Antibody-coated latex microsphere dispersion and preparation method thereof

Abstract

The present invention relates to the technical field of analysis and detection, and in particular to a latex microsphere dispersion coated with an antibody and a preparation method thereof. The preparation method comprises mixing latex microspheres with a buffer, then sequentially adding a coupling agent, a single antibody and a blocking agent for reaction, and not performing a centrifugal resuspension step after the single antibody and the blocking agent are added for reaction, and directly adding a storage liquid after the blocking agent reacts to obtain a latex microsphere dispersion coated with an antibody. The reagent prepared by the present invention has stable quality, and the performance of the reagent is equivalent to that of the reagent prepared by the centrifugation method, and the preparation cost is effectively reduced, which is conducive to increasing the maximum preparation amount of the reagent, so that the single output of the reagent is not limited, breaking the restriction of over-reliance on production equipment and production conditions in the original preparation method, providing a basis for the innovation of such a reagent preparation method, and having broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a latex microsphere dispersion liquid coated with an antibody and a preparation method thereof. Background Art

[0002] Latex immunoturbidimetric reagent is a type of immunoturbidimetric technology, which can be divided into common immunoturbidimetric and latex enhanced immunoturbidimetric. Common immunoturbidimetric refers to the method of analyzing turbidity by direct reaction between antigen and antibody, while latex enhanced immunoturbidimetric is a method of amplifying the signal by coupling carboxylated polystyrene microspheres with antibodies and then reacting with antigens.

[0003] Latex immunoturbidimetric kits generally contain three parts: detection reagents (divided into reagents R1 and R2), calibrators and quality control products. Reagent R1 is mainly a buffer salt system, which provides more suitable reaction conditions for antigen and antibody; the main component of reagent R2 is latex microspheres coated with antibodies. Latex immunoturbidimetric kits are widely used in the clinical diagnosis of cardiovascular and cerebrovascular diseases, tumors, kidney diseases, liver diseases, diabetes and other diseases.

[0004] At present, the preparation method of reagent R2 consists of the steps of washing, activation, coupling, centrifugation, resuspension, blocking, storage, etc. For example, CN113030489A discloses a method for preparing cystatin C rabbit polyclonal latex particles, comprising the following steps: (1) adding microspheres to water and buffer, and shaking and mixing at room temperature; (2) adding coupling agent, stirring at room temperature for 1 hour; (3) raising the reaction temperature, adding antibody solution; (4) centrifugally separating microspheres from unlabeled proteins, resuspending microspheres with blocking solution, and stirring at room temperature for 1 hour; (5) centrifugally separating microspheres from excess blocking agent, and resuspending microspheres with storage solution; (6) ultrasonically dispersing, diluting with storage solution to a certain concentration, and shaking and mixing at room temperature. Among them, due to the incomplete reaction between latex microspheres and antibodies, there are residual latex microspheres or antibodies free in the solution, so it is necessary to centrifuge to remove excess reaction components, resulting in overly cumbersome operation steps and limiting the maximum preparation amount of the reagent.

[0005] Therefore, how to simplify the preparation method of latex immunoturbidimetric reagent R2 and increase the maximum preparation amount of the reagent has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a dispersion of latex microspheres coated with antibodies, the raw materials of which include: latex microspheres with a particle size of 90-120 nm, a buffer, a coupling agent, a single antibody, a blocking agent and a storage solution; the weight ratio of the single antibody to the latex microspheres is (10-49) μg:1 mg.

[0007] The present invention finds that by controlling the particle size of latex microspheres, using a single antibody and reasonably controlling the ratio of the single antibody to the latex microspheres, the antibody and the latex microspheres can be fully reacted, so that the steps of centrifugation and resuspension are not required during the preparation process, and a dispersion of latex microspheres coated with antibodies can be prepared by a one-step method, thereby increasing the maximum preparation amount of the reagent.

[0008] Preferably, the weight ratio of the single antibody to the latex microspheres is (10-45) μg:1 mg.

[0009] More preferably, the weight ratio of the single antibody to the latex microspheres is (35-45) μg:1 mg.

[0010] Preferably, the latex microspheres are polystyrene carboxyl microspheres.

[0011] Preferably, the buffer is a HEPES buffer at pH 6.0-7.5, a MES buffer at pH 5.0-6.5 or a boric acid buffer at pH 7.5-10.0; and / or the concentration of the buffer is 10-100 mM.

[0012] Preferably, the coupling agents are EDC and NHS.

[0013] Preferably, the weight ratio of EDC to latex microspheres is (15-50) μg:1 mg, and the weight ratio of NHS to latex microspheres is (15-50) μg:1 mg.

[0014] Preferably, the single antibody is a polyclonal antibody that specifically binds to the target substance.

[0015] Preferably, the molecular weight of the single antibody is 150-160KD.

[0016] Preferably, the polyclonal antibody is derived from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

[0017] Preferably, the blocking agent is BSA.

[0018] Preferably, the concentration of BSA is (0.01-0.02) g / mL.

[0019] Preferably, after adding the coupling agent or the single antibody, the mixture is stirred and mixed at 25° C. to 37° C.

[0020] Preferably, after adding the blocking agent, the mixture is stirred and mixed at 37°C to 45°C.

[0021] Preferably, the final concentration of the latex microspheres after adding the storage solution is (0.1-0.5) mg / 100 mg.

[0022] Preferably, the storage solution comprises: 10~50mM HEPES buffer with a pH of 6.0~7.5, 0.5%~1% (w / v) BSA, 0.05%~0.1% (v / v) Proclin300, 1%~2% (w / v) NaCl, 1%~5% (w / v) glycerol, 1%~5% (w / v) mannitol, 0.5%~2% (w / v) Tween 20 and 1%~5% (w / v) sucrose.

[0023] The present invention further discovered that the antibody-coated latex microspheres have different compatibilities with different storage liquid systems. When the compatibility is poor, aggregation and even sedimentation are likely to occur. Therefore, during the resuspension and blocking process, it is often necessary to break up the aggregated particles by ultrasonic disruption, which makes the preparation steps more complicated, the use of ultrasonic equipment makes the preparation cost higher, and the control points are difficult to control, further limiting the maximum preparation amount of the reagent.

[0024] The present invention optimizes and adjusts the storage solution formula and finds that when the storage solution is used to store antibody-coated latex microspheres, the two have good compatibility, and will not produce agglutination and sedimentation of the latex microspheres. The ultrasonic crushing step can be omitted, further simplifying the preparation steps, reducing the production and preparation costs, and further increasing the maximum preparation amount of the reagent.

[0025] Furthermore, the present invention provides a method for preparing the antibody-coated latex microsphere dispersion, comprising: mixing latex microspheres with a particle size of 90 to 120 nm with a buffer, and then sequentially adding a coupling agent, a single antibody and a blocking agent for reaction, wherein no centrifugal resuspension step is performed after the single antibody and the blocking agent are added for reaction, and a storage solution is directly added after the blocking agent reaction to obtain the antibody-coated latex microsphere dispersion.

[0026] Furthermore, the present invention provides the use of the antibody-coated latex microsphere dispersion or the preparation method in preparing a latex immunoturbidimetric kit.

[0027] Furthermore, the present invention provides a latex immunoturbidimetric kit, which contains the antibody-coated latex microsphere dispersion or the antibody-coated latex microsphere dispersion prepared by the preparation method.

[0028] Preferably, the latex immunoturbidimetric kit comprises reagent R1 and reagent R2; the reagent R2 comprises the antibody-coated latex microsphere dispersion or the antibody-coated latex microsphere dispersion obtained by the preparation method; the reagent R1 comprises: 20-100 mM HEPES buffer (preferably pH 6.0-7.5) or PBS buffer or MES buffer (preferably pH 5.0-6.5), 1%-2% (w / v) NaCl, 0.5%-2% (w / v) sucrose, 0.5%-2% (w / v) Tween 20, 0.5%-2% (w / v) PEG6000 and 0.05%-0.1% (v / v) Proclin300.

[0029] In a specific implementation process, the target substance specifically bound by the single antibody includes but is not limited to cystatin C.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The invention provides an antibody-coated latex microsphere dispersion and a preparation method thereof. The preparation method has simple operation steps, and the antibody-coated latex microsphere dispersion can be prepared in a one-step method. The prepared reagent has stable quality, and the reagent performance is equivalent to that of a reagent prepared by a centrifugal method. The preparation cost is effectively reduced, and the maximum preparation amount of the reagent is increased, so that the single output of the reagent is not limited, breaking the restriction of the original preparation method that is too dependent on production equipment and production conditions, providing a basis for the innovation of such a reagent preparation method, and having broad application prospects. DETAILED DESCRIPTION

[0031] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, if no specific technology or conditions are indicated, the technology or conditions described in the literature in this field or the product instructions are used. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased through regular channels. The unit of w / v in the following embodiments is g / L, and the unit of v / v is mL / L.

[0032] Example 1 This embodiment provides a latex immunoturbidimetric kit for detecting cystatin C, comprising a reagent R1 and a reagent R2; the reagent R1 comprises: 100 mM HEPES buffer (pH 6.5), 2% (w / v) NaCl, 1% (w / v) sucrose, 1% (w / v) Tween 20, 1% (w / v) PEG6000 and 0.1% (v / v) Proclin 300, and the components are weighed and mixed uniformly to obtain the reagent R1; the preparation method of the reagent R2 is as follows: (1) Add 25 mg of latex microspheres (polystyrene carboxyl microspheres, particle size 100 nm) to 4.2 mL of 50 mM pH 8.0 boric acid buffer and swirl to mix evenly; (2) Dilute EDC and NHS with water to 1% (w / v), take 125 μL of EDC solution and 125 μL of NHS solution respectively and add them to the mixed solution in step (1), and rotate and stir for 20 minutes (25°C); (3) Add antibody solution (cystatin C antibody, purchased from Beijing Dacheng Biotechnology Co., Ltd., product number D008-01, molecular weight 150KD), antibody concentration is 10 mg / mL, added amount is 100 μL, that is, the weight ratio of antibody to latex microspheres is 40 μg:1 mg) and rotate and stir for 1 hour (25°C); (4) Add 50 μL of 2% BSA blocking agent and rotate and stir for 1 hour (42°C); (5) Finally, 5 mL of storage solution was added, the formula of which was: 20 mM HEPES buffer (pH 7.0), 0.5% (w / v) BSA, 0.1% (v / v) Proclin 300, 1% (w / v) NaCl, 1% (w / v) glycerol, 1% (w / v) ethylene glycol, 1% (v / v) Tween 20 and 1% (w / v) glucose. After mixing, visible agglomerated particles were found, so they were further dispersed by ultrasonication to make the final concentration of latex microspheres 0.26 mg / 100 mg, thus obtaining reagent R2 (latex microsphere dispersion coated with antibody).

[0033] Example 2 This embodiment provides a latex immunoturbidimetric kit for detecting cystatin C, which is different from the embodiment 1 in that the amount of antibody added is adjusted so that the weight ratio of antibody to latex microspheres is 10 μg:1 mg.

[0034] Example 3 This embodiment provides a latex immunoturbidimetric kit for detecting cystatin C, which differs from Embodiment 1 only in that: The storage solution in step (5) adopts the following formula: 20 mM HEPES buffer with pH 7.0, 0.5% (w / v) BSA, 0.1% (v / v) Proclin300, 1% (w / v) NaCl, 1% (w / v) glycerol, 1% (w / v) mannitol, 1% (v / v) Tween 20 and 1% (w / v) sucrose. After mixing, no agglomerated particles visible to the naked eye were found. No ultrasonic dispersion was required. The final concentration of latex microspheres was 0.26 mg / 100 mg, and reagent R2 (latex microsphere dispersion coated with antibody) was obtained.

[0035] Comparative Example 1 This comparative example provides a latex immunoturbidimetric kit for detecting cystatin C, which is different from Example 1 in that polystyrene carboxyl microspheres with a particle size of 150 nm are used.

[0036] Comparative Example 2 This comparative example provides a latex immunoturbidimetric kit for detecting cystatin C, which is different from Example 1 in that the amount of antibody added is adjusted so that the weight ratio of antibody to latex microspheres is 50 μg:1 mg.

[0037] Control group 1~5 Control groups 1 to 5 (centrifugation method) prepared reagent R2 according to the preparation conditions of Examples 1 to 3 and Comparative Examples 1 and 2, respectively, with the only difference being that the antibody solution was added for cross-linking and then centrifuged, the microspheres were resuspended, stirred at room temperature for 1 hour, the blocking agent was added for blocking and then centrifuged to separate the microspheres from the excess blocking agent, and finally the microspheres were resuspended in the storage solution and ultrasonically dispersed (control group 3 did not require ultrasonic dispersion) to obtain reagent R2.

[0038] Test Example 1 This test example uses the kits of the above-mentioned embodiment, comparative example and control group to measure the same quality control product, and the test method is as follows: The measurement was performed using a fully automatic biochemical analyzer with a dual reagent function, with a wavelength of 570 nm, a temperature of 37° C., and a cuvette light path of 1 cm. The operating steps are shown in Table 1.

[0039] Table 1

[0040] The test results are shown in Table 2, and the unit of the measured value is mg / L.

[0041] Table 2

[0042] It can be seen that Examples 1 to 3 can all achieve test results close to those of the corresponding control groups, while Comparative Examples 1 and 2 do not meet the reagent usage requirements.

[0043] Test Example 2 This test example tests the linearity of the kits of the above-mentioned embodiments, comparative examples and control groups, and the steps are as follows: a high concentration sample exceeding the upper limit of the linear range and a low concentration sample reaching the lower limit of the linear range are mixed into 10 dilutions (x i ). Using this as a sample, test each kit separately, test each dilution 3 times, and calculate the mean of the test results (y i ). i ) as the independent variable to determine the mean (y i ) as the dependent variable to find the linear regression equation. Calculate the linear regression correlation coefficient (r) according to formula (1), and then use the dilution (x i ) into the linear regression equation and calculate y i The estimated value of ), and calculate y according to formula (2) (3) i and relative or absolute deviation.

[0044] The results are shown in Table 3.

[0045] Table 3

[0046] It can be seen that the linearity of Examples 1 to 3 is close to that of the control group (centrifugation method), and can meet the reagent usage requirements.

[0047] Test Example 3 This test example evaluates the aggregation phenomenon of the kits of the above-mentioned examples, comparative examples and control groups, and the steps are as follows: Scan the reagent R2 sample at a wavelength of 570nm using a spectrophotometer and record the corresponding absorbance. The smaller the absorbance, the more uniform the dispersion of the latex microspheres in the solution.

[0048] The results are shown in Table 4.

[0049] Table 4

[0050] It can be seen that in Example 3, compared with Example 1, the latex microspheres in the reagent R2 are more evenly dispersed, indicating that the storage liquid formula of the present invention can effectively solve the problem of latex microsphere agglomeration.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dispersion of latex microspheres coated with an antibody, characterized in that: The raw materials include latex microspheres with a particle size of 90-120 nm, a buffer, a coupling agent, a single antibody, a blocking agent and a storage solution; the weight ratio of the single antibody to the latex microspheres is (10-49) μg: 1 mg.

2. The antibody-coated latex microsphere dispersion according to claim 1, characterized in that: The latex microspheres are polystyrene carboxyl microspheres.

3. The antibody-coated latex microsphere dispersion according to claim 1, characterized in that: The buffer is a HEPES buffer with a pH of 6.0 to 7.5, a MES buffer with a pH of 5.0 to 6.5, or a boric acid buffer with a pH of 7.5 to 10.0; and / or the concentration of the buffer is 10 to 100 mM.

4. The antibody-coated latex microsphere dispersion according to claim 1, characterized in that: The coupling agents are EDC and NHS.

5. The antibody-coated latex microsphere dispersion according to claim 1, characterized in that: The single antibody is a polyclonal antibody that specifically binds to a target substance; and / or the blocking agent is BSA.

6. The antibody-coated latex microsphere dispersion according to claim 1, characterized in that: The storage solution includes: 10~50mM HEPES buffer with pH 6.0~7.5, 0.5%~1% (w / v) BSA, 0.05%~0.1% (v / v) Proclin300, 1%~2% (w / v) NaCl, 1%~5% (w / v) glycerol, 1%~5% (w / v) mannitol, 0.5%~2% (w / v) Tween 20 and 1%~5% (w / v) sucrose.

7. The method for preparing the antibody-coated latex microsphere dispersion according to any one of claims 1 to 6, characterized in that: include: Latex microspheres with a particle size of 90-120 nm are mixed with a buffer solution, and then a coupling agent, a single antibody and a blocking agent are added in sequence for reaction, wherein no centrifugal resuspension step is performed after the single antibody and the blocking agent are added for reaction, and a storage solution is directly added after the blocking agent reaction to obtain the antibody-coated latex microsphere dispersion.

8. Use of the antibody-coated latex microsphere dispersion according to any one of claims 1 to 6 or the preparation method according to claim 7 in the preparation of a latex immunoturbidimetric kit.

9. A latex immunoturbidimetric kit, characterized in that: It contains the antibody-coated latex microsphere dispersion according to any one of claims 1 to 6 or the antibody-coated latex microsphere dispersion prepared by the preparation method according to claim 7.

10. The latex immunoturbidimetric kit according to claim 9, characterized in that: The invention comprises reagent R1 and reagent R2; the reagent R2 comprises the antibody-coated latex microsphere dispersion according to any one of claims 1 to 6 or the antibody-coated latex microsphere dispersion obtained by the preparation method according to claim 7; the reagent R1 comprises: 20-100 mM HEPES buffer or PBS buffer or MES buffer, 1%-2% (w / v) NaCl, 0.5%-2% (w / v) sucrose, 0.5%-2% (w / v) Tween 20, 0.5%-2% (w / v) PEG6000 and 0.05%-0.1% (v / v) Proclin300.

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