. Figure 4 shown.

[0362] Depend on Figure 4 It can be seen that the maximum concentration that can be measured by the conventional method is 200 mg / L. The maximum concentration that can be measured by Preparation Examples 1, 5 and 6 is 320 mg / L, the maximum concentration that can be measured by Preparation Example 7 is 240 mg / L, the maximum concentration that can be measured by Preparation Examples 3 and 4 is 160 mg / L, and the maximum concentration that can be measured by Preparation Example 2 is 120 mg / L; the maximum concentration that can be measured by Preparation Examples 21 to 23, 26 and 27 is 320 mg / L of BMG concentration, and the maximum concentration that can be measured by Preparation Examples 24, 25, and 28 is 240 mg / L.

[0363] Stability test

[0364] With reference to the description in the “Analysis Method” section above, the reagents obtained by the conventional method and the preparation examples of the present invention were subjected to stability tests at 2-8° C. for 12 months and 24 months. The results are shown in Table 20.

[0365] Table 20

[0366]

[0367] Note: “Milky white” means milky white suspension

[0368] It can be seen from Table 20 that the β2-microglobulin detection reagent prepared by the method of the present invention is superior to the reagent prepared by the conventional method in terms of stability.

[0369] Clinically relevant tests

[0370] The β2-microglobulin reagent prepared by the present invention was used to test 10 clinical samples, and the correlation analysis was performed using the β2-microglobulin assay kit (Japan Seiken), showing that the clinical correlation of each group was greater than 0.99. The results are shown in Table 21 below.

[0371] Table 21

[0372]

Claims

1. A method for preparing a reagent for detecting β2-microglobulin, the method comprising the following steps: 1) diluting the first microsphere and the first antibody with a buffer to obtain a mixed solution of the first microsphere and the first antibody, adding a crosslinking agent for incubation, and adding a blocking agent for blocking to obtain labeled first microspheres, wherein the crosslinking agent is EDC, and the buffer in step 1) is a Good's buffer with an ionic strength of 3 to 30 mM, or an amino acid buffer with an ionic strength of 10 to 500 mM; 2) diluting the second microsphere and the second antibody with a buffer to obtain a mixed solution of the second microsphere and the second antibody, adding a cross-linking agent for incubation, and adding a blocking agent for blocking to obtain a labeled second microsphere, wherein the cross-linking agent is EDC; 3) mixing the labeled first microspheres with the labeled second microspheres; Wherein, the first microsphere and the second microsphere are carboxyl microspheres with different diameters; the first antibody is different from the second antibody; the pH of the buffer is 6.3-9.5, wherein the method does not include any purification step and does not include any step for dispersing the microspheres; The weight ratio of the crosslinking agent to the microspheres is 0.5% to 4.0%; The weight ratio of the antibody to the microspheres is 14% to 16%.

2. The method according to claim 1, wherein: The pH of the buffer in step 1) is 7.0 to 9.

0. 3 . The method according to claim 1 , wherein the ionic strength of the Good's buffer is 5 mmol / L to 25 mmol / L.

4. The method according to claim 1, wherein: The amino acid buffer is a neutral aliphatic amino acid buffer and / or an alkaline amino acid buffer.

5. The method according to claim 1, wherein: The ionic strength of the amino acid buffer is 15 mmol / L to 300 mmol / L.

6. The method according to claim 1, wherein: The weight ratio of the crosslinking agent to the microspheres is 1.0% to 3.5%.

7. The method according to any one of claims 1 to 6, wherein: The diameter of the first microsphere is 30-120 nm; the diameter of the second microsphere is 140-400 nm.

8. The method according to claim 7, wherein: The diameter of the first microsphere is 40-110 nm.

9. The method according to claim 7, wherein: The diameter of the second microsphere is 160-300 nm.

10. A reagent for detecting β2-microglobulin prepared by the method according to any one of claims 1 to 9.

11. A kit for detecting β2-microglobulin, comprising the reagent according to claim 10.

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