Coupling method of carboxyl microspheres and antibody

Abstract

The invention belongs to the technical field of medical detection, and provides a coupling method of carboxyl microspheres and an antibody. The preparation method comprises the following steps: firstly, activating carboxyl microspheres by using an activating agent, then carrying out a coupling reaction with an antibody, and then carrying out a blocking reaction with a blocking agent, thereby finally preparing the carboxyl microsphere-antibody conjugate. The activated carboxyl microspheres are dispersed by adopting the buffer solution containing the nonionic surfactant, and then the coupling reaction is performed, so that the dispersity and the coupling efficiency of the carboxyl microspheres are effectively improved, the antibody coupling process can be optimized, the one-time coupling batch can reach 150mL, the batch-to-batch difference of the carboxyl microsphere reagent is further reduced, and the method is suitable for large-batch preparation and is suitable for industrial production. The method can effectively improve the detection performance of an immunochromatography reagent, is suitable for coupling carboxyl microspheres and various types of monoclonal antibodies, and is beneficial to large-scale preparation and production of immunochromatography test strips.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and more particularly to a method for coupling carboxyl microspheres with antibodies. Background Art

[0002] Immunochromatographic analysis is a large-scale in vitro diagnostic method widely used due to its simplicity, rapidity, and low cost. The more mature form is lateral immunochromatographic technology, which does not require professional operation or expensive equipment and can obtain test results in a short time (<30 minutes). Therefore, it is widely used in clinical diagnosis, food safety, environmental monitoring, and home testing. Lateral immunochromatographic test strips are typically assembled from a PVC backing, nitrocellulose membrane, absorbent paper, sample pad, and conjugate pad. Sandwich and competitive assays are commonly used to detect large and small molecule targets, respectively.

[0003] Common colorimetric markers include colloidal gold, colloidal silver, latex microspheres, composite nanomaterials, fluorescent microspheres, and the like. Among them, colloidal gold is the earliest and most widely used colorimetric marker, but it suffers from large batch-to-batch variability, low sensitivity, and uses physical adsorption to bind to antigens / antibodies, making the marker unstable. Latex microspheres, also known as polystyrene microspheres, have rich and bright colors, enable multiple detections, and have a uniform particle size. Even when the elementary particle size is between 100 and 1000 nm, they exhibit good monodispersity and stability. Compared to colloidal gold, latex microspheres have higher sensitivity and are more suitable for mass production. Latex microspheres can be further divided according to their groups into carboxyl latex microspheres, amino latex microspheres, and epoxy latex microspheres. Typically, the carboxyl microspheres carried on the surface of the latex microspheres covalently bind to antibodies to form a stable complex, which can be used for in vitro diagnostic analysis. However, the latex microsphere labeling process usually requires three or more refrigerated centrifugations in conjunction with a cell disruptor, and is usually completed using a smaller capacity (2mL, 5mL, and 15mL centrifuge tube) centrifuge. It is more suitable for routine experiments or small-batch production. For large-scale production, there are problems such as cumbersome operation, low fault tolerance, and large intra-batch / inter-batch differences, making large-scale production difficult.

[0004] Therefore, there is an urgent need to develop a stable labeling method for carboxyl microspheres and antibodies that is suitable for large-scale production. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for coupling carboxyl microspheres with antibodies. The labeling method of carboxyl microspheres and antibodies provided by the present invention is not only stable but also suitable for large-scale production. The single coupling batch can reach 150mL, which has good broadness and helps to expand the scope of use of carboxyl microspheres and enable large-scale preparation and production of immunochromatographic test strips.

[0006] The first aspect of the present invention provides a method for coupling carboxyl microspheres to antibodies.

[0007] Specifically, a method for coupling carboxyl microspheres with antibodies comprises the following steps:

[0008] (1) mixing carboxyl microspheres and an activator to perform an activation reaction to obtain activated carboxyl microspheres, centrifuging, collecting the solid, and adding a buffer solution containing a nonionic surfactant to obtain an activated carboxyl microsphere suspension;

[0009] (2) mixing the activated carboxyl microsphere suspension and the antibody to obtain a carboxyl microsphere-antibody mixed solution, performing a coupling reaction, and obtaining a post-reaction liquid;

[0010] (3) Mixing the post-reaction liquid and a blocking agent to obtain a blocking agent-containing mixed solution, performing a blocking reaction, and obtaining a carboxyl microsphere-antibody conjugate.

[0011] The present invention disperses the activated carboxyl microspheres using a buffer solution containing a nonionic surfactant and then performs a coupling reaction, thereby effectively improving the dispersibility and coupling efficiency of the carboxyl microspheres, optimizing the antibody coupling process, and increasing the single-coupling batch size (the volume of the activated carboxyl microsphere suspension can reach 150 mL, that is, the single-coupling batch size can reach 150 mL), thereby reducing the batch-to-batch difference of the carboxyl microsphere reagent, thereby enabling large-scale preparation, effectively improving the detection performance of the immunochromatographic reagent, and being suitable for coupling the carboxyl microspheres with various types of antibodies.

[0012] Preferably, in step (1), the carboxyl microspheres are at least one of red, green, blue or black carboxyl microspheres, and / or the carboxyl microspheres are polystyrene carboxyl latex microspheres.

[0013] Preferably, the average particle size of the carboxyl microspheres is 200-400 nm.

[0014] Preferably, in step (1), after the activation reaction is completed, ultrasonic dispersion is performed at 700-900W for 1-10 minutes, and then centrifugation is performed at 11000-13000rpm for 10-20 minutes. The solid is taken, a buffer solution containing a nonionic surfactant is added, and then ultrasonic dispersion is performed at 700-900W for 1-10 minutes to obtain an activated carboxyl microsphere suspension.

[0015] Preferably, in step (1), the volume of the activated carboxyl microsphere suspension is 100-150 mL. The coupling batch size of the present invention can reach 100 mL, or even 150 mL.

[0016] Preferably, in step (1), the buffer solution containing a nonionic surfactant is a boric acid buffer solution containing a nonionic surfactant, and / or the mass concentration of the nonionic surfactant in the buffer solution containing a nonionic surfactant is 1-3‰.

[0017] Further preferably, in step (1), the mass concentration of the non-ionic surfactant in the buffer solution containing the non-ionic surfactant is 2-3‰.

[0018] Preferably, in step (1), the carboxyl microspheres are pretreated before use, and the pretreatment step includes: first mixing 4-morpholineethanesulfonic acid buffer (MES buffer) and carboxyl microspheres, ultrasonic treatment at 700-900W for 2-8min, centrifugation at 11000-13000rpm for 10-20min, taking solid, and then adding the solid to 4-morpholineethanesulfonic acid buffer (MES buffer), dispersing the carboxyl microspheres into a monodisperse state to obtain a carboxyl microsphere suspension. The present invention utilizes MES buffer to clean the carboxyl microspheres, and further utilizes MES buffer to disperse the carboxyl microspheres.

[0019] Further preferably, in step (1), the carboxyl microsphere suspension and an activator are mixed to obtain an activated mixed solution, and an activation reaction is carried out.

[0020] Preferably, in step (1), the temperature of the activation reaction is 20-37° C., and / or the time of the activation reaction is 20-30 min.

[0021] Preferably, in step (1), the activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and / or N-hydroxysuccinimide (NHS).

[0022] Preferably, in step (1), the mass concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) in the activation mixture is 20-30%, and / or the mass concentration of N-hydroxysuccinimide (NHS) in the activation mixture is 10-20%.

[0023] Preferably, in step (2), the antibody is a monoclonal antibody, and / or the mass concentration of the antibody in the carboxyl microsphere-antibody mixture is 100-300 μg / mL.

[0024] Preferably, in step (2), the antibody and antibody buffer are first mixed, and then mixed with the activated carboxyl microsphere suspension, wherein the antibody buffer is a borate buffer with a solute molar concentration of 90-110 mmol / L.

[0025] The coupling reaction is carried out in boric acid buffer, and the antibody will have a net negative charge, maintaining a stable coupling environment, which is conducive to the binding of the antibody Fc end to the activated carboxyl microspheres.

[0026] Preferably, in step (2), the coupling reaction temperature is 20-37° C., and / or the coupling reaction time is 30-40 min.

[0027] Preferably, in step (3), the blocking agent is casein sodium salt and / or bovine serum albumin (BSA), and / or the mass concentration of the blocking agent in the blocking agent-containing mixture is 1-2%, and / or the temperature of the blocking reaction is 20-37° C., and / or the blocking reaction time is 60-90 min.

[0028] Further preferably, in step (3), the blocking agent is casein sodium salt and bovine serum albumin (BSA).

[0029] Preferably, in step (3), the blocking agent is first dissolved in water to obtain a blocking solution, which is then mixed with the reacted liquid.

[0030] Preferably, in step (3), after the blocking reaction, post-treatment is performed, and the post-treatment comprises the following steps: ultrasonic dispersion, centrifugation, taking solids, mixing the solids with a preservation solution, resuspending the solids, and ultrasonic dispersion to obtain a carboxyl microsphere-antibody conjugate resuspension.

[0031] Preferably, the centrifugation includes a first centrifugation and a second centrifugation, the speed of the first centrifugation is 10000-12000 rpm, and / or the time of the first centrifugation is 10-15 min, and / or the speed of the second centrifugation is 10000-12000 rpm, and / or the time of the second centrifugation is 10-15 min.

[0032] Preferably, in step (3), the preservation solution comprises the following components: tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), trehalose, a nonionic surfactant and a preservative.

[0033] Further preferably, in step (3), the preservation solution comprises the following components in percentage by weight: 1-2% tris(hydroxymethyl)aminomethane (Tris), 0.5-1.5% bovine serum albumin (BSA), 1-5% trehalose, 0.5-1‰ nonionic surfactant and 0.01-1‰ preservative.

[0034] Preferably, the nonionic surfactant is Tween-20.

[0035] Preferably, the preservative is KroVin950 preservative and / or ProClin300 preservative.

[0036] Preferably, in step (3), the pH value of the preservation solution is 8.5-10.5.

[0037] The buffer components, BSA, and trehalose in the preservation solution used in the present invention can protect the antibodies coupled to the carboxyl microspheres and prevent their denaturation. The low concentration of Tween-20 can ensure that the carboxyl microspheres are in a single dispersed state. In addition, a trace amount of preservative is added. The preservation solution can ensure that the carboxyl microspheres labeled with antibodies can be stably stored at 2-8°C for more than 3 months without any decrease in antibody activity.

[0038] Preferably, the mass concentration of 4-morpholineethanesulfonic acid in the 4-morpholineethanesulfonic acid (MES) buffer is 1%-1.5%, and / or the pH value of the 4-morpholineethanesulfonic acid buffer is 5.5-6.5.

[0039] The present invention uses MES buffer in the cleaning and activation process of the carboxyl microspheres, which can avoid the influence of complex factors such as foreign proteins and salt ions, avoid causing the carboxyl microspheres to precipitate and cause a decrease in coupling efficiency.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention first utilizes an activator to activate carboxyl microspheres, and then carries out a coupling reaction with an antibody, and then carries out a blocking reaction with a blocking agent, and finally obtains a carboxyl microsphere-antibody conjugate. The present invention disperses the activated carboxyl microspheres with a buffer solution containing a nonionic surfactant, and then carries out a coupling reaction, effectively improving the dispersibility and coupling efficiency of the carboxyl microspheres, and can optimize the antibody coupling process. A single coupling batch can reach 150mL (the activated carboxyl microsphere suspension having a volume of 150mL is used for the coupling reaction), and then reduces the batch difference of the carboxyl microsphere reagent, is suitable for large-scale preparation, can effectively improve the detection performance of the immunochromatographic reagent, and is suitable for the coupling of carboxyl microspheres with various types of monoclonal antibodies, has good broadness, helps to expand the scope of use of the carboxyl microspheres, and can achieve large-scale preparation and production of immunochromatographic test strips. DETAILED DESCRIPTION

[0042] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0043] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0044] The main raw materials used in the present invention are as follows:

[0045] Carboxyl microspheres: average particle size 400 nm, source: ML102-2 HeavyBio.

[0046] Antibody: Mouse anti-human influenza A monoclonal antibody, source: HeavyBio.

[0047] Boric acid buffer: Weigh 2.3 g sodium borate and 2.55 g boric acid, add deionized water, adjust the pH to 8.0, and dilute to 500 mL.

[0048] MES buffer: Weigh 5.331 g of 4-morpholineethanesulfonic acid, add deionized water, adjust the pH to 6.0, and dilute to 1 L.

[0049] Blocking solution: Weigh 10 g of Casein (casein sodium salt) and 10 g of BSA (bovine serum albumin), mix them, and add 80 mL of deionized water.

[0050] Preservative solution: Weigh 1.2 g Tris, 1 g BSA, 2 g trehalose, 0.1 g Tween-20, and 0.05 g KroVin950, dissolve them in deionized water and dilute to 100 mL. Adjust the pH to 8.5 with 5 mol / L HCl solution.

[0051] Example 1

[0052] A method for coupling carboxyl latex microspheres with antibodies comprises the following steps:

[0053] (1) Cleaning and dispersion of carboxyl microspheres: 4.5 mL of carboxyl microspheres with a solid content of 4% were placed in a 250 mL centrifuge tube, 150 mL of MES buffer (50 mmol / L MES) was added, and the tube was sonicated at 800 W for 5 min to completely disperse the carboxyl microspheres. The tube was centrifuged at 12000 rpm for 20 min, and the supernatant was discarded. 150 mL of MES buffer was added to the precipitate, and after pipetting and mixing, the tube was sonicated at 800 W for 5 min until the carboxyl microspheres were monodispersed, to obtain 150 mL of a carboxyl microsphere suspension.

[0054] (2) Activation of carboxyl microspheres: A magnetic stirrer was added to the centrifuge tube, and magnetic stirring was performed (the liquid was stirred until a vortex was just formed). 1.5 mL of 10 g / L NHS and 2.25 mL of 10 g / L EDC·HCl were added to 150 mL of the carboxyl microsphere suspension, and the mixture was quickly mixed. The activation reaction was carried out at 25°C for 25 min. After the activation reaction was completed, the magnetic stirrer was removed, and the mixture was ultrasonicated at 800 W for 5 min. The mixture was centrifuged at 12000 rpm for 20 min. The supernatant was discarded, and 150 mL of boric acid buffer containing 2‰ Tween-20 was added to the precipitate. The mixture was mixed by pipetting, and the mixture was ultrasonicated at 800 W for 5 min until the microspheres were in a monodisperse state. The activated carboxyl microsphere suspension was obtained.

[0055] (3) Coupling reaction between activated carboxyl microspheres and antibodies: 15 mg of mouse anti-human influenza A monoclonal antibody was taken, added to borate buffer, diluted to 10 mL, mixed thoroughly, and added to the activated microsphere suspension. The coupling reaction was carried out at 25°C for 30 min to obtain a microsphere-antibody complex.

[0056] (4) Blocking reaction and storage: After the coupling reaction is completed, ultrasonicate at 800W for 2 minutes, add 15mL of blocking solution to the carboxyl microsphere-antibody complex, stir magnetically, and block the reaction at 25°C for 90 minutes; after the blocking reaction is completed, ultrasonicate at 800W for 2 minutes, centrifuge at 12000rpm for 15 minutes, discard the supernatant, take the precipitate, add 150mL of preservation solution to resuspend the precipitate, and then repeat the previous centrifugation operation to obtain a resuspension, and finally store it at 2-8°C for use.

[0057] Example 2

[0058] A method for coupling carboxyl latex microspheres with antibodies is different from Example 1 in that, in step (1), 100 mL of a carboxyl microsphere suspension is prepared, and the specific steps are as follows: 3 mL of carboxyl microspheres with a solid content of 4% are placed in a 250 mL centrifuge tube, 100 mL of MES buffer (50 mmol / L MES) are added, 800 W ultrasonication is performed for 5 minutes to completely disperse the carboxyl microspheres, the mixture is centrifuged at 12000 rpm for 20 minutes, and the supernatant is discarded; 100 mL of MES buffer is added to the precipitate, the mixture is mixed by pipetting, and the mixture is ultrasonicated at 800 W for 5 minutes until the carboxyl microspheres are in a monodisperse state, thereby obtaining 100 mL of a carboxyl microsphere suspension.

[0059] Example 3

[0060] A method for coupling carboxyl latex microspheres with antibodies is different from Example 1 in that, in step (3), the coupling reaction temperature is 20° C. and the coupling reaction time is 35 min.

[0061] Comparative Example 1

[0062] A method for coupling carboxyl latex microspheres with antibodies is different from that of Example 1 in that, in step (2), after the activation reaction is completed, the magnetic stirrer is removed, ultrasound is applied at 800W for 5 minutes, centrifuged at 12000 rpm for 20 minutes, the supernatant is discarded, and 150 mL of boric acid buffer is added to the precipitate. The remaining operations are the same.

[0063] Product effect testing

[0064] The coupling products obtained in each embodiment and comparative example were tested for performance, and the test results are shown below:

[0065] Table 1 Test results of the embodiments and comparative examples

[0066]

[0067] Note: The color development level is L1 to L10. L1 is negative, and L2 to L10 are positive.

[0068] As can be seen from the above table, the coupling state of the coupling product prepared in Example 1 of the present invention is uniformly dispersed, PDI <0.2, and the nonspecific binding rate is as low as 0.7% ± 0.2%, indicating high selectivity and sensitivity. The blocking efficiency is as high as 99.2% ± 0.3%, indicating that the coupling reaction is successfully carried out.

[0069] Table 2 Test results of Example 1 and traditional coupling method (small capacity coupling method)

[0070]

[0071] Note: Example 1 and the traditional coupling method were repeated 5 times (ie, repeatability n=5).

[0072] As can be seen from the results in the above table, the method of Example 1 of the present invention can complete a coupling volume of 150 mL in a single batch, realizing large-scale production. Only one centrifuge tube is required to complete the coupling. The experiment was repeated 5 times, and the intra-batch / inter-batch differences were small. The inter-batch reagent loss rate was ≤3%, and the detection sensitivity reached 1.78±0.03, which improved the detection performance of the immunochromatographic reagent.

[0073] However, the traditional coupling method only has a single-batch coupling volume of 3 mL each time, which requires the use of 50 centrifuge tubes and 4 centrifuges at the same time, resulting in a high loss rate of reagents between batches and a significant decrease in average detection sensitivity.

Claims

1. A method for coupling carboxyl microspheres with antibodies, characterized in that: The steps include: (1) mixing carboxyl microspheres and an activator to perform an activation reaction to obtain activated carboxyl microspheres, centrifuging, collecting the solid, and adding a buffer solution containing a nonionic surfactant to obtain an activated carboxyl microsphere suspension; (2) mixing the activated carboxyl microsphere suspension and the antibody to obtain a carboxyl microsphere-antibody mixed solution, performing a coupling reaction, and obtaining a post-reaction liquid; (3) Mixing the post-reaction liquid and a blocking agent to obtain a blocking agent-containing mixed solution, performing a blocking reaction, and obtaining a carboxyl microsphere-antibody conjugate.

2. The coupling method according to claim 1, wherein In step (1), the carboxyl microspheres are at least one of red, green, blue or black carboxyl microspheres, and / or the carboxyl microspheres are polystyrene carboxyl latex microspheres.

3. The coupling method according to claim 1, wherein In step (1), the buffer solution containing a nonionic surfactant is a boric acid buffer solution containing a nonionic surfactant, and / or the mass concentration of the nonionic surfactant in the buffer solution containing a nonionic surfactant is 1-3‰.

4. The coupling method according to claim 1, wherein In step (1), the activation reaction temperature is 20-37° C., and / or the activation reaction time is 20-30 min.

5. The coupling method according to claim 1, wherein In step (1), the activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

6. The coupling method according to claim 1, wherein In step (2), the antibody is a monoclonal antibody, and / or the mass concentration of the antibody in the carboxyl microsphere-antibody mixture is 100-300 μg / mL.

7. The coupling method according to claim 1, wherein In step (2), the antibody and the antibody buffer are first mixed, and then mixed with the activated carboxyl microsphere suspension, and / or the antibody buffer is a borate buffer with a solute molar concentration of 90-110 mmol / L.

8. The coupling method according to claim 1, wherein In step (2), the coupling reaction temperature is 20-37° C., and / or the coupling reaction time is 30-40 min.

9. The coupling method according to claim 1, wherein In step (3), the blocking agent is casein sodium salt and / or bovine serum albumin, and / or the mass concentration of the blocking agent in the blocking agent-containing mixture is 1-2%, and / or the temperature of the blocking reaction is 20-37° C., and / or the blocking reaction time is 60-90 min.

10. Use of the method for coupling carboxyl microspheres with antibodies according to any one of claims 1 to 9 in immunochromatographic analysis.

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